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Interphase solubility and chromatographic retention

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Interphase solubility and chromatographic retention
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Sentell, Karen Belinda, 1957-
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Density ( jstor )
Dill ( jstor )
Liquid chromatography ( jstor )
Molecules ( jstor )
Silanes ( jstor )
Solutes ( jstor )
Solvents ( jstor )
Steepest descent method ( jstor )
Thermodynamics ( jstor )
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Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Glass -- Solubility ( lcsh )
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Silica -- Solubility ( lcsh )
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Thesis (Ph. D.)--University of Florida, 1987.
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Includes bibliographical references (leaves 164-173).
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Typescript.
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Vita.
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by Karen Belinda Sentell.

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INTERPHASE SOLUBILITY AND CHROMATOGRAPHIC RETENTION




By


KAREN BELINDA SENTELL













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1987




























This dissertation is dedicated to my fiance, Daniel Coffman.
I could never have done
this without you.














ACKNOWLEDGEMENTS



There are many people whom I wish to acknowledge for

their assistance with this work. Thanks are extended to Mel

Courtney for performing the numerous elemental analyses of my packings and to the technical staff in the departmental machine shop and glassblowing shop for their courteous assistance and advice. I am grateful to Dr. John Gerdes for suggesting the use of 4-dimethylaminopyridine (4-DMAP) in the bonded phase syntheses, to Nepera, Inc. for providing

the 4-DMAP to Dr. John Novak of the Aluminum Corporation of America for providing scanning electron micrographs of the silica and controlled pore glass supports and to Dr. Lane Sander of the National Bureau of Standards (NBS) for providing the NBS column evaluation test mixture.

I would like to express my gratitude to the Society for Analytical Chemists of Pittsburgh for funding my summer American Chemical Society (ACS) Analytical Division Graduate Fellowship and to Procter and Gamble for funding my fullyear ACS Analytical Division Graduate Fellowship.

Thanks are also due to my fellow Dorsey group members (both present and former) for their friendship, advice and support. The camaraderie within our group will be one of my warmest memories of graduate school. I look forward to i ii








continued association with them during iny postdoctoral tenure.

The love and moral support from my parents, Bobby and Ruth Sentell, have helped to sustain me throughout my education. They are responsible for instilling in me a love of reading, a respect for education and an unquenchable thirst for knowledge. I am grateful to them and to my sister, Michelle, for their encouragement during the

toughest times.

My deepest gratitude is extended to my graduate

research advisor, Dr. John G. Dorsey, for his advice and guidance. He is the epitome of what a research advisor should aspire to be and has served as an inspiration to me both as a research scientist and as a teacher. I have greatly enjoyed our conversations and I look forward to our continued professional interaction over the next year of my postdoctoral appointment. I also thank him for encouraging my oenophilic tendencies; after all, everyone needs to develop a new vice now and then.

Lastly, I want to thank my fiance, Daniel Coffman. His love, patience and support have sustained me even when I was discouraged and disheartened; without his help I could never have completed this work. In addition to his moral support, I would also like to thank him for his expert drafting and technical assistance as well as for accompanying me on my numerous midnight sorties to check on my reactions. He deserves my heartfelt gratitude now and forever for always being there when I need him.

iv













TABLE OF CONTENTS


P ag e

A CKNOWLED)GEMENTS...........................................1iii

ABSTRACT ................. . . ...................................... vii

CHAPTERS

I INTRODUCTION.. . . ..................................................... 1

Models of Reversed Phase Liquid Chromnatographic
Retention.. ............................................ ............1
Theories of Retention in RPLC ... .. .. .. .. .. .. .. .. .. ...17

II SYNTHESES OF SILICA-BASED RP STATIONARY PHASES ...... 29

Experimental Considerations in the Synthesis
of RP Stationary Phases...................................... 29
Experimental Procedure............................... 34
Syntheses Utilizing Ultrasonic Waves...............41
Effect of Subambient Temperature on the
Ultrasound Reaction ..............................44
The Use of 4-Dimethylaminopyridine *t~;* as. the'
Acid-Acceptor Catalyst.. ...................................... 45

III SYNTHESES OF CONTROLLED PORE GLASS-BASED RP STATIONARY PHASES.............................................. 52

Comparison of Controlled Pore Glass and Silica
as Supports for RP Stationary Phases ...............52
Experimental Procedure ............................56
Comparison of Silica and CPG'BondingDensities
via Reflux and Ultrasonic Syntheses .. .. .. .. .. .. .. ..60

IV CORRELATIONS BETWEEN CHROMATOGRAPHIC RETENTION
AND OCTADECYL BONDINGUDENSITY...................... 65

Chromatographic Determination of Thermodynamic
Partition Coefficients .............................65
Experimental Procedure ...............................76
Results............................................... 86




v








V CORRELATIONS BETWEEN CHROMATOGRAPHIC SELECTIVITY
AND OCTADECYL BONDING DENSITY .................... 105

Introduction ....................................... 105
Experimental Procedure ............................. 110
Results and Conclusions ............................ 113

VI CONCLUSIONS ........................................ 135

Syntheses of RP Stationary Phases .................. 135
Validity of Chromatographic Partition
Coefficient Measurements ......................... 138
The Effect of Octadecyl Bonding Density on the
the Chromatographic Partition Coefficient ........ 149
Suggestions for Future Work ........................ 154

REFERENCES ............................................... 164

BIOGRAPHICAL SKETCH ...................................... 174


































v i













Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy


INTERPHASE SOLUBILITY AND CHROMATOGRAPHIC RETENTION

By

KAREN BELINDA SENTELL

December, 1987


Chai rm an: John G. Dorsey
Major Department: Chemistry

The retention and selectivity behavior of small solutes on silica and controlled pore glass (CPG) reversed phase liquid chromatographic (RPLC) stationary phases was studied as a function of stationary phase alkyl bonding density. These monomeric octadecyl phases were synthesized by both reflux and ultrasound methods; high alkyl bonding densities (3.60 ipmol/m2) were obtained via low temperature ultrasound reactions using 4-dimethylaminopyridine as the acid-acceptor catalyst. Using an improved method for the calculation of the stationary phase volume, the chromatographic capacity factors for the solutes were divided by the volume phase ratio (stationary/mobile) to obtain the thermodynamic partition coefficients; their behavior as a function of stationary phase octaciecyl bonding density was examined in vi i







55/45 methanol/water and 85/15 acetonitrile/water mobile phase systems.

For the silica packings in both mobile phase systems, the partition coefficients linearly increased until a critical bonding density of about 3.1 imol/m2 was reached; after this point the partition coefficients began decreasing with increasing bonding density. This behavior supports a partitioning retention mechanism for RPLC. In this model, the driving force for retention is the creation of a solute sized cavity in the stationary phase interphase structure. Beyond the critical density, increased alkyl bonding density results in enhanced interphase chain packing constraints which increase the energy necessary for solute cavity formation, resulting in decreased chromatographic partition coefficients.

Methylene and phenyl selectivity were also examined as a function of octadecyl bonding density. Methylene selectivity was approximately constant, but phenyl selectivity increased linearly with bonding density. This further supports the partitioning theory; methylene selectivity is not expected to be affected by chain ordering but phenyl selectivity for the linear solutes used should increase as the interphase packing structure becomes more ordered. Identical selectivity and retention studies on the CPG bonded phases garnered inconclusive results, as no obvious trends were discernable in either study.

This work supports a partitioning mechanism for RPLC retention and as such gives insight into the retention v ii i







process on a molecular level. This theory is predictive without adjustable parameters and is relevant to partitioning behavior in organized assemblies, including micelles, membranes and vesicles.













































i x













CHAPTER I
I NTRODUCTION


Models of Reversed Phase Liquid Chromatographic Retention Retention Indices Based on Solute Descriptors

Reversed phase liquid chromatography (RPLC) is one of

the most popular and powerful analytical separation methods. In RPLC, the stationary phase support consists of silica particles which are typically 3 to 10 pm in diameter; alkyl chains, usually 8 or 18 carbons in length, are attached to oxygen atoms on the silica surface via covalent bonds, resulting in a nonpolar surface. The mobile phase consists of water and an organic modifier such as methanol, acetonitrile or tetrahydrofuran. Thus the mobile phase is much more polar than the stationary phase; mobile phase polarity is adjusted by varying the volume ratios of water and organic modifier. It has been estimated that 80-90% of the high performance liquid chromatography (HPLC) systems currently in use are reversed phase systems (Melander and Horvath, 1980). Yet many practitioners of RPLC view this technique as "black magic" because its retention mechanism is not well understood, especially at the molecular level. This makes the prediction of retention for new compounds of interest extremely difficult. A basic understanding of RPLC retention at the molecular level is a necessity for the

1







2

formulation of a predictive retention index system. Such a retention index system would allow accurate interlaboratory comparison of RPLC retention data.

Chromatographic retention is most often quantified by the capacity factor, k'. Thermodynamically, the capacity factor for a chromatographic solute is the ratio of the number of moles of solute in the stationary and mobile phases. The capacity factor is also a measure of solute retention which normalizes for the mobile phase flow rate and the physical dimensions of the chromatographic column, since k' = (Vr Vm)/Vm, where Vr is the solute retention volume and Vm is the retention volume of an unretained solute, often called the dead volume. Many different investigators have attempted to correlate RPLC retention data with topological, geometric and/or calculated physical property descriptors of chromatographic solutes in order to predict RP retention. Topological descriptors include molecular connectivity, molecular complexity and correlation factor; van der Waals volume, molecular surface area and length/breadth parameters are geometric decriptors. Physical property descriptors include hydrophobic substituent constants, UNIFAC models of activity coefficients, and octanol/water partition coefficients (D'Amboise and Bertrand, 1986; Funasaki et al., 1986; Jinno and Kawasaki, 1984a, 1984b and 1984c; Petrovic et al., 1985).







3
Topological descriptors such as molecular connectivity indices are used to correlate chromatographic retention with molecular structure. These indices are numerical values which quantitatively describe carbonaceous adjacency relationships in the molecular structure of a solute (Lehtonen, 1984). Molecular connectivity indices have been shown to be proportional to the cavity surface area of a molecule. When a nonpolar hydrocarbon solute is introduced into an aqueous or hydroorganic environment, a large negative entropy of solution results. It has been suggested

that this negative entropy is a result of structural ordering around the hydrocarbon molecule (Karger et al., 1976). This ordering comes about from the formation of a cavity of water molecules around the hydrocarbon molecule. To overcome the entropy loss, nonpolar molecule segments will try to remove themselves from the aqueous medium and/or they will group together. The term "hydrophobic effects" is used to decribe these two phenomena of cavity formation and nonpolar clustering. The calculated surface area of the water cavity is significant because it can be related to the solubility of hydrocarbons in water (Karger et al., 1976). Likewise molecular connectivity, since it is also proportional to the cavity surface area, has also been correlated to non-electrolyte water solubility; however in contrast to cavity surface area, simple first order molecular connectivity indices are quite easy to calculate. Since in some cases the logarithm of the aqueous solubility







4

of a solute is proportional to the logarithm of its capacity factor k', it was expected that molecular connectivity would also be proportional to k', allowing prediction of retention to be made from these molecular connectivity calculations (Karger et al., 1976).

In their comparisons of experimental capacity factors to those predicted via simple molecular connectivity calculations, Karger et al. (1976) found very good agreement for para-substituted phenols and primary alcohols. However, the predicted k' values were uniformly high for secondary alcohols. This is because the experimental log k' values for two of the primary alcohols were used to determine the slope and intercept in the presumed linear relationship between log k' and molecular connectivity index for both types of alcohols. The high predicted k' values for the secondary alcohols reflect that their steric environment is different from that of the primary alcohols, showing that simple molecular connectivity indices can only be used to predict relative retention for compounds with the same functional group as those used for standards (Karger et al., 1976) .

Lehtonen (1984) used molecular connectivity indices of different orders, which correct for complex branching as well as for the nature of atoms other than carbon which make up the solute framework, to predict retention behavior for 16 dansylamides. Predicted and experimental k' values could

be correlated very well by combinations of different order









connectivity indices. However, a computer program had to be used in order to find the best combination of indices to obtain good correlation and these combinations were often nonlinear, involving combinations of connectivity indices raised to powers ranging from -2 to +2. The choice of indices also varied according to which organic modifier was used in the mobile phase as well as its percent composition. Although Lehtonen obtained good correlations between predicted and experimental retention, his method requires extensive computer calculations as no general connectivity index combination was applicable even within the same class of solutes (dansylamides). These two types of examples point out the shortcomings in the use of molecular connectivity indices to predict retention: the capacity factors for at least two members of the same class of compounds must be determined in order to find the proportionality constant between capacity factor and molecular connectivity, these predictions are only valid for compounds of the same functional group as the standards, for complex molecules a computer program must be used in order to find the best combination of indices to predict retention and this combination for a particular type of molecule may change if the mobile phase composition is altered. This method is also unable to distinguish between geometric isomers (Funasaki et al., 1986).

Molecular complexity is a topological descriptor and

the general index of molecular complexity (GIMC) is an index







6
whose applicability is genera] and which does not require the use of experimental or empirical data (D'Amboise and Bertrand, 1986). The GIMC is derived from combinations of graph theory and statistical information theory. It is so named because it considers all of the features which make a molecule more or less complex such as size, symmetry, branching, ring structures, multiple bonds and atomic heterogeneity. Molecules are represented by their skeletal molecular graph whose complexity is determined from a statistical information theory derived formula. Since any observable behavior related to a molecule's complexity is a function of the GIMO, chromatographic retention should also correlate with the GIMC. O'Amboise and Bertrand (1986) point out that GIMG is able to make retention predictions for solutes such as alcohols or fatty acids, which are not well correlated with hyrophobicity. They poi nt out that GIMC is a structure sensitive parameter representing the various reactive attributes of a molecule; therefore it should be related to the interaction mechanism in retention. However, plots of log k' versus GIMC for alcohols show distinct curvature, especially for alcohols with five or less carbons. Other difficulties exist as well. The index does not seem to be well applicable to molecules with different heteroatoms that are similarly bonded or for molecules belonging to nonhomologous series. Correlation of data between different stationary phases has also proven to be a problem (D'Amboise and Bertrand, 1986).







7

Geometric descriptors are in general fairly easy to

calculate. Van der Waals volume and surface area are both calculated from the van der Waals radii of the atoms from which the molecule is composed (Jinno and Kawasaki, 1984a). Length to breadth ratio (L/B) is a shape parameter based on the rectangle with minimum area which could envelop a molecule (Wise et al., 1981). However, all of these descriptors considered alone or in combination were found to have poor direct correlation with RPLC retention for substituted benzene derivatives. dinno and Kawasaki (1984a) concluded that this indicates that molecular size and shape were not the dominant forces controlling retention for these molecules. However, they noted that size and shape are important contributors to retention for alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) (Jinno and Okamoto, 1984). Wise et al. (1981) have also found that L/B is useful for predicting PAH elution order; however this parameter is useless for establishing general PAH retention indices since these indices vary according to the type of octadecyl bonded phase column used, necessitating the determination of a retention index equation for each different octadecy] column.

Physical property descriptors have been the most successful for predicting RPLC solute retention. The physical properties on which these descriptors are based come about from the solute's solution behavior in the mobile and stationary phases (Karger et al., 1976). Since









retention is controlled by the thermodynamic equilibrium of the solute between the mobile phase and stationary phase, retention could theoretically be predicted from standard Gibbs free energies since AG0 = -RT ln K, where AGO is the standard Gibbs free energy, R is the gas constant, T the absolute temperature and K the equilibrium distribution constant for the solute between the stationary and mobile phases. Since k' =K(Vs/Vm) where Vs and Vm are the stationary and mobile phase volumes, the capacity factor for a solute (and thus its normalized retention) could easily be calculated were AG0, V5 and Vm known. Since experimental AG0 values for these systems are unavailable, they are often estimated using liquid mixture models with readily available physical parameters, such as Hildebrand solubility parameters and group contribution concepts. But Hildebrand solubility parameters are only useful for qualitative descriptions of chromatographic behavior; therefore the group contribution concept is often used, since it was developed to predict activity coefficients in nonelectrolyte liquid mixtures (Petrovic et al., 1985). This concept is the basis for the UNIFAC model for chromatographic retention, which combines a model based on extension of quasi-chemical theory of liquid mixtures (UNIQUAC) with the concept of functional group solubility. In this method solute activity coefficients in the mobile and stationary phases are calculated via structural and binary parameters characterizing the mutual interaction energy of the







9
functional groups in the system. The solute activity coefficient is the product of a combinatorial and residual contribution. The combinatorial contribution is dependent on the size and shape of the molecules in the system; the residual depends on the interaction energy of functional group pairs, as well as the fraction of the surfaces on these groups which are available for mutual interactions (Petrovic et al., 1985).

Assuming infinite dilution, the relationship between the capacity factor of a solute i and its activity coefficient (fi) in the mobile and stationary phases can be written as
ln ki' = In Ki + ln (VS/Vm)

therefore in ki' =In fim ln fijs + In (Vs/Vm). Petrovic et al. (1985) assume that fsand Vs/Vm are constants; therefore ln ki and in fim are linearly related with a slope of one. If the activity coefficients of the solute i in the chromatographic system and Vs/Vm are known, retention can then be predicted. Petrovic et al. (1985) calculated infinite dilution activity coefficients of solutes in the mobile phase from experimental gas-liquid chromatographic data and then correlated them with their RP retention values to see how well the UNIFAC method could predict chromatographic behavior. They assumed that Vs/Vm would be the same for any octadecyl RP column at any methanol/water mobile phase composition (an assumption that will be thoroughly disputed in Chapter IV of this tome) and that







10

in fis was zero (since stationary phase interactions were assumed to be very weak and nonselective). They found that the predicted values of k' were at best a rough estimate of actual chromatographic retention and concluded that therefore the solute interaction with the stationary phase could not be ignored in the prediction of RP retention. However they found the UNIFAC method to be useful for predicting changes in relative solute retention with varying mobile phase composition, since the solute activity coefficients in the mobile phase could be accurately calculated using UNIFAC parameters (Petrovic et al., 1985).

Hydrophobicity is the physical descriptor which has most accurately been used to predict RPLC retention. Hydrophobic effects between solutes and hydroorganic mobile phases were described earlier in this chapter. Solute hydrophobicity is usually described in terms of the pi scale developed by H-ansch and Leo (1979). By evaluation of solute partition coefficients between n-octanol and water (P) they were able to establish substituent hydrophobicity parameters. The logarithm of the partition coefficient is determined for both a compound containing the substituent group and the parent compound; the difference in these two log P values is pi, the hydrophobicity parameter for the substituent (Melander and Horvath, 1980). Jinno (1982) and dinno and Kawasaki (1984a and 1984c) use the descriptors pi, HA and HD, where HA is the number of electron acceptor groups and H-D is the number of electron donor groups, to









correlate with RPLC retention for substituted benzenes (excluding phenol). They found a very good correlation between ln k' and pi, and poor correlations between in k' and molecular connectivity, correlation factor (number of double bonds plus number of primary and secondary carbons minus 0.5 for nonaromatic rings) and van der Waals volume and surface area. They interpret this to mean that the size and shape of these molecules were not dominant for controlling their retention. However, when the size and shape of the solute molecules are a dominant retention force such as for PAHs and hydroaromatics, the correlation factor

(F) has been found to correlate quite well with log k' (Hurtubise et al., 1982). If a linear combination of HA, HD and pi descriptors was used, they found an even better correlation between predicted and experimental k' values. For phenol solutes, hydrophobicity alone was not an adequate retention descriptor so they added Hammett's acidity parameters (sigma) to account for the strong hydrogen bonding ability of the phenols (Jinno and Kawasaki, 1984a and 1984c). Linear combinations of pi and sigma for the phenol solutes resulted in excellent correlations with retention behavior. However, for both sets of solutes the parameters had to be multiplied by certain proportionality constants in order to correctly predict retention and these constants were different not only for different organic modifiers in the mobile phase but also for each volume composition. This requires that at every different mobile







12

phase composition a least squares fitting for a large data set must take place in order to determine these proportionality constants, requiring a tremendous amount of data both in terms of pi (and possibly sigma) values for each solute and in terms of retention values for these solutes at each mobile phase composition. Jinno and Kawasaki (1984a and 1984c) also have not shown that this model is applicable for larger more complex molecules.

Funasaki et al. (1986) examined retention behavior for alcohols and ethers with positional and geometric isomers and examined the correlations between the solute's log k' and molecular cavity surface area (S), the logarithm of the aqueous solubility (log Cw) and the logarithm of the octanol-water partition coefficient (log P). As previously mentioned in this chapter, it was found that S and k' were very well correlated, even in the case of conformational isomers. This is because S for a molecule in water is defined as the area of the surface traced out by the center of a water molecule rolling over the van der Waals surface of the solute molecule (Funasaki et al., 1986). However S is very difficult to accurately calculate, requiring the construction of solute molecular models for rigorous work, as well as detailed knowledge of the molecular conformation of the molecule of interest. If S is calculated from group surface areas, an easier but less rigorous approach, the molecular surface area is usually overestimated for very crowded molecules. Using S to predict retention does have







13

one very strong merit--since the slope of a log k' versus S plot is related to interfacial tension, the dependence of log k' on the organic modifier content of the mobile phase can be predicted (Funasaki et al., 1986).

Funasaki et al. (1986) found the correlation between log k' and the logarithm of solute aqueous solubility, Cw, to be rough at best. The main drawbacks to this method are that the extent of correlation will depend on whether Cw was measured for the compound in the gas, liquid or solid state, that some compounds are infinitely water soluble and that the correlation is particularly poor for branched solutes. In contrast, they found that the correlation between the logarithm of the solute octanol-water partition coefficient (log P) and log k' was particularly good for the solutes examined (alcohols and ethers) in methanol/water mobile phases. Braumann (1986) reports that other workers have found good correlations between log k' and log P for a variety of compounds including PAHs, alkylbenzenes, substituted benzenes, pesticides, phenols and barbiturates; again correlations were much better with methanolic mobile phases than with those containing acetonitrile, due to methanol's hydrogen bonding properties. One drawback to this method is that octanol-water partition coefficients are measured via shake-flask methods, which are very time consuming. However, P values are tabulated for many compounds and additivity rules using Hansch's pi parameters may be used to estimate P for other compounds although







14

estimated P values do not in general correlate as well with retention as measured ones (Braumann, 1986; Funasaki et a]., 1986). This approach is very much like that of Jinno and Kawasaki (1984a and 1984c) previously discussed except that Jinno and Kawasaki examined substituent group hydrophobicity (pi parameters) in relation to retention whereas Funasaki et al. (1986) examined its relation with overall molecular hydrophobicity (log P).

Funasaki et al. (1986) also examined the effect of

temperature and mobile phase composition on the degree of correlation between log k' and log P. Not surprisingly, they found that they were better correlated when both were measured at the same temperature than when they were measured at different temperatures. They also found that the log k' value estimated from extrapolation to zero percent methanol (i.e. totally aqueous) mobile phase gave a better correlation with log P than those obtained with hydroorganic mobile phases. As in all other cases previously discussed, the extent of correlation will be dependent upon the test solutes, the chromatographic column and the experimental conditions used (Funasaki et al.,

1986).

In summary, most of the retention indices based on

solute descriptors tend to accurately predict retention for certain small sets of similar compounds; they are by no means universal. Additionally, some of them require extensive calculations and/or experimental data in order to







15
predict retention. At present, none of these solute descriptor index systems is adequate for reliable prediction of RPLC retention.

Empirical Prediction of Retention

Jandera and coworkers (Colin et al., 1983a; Jandera, 1986; Jandera et al., 1982; Jandera and Spacek, 1986) have developed an empirical model to predict absolute or relative retention. They assumed that the stationary phase contribution to retention is very small compared to that of the mobile phase and that nonpolar interactions between the solute, stationary phase and mobile phase cancel each other. If this is true, the energy of transfer of the solute from the mobile to the stationary phase will depend on the interaction energy (energy of cohesion) between mobile phase molecules and the interaction energy between the mobile phase and solute. They defined an interaction index, determined from retention data in hydroorganic mobile phase systems, which describes polar interactions between solute molecules and the mobile phase components.

The interaction index for a solute (Ix) can be

determined if the volume of interaction (Vx) for the solute and the column phase ratio (Vs/Vm) are known, since in this model,

log (kx'/Vx) log ((Vs/VmT)/Vx) = A BIx (1)

where A and B are constants which depend on the stationary and mobile phases used. Jandera et al. (1982) plotted (log kx' log (Vs/Vm))/Vx versus solute polarity (based on







16

Snyder's polarity index) to determine the value of Ix for "standard" solutes with different functional groups, and then found the average value of these Ix's for a given solute in many different mobile and stationary phases. They then assumed that IX will be constant for compounds which undergo the same type of interactions with the mobile phase as the "standard" solute does. Therefore if VX for a similar compound is known, its retention can be predicted from equation 1, since A, B and Ix are already known from the standard data Wandera et al., 1982).

Although this model is useful in a practical sense

since it is based on empirical considerations, it cannot be completely justified in a theoretical sense. Although the solute experiences stronger interactions with the mobile phase than with the stationary phase, Jandera et al. (1982) do not prove that stationary phase interactions can be completely ignored. They also state that the standard compounds must be chosen "correctly" or else the predictive power of the model fails; "correct" solutes will be those with little or no specific interactions (such as hydrogen bonding) between the solutes and mobile phase components. This severely limits the types of compounds whose retention can be predicted from this model. Finally, at present the model cannot give very accurate (about 5 to 20% accuracy) retention predictions (Jandera et al., 1982).






17

Theories of Retention in RPLC

Sol vophobi c Theory

In order to truly understand the retention process in RPLC and thereby be able to predict solute retention, the retention process must be examined at the molecular level. At present there are two main schools of thought on the retention mechanism of RPLC at the molecular level. The solvophobic theory espoused by Melander and Horvath (1980) states that RP retention comes about from solute binding onto the stationary phase fromn the mobile phase and is mainly due to hydrophobic interactions between the solute and the mobile phase. Other workers have utilized statistical mechanical analysis based on mean field lattice theory to show that RPLC solute retention is due to solute partitioning from the mobile phase into the bonded stati onary phase chains (Dill, 1987a and 1987b; Marqusee and Dill, 1986; Martire and Boehm, 1983). The main tenets of both of these proposed theories will be outlined below.

Melander and Horvath's (1980) solvophobic theory

assumes that the mobile phase plays the dominant role in the RPLC retention process. This is because the stationary phase is nonpolar; therefore the only attractive forces occurring between the stationary phase and a nonpolar solute will be van der Waals forces, which are weak and nonspecific. They attribute the interactions between the solute and the mobile phase to a type of hydrophobic effect, which was discussed earlier in this chapter. In the








specialized RPLC environment, they have adopted a variation of this effect, termed the "solvophobic" theory, since the hydrophobic theory assumes a totally aqueous environment and RPLC mobile phases are generally a mixture of aqueous and organic components. Solvophobic theory is based on a theory of solvent effects on chemical equilibria developed by Sinanoglu (1968). The theory states that chromatographic retention is based on the free energy change as the solute is transferred from a hypothetical gas phase at atmospheric pressure to the mobile phase. The energy involved in this process is calculated in two steps. In the first, a cavity of the proper shape and size for the solute molecule is formed in the solvent. In the second, the solute enters the cavity and interacts with the surrounding solvent molecules via van der Waals and electrostatic interactions (Melander and Horvath, 1980).

The free energy change accompanying the mobile phase

cavity formation comes about from the fact that the solvent surface area will increase by the molecular surface area of the solute (Melander and Horvath, 1980). Therefore the mobile phase free energy will increase by an amount proportional to the solvent surface tension and the increase in area. The change in free energy due to the interaction of the solute with the surrounding solvent molecules will be due to chemical and entropic effects. The chemical effects are van der Waals interactions and electrostatic effects. The van der Waals interaction energies are a function of the







19

polarizability and ionization potentials of the solute and solvent species. Electrostatic interactions consider both dipole and ionic effects; dipole effects are calculated from the solute dipole moment, polarizability and molecular radius as well as from the dielectric constant of the solvent. Ionic effects are calculated from conventional electrostatic theories such as Debye-Huckel treatments. The entropic term is a measure of solute "free volume," which is the volume that the molecule encounters before colliding with another molecule. The "free volume" is assumed to be proportional to the solute molar volume (Melander and Horvath, 1980).

Although the solvophobic theory outlined above is

pertinent to a liquid-liquid system, the bonded stationary phase has not been considered in this process. Melander and Horvath (1980) regard the change in free energy for retention to be a combination of the mobile phase effects just described and a small contribution from the adsorption of the solute onto the stationary phase surface. This

adsorption is viewed as a reversible reaction between the solute and stationary phase to form an associated complex. The free energy of adsorption is quantified as the van der Waals interaction energy between the solute and stationary phase in the absence of solvent molecules. Althouyh Melander and Horvath (1980) mention that an entropic term should be introduced to account for the restricted translational freedom of the bonded chains at the silica






20

surface, they ignore this effect because they feel its contribution is negligible. In summary, in the so] vophobic theory of RPLC retention, retention is mainly dependent upon the free energy of creation of a solute sized cavity in the mobile phase; stationary phase effects are considered to be weak and therefore rather negligible. Partitioning Theory

One of the most severe drawbacks to Melander and

Horvath's (1980) solvophobic theory is that it is based on a one phase model--that of the mobile phase. But RPLG involves two phases, the stationary and mobile phases; therefore a one phase model is not completely applicable for such a system. Melander and Horvath (1980) view the retention process as if there is no true transfer of the solute from the mobile phase to the stationary phase; the solute is merely associated with the stationary phase through weak adsorptive effects. Melander and Horvath (1980) also account for the stationary and mobile phase

interactions by viewing them as bulk phases with homogeneous properties throughout. In reality, the stationary phase/mobile phase boundary is a highly heterogeneous area consisting of the core silica particles, the alkyl chains bonded to the silica surface, residual silanol groups remaining on the silica surface and the mobile phase solvating these silanols and the bonded chains. In such a heterogeneous system, it is highly unlikely that bulk phase thermodynamic considerations based on ideal solution behavior are applicable (Marqusee and Dill, 1986).







21
In their adsorption model, Melander and Horvath (1980) use very simple descriptions of the stationary phase surface which contain two gross oversimplifications. They imply first of all that the bonded chains are rigid rods containing no internal degrees of freedom. But at the temperatures commonly used in RPLC, the bonded alkyl chains are quite disordered (Dill, 1987a). Melander and Horvath (1980) also use stationary phase models wherein the bonded alkyl chains are fully exposed to the mobile phase. However, in a hydroorganic mobile phase system the chains cannot be fully exposed to such a highly aqueous environment; such a configuration would be prohibitively expensive in free energy terms (Dill, 1987a).

Dill (1987a) has proposed an alternative model of the RPLC stationary phase surface which regards the grafted phase as an organized "interphase" similar to those found in surfactant aggregates such as monolayers, bilayers, micelles and microemulsions (Marqusee and Dill, 1986). Interphases are composed of alkyl chains that have one end anchored at an interface; their thickness is on the order of a few molecular dimensions. The anchored chain density (i.e. the number of chains anchored per unit silica surface area) is sufficiently high as to cause severe configurational constraints. Two important properties distinguish this system as an interphase: its surface area/volume ratio is high and its properties vary with the distance from the anchored end. The relationship between orientational order







22
and distance from the silica surface is termed a "disorder gradient"; the bonded chains have much greater orientational order at the anchored ends and this order decreases with the distance from the attached end. This is in contrast to bulk matter phases in which by definition properties are invariant with spatial position (Dill, 1987a).

In Dill's (1987a) retention theory, the nature of the retention process is dependent upon the nature of the molecular organization within the interphase. There are three factors which determine this organization: the first is those constraints imposed by the surface density and chain lengths of the alkyl groups bonded to the surface and by the surface's geometry; the second requirement is that in highly aqueous mobile phases the interphase region must largely exclude the solvent due to hydrophobic effects; the interphase volume will be filled by chain segments and solute molecules. The final requirement is that the chains adopt as much disorder as is consistent with the other two constraints in order to conform to the second law of thermodynamics. This approach allows the consideration of any possible geometry of the silica surface onto which the chains are bonded. Dill (1987a) assumes that the silica surface is approximately planar; in terms of molecular dimensions this should be a good approximation for chromatographic silicas, which commonly have pore diameters of 60 to 100 Angstroms or more; the effects of curvature will be small unless the radii of curvature are a few molecular chain lengths or less (Marqusee and Dill, 1986).






23

Dill (1987a) considers partition and adsorption

separately as alternative RPLC retention mechanisms. In both cases a lattice interphase mode] is used for the bonded phase surface and statistical thermodynamic calculations are used to predict solute retention in the system (Dill, 1987a; Marqusee and Dill, 1986; Martire and Boehm, 1983). Dill (1987a) predicts the equilibrium partition coefficient for a solute from the mobile phase to the stationary phase from the chemical potentials of the solute in the mobile phase system and in the bonded chain interphase. These calculations include the entropy of mixing of the solute and solvent in the mobile phase, the decrease in configurational entropy of the bonded chains when the solute is inserted within the interphase and the total contact free energy of the system, which will be due to intermolecular interactions of the molecules with their neighbors. After careful consideration of both the partitioning and adsorption retention mechanisms in conjunction with his interphase model and available experimental evidence, Dill (1987a) concludes that the principal retention mechanism in RPLC is partitioning due to two lines of evidence. Partitioning will be affected by the surface density of the bonded alkyl chains; adsorption will not. Therefore if partitioning is dominant, after a certain critical bonding density, solute retention should decrease with increasing alkyl chain surface density. It has been observed that there is less solute retention in bonded alkyl phase stationary phase







24

systems than in the corresponding bulk alkane systems (Colin et al., 1983b). Although Melander and Horvath (1980) have interpreted this as favoring an adsorption mechanism, Dill (1987a) interprets this in terms of partial chain ordering in the stationary phase, leading to less retention than in the completely disordered bulk alkane. This is supported by the work of Lochmuller and Wilder (1979) since solute methylene selectivities should be unaffected by the molecular organization of the interphase (Dill, 1987a).

The other line of evidence is that ln k' for congeneric sets of molecules can be linearly correlated with ln P (octanol-water partition coefficient) with a slope of one. A slope of one is expected for the partitioning mechanism, since all of the solute surface area would be available for partitioning within the interphase. The slope for the adsorption mechanism is expected to be considerably less (about 1/6) because only a small fraction of the solute surface area would contact the hydrocarbon chains, giving a smaller driving force for retention (Dill, 1987a). Experimental evidence has resulted in linear plots of ln k' versus ln P with a slope of one (Melander and Horvath, 1980) .

Based on the partitioning mechanism of retention, Dill (1987b) predicts that at low surface densities (less than 2.1 micromoles of bonded alkyl chains per square meter of silica surface) nonpolar solute retention will increase linearly with increasing surface density. At these low







25

densities, chain configurational constraints are very small and interphase chain packing will have no effect on solute retention; solute retention will increase as the volume of chains increases since there will be more alkyl phase for the solutes to partition into. At a chain density of zero (bare silica) the nonpolar solute will be unretained (Dill, 19 8 7b) .

Once a critical bonding density (predicted to be about 2.7 wPmol/m2) is reached, the bonding density is high enough for severe configurational constraints to result. In the Dill (1987a) retention model, the free energy involved in retention is determined by the differences in the free energy between the creation of a solute sized cavity in the interphase region and the destruction of a solute sized cavity in the mobile phase. As alkyl bonding density is increased past the critical bonding density, the chain packing constraints become more and more severe, requiring larger amounts of energy to create a solute cavity in the interphase structure. Thus in the high density region solute partitioning is predicted to decrease with increasing alkyl bonding density due to the increasingly prohibitive expenditure necessary for interphase cavity creation to accomodate the solute. Dill (1987a) predicts that at 8.1 Pmol/m2, the maximum achievable bonding density if every surface hydroxyl group on the silica is derivatized with an alkyl ligand, solute retention would be zero and the solute would be completely excluded from the interphase chain packing structure.






26

Some researchers have examined solute retention as a function of increasing alkyl chain length of the bonded ligands. Colin et al. (1983b) and Jinno and Kawasaki (1984c) found that log k' increased with increasing bonded chain length, while Melander and Horvath (1980) have stated that more or less contradictory results have appeared in the literature on the influence of chain length. Spacek et al. (1980) and Berendsen and de Galan (1978b) have also noted a general trend of increased retention with increasing chain length of the bonded ligand, but their results were less conclusive than those of Jinno and Kawasaki (1984c). It is not clear from any of these studies whether this trend is due to the actual increased partitioning of the solute into the longer alkyl chains or whether this trend is an artifact of the retention parameter measured. This is because in all of these studies, it is the capacity factor, k', that is used to quantitate retention. But k' = K(Vs/Vm) where K is the chromatographic partition coefficient and Vs/Vm is the volume phase ratio of the stationary and mobile phases. It is obvious that as the chain length of the bonded alkyl ligand is increased, a corresponding increase in V5 will occur, as pointed out by Colin et al. (1983b). Therefore it is unclear as to whether solute retention increased because of increasing partition coefficient or merely because of the phase ratio increase. In order to reliably determine the cause of changes in actual solute retention as stationary phase parameters are changed, the changes in the solute partition coefficient must be examined.







27

In this study, wie examined the effects of octadecyl

alkyl chain bonding density on both retention and selectvity of small nonpolar salutes. We were particularly interested in experimental verification of the molecular mechanisms of RPLC retention proposed by Dill (1987a and 1987b). Although Sander and Wise (1984a and 1984b; Wise and Sander, 1985) and Wise and May (1983) have extensively examined the effect of alkyl bonding density on retention and selectivity for PAHs, they have mainly examined polymeric stationary phases, which are not as well structurally characterized as the monomeric stationary phases used in our study. Another problem with their work is that they examined capacity factor (k') behavior in their retention studies, which fails to account for phase ratio changes. Additionally, PA~s are not ideal salutes for such a study, since their large sizes and unusual shapes are not typical of most chromatographic s o1 ut es .

In order to determine the effect of interphase chain packing on solute partitioning, the behavior of the chromatographic partition coefficient was examined as a function of octadecyl bonding density. Chromatographic selectivity was also studied as a function of bonding density for salutes of different sizes and shapes. Novel synthetic methods utilizing ultrasound as a reaction driving force were devised to obtain stationary phases with high bonding densities. In this manner, we were able to see if Dill 's (1987a and 1987b) proposed RPLC retention mechanism







28

was verified by actual chromatographic behavior. These experiments were carried out using both silica and controlled pore glass substrates in order to compare the two materials as stationary phase supports.













CHAPTER II
SYNTHESES OF SILICA-BASED RP STATIONARY PHASES


Experimental Considerations in the Synthesis of RP
Stati onary Phases

Reversed phase bonded silicas are the most popular packings used in high performance liquid chromatography (HPLC). Although the role of the mobile phase in chromatographic retention and selectivity has been extensively studied, that of the stationary phase has only come under intense scrutiny recently and as a result the effects of the stationary phase on these chromatographic properties is not yet fully understood. One reason for this dearth of knowledge is the lack of precise and reliable methods for determining bonded phase characteristics such as the density, homogeneity and topographical distribution of the bonded alkyl ligands and the residual hydroxyl groups on the support surface. These properties are a direct consequence of the bulk silica medium and the reagent and reaction conditions for the silanization process (Kinkel and Unger, 1984). In order to obtain reversed phase packings with reproducible surface characteristics, the silanization reaction conditions must be painstakingly controlled.

In the preparation of reversed phase packings, one

objective is the modification of as many surface hydroxyl


29






30

groups on the silica as possible, especially the highly acidic isolated silanols. These residual isolated silanol groups have been shown to be the main cause of tailing of chromatographic peaks for basic compounds, of mechanical instability of the packing, and of low sample capacity of the column (Kohler et al., 1986; Kohler and Kirkland, 1987). Di- or trireactive alkylsilanes had previously found favor over monoreactive silanes because of their greater reactivity and the possibility of reacting simultaneously with two or three hydroxyl groups. However, any unreacted sites on the bonded functional groups will be hydrolyzed upon contact with water (i.e. from the mobile phase), forming additional undesirable silanol groups (Berendsen and de Galan, 1978b; Snyder and Kirkland, 1979). Di- and trireactive silane reagents also often result in nonreproducible stationary phases since the degree of polymerization is highly dependent on the residual water content of the silica and the reagents used in the bonding reaction (Snyder and Kirkland, 1979). Another drawback of polymeric stationary phases is their lower chromatographic efficiency, which results from poor solute mass transfer in these relatively thick stationary phases. Therefore many investigators now advocate the use of monofunctional silanes for the silica derivatization reaction, since this results in a reproducible and well defined chemically bonded phase. Additionally, monomeric stationary phases generally exhibit superior column performance to polymeric phases due to their







31

faster solute mass transfer kinetics (Cooke and Olsen, 1980). For octadecyldimethylchlorosilane, the most commonly used monoreactive silane, the resulting bonding reaction is depicted in Figure 2-1.

Kinkel and Unger (1984) have studied the roles of the solvent and the base in these monofunctional bonding reactions and have found their choice to be crucial. When alkylhalosilanes are reacted with silica, a base is added to serve as the acid-acceptor catalyst, binding the haloacid formed during the reaction and driving the equilibrium to the product side. In addition, the base favorably affects the kinetics of the silanization reaction. Mechanistic studies of these types of reactions (Corriu and Guerin, 1980) have shown that two molecules of base attack one molecule of silane, activating the Si-X bond such that a reactive intermediate and a hydrohalide are formed. Formation of this reactive intermediate greatly increases the kinetics of the bonding reaction; indeed, the addition of the acid-acceptor catalyst results in approximately 90% of the total conversion taking place within the first hour of the reaction. In their study, Kinkel and Unger (1984) found that the two most effective acid-acceptor catalysts for organohalosilanes were imidazole and 2,6-lutidine.

The reaction solvent must also be carefully chosen.

The solvent can interact specifically with the silane, the base and the surface silanol groups on the silica. When the solvent interacts with a silanol group, there is a












CH3 CH3
Si -0-H + CI-SH-CH)FCH3 --o Si-0-SCH)rTCH3 + HCI

CH3 CH3


Figure 2-1. Bonding reaction for monomeric octadecyl
reversed phase packings.






33
considerable effect on the strength of the bond between the silicon and oxygen atoms. Solvents which have both a pronounced Lewis acid and Lewis base character cause the Si-O bond strength to be weakened and facilitate the bonding reaction. The solvent can also activate the silicon atom of the organohalosilane by forming a pentacoordinated intermediate through nucleophilic attack. The resultant bond lengthening causes nucleophilic activation to occur, favoring attack by a second nucleophile (such as the base). The solvent may influence the base as well, as it is known that in aprotic polar solvents the nucleophilic character of reactants is more pronounced. All of these considerations may have a synergistic relationship as well. Based on their experimental work with organohalosilanes, Kinkel and Unger (1984) found that methylene chloride and N,N-dimethylformamide were the most effective solvents for the bonding reaction.

Many organic reactions have been shown to be enhanced

by ultrasound (Boudjouk, 1986; Bremner, 1986; Clough et al., 1986; Han and Boudjouk, 1982 and 1983; Suslick, 1986). Boudjouk and Han (1981) have shown that in the presence of ultrasonic waves both alkyl and aryl chlorosilanes could be coupled over lithium wire; without ultrasonification, this reaction occurred to no appreciable extent. Reactions at solid-liquid interfaces are also particularly enhanced by ultrasound (Bremner, 1986; Suslick, 1986). It is then reasonable to assume that reversed phase bonding reactions






34
might be facilitated under ultrasonification. The use of ultrasound has two distinct advantages over traditional reflux methods. The ultrasonic waves serve as a driving force which is controlled independently of temperature, allowing reaction temperatures to be varied over any desired range. Secondly, the power of the ultrasonic driving force can be varied by using a variable power ultrasonic probe.

We have investigated the effect of ultrasound on the

silane bonding reaction, including the effects of subambient and superambient temperatures on the ultrasonic reaction. In addition to these investigations, a novel base, 4-dimethylaminopyridine, was utilized as the acid-acceptor catalyst, in hopes that it might prove superior to 2,6-1utidine.

Experimental Procedure

Reagents

All of the organic solvents used were supplied by Fisher Scientific (Fairlawn, NJ). Water was deionized, passed through a Barnstead Nanopure (Boston, MA) purification system, irradiated in a Photronix Model 816 HPLC reservoir with a UV source (Photronix Corp., Medway, MA) for at least 48 hours, and filtered through a 0.45 im Nylon 66 membrane (Rainin, Woburn, MA). The methanol used was HPLC grade; the chloroform, methylene chloride, and diethyl ether were reagent grade. Methylene chloride was dried by stirring over phosphorus pentoxide (Fisher Scientific) for 24 hours, followed by distillation under a dry nitrogen atmosphere.






35

Dimethyloctadecylchlorosi lane, n-octyldimethylchlorosilane and trimethylchlorosilane (99.9%) were used as received from Petrarch Systems (Bristol, PA). The 2,6-lutidine (Sigma Chemical Co., St. Louis, MO) was stirred for 24 hours over barium oxide (Fisher Scientific) prior to distillation under dry nitrogen atmosphere; 4-dimethylaminopyridine (4-DMAP; Nepera Inc., Harriman, NY) was oven dried at 80 C for 24 hours before use.

The chromatographic silica was from a single lot of Davisil (W. R. Grace, Baltimore, MD) synthetic amorphous silica, grade 641LCOX1823. The silica had an average pore diameter of 147 Angstroms, an absolute surface area (SBET, as measured by BET analysis) of 300 m2/g, a particle size range of 20-30 pm with an 80% distribution of 23 + 10 pm and a nitrogen pore volume of 1.10 cm3/g (Grace, 1984). As recommended by Snyder and Kirkland (1979) the silica was acid leached in 0.1 M nitric acid at 90 C for 24 hours in order to fully hydroxylate the surface and to remove any metal contaminants remaining from the manufacturing process. The silica was then washed thoroughly with water until all traces of the nitric acid had been removed and dried under vacuum at 240 'C for 24 hours prior to use in order to remove physically adsorbed water from the surface (Unger, 1979). Scanning electron micrographs of the acid-leached silica (Figures 2-2 and 2-3) show that this type of chromatographic silica exhibits an irregular shape as well as an irregular surface. This surface irregularity is reflected in the silica's high surface area.





































Figure 2-2. Scanning electron micrograph of acid-leached Davisil
silica; 500X magnification.

























ip










Figure 2-3. Scanning electron micrograph of acid-leached Davisil
silica; 3000X magnification.






38

Silane Bonding Reaction

It is essential that the silane bonding reaction be carried out under scrupulously dry conditions in order to prevent the water-initiated dimerization of the silane reagent. Glassware used in the derivatization reaction was presilanized by etching the surface with a 10% (v/v) hydrofluoric acid (Fisher Scientific) solution, drying, and then soaking the glassware for an hour in a 5% (v/v) trimethylchlorosilane in chloroform solution. Immediately prior to use, the glassware was oven dried at 125 'C for at least 4 hours in order to remove trace moisture and allowed to cool in a dry box under nitrogen atmosphere. The reagents were mixed together in the dry box and the reaction flasks kept under dry nitrogen atmosphere at all times. Based on Kinkel and Unger's (1984) estimation of a maximum of five micromoles of reactive hydroxyl sites per square meter of silica surface, a twofold excess of the silane reagent was added to achieve exhaustive derivatization of the silica surface. A fourfold excess of the base (2,6-lutidine or 4-DMAP) was added both to serve as an acid-acceptor catalyst for the HCI produced in the reaction and to act as a reactive intermediate at the silica-solution interface. Dry methylene chloride was used as the reaction solvent, using a ratio of 10 ml of inethylene chloride per gram of base

silica.

The reaction flasks were sonicated by immersion to the flask neck in an ultrasonic cleaning bath (Bransonic model







39
B-2200R-1, Branson Cleaning Equipment Co., Shelton, CT) with a power rating of 100 W and a frequency of 55 kHz. Stirring of the reagents within the flasks was accomplished by rotating a magnetic bar submerged in the bath adjacent to the reaction flask, resulting in the corresponding rotation of a magnetic stirring bar within the flask. Temperature control of the ultrasonic bath was accomplished by passing a thermostatted solution of ethylene glycol and water through coiled copper tubing lining the inner perimeter of the bath. The solution was thermostatted by an Endocal or Exacal water bath (Neslab Instruments, Portsmouth, NH). Refluxed reactions were carried out at 50 'C using an oil bath and magnetic stirrer. Control reactions were carried out by stirring the reaction mixture at room temperature.

Refluxed reactions utilizing n-octyldimethylchlorosilane as the reactive silane, 2,6-lutidine as the acidacceptor catalyst and methylene chloride as the reaction solvent were carried out for reaction times of 24, 36 and 48 hours in order to ascertain whether the differences in reaction time would make a statistically significant difference in the reaction yield. Nine replicate reactions were performed for each reaction time. Student's t (tcalc) was calculated from the pooled standard deviation of percent carbon for all 27 reaction yields, the differences in the mean percent carbon at each reaction time, and the number of replicates at each time in order to determine if the mean yield for each reaction time was statistically different






40

from those at the other reaction times. In the comparison of the 24 hour reactions to the 36 hour reactions, tcalc was

0.984; for the 24 hour reaction time versus the 48 hour reaction time, tcalc was 0.148 and for the 36 hour reaction time versus the 48 hour reaction time, tcalc was 0.835. Since all of the calculated t values are less than the critical t value (tcrit) at the 80% confidence level (tcrit,80%= 1.34 for 16 degrees of freedom), there is no significant statistical difference in the reaction yields for reaction times of 24, 36 and 48 hours at the 80 confidence level (Peters et a]., 1974). Therefore, reactions were carried out for 24 hours (as also recommended by Kinkel and Unger (1984)) unless otherwise noted.

Once the reaction time was complete, the product was washed in order to remove excess reagents. Each bonded phase product was washed three times with each solvent using the rinse sequence methylene chloride, methanol, 50/50 (v/v) methanol/water, methanol and diethyl ether. After the ether was allowed to evaporate from the product, the derivatized silica was dried in a vacuum oven at 125 'C for 16-24 hours. (Caution: It is imperative that the ether be completely evaporated from the product prior to drying the product in the vacuum oven in order to avoid a possible explosion.) Products were analyzed by in-house elemental analysis performed at least in duplicate for each sample. Reliability of the elemental analysis was confirmed by repeated submission of a standard packing material over a







41

two year period; for 66 measurements the resultant standard deviation was + 0.20% carbon.

Syntheses Utilizing Ultrasonic Waves Use of Ultrasound as a Reaction Catalyst

The region of frequencies above 16 kHz is beyond the sensitivity of the human ear; it is therefore termed the ultrasound region. The first reported use of ultrasound in organic chemistry was in 1938, but it was not until the late 1970s that ultrasound was used to speed reactions in nonaqueous media; indeed the use of ultrasound as a reaction catalyst is still in its infancy (Boudjouk, 1986; Bremner, 1986). Ultrasonic radiation can be introduced to the reaction medium either by immersion of the reaction vessel into the liquid of a common laboratory ultrasonic cleaning bath or by introduction of an ultrasonic generating probe directly into the reaction medium.

Ultrasonic frequencies span the range of 20 kHz to 10 MHz, with associated acoustic wavelengths of 7.6 to 0.015 cm. Therefore sonochemistry cannot be accounted for in terms of direct coupling of the acoustic field with chemical species on a molecular level (Suslick, 1986). However, the effects of ultrasound can be attributed to three different phenomena. The variation of sonic pressure causes the rapid movement (oscillation) of fluids, subjecting them to compression and rarefaction. Negative pressure in the rarefaction region gives rise to cavitation, the formation and collapse of microbubbles. The violent implosion of






42
these microbubbles generates powerful shock waves with a considerable energy output (Boudjouk, 1986; Bremnner, 1986; Suslick, 1986). Pressures in the kilobar range and temperatures of 2000-3000 'C have been estimated in the region of the collapsing bubble for time periods in the nanosecond range (Sehgal et al., 1980). The third contributing phenomenon is microstreaming, where a large amount of vibrational energy is put into small volumes with little heating (Bremner, 1986). The extremes of temperature and pressure generated by ultrasonic waves cause the generation of free radicals and ions, the dispersion of chemical layers and the promotion of intimate contact between reactants. Emulsification of immiscible liquids and enhanced mass transfer at solid-liquid interfaces are secondary effects of ultrasonification. All of these effects can contribute to the promotion of chemical reactions (Bremner, 1986; Suslick, 1986). Effect of Ultrasound on the Bonding Reaction

In order to define the surface coverage of the bonded silica in an unambiguous and pertinent manner, the surface coverage should be expressed as the number of silane molecules attached to the surface, usually as micromoles of bonded silane molecules per square meter of silica surface, taking into account the increase in weight of the silica after the bonding reaction. These surface coverages are calculated from the percentage of carbon as obtained from elemental analysis of the bonded phase (Berendsen and de







43
Galan, 1978b). This calculation is quite straightforward for monoreactive silanes and for monochlorosilanes (the most commonly used monoreactive reagents) can be expressed by = (%C) (106) (1)
(12.011) (nc) (S) (lO0-L(%C/(12.011)(nc)](M-36.5)) where a is the surface coverage (pmoles/m2); %C is grams carbon per 100 grams bonded silica, as obtained from elemental analysis; nc is the number of carbon atoms per mole silane; M is the molecular weight of the silane; and S is the surface area of the native silica in m2/g. Although

the typical value for the average surface hydroxyl concentration of amorphous silica is 8 imol/m2 (Cheng and McCown, 1985), in practice octadecyl bonded phase coverages are limited to about 3 Pmol/m2 due to steric considerations (Berendsen et al. 1980a; Berendsen and de Galan, 1978a and 1978b; Cheng and McCown, 1985; Snyder and Kirkland, 1979).

Three sets of experiments were compared in order to determine the effect of ultrasound on the silica bonding reaction. In all cases dimethyloctadecylchlorosilane was the reactive silane, methylene chloride was the reaction solvent and 2,6-lutidine the acid-acceptor catalyst; all reaction mixtures were stirred during the reaction time period (24 hours). In the first set of experiments, the reaction mixture was stirred at ambient temperature (22.0 'C); in the second set the reaction mixture was refluxed at 50.0 'C. The third set of experiments was performed at 28.5 'C, but the reaction vessels were immersed in an ultrasonic






44
cleaning bath. The refluxed stationary phases had an average bonding density (+ one standard deviation over three trials) of 2.82 + 0.02 pmol/m2. The room temperature reaction resulted in a bonding density of 2.69 pmol/m2 with a range of + 0.03 Pmol/m2 over two trials; the ultrasound reaction gave a bonded phase (over two trials) with an average bonding density of 2.71 + 0.01 umol/m2. The small bonding density difference between the stirred reaction at ambient temperature and the one at reflux temperature is not surprising as Lork et al. (1986) have shown that the bonding density increases slightly and in a linear fashion with increasing reaction temperature when monochlorosilanes are used as the silanizing reagent. These experimental results show that ultrasound is indeed a viable method for the bonded phase synthesis, giving results which are comparable to those obtained using traditional reflux techniques.

Effect of Subambient Temperature on the Ultrasound Reaction
Two sets of experiments were performed using ultrasound in conjunction with subambient reaction temperatures. In achieving high bonding densities one of the greatest obstacles is increasing steric hindrance at the silica surface as more and more bulky dimethyloctadecylsilyl groups are bonded to the surface. It is possible that at low temperatures the bonding density might be enhanced due to the increased order (decreased entropy) in a lower temperature system. It is here that the ultrasound reactions are most unique, as they allow the temperature of the







45
reaction to be controlled independently of the ultrasonic driving force. Additionally, low reaction temperatures have often been found to enhance reaction yields for ultrasonically catalysed chemical reactions. One explanation for this phenomenon is that low temperatures cause the vapor pressures of the reactants to be decreased, enabling increased efficiency of ultrasonically produced cavitation (Boudjouk, 1986; Bremner, 1986; Suslick, 1986). In order to overcome the slower kinetics expected at lower temperatures, reaction times were increased beyond the usual 24 hour time period.
In the first set of experiments, two reaction vessels were sonicated and stirred at 15.0 'C for 48 hours with a resultant average bonding density (+ the range) of 2.74 +

0.00 Pmol/m2. Since this result was little different from that at room temperature, it was decided to increase the reaction time as well as to decrease the reaction temperature. In this set of experiments, two reaction flasks were sonicated and stirred at 8.5 0C for 101 hours with a resultant average bonding density of 2.84 + 0.01 pmol/m2, a slightly higher value than for those ultrasound reactions run at higher temperatures. These preliminary results indicated that subambient temperatures could indeed enhance the ultrasonic silica bonding reaction. The Use of 4-Dimethylaminopyridine as the Acid-Acceptor Catalyst
There are also advantages in the use of 4-dimethylaminopyridine (4-DMAP) as the acid-acceptor catalyst. The






46

presence of the dimethylamino group should serve to make this base better at forming a reactive intermediate with the silane than 2,6-lutidine. In addition it has a relatively high melting point (108-110 'C) which allows it to be oven-dried rather than necessitating distillation to remove adsorbed water. The odor of 4-DMAP is also quite mild in comparison to that of 2,6-lutidine.

The first set of experiments using 4-DMAP as the acid-acceptor catalyst was performed using methylene chloride as the solvent and dimethyloctadecylchlorosilane as the reactive silane. The reaction mixture was refluxed and stirred at 50.0 0C for 24 hours. The bonded phase product had an average bonding density (+ the range for two trials) of 3.44 + 0.02 imol/m2, much higher than that achieved in our previous syntheses using 2,6-lutidine (2.82 + 0.02 Pmol/m2). This bonding density is also greater than that (3.34 imol/m2) achieved by Kinkel and Unger (1984) under reflux conditions using methylene chloride and 2,6-lutidine. This is especially significant because the silane in our experiments was used as received from a commercial source; Kinkel and Unger synthesized and then distilled their silane under reduced pressure in order to obtain a reactive silane of the utmost purity. Silane purity has been shown to be a very important factor in obtaining high bonding densities (Kinkel and Unger, 1984).

The second set of experiments using 4-DMAP as the

acid-acceptor catalyst was run with the same reagents as







47
described above; the reaction mixture was immersed in the ultrasonic bath and stirred at a temperature of 31.0 'C for 24 hours. The average bonding density of the resultant bonded phases (+ the range for two trials) was 3.35 + 0.05 i mol/m2, again much higher than that achieved under similar circumstances using 2,6-1 uti dine as the aci d-acceptor catalyst (2.71 + 0.01 p.mol/m2).

A low temperature ultrasound reaction was then carried out under the same conditions as stated above, with a reaction temperature of 4.0 'C for a duration of 97 hours. For the two trials, an average bonding density of 3.24 +

0.01 ijmol/m2 was obtained. A second set of low temperature ultrasound reactions was performed under analogous conditions with a reaction temperature of 3.0 'C for 144 hours. The bonded phase resulting from this experiment had a higher bonding density than achieved in any of our previous attempts; the average + the range for the set was

3.60 + 0.01 ,imol/m2. To our knowledge, this bonding density is higher than any previously reported in the literature using dimethyloctadecylchlorosilane as the reactive silane (Berendsen et al., 1980a; Cheng and McCown, 1985; Kinkel and Unger, 1984).

To ensure that the high bonding density in this second low temperature experiment was a result of the ultrasonic driving force as well as the lengthy reaction time, two other reactions were carried out. In one, the reaction was performed exactly as above (at 3.0 0C for 144 hours with






48
stirring) except that the reaction flask was not sonicated. In the other, the reaction was stirred for 144 hours, but the reaction mixture was refluxed at 50.0 'C rather than sonicated. From duplicate elemental analyses of each of these two materials, the average bonding density (+ the range) for the silica stirred (but not sonicated) at 3.0 C for 144 hours was 3.48 + 0.00 Pmol/m2; that for the silica refluxed and stirred at 50.0 'C for 144 hours was 3.44 + 0.03 Pmol/m2. Since the absolute error in the elemental analysis is + 0.20% carbon, which corresponds to + 0.03 Pmol/m2 for the octadecyl packings, the differences in bonding density between these two materials and the silica which was sonicated at 3.0 0C for 144 hours (3.60 pmol/m2) is both real and significant. Therefore it can be concluded that subambient ultrasound reactions are especially efficacious for synthesizing stationary phases with very high alkyl bonding densities.

In order to investigate the effect of superambient

temperatures on the ultrasonic bonding reaction, two types of experiments were performed. In the first, the reaction was carried out by stirring with the same reagents as previously described for a reaction time of 24 hours and with the ultrasonic bath maintained at a temperature of 50.0 'C. For two trials, the average + the range was 3.34 +

0.04 Pmol/m2, virtually identical to that achieved under ambient ultrasonic conditions (3.35 + 0.05 pmol/m2). In the second experiment, the reagents were stirred and sonicated







49
at 31.0 'C for 1 hour and then refluxed and stirred at 50.0 C for an additional 23 hours, in hopes that the preliminary sonication of the reagents would permit greater accessibility of the reactive silane to silanols located deep within the silica pores. The resulting bonding density (for two trials) of 3.42 + 0.03 jmol/m2 is comparable to that achieved under reflux conditions alone (3.44 + 0.02 Pmol/m2). These results indicate that silica bonding reactions performed in an ultrasonic bath are not affected by superambient temperatures; this is in contrast to those performed at subambient temperatures, which were found to give an increasing yield as the temperature was decreased.

Experiments were also carried out in the ultrasonic bath at 28.0 0C for 24 hours using trimethylchlorosilane (TMCS) as the reactive silane, 4-DMAP as the base and methylene chloride as the reaction solvent. TMCS is a much smaller molecule than the octadecyl silane and therefore should approximate the maximum bonding density obtainable in these reactions when steric hindrance is minimized. The average bonding density achieved in the two trials was 3.51 + 0.01 Pmol/m2; the octadecyl bonding densities achieved in the above reactions show that the TMCS bonding density at ambient temperatures can be exceeded under subambient conditions even with bulky octadecyl reagents. The results of these 4-DMAP experiments, as summarized in Table 2-1, demonstrate that it is indeed a superior acid-acceptor catalyst to 2,6-lutidine for reversed phase bonding reactions.












Table 2-1. Comparison of silica octadecyl bonding densities using
4-DMAP and 2,6-lutidine as acid-acceptor catalysts.

Reaction Reaction C18 Bonding Density (pmol/m2)
Conditions Temperature (oC) Time (h) 4-DMAP 2,6-lutidine

Refluxed 50.0 24 3.44 2.82

Ultrasound 28.0 24 2.71

Ultrasound 31.0 24 3.35

Ultrasound 8.5 101 2.84

Ultrasound 4.0 97 3.24

Ultrasound 3.0 144 3.60

Stirred Only 3.0 144 3.48

Refluxed 50.0 144 3.44










0o







51
The use of ultrasound as a driving force for reversed

phase syntheses has been shown to be a viable synthetic procedure. Ultrasonic syntheses performed at subambient temperatures have proven to be especially effective for the production of high alkyl bonding density stationary phases. The use of 4-dimethylaminopyridine as the acid-acceptor catalyst is recommended due to its ease of use and the resulting high bonding densities. The small ranges of the bonding density for duplicate syntheses show that reversed phase packings with reproducible bonding densities can be synthesized by these methods.













CHAPTER III
SYNTHESES OF CONTROLLED PORE GLASS-BASED RP STATIONARY PHASES


Comparison of Controlled Pore Glass and Silica as Supports for RP Stationary Phases

The most commonly used column packing materials for reversed phase liquid chromatography (RPLC) are based on microparticulate silica. As has been described in Chapter II, this material is modified by chemically bonding alkyl chains of the desired length onto the silica surface. The use of such siliceous supports is widespread due to their high reactivity and relatively low cost. S ilIica-based RP bonded phases also exhibit good column stability within the pH range of 2.5 to 7.5 (Melander and Horvath, 1980).

However, such materials are not without problems. The surface of silica gel is very porous in nature and there is a wide distribution in the size of these pores. This can affect chromatographic selectivity by causing size exclusion effects. This broad pore size distribution is one of the contributors to the problem of inhoinogeneous energies of transfer between the stationary and mobile phases for solutes in RPLC, leading to distorted peak shapes and decreasing chromatographic efficiency. The effects of pore size and structure have received much attention in size exclusion chromatography, but these parameters have garnered 52







53
little attention in reversed phase systems (Sander and Wise, 1984b). At present, there is no satisfactory explanation of the effect of pore size distribution on the properties of hydrocarbonaceous bonded phases; however differences in the pore structure of the support material may account for some of the differences observed in the RPLC behavior of various commercial bonded phases having the same alkyl chain length but different siliceous substrates (Melander and Horvath,

1980).

Controlled pore glass (CPGY) offers an ideal medium for investigating the effects of pore size and structure on RP retention and selectivity. CPG, which has mainly been used in size exclusion chromatography, consists of nearly pure quartz glass with pores of uniform size. In CPG, the pore diameter is the same at the surface as it is in the interior of the particle; 80% of the pores show a deviation of less than + 10% from the nominal pore diameter (Fluka). Chemical modification of the surface of CPG is accomplished by reacting surface silanol groups with the appropriate reactive silane, as has been described for silica in Chapter II. Although such reactions have been performed to prepare CPG RPLC bonded phases (Dawidowicz et al., 1983; Dawidowicz and Rayss, 1985; Rayss et al., 1983; Suprynowicz et al., 1978 and 1985) commercial bonded phases based on CPG are currently impractical due to its much greater expense compared to that of silica.







54
The differences in the pore structures of silica and CPG come about from the differences in their chemical compositions and in their manufacturing processes. The manufacture of chromatographic silica is described in detail by Unger (1979). The starting materials in the manufacture of porous silica are soluble silicates such as sodium silicates, silicon tetrachloride or tetraalkoxysilanes. By adjusting the pH of an aqueous solution of the starting material within a range of 8 to 9, silica sols are made. In the sol, polysilicic acids are formed by polycondensation and polymerization, growing into colloidal particles ranging from 1 to 100 nm. The sol consists of spherically shaped, nonporous and amorphous discrete silica particles. Unless stabilized, the discrete particles in the sol aggregate, mainly due to gelling. The particles become linked together to eventually form a three dimensional packing of silica particles that is a gelatinous mass called silica hydrogel. The hydrogel is washed and water is then removed by heating. This dehydration results in shrinkage from the partial collapse of the globular hydrogel structure; the resultant xerogel consists of hard porous grains. The silica particles are also cemented together by dissolutiondeposition processes. The conversion of the hydrogel to the xerogel is the origin of the porosity of chromatographic silica. This porosity comes about from compaction of the dispersed silica in the hydrogel; the pore space is made up of the interparticle interstices and voids. This results in







55
a totally porous structure; moreover these pores are quite irregularly shaped. Factors which can be varied to control

the final pore structure include changes in the sol and/or hydrogel pH, changes in the duration of the hydrogel ripening (via stabilization of the sol), variation of pH during washing of the hydrogel, and substitution of an organic liquid wash for the hydrogel rather than an aqueous one (Unger, 1979). The chemical composition of the final amorphous silica can be exemplified by the composition of the Davisil silica used in our experiments; it consists of 99.60% by weight of SiO2 and 0.10% Na20 with the remaining

0.30% made up of other metal oxides (Grace, 1984).

The procedure used to produce controlled pore glasses was first reported by Wolfgang Haller in 1965 (1965a and 1965b). The starting material consists of a \lycor type glass consisting of 7% Na2O, 23% B203 and 70% Si02. The glass is crushed, fractionated to the desired particle size distribution by sieving, and then heated at approximately 600 'C for the desired number of hours. This heating period causes fusion to take place within the glass, resulting in the formation of microheterogeneous regions in the continuous silica network. This alkali borate-rich microphase is then removed from the glass by a series of acidic and basic leachings, resulting in a finished material which is porous throughout its entire volume (Dawidowicz et al., 1983). The diameter of the pores is determined by the length and temperature of the heat treatment (Haller,






56
1965a). The chemical composition of controlled pore glass is also different from that of amorphous silica; the composition of the finished product is typically 96% by weight of Si02, 3% B203, less than 1% Na20, and a trace amount of other metal oxides (Electro-nucleonics Inc., 1987).

Experimental Procedure

Reagents

All of the reagents used in the preparation of the

reversed phase CPG packings were as described in Chapter II, with the exception of CPG being used as the support material instead of silica. All CPG was manufactured by Electronucleonics Inc. (Fairfield, NJ). The CPG denoted as CPG-86 was from a single lot of CPG-10-75A (Fluka Chemical Corp,; Hauppauge, NY) and had a mean pore diameter of 86 Angstroms with a pore size distribution of + 9.8%. The absolute surface area (SBET, as measured by BET analysis) was 153.1 m2/g; the particle size range was 37-74 jim and the nitrogen pore volume was 0.48 cm3/g. The CPG denoted as CPG-167 was from a single lot of PG-170-400 (Sigma Chemical Co., St. Louis, MO) with a mean pore diameter of 167 Angstroms and a pore size distribution of + 9.6%. The absolute surface area was 161 m2/g, the particle size distribution was 37-74 pm and the nitrogen pore volume was 1.0 cm3/g. Bonded Phase Preparation

Both CPG's were acid leached, dried and reacted with the appropriate silane reagents for derivatization as







57
described in Chapter II. The only difference in the procedure between the CPG and silica was in t'he method of agitation. CPG is more mechanically fragile than silica; therefore direct stirring via a magnetic stirring bar is inadvisable. Agitation was accomplished by a rotary evaporator; a nitrogen gas line was attached to what is normally the vacuum outlet in order to maintain a dry atmosphere. An evacuated glass Dewar-type condenser (fabricated in-house) was used to join the reaction flask to the rotary evaporator; this piece of glassware was necessary in order to prevent the escape of the reaction solvent, especially under reflux conditions.

Reactions were performed under ambient as well as

reflux conditions; ultrasonic reactions were also carried out. The reaction products were washed and vacuum dried as described in Chapter II. Evaluation of the bonding procedure was performed via in-house elemental analysis. Scanning electron micrographs of the acid-leached CPG-86 (Figures 3-1 and 3-2) show that like the silica used in previous reactions, the CPG also exhibits an irregular particle shape as well as an irregular surface. This irregularity is not surprising considering that CPG particles of the desired size range are obtained by mechanically crushing and sieving bulk Vycor glass (Haller,

19 6 5 a ) .









































Figure 3-1. Scanning electron micrograph of acid-leached CPG-86;
820X magnification.



00














L/

























Figure 3-2. Scanning electron micrograph of acid-leached CPG-86;
3010X magnification.







60
Comparison of Silica and CPG Bonding Densities via Reflux and Ultrasonic Syntheses
The controlled pore glasses of both pore sizes (denoted CPG-86 and CPG-167) were derivatized under the same types of reaction conditions as for the Davisil silica. Two aspects in particular were to be examined by these experiments; the first was to determine the reactivity of CPG compared to that of amorphous silica and the second was to determine the effect of pore size on CPG reactivity. From pore shape indices, it has been found that the pore shape of siliceous pores is not usually cylindrical; therefore the cylindrical pore model is not a good approximation for silica (Nikolov, 1986). Since CPG has a very uniform pore diameter compared to that of silica and the CPG pores are much larger than the molecular dimensions of the reactive silane (the average molecular cross-sectional area for the octadecylchlorosilane is 50.95 (Angstroms)2 per molecule and its length is 24.72 Angstroms (Cheng and McCown, 1985)) it was expected that the CPG's would exhibit higher reactivity and therefore result in alkyl bonding densities higher than those achieved with silica. In addition, it was expected that CPG-167 would be more reactive than CPG-86 due to its larger pore size. Other workers have found that octadecyl bonding density increases with increasing pore size for silica substrates (Engelhardt et al., 1982; Sander and Wise, 1984b; Sands et al., 1986; Staroverov et al., 1986).

In the first set of reactions, both CPG-86 and CPG-167 were rotated at ambient temperature (26.0 'C) for 24 hours







61
using dimethyloctadecylchlorosilane as the reactive silane, 2,6-lutidine as the acid-acceptor catalyst and methylene chloride as the reaction solvent. All bonding densities are calculated from duplicate elemental analyses as described in Chapter II and the mean value + the range is reported in all cases. The bonding densities for the CPG-86 and the CPG-167 were 2.56 + 0.03 Pmol/m2 and 2.07 + 0.02 imol/m2 respectively. In the second set of experiments, the same reagents and reaction time as above were used to react both CPG's, but the reactions were performed under reflux conditions at a temperature of 50.0 0C. The CPG-86 bonding density was 2.63 + 0.00 pmol/m2 and that for the CPG-167 was

2.28 + 0.00 Pmol/m2. As expected from previous work with silica (Chapter II), reflux temperatures resulted in a higher bonding density than ambient temperatures.

The next three sets of experiments were run for 24

hours with rotation under ultrasound conditions as described in Chapter II. The first set of experiments used the same reagents as described above at a temperature of 28.0 C. The CPG-86 had a resultant bonding density of 2.55 + 0.01 Pmol/m2; that for the CPG-167 was 2.04 + 0.03 pmol/m2. As in the case for the silica, bonding densities achieved under ultrasonic conditions with 2,6-lutidine as the acid-acceptor catalyst were comparable to those achieved at ambient temperatures.

The second set of ultrasound experiments was run under the same conditions as the first set and at a temperature of






62
28.5 'C, except that 4-dimethylaminopyridine (4-DMAP) was used as the acid-acceptor catalyst instead of 2,6-lutidine. The bonding densities for CPG-86 and CPG-167 were 3.30 +

0.02 and 3.04 + 0.08 Pmol/m2 respectively. The 4-DMAP had again proven to be a superior acid-acceptor catalyst to the 2,6-lutidine as had been the case for silica. The third set of ultrasound experiments was performed at 28.0 C using 4-DMAP as the acid-acceptor catalyst, methylene chloride as the reaction solvent and trimethylchlorosilane (TMCS) as the reactive silane. TMCS was used in order to approximate the maximum bonding density achievable under minimum steric hindrance conditions, as explained in Chapter II. The TMCS bonding density was 4.19 + 0.02 pmol/m2 for CPG-86 and 5.15 + 0.10 Pmol/m2 for CPG-167. As expected, use of a less bulky silane reagent resulted in a higher alkyl bonding density, since steric hindrances are minimized.

A comparison of the bonding densities of the 147
Angstrom pore size silica, 86 Angstrom CPG and 167 Angstrom CPG achieved under all sets of conditions is tabulated in Table 3-1. As seen from these results, neither of our expectations was realized. In all of the octadecyl silane reactions, reactivity of the amorphous silica was greater than that of either CPG. Even more puzzling, the smaller pore CPG (CPG-86) exhibited greater reactivity than the wider pore CPG (CPG-167). In the case where TMCS was the reactive silane, these trends were reversed. This seems to indicate that the reactivity trends for the silane bonding












Table 3-1. Comparison of octadecyl bonding densities for 147 Angstrom (pore size)
silica, 86 Angstrom controlled pore glass (CPG-86) and 167 Angstrom
controlled pore glass (CPG-167).

Reaction 1 C18 Bonding Density (pmol/m2)
Conditions Temperature _(C) silica CPG-86 CPG-167

Refluxed/ 50.0 2.82 2.63 2.28
2,6-lutidine

Ultrasound/ 28.0 2.71 2.55 2.04
2,6-1utidine

Ultrasound/ 28.5 3.35 3.30 3.04
4-DMAP

Ambient/ 26.0 2.69 2.56 2.07
2,6-lutidine

C silane2/ 28.0 3.51 4.19 5.15
trasound/
4-DMAP

1 Reaction method/acid-acceptor catalyst.
2 C1 silane used instead of C18 silane in order to estimate bonding density
achievable using a less bulky silane reagent.





(A






64
reactions could be due to steric problems, since TMCS is much smaller than dimethyloctadecylchlorosilane, but the lower reactivity of the CPG-167 compared to that of CPG-86 for the octadecyl reaction contradicts this theory. The chemical composition of the CPG's may be implicated in this problem, since CPG contains 3% B203 and silica contains no more than trace amounts. Other workers have found that heating CPG to a temperature of 700 0C for 5 to 100 hours results in migration of boron atoms to the glass surface and consequent enrichment of boron on the surface of the porous glass. They further found that this surface boron enrichment resulted in higher octadecyl bonding densities than achieved on untreated CPG (Dawidowicz et al., 1983; Dawidowicz et al., 1986; Dawidowicz and Rayss, 1985; Rayss et al., 1983; Rayss and Dawidowicz, 1986; Suprynowicz et al., 1985). At present, we are unable to explain the anomalous octadecyl bonding behavior of the controlled pore

glasses.














CHAPTER IV
CORRELATIONS BETWEEN CHROMATOGRAPHIC RETENTION AND OCTADECYL BONDING DENSITY



Chromatographic Determination of Thermodynamic Partition
C o e f f i c i e n t s

Retention in any chromatographic process occurs when

the solute of interest is transferred from the mobile phase to the stationary phase. The process of transfer of the solute between the mobile phase and the stationary phase is characterized by the thermodynamic distribution or partition coefficient, K, which is the ratio of the concentration of the solute in the stationary and mobile phase. Chromatographic retention is a function of the distribution coefficient and the volumes of the respective phases, and is most often described by the capacity factor, k', the ratio of the number of moles of solute in the stationary phase and in the mobile phase. The capacity factor also expresses the ratio between the amount of time the solute spends in the stationary phase and in the mobile phase. It is the commonly used measure to describe retention because it accounts for differences in column dimensions and mobile phase flow rates; it is also easy to measure as k' = (Vr Vm)/Vm where Vr is the solute retention volume and Vm is the mobile phase void volume. Measurement of the capacity factor provides valuable thermodynamic information

65







66
about solute retention in a particular chromatographic system since retention is related to the thermodynamic distribution coefficient through the volume phase ratio Vs/Vm (stationary/mobile) in that k' = K(VS/Vm). Therefore for rigorous theoretical treatment of chromatographic retention, the phase ratio must be accurately known in order to determine the partition coefficient. Measurement of the Mobile Phase Volunme in RPLC

The determination of the mobile phase volume, Vm, in liquid chromatographic systems is a problem that has generated great interest as well as considerable controversy. Its value is an essential component for the calculation of the capacity factor k' as well as for the thermodynamic distribution coefficient K. Many workers have addressed this dilemma, yet there is little consensus on a generally applicable convention for measuring Vm (Berendsen et al. 1980b; Engelhardt et al., 1984; Gutnikov and Hung, 1984; Knox and Kaliszan, 1985; Le Ha et al., 1982; McCormick and Karger, 1980; Melander et al., 1983a and 1983b; Slaats et al., 1981; Wainwright et al., 1985; Wells and Clark, 1981). The problem is especially complex for RPLC, since preferential sorption of mobile phase components by the stationary phase results in the formation of a solvation layer on this surface. The thickness and composition of the solvation layer varies with the bulk composition of the mobile phase and the local concentration of organic modifier in the solvation layer may be greater than that in the bulk







67
mobile phase due to the hydrophobicity of the stationary phase. The composition of the solvation layer also varies with its distance from the anchored ends of the RP chains; therefore its presence results in an ill-defined boundary between the stationary and mobile phases (Berendsen et al. 1980b; Gutnikov and Hung, 1984; Le Ha et al., 1982, Knox and Kaliszan, 1985).

There are three general categories of procedures used to determine Vm: the use of unretained compounds, the linearization of the net retention time for a homologous series and static methods (Berendsen et al., 1980b). The choice of an "unretained5 compound for V. measurements in RPLC systems is a difficult one. In any case, neither its heat of sorption nor its size should differ from those of the mobile phase components. For this reason, mobile phase constituents and especially their deuterated analogs are often used (Engelhardt et al., 1984). However, this choice is not without its problems. Since these compounds are transparent in the UV region, sensitive detection of them requires a refractive index detector. The use of deuterated organic modifier as an unretained compound is only valid when there is a large amount of organic modifier present in the mobile phase since in organic-lean mobile phases the marker will be slightly retained, especially in the case of methanol/water mobile phase systems (Engelhardt et al., 1984). The same is true for 020 in organic-rich mobile phase systems; this phenomenon is attributed to D20







68
adsorption onto the residual silanol groups on the stationary phase surface (McCormick and Karger, 1980). Melander et al. (1983a, p. 213) suggest that . the most weakly bound solvent component is not present in the salvation layer." and that this component should be used for the determination of Vm. They concur with McCormick and Karger (1980) that D20 is a useful probe for mobile phase volume determination unless the mobile phase is water-lean. As secondary probes of Vm, fructose and urea have been suggested for all compositions of methanol/water mobile phases and for acetonitrile volume fractions from 0 to 0.75 for acetonitrile/water mobile phase (Melander et al., 1983a). Gutnikov and Hung (1984) have also proposed the use of oxalohydroxamic acid as a UV-detectable Vm probe.

The use of UV-active inorganic salts such as nitrates has also been recommended for determination of RPLC dead volumes; however in unbuffered mobile phases the dead volumes obtained increase with increasing amount of salt injected. Nitrate is also prevented from penetrating the stationary phase pores by the Donnan potential which comes about from the negatively charged silicate ions present on the stationary phase surface (Berendsen et al., 1980b; Engelhardt et al., 1984; Wells and Clark, 1981). Knox and Kaliszan (1985) suggest using a volume fraction-weighted average of the retention volumes of isotopically labelled forms of the mobile phase components. However, besides the aforementioned problems associated with the detection of







69

these species, this method adopts the convention that Vm is the total volume of all mobile phase components within the column bed; the solvation layer adsorbed on the stationary phase is thereby included in the mobile phase volume.

The linearization of a homologous series of compounds

in order to find the column dead volume has been widely used in gas chromatography; therefore it is not surprising that this method has also been applied for LC systems. This method assumes that there is a linear relationship between the logarithm of the net retention time tr and the carbon number of a homologous series (Berendsen et al., 1980b; Laub and Madden, 1985; Wainwright et al., 1985). The mobile phase volume can be obtained by comparing the retention times for two consecutive homologs, termed n and n+1. For a

homologous series the ratio of capacity factors for consecutive homologs is assumed to be constant and

trn+l = A(trn) (A I)t0.

By plotting tr,n+1 versus tr,n, the slope A can be obtained and to can be determined from the intercept and multiplied by the mobile phase volume flow rate to obtain Vm. The results obtained can be precise to within 1% if alkylbenzenes are the homologous series used (Berendsen et al., 1980b); however the use of homologous aromatic alcohols gives inconsistent data due to their interactions with silanol groups on the stationary phase (Laub and Madden, 1985) .

The linearization method is not without criticism. It assumes that the relationship between the logarithm of the







70
retention times for a homologous series is a linear function of the number of carbons in the series and therefore that the change in free energy of partitioning per methylene group is constant. This implies that as the number of methylene groups increase, the rest of the molecule has a constant effect on stationary phase interactions. Yet in some cases the relationship between the logarithm of retention and carbon number is not linear, implying that this assumption is invalid. The linearization method is also very time consuming, since the retention time measurements must be determined very precisely in order to obtain precision in the Vm value (Knox and Kaliszan, 1985).

Determination of Vm by the static method is a

gravimetric procedure. A thermostatted packed LC column is filled successively with two pure liquids with greatly different densities and weighed. From the differences in the column masses, w, and the density of each liquid, d, the total mobile phase volume can be calculated, since m=W a Wbt
d d
a b
(Berendsen et al., 1980b; Knox and Kaliszan, 1985; McCormick and Karger, 1980). The mobile phase volume as determined by this method is the maximum volume within the column that is accessible to a molecule comparable in size to those used in the procedure (McCormick and Karger, 1980). However, this method ignores the possibility of a salvation layer on the stationary phase and therefore can overestimate the value of a dynamic Vm by as much as 15% for a pure methanol mobile







71
phase (Berendsen et al., 1980b). The volume of the mobile phase as determined by this method is useful for a reference point, both in terms of whether or not a compound experiences retention in a chromatographic system and in understanding the changes in the salvation layer which occur when the bulk mobile phase composition is changed (McCormick and Karger, 1980). Knox and Kaliszan (1985) even argue that in the theoretical treatment of thermodynamic aspects of chromatography that the maximum Vm value is the pertinent one. They argue that since the thickness of the boundary between the bulk stationary phase and bulk mobile phase is on the order of one nanometer that its position cannot be sufficiently well defined to give an accurate measure of the two volumes and that calculational methods for the volume of the solvation layer are very arbitrary. Because the gravimetric procedure gives a precise and reproducible value for Vm that is unambiguous and convenient to measure, this convention was chosen to determine the mobile phase volume for the chromatographic thermodynamic distribution coefficients calculated in this work. Measurement of the Stationary Phase Volume in RPLC

Although much work has been done on measuring the

volume of the mobile phase, measurement of Vs,' the volume of the stationary phase has not been as thoroughly investigated (Berendsen et al., 1980b; Jandera et al, 1982;, McCormick and Karger, 1980; Melander et al., 1980; Sander and Field, 1980; Slaats et al., 1981). In determining a method for the






72

measurement of Vs, a convention for defining V. must be chosen, since the choice of the phase ratio must be compatible with the definition of K that is in agreement with the molecular mechanism of retention. Jandera et al. (1982) have defined the stationary phase volume as that fraction of the column volume that is not occupied by the mobile phase. While this choice is certainly convenient and can be readily determined, it is at best a crude measure, as similar (or even identical) values of VS would be obtained for stationary phases made from the same bulk silica but with different bonding densities of alkyl chains, or possibly even of different chain lengths. Any determination of stationary phase volume based solely on mobile phase volume measurements is doomed to failure, as such a measurement cannot be sufficiently sensitive to ascertain bonding density or small chain length differences.

Melander and Horvath (1980) have suggested defining the phase ratio as the ratio of the surface area of the adsorbent (m2) divided by the column dead volume (cm3). While this approach is an improvement in definition, it again fails to account for certain variations in the structure of the bonded phase and it implies that adsorption is the sole mechanism in RPLC retention. The major drawback to this proposed phase ratio convention, however, lies in the accurate measurement of the two parameters involved. As previously mentioned, chromatographers have been unable to embrace any one of the commonly used methods for determining







73
column dead volumes as being accurate and consistent enough for precise work (Engelhardt et al., 1984; Melander et al., 1982; Smith et al., 1986). Melander and Horvath (1980) state that a small relative error in the determination of the column dead volume results in a commensurate relative error in calculating both the capacity factor and the Gibbs free energy of the solute transfer.

Other problems exist as well. The surface area of the adsorbent is usually found by use of the BET analysis method. It should be noted that the surface area of the adsorbent must be determined after derivatization with the alkyl ligand, as the surface area of the derivatized silica will be significantly different from that of the underivatized support. Although use of the BET method for surface area determination is widespread, this method is inappropriate in assessing that surface area of derivatized silica packings which is chromatographically significant. The BET method measures the area of surface that is accessible to a small molecular probe such as nitrogen. Yet in an irregular surface such as porous silica, there may exist many pores which are large enough to allow nitrogen in, but which are too small to allow the passage of any larger molecules of chromatographic interest. Chromatographic support surface area data based on BET analysis is usually overestimated, and the amount of overestimation is by no means a constant, depending on the base silica structure and the derivatization method.







74

Melander and Horvath (1980, p. 270) state that any estimation of "stationary phase volume" on the basis of BET surface area of the support is likely to be inaccurate." Due to the errors in determining both adsorbent surface area and column dead volume, there will consequently be a large error propagated in the subsequent calculation of the phase ratio if Melander and Horvath's convention is used.

Sander and Field (1980) have estimated the phase ratio by constructing physical models of the bonded phase using manufacturer's data regarding silanol surface coverage and percent carbon loading. This approach is quite reasonable from a theoretical standpoint, as it accounts for variation in bonding density and alkyl chain length. However because it is based on models it can only be an estimate of VS; the construction of such physical models is also time consuming.

In determining the stationary phase volume, the

pertinent volume should be the volume of the alkyl chains bonded to the silica surface. Dill (1987a) has performed statistical mechanical calculations based on a lattice interphase model of RPLC stationary phases which describe chromatographic retention in reversed phase systems. These calculations have shown that in a well endcapped column, chain interactions with solutes are the most important stationary phase contribution to solute retention. Therefore the calculation of Vs should give only the actual volume of the alkyl chains bonded to the support surface. The assumption here is that all of the bonded stationary







75

phase volume is accessible to the solute.

A simple method for calculating the stationary phase volume has been devised in our laboratory. The only measurements necessary are the carbon load of the packing and the actual weight of packing contained in the chromatographic column. Wise and May (1983) proposed that the surface density of a bonded alkyl ligand (in micromoles of alkyl ligand per square meter of packing surface) can be calculated by
Cs : %C (106) (4-1)
1200 nc SBET

where Cs is the bonding density (pmol/m2), %C is the carbon loading of the packing as determined from elemental analysis or by a gravimetric procedure (Cheng, 1985), nc is the number of carbons in the alkyl ligand, and SBET is the surface area of the derivatized packing as determined by BET analysis. But the volume of the stationary phase, Vs, can be expressed as

V = (Cs)(SBET)(v)(Wp)(10-6) (4-2)
Vs

where v is the molar volume of the bonded alkyl group in cm3/mole and W is the weight of the bonded packing contained in the chromatographic column. Molar volume, v,

is

v = M/d (4-3)

where M is the weight of the bonded phase alkyl group and d is the density of the bonded alkyl group. Cheng (1985) has experimentally determined the pertinent densities of commonly used bonded silanes and reported values of 0.8607







76

g/cm3, 0.8625 g/cm3, and 0.8638 g/cm3 respectively for the octadecyldimethylsilyl, octyldimethylsilyl, and trimethylsilyl bonded groups. Substitution of equations 4-1 and 4-3 into equation 4-2 results in the volume of the stationary phase, Vs (in cm3), as expressed by the following formula

(10%C M) d) (4-4).
(100)(12.011) nc)(d)

This method provides a much more accurate calculation of Vs than has been previously possible. A principal advantage of this method is that the surface area of the packing is not used in determining Vs, which eliminates the errors associated with this measurement. The stationary phase volume that is calculated in this method is the volume that is important in the chromatographic process, i.e. the actual volume of the bonded alkyl chains themselves. The precision is limited only by the carbon loading determination (+ 0.20 %C for our departmental elemental analysis) and by the measurement of the mass of packing in the column (+ 0.1 mg on any analytical balance). Calculation of the volume of the stationary phase by this method provides the means for a more accurate and uniform determination of the phase ratio.

Experimental Procedure

Preparation of Bonded Phases of Varied Bonding Densities

All of the reagents used in the preparation of the

silica and CPG bonded phases are described in Chapters II and III. Silica bonded phases with octadecyl bonding







77
densities greater than or equal to 2.75 pmol/m2 were prepared under traditional reflux conditions as well as under ambient, subambient and superambient ultrasonic conditions, using 2,6-1utidine or 4-DMAP as the acid-acceptor catalyst as described in Chapter II. Controlled pore glass bonded phases with octadecyl bonding densities of 3.30 and 2.63 pmol/m2 were prepared from the 86 Angstrom CPG (CPG-86) under ambient ultrasonic conditions using 4-DMAP as the acid-acceptor catalyst or under reflux conditions using 2,6-1utidine as the acid-acceptor catalyst respectively as described in Chapter III. Table 4-1 lists the experimental conditions, acid-acceptor catalyst and resultant octadecyl bonding density for each of these stationary phases.

In order to prepare bonded phases with octadecyl
bonding densities less than 2.6 pmol/m2, the experimental conditions of the bonding reaction must be altered so that a less than maximal bonding density is achieved. One strategy that can be used to accomplish this is to use a less than stoichiometric amount of the reactive silane. Another approach is to partially cover some of the reactive silanols with trimethylsilane before exhaustive derivatization with the octadecyl silane reagent (Marshall et al., 1984 and 1986). By varying the amount of trimethylsilane precoverage and then reacting the precovered silicas with an excess of the octadecylsilane, lower coverage octadecyl bonded phases of varying bonding densities can be synthesized.









Table 4-1. High octadecyl bonding density reversed phase packings. Packing Base Reaction Temperature Reaction Acid-Acceptor C18 Bonding Density
Identifier Packing Conditions (oC) Time (h) Catalyst (pmol/m2)

C18-2 silica reflux 50.0 24 2,6-lutidine 2.75

LT2 silica ultrasound 8.5 101 2,6-1utidine 2.84

DMAP1 silica ultrasound 28.0 24 4-DMAP 3.06

DMAP1/31 silica ultrasound 28.0/4.0 24/97 4-DMAP 3.15

DMAP3 silica ultrasound 4.0 97 4-DMAP 3.24

US/refl silica ultrasound 50.0 24 4-DMAP 3.34

ref/DMAP silica reflux 50.0 24 4-DMAP 3.43

rederDMAPl2 silica ultrasound 28.0 24 4-DMAP 3.56

DMAP5 silica ultrasound 3.0 144 4-DMNAP 3.60

CPG2 CPG-86 reflux 50.0 24 2,6-1utidine 2.68

CPG4 CPG-86 ultrasound 28.5 24 4-DMAP 3.30

1 Packing DMAP1/3 is a 50/50 (weight/weight) mixture of packings DMAPI and DMAP3.
2 Packing rederDMAPl was obtained by reacting packing DMAPI with the octadecyl silane
under identical conditions as for DMAP1.




0O







79

Based on Kinkel and Unger's (1984) estimation of a

maximum of five micromoles of reactive hydroxyl sites per square meter of silica surface, amounts of trimethylchlorosilane (TMCS) corresponding to approximately 5%, 10%, 15%, 30% and 40% coverage of these hydroxyl sites were reacted as described in Chapters II and III under ambient conditions (at a temperature of 26.5 OC) for 24 hours to partially precover the silica and controlled pore glass supports. The reaction solvent was dry methylene chloride and 2,6-lutidine was used as the acid-acceptor catalyst. It should be noted that it is unlikely that exactly 5%, 10%, 15%, 30% or 40% of the surface hydroxyl groups on the supports were reacted; the amounts of TMCS used merely represent some fraction of the amount necessary for total coverage of the surface (Marshall et al., 1984). After TMCS precoverage, the supports were washed and dried as described in Chapters II and III. The precovered supports were then reacted with a twofold excess of octadecyldimethylchlorosilane and a fourfold excess of 2,6-lutidine with methylene chloride at a temperature of 26.5 'C for 24 hours and washed and dried as described in Chapters II and III. The resultant C1 and C18 bonding densities for these precovered bonded phases are summarized in Tables 4-2 and 4-3. HPLC Column Packing Procedures

HPLC columns were assembled from 15 cm lengths of 1/4" outer diameter and 4.6 mm inner diameter seamless precision bore polished HPLC tubing (Alltech Associates, Inc.,







80







Table 4-2. Bonding densities for precovered silica reversed
phase packings.

Packing C1 Bonding density C18 Bonding density
Identifier (2mol/m ) (mol/m )

5% 0.63 2.07

10% 0.98 2.09

15% 1.50 1.98

30% 1.38 1.74

40% 1.90 1.60







81







Table 4-3. Bonding densities for precovered 86 Angstrom
controlled pore glass reversed phase packings. Packing C1 Bonding Pensity C18 Bonding ensity
Identifier (Pmol/m ) (umol/m )

5% CPG 0.55 3.21

10% CPG 1.08 2.83

15% CPG 2.25 1.70

30% CPG 1.27 2.72

40% CPG 1.34 2.59







82
Deerfield, IL) and from Swagelok 1/4" to 1/16" zero dead volume reducing union chromatographic end fittings (Crawford Fitting Company, Solon, OH) which had been fitted with 2 pm passivated 316 stainless steel frits (Alltech Associates, Inc., Deerfield, IL). The column tubing and end fittings were made of 316 stainless steel and were passivated prior to use by ultrasonication for 30 minutes in 3 M nitric acid, followed by an aqueous and a methanol rinse.

Silica columns were packed using a Shandon high

pressure HPLC column packer with a 33 ml slurry reservoir (Shandon Southern Instruments, Inc., Sewickley, PA). Approximately 1.5 grams of derivatized silica were slurried in 30 ml of chloroform and sonicated for 10 minutes. The column was then packed at a packing pressure of 6000 psi in the downward position using a sequence of 150 ml each of 50/50 (v/v) chloroform/methanol, methanol and 50/50 methanol/water. The column was then removed from the packer fittings, the packing leveled with a spatula and the remaining column end fitting installed. Controlled pore glass columns were packed in an identical manner except for the type of packer used. Since CPG is mechanically fragile and brittle (Fluka), it cannot be packed using a high pressure packer. Therefore a Beckman Model 100A HPLC pump(Beckman Instruments Inc., San Ramon, CA) running at a flow rate of 9.9 ml/min was used in conjunction with continuous mechanical vibration to pack the CPG columns. Packing in such a manner generated a packing pressure of







83
200-300 psi, preserving the integrity of the CPG packings and resulting in a stable packing bed. All columns were equilibrated with the desired chromatographic mobile phase by passing approximately 125 ml of the mobile phase through the column immediately prior to use. Chromatographic Measurements

The liquid chromatographic system used for

chromatographic measurements consisted of a Valco C6W injector (Valco Instrument Company, Inc., Houston, TX) with a 10 microliter sample loop, a Beckman Model 100A isocratic HPLC pump, a Beckman Model 153 fixed wavelength 254 nm UV detector and a Fisher Recordall chart recorder (Fisher Scientific, Fairlawn, NJ). Samples were loaded in the injection loop via a Hamilton 705 SNR syringe (Reno, NV). Temperature control of the chromatographic column was accomplished by passing thermostatted water through a water jacket fitted around the column. Superambient temperatures were controlled using a Lauda Model MT heater/circulator (Brinkmann Instruments Company, Westbury, NY). Subambient temperatures were brought about by a Neslab Endocal 800 water bath (Neslab Instruments, Portsmouth, NH). Mobile phases were made of HPLC grade methanol or acetonitrile (Fisher Scientific, Fairlawn, NJ) mixed with water which had been prepared as described in Chapter II; mobile phase compositions are designated by volume ratios of organic modifier to water. Mobile phases were premixed by adding the appropriate volume of organic modifier to the







84
appropriate volume of water; these mobile phases were then mixed well and placed in an ultrasonic bath for 15-30 minutes in order to degas them. The flow rate of the mobile phase in all cases was 1.5 ml/min. Naphthalene (Eastman Organic Chemicals, Rochester, NY) was chosen as a small nonpolar test solute; standards were made up in HPLC grade methanol for use in the retention studies. Solute retention and column holdup volumes were measured from the chart recorder tracings. The solvent disturbance peak was used to determine column mobile phase volumes for the calculation of capacity factors; since this disturbance comes about from the methanol in which the test solute is dissolved, its choice for V. falls under the category of using an unretained compound to determine Vm. This convention was chosen in order to account for variances in the individual column dead volumes resulting from differences in the stationary phase packing density within the chromatographic

c o 1 u m n s .

Measurement of V,,, and V

The gravimetric procedure previously described was used to calculate Vm. While an ambient temperature of 25.0 'C was maintained, 150 ml of methylene chloride was passed through a 15 cm LC column packed with either silica or CPG. The column was then capped and weighed on an analytical balance. The procedure was duplicated using methanol as the mobile phase and by dividing the difference in column masses by the difference in the solvent densities at 25.0 OC (1.318







85

and 0.7866 g/cm3 for methanol and methylene chloride respectively) the silica column dead volume was determined to be 1.805 ml while Vm for the CPG column was 1.752 ml. It was assumed that the mobile phase volumes measured by this procedure will be constant (within experimental error) for any of the silica or CPG reversed phase columns used in this work, since in each case the packings were based on the same starting material.

The stationary phase volume for each LC column was

calculated using Equation 4-4. For the precovered bonded phases the total volume of both the trimethyl- and octadecylsilyl alkyl groups was used for Vs. Percent carbon for each column packing was obtained from in-house elemental analysis and the densities used for the trimethylsilyl and octadecylsilyl groups were 0.8638 and 0.8607 g/cin3 respectively as reported by Cheng (1985). The weight of the packing contained in the chromatographic column was determined by weighing an empty chromatographic column, packing it as described previously in this chapter and drying it at 100 0C to constant weight in a gas chromatograph with a constant helium flow through the column. From the mass differences in the two weighings the weight of the column packing was determined to be 1.1705 g for the silica columns and 1.2898 for the CPG columns. The capacity factors for the naphthalene solute were determined by triplicate injections onto each of the chromatographic columns of different alkyl bonding density using mobile







86

phases consisting of 55/45 methanol/water and 85/15 acetonitrile/water. Thermodynamic partition coefficients for the transfer of the naphthalene solute from the stationary phase to the mobile phase were calculated by dividing the capacity factor by the volume phase ratio.

Re s ult s

Silica-based Stationary Phases

Thermodynamic partition coefficients for the

naphthalene solute as a function of silica stationary phase octadecyl bonding density are listed in Tables 4-4, 4-5 and 4-6 respectively for the 55/45 methanol/water mobile phase system at 20.0 CC and 35.0 0C and for the 85/15 acetonitrile/water mobile phase system at 35.0 'C. Graphical representations of these data are shown in Figures 4-1, 4-2 and 4-3.

In all cases the partition coefficient increased as a linear function of bonding density until a bonding density of 3.1 pmol/m2 was reached. The best fit line for this linear region of each plot was calculated using least squares linear regression. For 55/45 methanol/water at 20.0 OC the slope and y-intercept for the best fit line were 30.1 and 12.3 respectively with a coefficient of correlation of 0.989. At 35.0 'C for 55/45 methanol/water the slope was 21.0, the y-intercept 12.7 and the coefficient of correlation 0.973. For the 85/15 acetonitrile/water mobile phase at 35.0 'C the slope was 1.20, the y-intercept 1.54 and the coefficient of correlation 0.982. In all cases the







87








Table 4-4. Naphthalene thermodynamic partition
coefficients at 20.0 0C as a function of
silica octadecyl bonding density for
55/45 methanol/water mobile phase.


C18 Bonding ensity Naphthalene Thermodynamic Partition
(hirol/m )Coefficient at 20.0 0C


1 .60 57 .6

1 .74 66.7

1 .98 69 .6

2 .07 78.6

2 .09 75.8

2 .75 94.3

2 .84 96.2

3.06 104

3 .15 97 .2

3 .24 93.4

3 .34 89 .6

3.43 87 .2

3 .56 86.1

3 .60 85.9







88








Table 4-5. Naphthalene thermodynamic partition
coefficients at 35.0 'C as a function of
silica octadecyl bonding density for
55/45 methanol/water mobile phase.

C18 Bonding ensity Naphthalene Thermodynamic Partition
jjMo jm Ceficient at 35.0 0C

1 .60 43.4

1 .74 50.7

1 .98 51 .3

2 .07 60.5

2 .16 59 .5

2 .75 69 .8

2 .84 71 .3

3.06 76.1

3 .15 70 .9

3.24 69.2

3.34 66.8

3.43 65 .0

3.56 63.9

3.60 63.7







89








Table 4-6. Naphthalene thermodynamic partition
coefficients at 35.0 'C as a function of
silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.

C18 Bonding ensity Naphthalene Thermodynamic Partition

(4mol/m ) Coefficient at 35.0 0C

1.60 3.28

1 .74 3.69

1.98 3.79

2.07 4.20

2.09 4.16

2.75 4.90

2.84 4.95

3.06 5.09

3.15 4.88

3.24 4.73

3.34 4.66

3.56 4.48

3.60 4.41














147 A Silica at 20 C 120


100


80
Fbrtltion
Coefficient,
K
K 6040


20


I I I I I I I I0.5 1D 1.5 2.0 2.5 3.0 3.5 4.0
Cme Bonding Density (xmol/ n2)


Figure 4-1. Naphthalene thermodynamic partition coefficient at 20.0 oC
as a function of silica octadecyl bonding density for
55/45 methanol/water mobile phase.














147 A Silica at 35 C


90



70
Partition
Coefficient,
K
50



30



I0

I I I I I I I I
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Ci Bonding Density (jpmrnol/ rn2)


Figure 4-2. Naphthalene thermodynamic partition coefficient at 35.0 oC
as a function of silica octadecyl bonding density for
55/45 methanol/water mobile phase.




Full Text
This dissertation is dedicated
to my fiance, Daniel Coffman.
I could never have done
this without you.


CHAPTER IV
CORRELATIONS BETWEEN CHROMATOGRAPHIC RETENTION AND OCTADECYL
BONDING DENSITY
Chromatographic Determination of Thermodynamic Partition
Coefficients
Retention in any chromatographic process occurs when
the solute of interest is transferred from the mobile phase
to the stationary phase. The process of transfer of the
solute between the mobile phase and the stationary phase is
characterized by the thermodynamic distribution or partition
coefficient, K, which is the ratio of the concent ration of
the solute in the stationary and mobile phase.
Chromatographic retention is a function of the distribution
coefficient and the volumes of the respective phases, and is
most often described by the capacity factor, k', the ratio
of the number of moles of solute in the stationary phase and
in the mobile phase. The capacity factor also expresses the
ratio between the amount of time the solute spends in the
stationary phase and in the mobile phase. It is the
commonly used measure to describe retention because it
accounts for differences in column dimensions and mobile
phase flow rates; it is also easy to measure as
k' = (Vr Vm)/Vm where Vr is the solute retention volume
and Vm is the mobile phase void volume. Measurement of the
capacity factor provides valuable thermodynamic information
65


148
Finally, it was predicted in Chapter IV that the
chromatographic partition coefficient was probably
overestimated since the solvation layer volume could not be
accurately estimated. This would result in too small a
value for Vs and too large a value of Vm, resulting overall
in an overestimation of Kc|nromat0grap|11- c. The comparison of
the results for the benzene system are encouraging in this
respect; there is about a factor of two difference in the
chromatographic and dynamic partition coefficients and this
is quite good when all sources of error are considered. The
acetonitrile system showed a much larger discrepancy, but
this is not unexpected. The k values in the 85/15
acetonitri 1 e/water system have much more error associated in
their measurement than in the methanolic mobile system since
the retention times in the acetonitrile system were shorter
for the solute (less than 1.5 minutes) as was the system
"dead time" due to a thicker solvation layer in acetonitrile
systems than in methanolic ones (McCormick and Karger,
1980). Additionally, the molar amount of naphthalene in the
acetonitrile dynamic system was eight times less (and the
mass amount 13 times less) than the amount of benzene put
into the methanolic system; this was necessary because of
the large differences in their molar absorptivi ties at 254
nm. If the absolute amount of error is constant in both
systems, the relative amount of error in the naphthalene
system will be much larger than that in the benzene system
due to the much smaller amount of solute involved. In


46
presence of the dimethy lamino group should serve to make
this base better at forming a reactive intermediate with the
silane than 2,6-1utidine. In addition it has a relatively
high melting point (108-110 C) which allows it to be
oven-dried rather than necessitating distillation to remove
adsorbed water. The odor of 4-DMAP is also quite mild in
comparison to that of 2,6-1utidine.
The first set of experiments using 4-DMAP as the
acid-acceptor catalyst was performed using methylene
chloride as the solvent and dimethy1octadecy1ch1 oros i 1 ane as
the reactive silane. The reaction mixture was refluxed and
stirred at 50.0 C for 24 hours. The bonded phase product
had an average bonding density (+ the range for two trials)
of 3.44 + 0.02 umol/m^, much higher than that achieved in
our previous syntheses using 2,6-lutidine (2.82 + 0.02
nmol/m^). This bonding density is also greater than that
(3.34 ymol/m2) achieved by Kinkel and Unger (1984) under
reflux conditions using methylene chloride and 2,6-lutidine.
This is especially significant because the silane in our
experiments was used as received from a commercial source;
Kinkel and Unger synthesized and then distilled their silane
under reduced pressure in order to obtain a reactive silane
of the utmost purity. Silane purity has been shown to be a
very important factor in obtaining high bonding densities
(Kinkel and Unger, 1984).
The second set of experiments using 4-DMAP as the
acid-acceptor catalyst was run with the same reagents as


170
Marqusee, J. A.; Dill, K. A. "Solute Partitioning into Chain
Molecule Interphases: Monolayers, Bilayer Membranes, and
Micelles," J. Chem. Ph.ys. 1986, 5., 434-444.
Marshall, D. B.; Cole, C. L .; Connolly, D. E. "Variable
Reactivity in the Chemical Modification of Silica. Effects
of Initial Deactivation on High-Performance Liquid
Chromatographic Performance," J. Chromatoqr. 1986, 361,
71-82. ~
Marshall, D. B.; Stutier, K. A.; Lochmuller, C. H.
"Synthesis of LC Reversed Phases of Higher Efficiency by
Initial Partial Deactivation of the Silica Surface," J.
Chromatoqr. Sci 1984, 2_2 217-220.
Mart ire, D. E.; Boehm, R. E. "Unified Theory of Retention
and Selectivity in Liquid Chromatography. 2. Reversed-
Phase Liquid Chromatography with Chemically Bonded Phases,"
J. Ph.ys. Chem. 1983 87 1045-1062 .
McCormick, R. M.; Karger, B. L. "Distribution Phenomena of
Mobile-Phase Components and Determination of Dead Volume in
Reversed-Phase Liquid Chromatography," Anal. Chem. 1980,
52, 2249-2257.
Melander, W. R.; Erard, J. F.; Horvath, Cs. "Movement of
Components in Reversed-Phase Chromatography I. Mobile Phase
Space with Mu 11i-Component Eluents," J. Chromatoqr. 1983a,
282 21 1-223 .
Melander, W. R.; Erard, J. F.; Horvath, Cs. "Movement of
Components in Reversed-Phase Chromatography II. Eigenpeaks
in Reversed-Phase Chromatography with Silica-Bound
Hydrocarbonaceous Stationary Phases: Effect of the Eluite
Structure," J. Chromatoqr. 1983b, 282 229-248 .
Melander, W.; Horvath, Cs. In High-Performance Liquid
Chromatography: Advances and Perspectives. Horvath, Cs.,
Ed.; Academic Press: New York", 1980 Vol. 2, 113-319.
Melander, W. R.; Horvath, Cs. "Stationary Phase Effects in
Reversed-Phase Chromatography II. Substituent Selectivities
for Retention on Various Hydrocarbonaceous Bonded Phases,"
Chromatographia 1982, 15, 86-90.
Melander, W. R.; Mannan, C. A.; Horvath, Cs. "Mobile Phase
Effects in Reversed-Phase Chromatography IV. Retention by
n-Alkylbenzenes as a Function of Column Temperature and the
Nature and Concentration of Organic Co-Solvent,"
Chromatographia 1982, 15, 611-615.
Melander, W.; Stoveken, J.; Horvath, Cs. "Stationary Phase
Effects in Reversed-Phase Chromatography I. Comparison of
Energetics of Retention on Alkyl-Silica Bonded Phases," J
Chromatogr. 1980 199 35-56 .


INTERPHASE SOLUBILITY AND
CHROMATOGRAPHIC RETENTION
By
KAREN BELINDA SENTELL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987

This dissertation is dedicated
to my fiance, Daniel Coffman.
I could never have done
this without you.

ACKNOWLEDGEMENTS
There are many people whom I wish to acknowledge for
their assistance with this work. Thanks are extended to Mel
Courtney for performing the numerous elemental analyses of
my packings and to the technical staff in the departmental
machine shop and glassblowing shop for their courteous
assistance and advice. I am grateful to Dr. John Gerdes for
suggesting the use of 4-dimethyl aminopyridine (4-DMAP) in
the bonded phase syntheses, to Nepera, Inc. for providing
the 4-DMAP, to Dr. John Novak of the Aluminum Corporation of
America for providing scanning electron micrographs of the
silica and controlled pore glass supports and to Dr. Lane
Sander of the National Bureau of Standards (NBS) for
providing the NBS column evaluation test mixture.
I would like to express my gratitude to the Society for
Analytical Chemists of Pittsburgh for funding my summer
American Chemical Society (ACS) Analytical Division Graduate
Fellowship and to Procter and Gamble for funding my full-
year ACS Analytical Division Graduate Fellowship.
Thanks are also due to my fellow Dorsey group members
(both present and former) for their friendship, advice and
support. The camaraderie within our group will be one of my
warmest memories of graduate school. I look forward to
i i i

continued association with them during my postdoctoral
tenure.
The love and moral support from my parents, Bobby and
Ruth Sentell, have helped to sustain me throughout my
education. They are responsible for instilling in me a love
of reading, a respect for education and an unquenchable
thirst for knowledge. I am grateful to them and to my
sister, Michelle, for their encouragement during the
toughest times.
My deepest gratitude is extended to my graduate
research advisor, Dr. John G. Dorsey, for his advice and
guidance. He is the epitome of what a research advisor
should aspire to be and has served as an inspiration to me
both as a research scientist and as a teacher. I have
greatly enjoyed our conversations and I look forward to our
continued professional interaction over the next year of my
postdoctoral appointment. I also thank him for encouraging
my oenophilic tendencies; after all, everyone needs to
develop a new vice now and then.
Lastly, I want to thank my fiance, Daniel Coffman. His
love, patience and support have sustained me even when I was
discouraged and disheartened; without his help I could never
have completed this work. In addition to his moral support,
I would also like to thank him for his expert drafting and
technical assistance as well as for accompanying me on my
numerous midnight sorties to check on my reactions. He
deserves my heartfelt gratitude now and forever for always
being there when I need him.

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i i i
ABSTRACT. vi i
CHAPTERS
I INTRODUCTION 1
Models of Reversed Phase Liquid Chrornatographi c
Retenti on 1
Theories of Retention in RPLC 17
II SYNTHESES OF SILICA-BASED RP STATIONARY PHASES 29
Experimental Considerations in the Synthesis
of RP Stationary Phases 29
Experimental Procedure 34
Syntheses Utilizing Ultrasonic Waves 41
Effect of Subambient Temperature on the
Ultrasound Reaction 44
The Use of 4-Dimethylamino pyridine as the
Aci d-Acceptor Catalyst 45
III SYNTHESES OF CONTROLLED PORE GLASS-BASED RP
STATIONARY PHASES 52
Comparison of Controlled Pore Glass and Silica
as Supports for RP Stationary Phases 52
Experimental Procedure 56
Comparison of Silica and CPG Bonding Densities
via Reflux and Ultrasonic Syntheses 60
IV CORRELATIONS BETWEEN CHROMATOGRAPHIC RETENTION
AND OCTADECYL BONDING DENSITY 65
Chromatographic Determination of Thermodynamic
Partition Coefficients 65
Experimental Procedure 76
Resul ts . 86
v

V CORRELATIONS BETWEEEN CHROMATOGRAPHIC SELECTIVITY
AND OCTADECYL BONDING DENSITY 105
I ntroducti on 105
Experimental Procedure ...110
Results and Conclusions 113
VI CONCLUSIONS 135
Syntheses of RP Stationary Phases 135
Validity of Chromatographic Partition
Coefficient Measurements 138
The Effect of Octadecyl Bonding Density on the
the Chromatographic Partition Coefficient 149
Suggestions for Future Work 154
REFERENCES 164
BIOGRAPHICAL SKETCH
174

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INTERPHASE SOLUBILITY AND
CHROMATOGRAPHIC RETENTION
By
KAREN BELINDA SENTELL
December, 1987
Chairman: John G. Dorsey
Major Department: Chemistry
The retention and selectivity behavior of small solutes
on silica and controlled pore glass (CPG) reversed phase
liquid chromatographic (RPLC) stationary phases was studied
as a function of stationary phase alkyl bonding density.
These monomeric octadecyl phases were synthesized by both
reflux and ultrasound methods; high alkyl bonding densities
(3.60 pmol/m^) were obtained via low temperature ultrasound
reactions using 4-dimethylamino pyridine as the acid-acceptor
catalyst. Using an improved method for the calculation of
the stationary phase volume, the chromatographic capacity
factors for the solutes were divided by the volume phase
ratio (stationary/mobile) to obtain the thermodynamic
partition coefficients; their behavior as a function of
stationary phase octadecyl bonding density was examined in
vi i

55/45 methanol/water and 85/15 acetonitrile/water mobile
phase systems.
For the silica packings in both mobile phase systems,
the partition coefficients linearly increased until a
critical bonding density of about 3.1 pmol/m^ was reached;
after this point the partition coefficients began decreasing
with increasing bonding density. This behavior supports a
partitioning retention mechanism for RPLC. In this model,
the driving force for retention is the creation of a solute
sized cavity in the stationary phase interphase structure.
Beyond the critical density, increased alkyl bonding density
results in enhanced interphase chain packing constraints
which increase the energy necessary for solute cavity
formation, resulting in decreased chromatographic partition
coefficients.
Methylene and phenyl selectivity were also examined as
a function of octadecyl bonding density. Methylene
selectivity was approximately constant, but phenyl
selectivity increased linearly with bonding density. This
further supports the partitioning theory; methylene
selectivity is not expected to be affected by chain ordering
but phenyl selectivity for the linear solutes used should
increase as the interphase packing structure becomes more
ordered. Identical selectivity and retention studies on the
CPG bonded phases garnered inconclusive results, as no
obvious trends were discernable in either study.
This work supports a partitioning mechanism for RPLC
retention and as such gives insight into the retention
v i i i

process on a molecular level. This theory is predictive
without adjustable parameters and is relevant to
partitioning behavior in organized assemblies,
micelles, membranes and vesicles.
including

CHAPTER I
INTRODUCTION
Models of Reversed Phase Liquid Chromatographic Retention
Retention Indices Based on Solute Descriptors
Reversed phase liquid chromatography (RPLC) is one of
the most popular and powerful analytical separation methods.
In RPLC, the stationary phase support consists of silica
particles which are typically 3 to 10 pm in diameter; alkyl
chains, usually 8 or 18 carbons in length, are attached to
oxygen atoms on the silica surface via covalent bonds,
resulting in a nonpolar surface. The mobile phase consists
of water and an organic modifier such as methanol,
acetonitrile or tetrahydrofuran. Thus the mobile phase is
much more polar than the stationary phase; mobile phase
polarity is adjusted by varying the volume ratios of water
and organic modifier. It has been estimated that 80-90% of
the high performance liquid chromatography (HPLC) systems
currently in use are reversed phase systems (Melander and
Horvath, 1980). Yet many practitioners of RPLC view this
technique as "black magic" because its retention mechanism
is not well understood, especially at the molecular level.
This makes the prediction of retention for new compounds of
interest extremely difficult. A basic understanding of RPLC
retention at the molecular level is a necessity for the
1

2
formulation of a predictive retention index system. Such a
retention index system would allow accurate interlaboratory
comparison of RPLC retention data.
Chromatographic retention is most often quantified by
the capacity factor, k'. Thermodynamically, the capacity
factor for a chromatographic solute is the ratio of the
number of moles of solute in the stationary and mobile
phases. The capacity factor is also a measure of solute
retention which normalizes for the mobile phase flow rate
and the physical dimensions of the chromatographic column,
since k' = (Vr Vm)/Vm, where Vr is the solute retention
volume and Vm is the retention volume of an unretained
solute, often called the dead volume. Many different
investigators have attempted to correlate RPLC retention
data with topological, geometric and/or calculated physical
property descriptors of chromatographic solutes in order to
predict RP retention. Topological descriptors include
molecular connectivity, molecular complexity and correlation
factor; van der Waals volume, molecular surface area and
length/breadth parameters are geometric decriptors.
Physical property descriptors include hydrophobic
substituent constants, UNIFAC models of activity
coefficients, and octanol/water partition coefficients
(D'Amboise and Bertrand, 1986; Funasaki et al., 1986; Jinno
and Kawasaki, 1984a, 1984b and 1984c; Petrovic et al.,
1985 ) .

Topological descriptors such as molecular connectivity
indices are used to correlate chromatographic retention with
3
molecular structure. These indices are numerical values
which quantitatively describe carbonaceous adjacency
relationships in the molecular structure of a solute
(Lehtonen, 1984). Molecular connectivity indices have been
shown to be proportional to the cavity surface area of a
molecule. When a nonpolar hydrocarbon solute is introduced
into an aqueous or hydroorganic environment, a large
negative entropy of solution results. It has been suggested
that this negative entropy is a result of structural
ordering around the hydrocarbon molecule (Karger et al.,
1976). This ordering comes about from the formation of a
cavity of water molecules around the hydrocarbon molecule.
To overcome the entropy loss, nonpolar molecule segments
will try to remove themselves from the aqueous medium and/or
they will group together. The term "hydrophobic effects" is
used to decribe these two phenomena of cavity formation and
nonpolar clustering. The calculated surface area of the
water cavity is significant because it can be related to the
solubility of hydrocarbons in water (Karger et al., 1976).
Likewise molecular connectivity, since it is also
proportional to the cavity surface area, has also been
correlated to non-electrolyte water solubility; however in
contrast to cavity surface area, simple first order
molecular connectivity indices are quite easy to calculate.
Since in some cases the logarithm of the aqueous solubility

4
of a solute is proportional to the logarithm of its capacity
factor k1, it was expected that molecular connectivity would
also be proportional to k1, allowing prediction of retention
to be made from these molecular connectivity calculations
(Karger et al., 1976 ).
In their comparisons of experimental capacity factors
to those predicted via simple molecular connectivity
calculations, Karger et al. (1976) found very good agreement
for para-substituted phenols and primary alcohols. However,
the predicted k' values were uniformly high for secondary
alcohols. This is because the experimental log k' values
for two of the primary alcohols were used to determine the
slope and intercept in the presumed linear relationship
between log k' and molecular connectivity index for both
types of alcohols. The high predicted k1 values for the
secondary alcohols reflect that their steric environment is
different from that of the primary alcohols, showing that
simple molecular connectivity indices can only be used to
predict relative retention for compounds with the same
functional group as those used for standards (Karger et al.,
1976 ).
Lehtonen (1984) used molecular connectivity indices of
different orders, which correct for complex branching as
well as for the nature of atoms other than carbon which make
up the solute framework, to predict retention behavior for
16 dansy1 ami des. Predicted and experimental k' values could
be correlated very well by combinations of different order

5
connectivity indices. However, a computer program had to be
used in order to find the best combination of indices to
obtain good correlation and these combinations were often
nonlinear, involving combinations of connectivity indices
raised to powers ranging from -2 to +2. The choice of
indices also varied according to which organic modifier was
used in the mobile phase as well as its percent composition.
Although Lehtonen obtained good correlations between
predicted and experimental retention, his method requires
extensive computer calculations as no general connectivity
index combination was applicable even within the same class
of solutes (dansy1 ami des). These two types of examples
point out the shortcomings in the use of molecular
connectivity indices to predict retention: the capacity
factors for at least two members of the same class of
compounds must be determined in order to find the
proportionality constant between capacity factor and
molecular connectivity, these predictions are only valid for
compounds of the same functional group as the standards, for
complex molecules a computer program must be used in order
to find the best combination of indices to predict retention
and this combination for a particular type of molecule may
change if the mobile phase composition is altered. This
method is also unable to distinguish between geometric
isomers (Funasaki et al., 1986).
Molecular complexity is a topological descriptor and
the general index of molecular complexity (GIMC) is an index

6
whose applicability is general and which does not require
the use of experimental or empirical data (D'Amboise and
Bertrand, 1986). The GIMC is derived from combinations of
graph theory and statistical information theory. It is so
named because it considers all of the features which make a
molecule more or less complex such as size, symmetry,
branching, ring structures, multiple bonds and atomic
heterogeneity. Molecules are represented by their skeletal
molecular graph whose complexity is determined from a
statistical information theory derived formula. Since any
observable behavior related to a molecule's complexity is a
function of the GIMC, chromatographic retention should also
correlate with the GIMC. D'Amboise and Bertrand (1986)
point out that GIMC is able to make retention predictions
for solutes such as alcohols or fatty acids, which are not
well correlated with hyrophobicity. They point out that
GIMC is a structure sensitive parameter representing the
various reactive attributes of a molecule; therefore it
should be related to the interaction mechanism in retention.
However, plots of log k' versus GIMC for alcohols show
distinct curvature, especially for alcohols with five or
less carbons. Other difficulties exist as well. The index
does not seem to be well applicable to molecules with
different heteroatoms that are similarly bonded or for
molecules belonging to nonhomologous series. Correlation of
data between different stationary phases has also proven to
be a problem (D'Amboise and Bertrand, 1986).

7
Geometric descriptors are in general fairly easy to
calculate. Van der Waals volume and surface area are both
calculated from the van der Waals radii of the atoms from
which the molecule is composed (Jinno and Kawasaki, 1984a).
Length to breadth ratio (L/B) is a shape parameter based on
the rectangle with minimum area which could envelop a
molecule (Wise et al., 1981). However, all of these
descriptors considered alone or in combination were found to
have poor direct correlation with RPLC retention for
substituted benzene derivatives. Jinno and Kawasaki (1984a)
concluded that this indicates that molecular size and shape
were not the dominant forces controlling retention for these
molecules. However, they noted that size and shape are
important contributors to retention for a 1ky1benzenes and
polycyclic aromatic hydrocarbons (PAHs) (Jinno and Okamoto,
1984). Wise et al. (1981) have also found that L/B is
useful for predicting PAH elution order; however this
parameter is useless for establishing general PAH retention
indices since these indices vary according to the type of
octadecyl bonded phase column used, necessitating the
determination of a retention index equation for each
different octadecyl column.
Physical property descriptors have been the most
successful for predicting RPLC solute retention. The
physical properties on which these descriptors are based
come about from the solute's solution behavior in the mobile
and stationary phases (Karger et al., 1976). Since

8
retention is controlled by the thermodynamic equilibrium of
the solute between the mobile phase and stationary phase,
retention could theoretically be predicted from standard
Gibbs free energies since aG = -RT In K, where AG is the
standard Gibbs free energy, R is the gas constant, T the
absolute temperature and K the equilibrium distribution
constant for the solute between the stationary and mobile
phases. Since k' = K(Vs/Vm) where V$ and Vm are the
stationary and mobile phase volumes, the capacity factor for
a solute (and thus its normalized retention) could easily be
calculated were aG, Vs and Vm known. Since experimental
aG values for these systems are unavailable, they are often
estimated using liquid mixture models with readily available
physical parameters, such as Hildebrand solubility
parameters and group contribution concepts. But Hildebrand
solubility parameters are only useful for qualitative
descriptions of chromatographic behavior; therefore the
group contribution concept is often used, since it was
developed to predict activity coefficients in nonelectrolyte
liquid mixtures (Petrovic et a 1 ., 1985 ). This concept is
the basis for the UNIFAC model for chromatographic
retention, which combines a model based on extension of
quasi-chemical theory of liquid mixtures (UNIQUAC) with the
concept of functional group solubility. In this method
solute activity coefficients in the mobile and stationary
phases are calculated via structural and binary parameters
characterizing the mutual interaction energy of the

9
functional groups in the system. The solute activity
coefficient is the product of a combinatorial and residual
contribution. The combinatorial contribution is dependent
on the size and shape of the molecules in the system; the
residual depends on the interaction energy of functional
group pairs, as well as the fraction of the surfaces on
these groups which are available for mutual interactions
(Petrovic et al. 1985 ) .
Assuming infinite dilution, the relationship between
the capacity factor of a solute i and its activity
coefficient (f-j) in the mobile and stationary phases can be
written as
In ki1 = In i + In (Vs/Vm)
therefore In k ^ 1 = In f-¡m In f-¡ s + In (Vs/Vm). Petrovic
et al. ( 1985 ) assume that f^s and Vs/Vm are constants;
therefore In k^1 and In f-¡m are linearly related with a
slope of one. If the activity coefficients of the solute i
in the chromatographic system and Vs/Vm are known, retention
can then be predicted. Petrovic et al. (1985) calculated
infinite dilution activity coefficients of solutes in the
mobile phase from experimental gas-liquid chromatographic
data and then correlated them with their RP retention values
to see how well the UNIFAC method could predict
chromatographic behavior. They assumed that Vs/Vm would be
the same for any octadecyl RP column at any methanol/water
mobile phase composition (an assumption that will be
thoroughly disputed in Chapter IV of this tome) and that

10
In f.js was zero (since stationary phase interactions were
assumed to be very weak and nonselective). They found that
the predicted values of k1 were at best a rough estimate of
actual chromatographic retention and concluded that
therefore the solute interaction with the stationary phase
could not be ignored in the prediction of RP retention.
However they found the UNIFAC method to be useful for
predicting changes in relative solute retention with varying
mobile phase composition, since the solute activity
coefficients in the mobile phase could be accurately
calculated using UNIFAC parameters (Petrovic et al., 1985).
Hydrophobicity is the physical descriptor which has
most accurately been used to predict RPLC retention.
Hydrophobic effects between solutes and hydroorganic mobile
phases were described earlier in this chapter. Solute
hydrophobicity is usually described in terms of the pi scale
developed by Hansch and Leo (1979). By evaluation of solute
partition coefficients between n-octanol and water (P) they
were able to establish substituent hydrophobicity
parameters. The logarithm of the partition coefficient is
determined for both a compound containing the substituent
group and the parent compound; the difference in these two
log P values is pi, the hydrophobicity parameter for the
substituent (Melander and Horvath, 1980). Jinno (1982) and
Jinno and Kawasaki (1984a and 1984c) use the descriptors pi,
HA and HD, where HA is the number of electron acceptor
groups and HD is the number of electron donor groups, to

11
correlate with RPLC retention for substituted benzenes
(excluding phenol). They found a very good correlation
between In k and pi, and poor correlations between In k1
and molecular connectivity, correlation factor (number of
double bonds plus number of primary and secondary carbons
minus 0.5 for nonaromatic rings) and van der Waals volume
and surface area. They interpret this to mean that the size
and shape of these molecules were not dominant for
controlling their retention. However, when the size and
shape of the solute molecules are a dominant retention force
such as for PAHs and hydroaromatics, the correlation factor
(F) has been found to correlate quite well with log k1
(Hurtubise et a 1., 1982). If a linear combination of HA, HD
and pi descriptors was used, they found an even better
correlation between predicted and experimental k1 values.
For phenol solutes, hydrophobicity alone was not an adequate
retention descriptor so they added Hammett's acidity
parameters (sigma) to account for the strong hydrogen
bonding ability of the phenols (Jinno and Kawasaki, 1984a
and 1984c). Linear combinations of pi and sigma for the
phenol solutes resulted in excellent correlations with
retention behavior. However, for both sets of solutes the
parameters had to be multiplied by certain proportionality
constants in order to correctly predict retention and these
constants were different not only for different organic
modifiers in the mobile phase but also for each volume
composition. This requires that at every different mobile

12
phase composition a least squares fitting for a large data
set must take place in order to determine these
proportionality constants, requiring a tremendous amount of
data both in terms of pi (and possibly sigma) values for
each solute and in terms of retention values for these
solutes at each mobile phase composition. Jinno and
Kawasaki (1984a and 1984c) also have not shown that this
model is applicable for larger more complex molecules.
Funasaki et a 1 (1986) examined retention behavior for
alcohols and ethers with positional and geometric isomers
and examined the correlations between the solute's log k1
and molecular cavity surface area (S), the logarithm of the
aqueous solubility (log Cw) and the logarithm of the
octanol-water partition coefficient (log P). As previously
mentioned in this chapter, it was found that S and k' were
very well correlated, even in the case of conformational
isomers. This is because S for a molecule in water is
defined as the area of the surface traced out by the center
of a water molecule rolling over the van der Waals surface
of the solute molecule (Funasaki et a 1 ., 1986 ). However S
is very difficult to accurately calculate, requiring the
construction of solute molecular models for rigorous work,
as well as detailed knowledge of the molecular conformation
of the molecule of interest. If S is calculated from group
surface areas, an easier but less rigorous approach, the
molecular surface area is usually overestimated for very
crowded molecules. Using S to predict retention does have

13
one very strong merit--since the slope of a log k1 versus S
plot is related to interfacial tension, the dependence of
log k on the organic modifier content of the mobile phase
can be predicted (Funasaki et a!., 1986).
Funasaki et al. (1986) found the correlation between
log k' and the logarithm of solute aqueous solubility, Cw,
to be rough at best. The main drawbacks to this method are
that the extent of correlation will depend on whether Cw was
measured for the compound in the gas, liquid or solid state,
that some compounds are infinitely water soluble and that
the correlation is particularly poor for branched solutes.
In contrast, they found that the correlation between the
logarithm of the solute octanol-water partition coefficient
(log P) and log k' was particularly good for the solutes
examined (alcohols and ethers) in methanol/water mobile
phases. Braumann (1986) reports that other workers have
found good correlations between log k' and log P for a
variety of compounds including PAHs, alkylbenzenes,
substituted benzenes, pesticides, phenols and barbiturates;
again correlations were much better with methanolic mobile
phases than with those containing acetonitrile, due to
methanol's hydrogen bonding properties. One drawback to
this method is that octanol-water partition coefficients are
measured via shake-flask methods, which are very time
consuming. However, P values are tabulated for many
compounds and additivity rules using Hansch's pi parameters
may be used to estimate P for other compounds although

14
estimated P values do not in general correlate as well with
retention as measured ones (Braumann, 1986; Funasaki et al.,
1986). This approach is very much like that of Jinno and
Kawasaki (1984a and 1984c) previously discussed except that
Jinno and Kawasaki examined substituent group hydrophobicity
(pi parameters) in relation to retention whereas Funasaki et
al ( 1986 ) examined its relation with overall molecular
hydrophobicity (log P).
Funasaki et al. (1986) also examined the effect of
temperature and mobile phase composition on the degree of
correlation between log k' and log P. Not s u rpri s i ng 1 y ,
they found that they were better correlated when both were
measured at the same temperature than when they were
measured at different temperatures. They also found that
the log k' value estimated from extrapolation to zero
percent methanol (i.e. totally aqueous) mobile phase gave a
better correlation with log P than those obtained with
hydroorganic mobile phases. As in all other cases
previously discussed, the extent of correlation will be
dependent upon the test solutes, the chromatographic column
and the experimental conditions used (Funasaki et al.,
1986 ).
In summary, most of the retention indices based on
solute descriptors tend to accurately predict retention for
certain small sets of similar compounds; they are by no
means universal. Additionally, some of them require
extensive calculations and/or experimental data in order to

15
predict retention. At present, none of these solute
descriptor index systems is adequate for reliable prediction
of RPLC retention.
Empirical Prediction of Retention
Jandera and coworkers (Colin et al., 1983a; Jandera,
1986; Jandera et al., 1982; Jandera and Spacek, 1986) have
developed an empirical model to predict absolute or relative
retention. They assumed that the stationary phase
contribution to retention is very small compared to that of
the mobile phase and that nonpolar interactions between the
solute, stationary phase and mobile phase cancel each other.
If this is true, the energy of transfer of the solute from
the mobile to the stationary phase will depend on the
interaction energy (energy of cohesion) between mobile phase
molecules and the interaction energy between the mobile
phase and solute. They defined an interaction index,
determined from retention data in hydroorganic mobile phase
systems, which describes polar interactions between solute
molecules and the mobile phase components.
The interaction index for a solute (Ix) can be
determined if the volume of interaction (Vx) for the solute
and the column phase ratio (Vs/Vm) are known, since in this
model ,
log (kx'/Vx) log ((Vs/Vm)/Vx) = A BIx (1)
where A and B are constants which depend on the stationary
and mobile phases used. Jandera et al. (1982) plotted
(log kx log (Vs/Vm))/Vx versus solute polarity (based on

16
Snyder's polarity index) to determine the value of Ix for
"standard" solutes with different functional groups, and
then found the average value of these Ix's for a given
solute in many different mobile and stationary phases. They
then assumed that Ix will be constant for compounds which
undergo the same type of interactions with the mobile phase
as the "standard" solute does. Therefore if Vx for a similar
compound is known, its retention can be predicted from
equation 1, since A, B and I are already known from the
standard data (Jandera et al., 1982).
Although this model is useful in a practical sense
since it is based on empirical considerations, it cannot be
completely justified in a theoretical sense. Although the
solute experiences stronger interactions with the mobile
phase than with the stationary phase, Jandera et al. (1982)
do not prove that stationary phase interactions can be
completely ignored. They also state that the standard
compounds must be chosen "correctly" or else the predictive
power of the model fails; "correct" solutes will be those
with little or no specific interactions (such as hydrogen
bonding) between the solutes and mobile phase components.
This severely limits the types of compounds whose retention
can be predicted from this model. Finally, at present the
model cannot give very accurate (about 5 to 20% accuracy)
retention predictions (Jandera et al., 1982).

17
Theories of Retention in RPLC
Solvophobic Theory
In order to truly understand the retention process in
RPLC and thereby be able to predict solute retention, the
retention process must be examined at the molecular level.
At present there are two main schools of thought on the
retention mechanism of RPLC at the molecular level. The
solvophobic theory espoused by Melander and Horvath (1980)
states that RP retention comes about from solute binding
onto the stationary phase from the mobile phase and is
mainly due to hydrophobic interactions between the solute
and the mobile phase. Other workers have utilized
statistical mechanical analysis based on mean field lattice
theory to show that RPLC solute retention is due to solute
partitioning from the mobile phase into the bonded
stationary phase chains (Dill, 1987a and 1987b; Marqusee and
Dill, 1986; Martire and Boehm, 1983). The main tenets of
both of these proposed theories will be outlined below.
Melander and Horvath's (1980) solvophobic theory
assumes that the mobile phase plays the dominant role in the
RPLC retention process. This is because the stationary
phase is nonpolar; therefore the only attractive forces
occurring between the stationary phase and a nonpolar solute
will be van der Waals forces, which are weak and
nonspecific. They attribute the interactions between the
solute and the mobile phase to a type of hydrophobic effect,
which was discussed earlier in this chapter. In the

18
specialized RPLC environment, they have adopted a variation
of this effect, termed the "solvophobic" theory, since the
hydrophobic theory assumes a totally aqueous environment and
RPLC mobile phases are generally a mixture of aqueous and
organic components. Solvophobic theory is based on a theory
of solvent effects on chemical equilibria developed by
Sinanoglu (1968). The theory states that chromatographic
retention is based on the free energy change as the solute
is transferred from a hypothetical gas phase at atmospheric
pressure to the mobile phase. The energy involved in this
process is calculated in two steps. In the first, a cavity
of the proper shape and size for the solute molecule is
formed in the solvent. In the second, the solute enters the
cavity and interacts with the surrounding solvent molecules
via van der Waals and electrostatic interactions (Melander
and Horvath, 1980) .
The free energy change accompanying the mobile phase
cavity formation comes about from the fact that the solvent
surface area will increase by the molecular surface area of
the solute (Melander and Horvath, 1980). Therefore the
mobile phase free energy will increase by an amount
proportional to the solvent surface tension and the increase
in area. The change in free energy due to the interaction
of the solute with the surrounding solvent molecules will be
due to chemical and entropic effects. The chemical effects
are van der Waals interactions and electrostatic effects.
The van der Waals interaction energies are a function of the

19
polarizability and ionization potentials of the solute and
solvent species. Electrostatic interactions consider both
dipole and ionic effects; dipole effects are calculated from
the solute dipole moment, polarizability and molecular
radius as well as from the dielectric constant of the
solvent. Ionic effects are calculated from conventional
electrostatic theories such as Debye-Huckel treatments. The
entropic term is a measure of solute "free volume," which is
the volume that the molecule encounters before colliding
with another molecule. The "free volume" is assumed to be
proportional to the solute molar volume (Melander and
Horvath, 1980).
Although the solvophobic theory outlined above is
pertinent to a 1 iquid-1 iquid system, the bonded stationary
phase has not been considered in this process. Melander and
Horvath (1980) regard the change in free energy for
retention to be a combination of the mobile phase effects
just described and a small contribution from the adsorption
of the solute onto the stationary phase surface. This
adsorption is viewed as a reversible reaction between the
solute and stationary phase to form an associated complex.
The free energy of adsorption is quantified as the van der
Waals interaction energy between the solute and stationary
phase in the absence of solvent molecules. Although
Melander and Horvath (1980) mention that an entropic term
should be introduced to account for the restricted
translational freedom of the bonded chains at the silica

20
surface, they ignore this effect because they feel its
contribution is negligible. In summary, in the solvophobic
theory of RPLC retention, retention is mainly dependent upon
the free energy of creation of a solute sized cavity in the
mobile phase; stationary phase effects are considered to be
weak and therefore rather negligible.
Partitioning Theory
One of the most severe drawbacks to Melander and
Horvath's (1980) solvophobic theory is that it is based on a
one phase model--that of the mobile phase. But RPLC
involves two phases, the stationary and mobile phases;
therefore a one phase model is not completely applicable for
such a system. Melander and Horvath (1980) view the
retention process as if there is no true transfer of the
solute from the mobile phase to the stationary phase; the
solute is merely associated with the stationary phase
through weak adsorptive effects. Melander and Horvath
(1980) also account for the stationary and mobile phase
interactions by viewing them as bulk phases with homogeneous
properties throughout. In reality, the stationary
phase/mobile phase boundary is a highly heterogeneous area
consisting of the core silica particles, the alkyl chains
bonded to the silica surface, residual silanol groups
remaining on the silica surface and the mobile phase
solvating these silanols and the bonded chains. In such a
heterogeneous system, it is highly unlikely that bulk phase
thermodynamic considerations based on ideal solution
behavior are applicable (Marqusee and Dill, 1986).

21
In their adsorption model, Melander and Horvath (1980)
use very simple descriptions of the stationary phase surface
which contain two gross oversimplifications. They imply
first of all that the bonded chains are rigid rods
containing no internal degrees of freedom. But at the
temperatures commonly used in RPLC, the bonded alkyl chains
are quite disordered (Dill, 1987a). Melander and Horvath
(1980) also use stationary phase models wherein the bonded
alkyl chains are fully exposed to the mobile phase.
However, in a hydroorganic mobile phase system the chains
cannot be fully exposed to such a highly aqueous
environment; such a configuration would be prohibitively
expensive in free energy terms (Dill, 1987a).
Dill (1987a) has proposed an alternative model of the
RPLC stationary phase surface which regards the grafted
phase as an organized "interphase" similar to those found in
surfactant aggregates such as monolayers, bilayers, micelles
and microemulsions (Marqusee and Dill, 1986). Interphases
are composed of alkyl chains that have one end anchored at
an interface; their thickness is on the order of a few
molecular dimensions. The anchored chain density (i.e. the
number of chains anchored per unit silica surface area) is
sufficiently high as to cause severe configurational
constraints. Two important properties distinguish this
system as an interphase; its surface area/volume ratio is
high and its properties vary with the distance from the
anchored end. The relationship between orientational order

22
and distance from the silica surface is termed a "disorder
gradient"; the bonded chains have much greater orientational
order at the anchored ends and this order decreases with the
distance from the attached end. This is in contrast to bulk
matter phases in which by definition properties are
invariant with spatial position (Dill, 1987a).
In Dill's (1987a) retention theory, the nature of the
retention process is dependent upon the nature of the
molecular organization within the interphase. There are
three factors which determine this organization: the first
is those constraints imposed by the surface density and
chain lengths of the alkyl groups bonded to the surface and
by the surface's geometry; the second requirement is that in
highly aqueous mobile phases the interphase region must
largely exclude the solvent due to hydrophobic effects; the
interphase volume will be filled by chain segments and
solute molecules. The final requirement is that the chains
adopt as much disorder as is consistent with the other two
constraints in order to conform to the second law of
thermodynamics. This approach allows the consideration of
any possible geometry of the silica surface onto which the
chains are bonded. Dill (1987a) assumes that the silica
surface is approximately planar; in terms of molecular
dimensions this should be a good approximation for
chromatographic silicas, which commonly have pore diameters
of 60 to 100 Angstroms or more; the effects of curvature
will be small unless the radii of curvature are a few
molecular chain lengths or less (Marqusee and Dill, 1986).

23
Dill (1987a) considers partition and adsorption
separately as alternative RPLC retention mechanisms. In
both cases a lattice interphase model is used for the bonded
phase surface and statistical thermodynamic calculations are
used to predict solute retention in the system (Dill, 1987a;
Marqusee and Dill, 1986; Martire and Boehm, 1983). Dill
(1987a) predicts the equilibrium partition coefficient for a
solute from the mobile phase to the stationary phase from
the chemical potentials of the solute in the mobile phase
system and in the bonded chain interphase. These
calculations include the entropy of mixing of the solute and
solvent in the mobile phase, the decrease in configurational
entropy of the bonded chains when the solute is inserted
within the interphase and the total contact free energy of
the system, which will be due to intermolecular interactions
of the molecules with their neighbors. After careful
consideration of both the partitioning and adsorption
retention mechanisms in conjunction with his interphase
model and available experimental evidence, Dill (1987a)
concludes that the principal retention mechanism in RPLC is
partitioning due to two lines of evidence. Partitioning
will be affected by the surface density of the bonded alkyl
chains; adsorption will not. Therefore if partitioning is
dominant, after a certain critical bonding density, solute
retention should decrease with increasing alkyl chain
surface density. It has been observed that there is less
solute retention in bonded alkyl phase stationary phase

24
systems than in the corresponding bulk alkane systems (Colin
et a 1 1983b). Although Melander and Horvath (1980) have
interpreted this as favoring an adsorption mechanism, Dill
(1987a) interprets this in terms of partial chain ordering
in the stationary phase, leading to less retention than in
the completely disordered bulk alkane. This is supported by
the work of Lochrnuller and Wilder ( 1979 ) since solute
methylene selectivities should be unaffected by the
molecular organization of the interphase (Dill, 1987a).
The other line of evidence is that In k1 for congeneric
sets of molecules can be linearly correlated with In P
(octanol-water partition coefficient) with a slope of one.
A slope of one is expected for the partitioning mechanism,
since all of the solute surface area would be available for
partitioning within the interphase. The slope for the
adsorption mechanism is expected to be considerably less
(about 1/6) because only a small fraction of the solute
surface area would contact the hydrocarbon chains, giving a
smaller driving force for retention (Dill, 1987a).
Experimental evidence has resulted in linear plots of In k1
versus In P with a slope of one (Melander and Horvath,
1980).
Based on the partitioning mechanism of retention, Dill
(1987b) predicts that at low surface densities (less than
2.7 micromoles of bonded alkyl chains per square meter of
silica surface) nonpolar solute retention will increase
linearly with increasing surface density. At these low

25
densities, chain configurational constraints are very small
and interphase chain packing will have no effect on solute
retention; solute retention will increase as the volume of
chains increases since there will be more alkyl phase for
the solutes to partition into. At a chain density of zero
(bare silica) the nonpolar solute will be unretained (Dill,
1987b).
Once a critical bonding density (predicted to be about
2.7 umol/rn^) is reached, the bonding density is high enough
for severe configurational constraints to result. In the
Dill (1987a) retention model, the free energy involved in
retention is determined by the differences in the free
energy between the creation of a solute sized cavity in the
interphase region and the destruction of a solute sized
cavity in the mobile phase. As alkyl bonding density is
increased past the critical bonding density, the chain
packing constraints become more and more severe, requiring
larger amounts of energy to create a solute cavity in the
interphase structure. Thus in the high density region
solute partitioning is predicted to decrease with increasing
alkyl bonding density due to the increasingly prohibitive
expenditure necessary for interphase cavity creation to
accomodate the solute. Dill (1987a) predicts that at 8.1
umol/m^, the maximum achievable bonding density if every
surface hydroxyl group on the silica is derivatized with an
alkyl ligand, solute retention would be zero and the solute
would be completely excluded from the interphase chain
packing structure.

26
Some researchers have examined solute retention as a
function of increasing alkyl chain length of the bonded
ligands. Colin et al. (1983b) and Jinno and Kawasaki
(1984c) found that log k' increased with increasing bonded
chain length, while Melander and Horvath (1980) have stated
that more or less contradictory results have appeared in the
literature on the influence of chain length. Spacek et al.
(1980) and Berendsen and de Galan (1978b) have also noted a
general trend of increased retention with increasing chain
length of the bonded ligand, but their results were less
conclusive than those of Jinno and Kawasaki (1984c). It is
not clear from any of these studies whether this trend is
due to the actual increased partitioning of the solute into
the longer alkyl chains or whether this trend is an artifact
of the retention parameter measured. This is because in all
of these studies, it is the capacity factor, k' that is
used to quantitate retention. But k = K(Vs/Vm) where K is
the chromatograpnic partition coefficient and Vs/Vm is the
volume phase ratio of the stationary and mobile phases. It
is obvious that as the chain length of the bonded alkyl
ligand is increased, a corresponding increase in V $ will
occur, as pointed out by Colin et al. (1983b). Therefore it
is unclear as to whether solute retention increased because
of increasing partition coefficient or merely because of the
phase ratio increase. In order to reliably determine the
cause of changes in actual solute retention as stationary
phase parameters are changed, the changes in the solute
partition coefficient must be examined.

27
In this study, we examined the effects of octadecyl
alkyl chain bonding density on both retention and selectvity
of small nonpolar solutes. We were particularly interested
in experimental verification of the molecular mechanism of
RPLC retention proposed by Dill (1987a and 1987b). Although
Sander and Wise (1984a and 1984b; Wise and Sander, 1985) and
Wise and May (1983) have extensively examined the effect of
alkyl bonding density on retention and selectivity for PAHs,
they have mainly examined polymeric stationary phases, which
are not as well structurally characterized as the monomeric
stationary phases used in our study. Another problem with
their work is that they examined capacity factor (k1)
behavior in their retention studies, which fails to account
for phase ratio changes. Additionally, PAHs are not ideal
solutes for such a study, since their large sizes and
unusual shapes are not typical of most chromatographic
solutes.
In order to determine the effect of interphase chain
packing on solute partitioning, the behavior of the
chromatographic partition coefficient was examined as a
function of octadecyl bonding density. Chromatographic
selectivity was also studied as a function of bonding
density for solutes of different sizes and shapes. Novel
synthetic methods utilizing ultrasound as a reaction driving
force were devised to obtain stationary phases with high
bonding densities. In this manner, we were able to see if
Dill's (1987a and 1987b) proposed RPLC retention mechanism

23
was verified by actual chromatographic behavior. These
experiments were carried out using both silica and
controlled pore glass substrates in order to compare the two
materials as stationary phase supports.

CHAPTER II
SYNTHESES OF SILICA-BASED RP STATIONARY PHASES
Experimental Considerations in the Synthesis of RP
Stationary Phases
Reversed phase bonded silicas are the most popular
packings used in high performance liquid chromatography
(HPLC). Although the role of the mobile phase in
chromatographic retention and selectivity has been
extensively studied, that of the stationary phase has only
come under intense scrutiny recently and as a result the
effects of the stationary phase on these chromatographic
properties is not yet fully understood. One reason for this
dearth of knowledge is the lack of precise and reliable
methods for determining bonded phase characteristics such as
the density, homogeneity and topographical distribution of
the bonded alkyl ligands and the residual hydroxyl groups on
the support surface. These properties are a direct
consequence of the bulk silica medium and the reagent and
reaction conditions for the silanization process (Kinkel and
Unger, 1984). In order to obtain reversed phase packings
with reproducible surface characteristics, the silanization
reaction conditions must be painstakingly controlled.
In the preparation of reversed phase packings, one
objective is the modification of as many surface hydroxyl
29

30
groups on the silica as possible, especially the highly
acidic isolated si lands. These residual isolated silanol
groups have been shown to be the main cause of tailing of
chromatographic peaks for basic compounds, of mechanical
instability of the packing, and of low sample capacity of
the column (Kohler et al., 1986; Kohler and Kirkland, 1987).
Di- or trireactive alkylsilanes had previously found favor
over monoreactive silanes because of their greater
reactivity and the possibility of reacting simultaneously
with two or three hydroxyl groups. However, any unreacted
sites on the bonded functional groups will be hydrolyzed
upon contact with water (i.e. from the mobile phase),
forming additional undesirable silanol groups (Berendsen and
de Galan, 1978b; Snyder and Kirkland, 1979). Di- and
trireactive silane reagents also often result in
nonreproducible stationary phases since the degree of
polymerization is highly dependent on the residual water
content of the silica and the reagents used in the bonding
reaction (Snyder and Kirkland, 1979). Another drawback of
polymeric stationary phases is their lower chromatographic
efficiency, which results from poor solute mass transfer in
these relatively thick stationary phases. Therefore many
investigators now advocate the use of monofunctional silanes
for the silica derivatization reaction, since this results
in a reproducible and well defined chemically bonded phase.
Additionally, monomeric stationary phases generally exhibit
superior column performance to polymeric phases due to their

faster solute mass transfer kinetics (Cooke and Olsen,
1980). For octadecy1 dimethy1ch1 oros i 1ane, the most commonly
used monoreactive silane, the resulting bonding reaction is
depicted in Figure 2-1.
Kinkel and Unger (1984) have studied the roles of the
solvent and the base in these monofunctional bonding
reactions and have found their choice to be crucial. When
al ky 1 halosi 1anes are reacted with silica, a base is added to
serve as the acid-acceptor catalyst, binding the haloacid
formed during the reaction and driving the equilibrium to
the product side. In addition, the base favorably affects
the kinetics of the silanization reaction. Mechanistic
studies of these types of reactions (Corriu and Guerin,
1980) have shown that two molecules of base attack one
molecule of silane, activating the Si-X bond such that a
reactive intermediate and a hydrohalide are formed.
Formation of this reactive intermediate greatly increases
the kinetics of the bonding reaction; indeed, the addition
of the acid-acceptor catalyst results in approximately 90%
of the total conversion taking place within the first hour
of the reaction. In their study, Kinkel and Unger (1984)
found that the two most effective acid-acceptor catalysts
for organohalosilanes were imidazole and 2,6-lutidine.
The reaction solvent must also be carefully chosen.
The solvent can interact specifically with the silane, the
base and the surface silanol groups on the silica. When the
solvent interacts with a silanol group, there is a

CHs
i
Si-O-H + CI-SMCI-WirCHs
i
ch3
Figure 2-1. Bonding reaction for monomeric
reversed phase packings.
CHs
Si-0-Si-(CH2)i7 CH3 + HCI
CH3
octadecy1
CO
IX}

33
considerable effect on the strength of the bond between the
silicon and oxygen atoms. Solvents which have both a
pronounced Lewis acid and Lewis base character cause the
Si-0 bond strength to be weakened and facilitate the bonding
reaction. The solvent can also activate the silicon atom of
the organohalosilane by forming a pentacoordinated
intermediate through nucleophilic attack. The resultant
bond lengthening causes nucleophilic activation to occur,
favoring attack by a second nucleophile (such as the base).
The solvent may influence the base as well, as it is known
that in aprotic polar solvents the nucleophilic character of
reactants is more pronounced. All of these considerations
may have a synergistic relationship as well. Based on their
experimental work with organoha1 os i 1anes, Kinkel and Unger
(1984) found that methylene chloride and N,N-dimethyl-
formamide were the most effective solvents for the bonding
reaction.
Many organic reactions have been shown to be enhanced
by ultrasound (Boudjouk, 1986; Brernner, 1986; Clough et al.,
1986; Han and Boudjouk, 1982 and 1983; Suslick, 1986).
Boudjouk and Han (1981) have shown that in the presence of
ultrasonic waves both alkyl and aryl ch1 oros i 1anes could be
coupled over lithium wire; without ultrasonification, this
reaction occurred to no appreciable extent. Reactions at
solid-liquid interfaces are also particularly enhanced by
ultrasound (Brernner, 1986; Suslick, 1986). It is then
reasonable to assume that reversed phase bonding reactions

might be facilitated under ultrasonification. The use of
ultrasound has two distinct advantages over traditional
reflux methods. The ultrasonic waves serve as a driving
force which is controlled independently of temperature,
allowing reaction temperatures to be varied over any desired
range. Secondly, the power of the ultrasonic driving force
can be varied by using a variable power ultrasonic probe.
We have investigated the effect of ultrasound on the
silane bonding reaction, including the effects of subambient
and superambient temperatures on the ultrasonic reaction.
In addition to these investigations, a novel base,
4-dimethyl aminopyridine, was utilized as the acid-acceptor
catalyst, in hopes that it might prove superior to
2,6-lutidine.
Experimental Procedure
Reagents
All of the organic solvents used were supplied by
Fisher Scientific (Fair!awn, NJ). Water was deionized,
passed through a Barnstead Nanopure (Boston, MA)
purification system, irradiated in a Photronix Model 816
HPLC reservoir with a UV source (Photronix Corp., Medway,
MA) for at least 48 hours, and filtered through a 0.45 pm
Nylon 66 membrane (Rainin, Woburn, MA). The methanol used
was HPLC grade; the chloroform, methylene chloride, and
diethyl ether were reagent grade. Methylene chloride was
dried by stirring over phosphorus pentoxide (Fisher
Scientific) for 24 hours, followed by distillation under a
dry nitrogen atmosphere.

35
Dimethyloctadecylchlorosilane, n-octyldimethylchloro-
silane and trimethy1ch1 oros i 1 ane (99.9%) were used as
received from Petrarch Systems (Bristol, PA). The
2,6-lutidine (Sigma Chemical Co., St. Louis, MO) was stirred
for 24 hours over barium oxide (Fisher Scientific) prior to
distillation under dry nitrogen atmosphere; 4-dimethy1 ami no
pyridine (4-DMAP; Nepera Inc., Harriman, NY) was oven dried
at 80 C for 24 hours before use.
The chromatographic silica was from a single lot of
Davisil (W. R. Grace, Baltimore, MD) synthetic amorphous
silica, grade 641LC0X1823. The silica had an average pore
diameter of 147 Angstroms, an absolute surface area (SB£j,
as measured by BET analysis) of 300 m^/g, a particle size
range of 20-30 pm with an 80% distribution of 23 + 10 pm and
a nitrogen pore volume of 1.10 cm3/g (Grace, 1984). As
recommended by Snyder and Kirkland (1979) the silica was
acid leached in 0.1 M nitric acid at 90 C for 24 hours in
order to fully hydroxylate the surface and to remove any
metal contaminants remaining from the manufacturing process.
The silica was then washed thoroughly with water until all
traces of the nitric acid had been removed and dried under
vacuum at 240 C for 24 hours prior to use in order to
remove physically adsorbed water from the surface (Unger,
1979). Scanning electron micrographs of the acid-leached
silica (Figures 2-2 and 2-3) show that this type of
chromatographic silica exhibits an irregular shape as well
as an irregular surface. This surface irregularity is
reflected in the silica's high surface area.

Figure 2-2. Scanning electron micrograph of acid-leached Davisil
silica; 500X magnification.
CO
Ch

Figure 2-3. Scanning electron micrograph of acid-leached Davisil
silica; 3000X magnification.
CO

Silane Bonding Reaction
It is essential that the silane bonding reaction be
carried out under scrupulously dry conditions in order to
prevent the water-initiated dimerization of the silane
reagent. Glassware used in the derivatization reaction was
presilanized by etching the surface with a 10% (v/v)
hydrofluoric acid (Fisher Scientific) solution, drying, and
then soaking the glassware for an hour in a 5% (v/v)
trimethylchlorosilane in chloroform solution. Immediately
prior to use, the glassware was oven dried at 125 C for at
least 4 hours in order to remove trace moisture and allowed
to cool in a dry box under nitrogen atmosphere. The reagents
were mixed together in the dry box and the reaction flasks
kept under dry nitrogen atmosphere at all times. Based on
Kinkel and Unger's (1984) estimation of a maximum of five
micromoles of reactive hydroxyl sites per square meter of
silica surface, a twofold excess of the silane reagent was
added to achieve exhaustive derivatization of the silica
surface. A fourfold excess of the base (2 ,6-1utidine or
4-DMAP) was added both to serve as an acid-acceptor catalyst
for the HC1 produced in the reaction and to act as a
reactive intermediate at the silica-solution interface. Dry
methylene chloride was used as the reaction solvent, using a
ratio of 10 ml of methylene chloride per gram of base
silica.
The reaction flasks were sonicated by immersion to the
flask neck in an ultrasonic cleaning bath (Bransonic model

39
B-2200R-1, Branson Cleaning Equipment Co., Shelton, CT) with
a power rating of 100 W and a frequency of 55 kHz. Stirring
of the reagents within the flasks was accomplished by
rotating a magnetic bar submerged in the bath adjacent to
the reaction flask, resulting in the corresponding rotation
of a magnetic stirring bar within the flask. Temperature
control of the ultrasonic bath was accomplished by passing a
thermostatted solution of ethylene glycol and water through
coiled copper tubing lining the inner perimeter of the bath.
The solution was thermostatted by an Endocal or Exacal water
bath (Neslab Instruments, Portsmouth, NH). Refluxed
reactions were carried out at 50 C using an oil bath and
magnetic stirrer. Control reactions were carried out by
stirring the reaction mixture at room temperature.
Refluxed reactions utilizing n-octyldimethylchloro-
silane as the reactive silane, 2,6-lutidine as the acid-
acceptor catalyst and methylene chloride as the reaction
solvent were carried out for reaction times of 24, 36 and 48
hours in order to ascertain whether the differences in
reaction time would make a statistically significant
difference in the reaction yield. Nine replicate reactions
were performed for each reaction time. Student's t (tca]c)
was calculated from the pooled standard deviation of percent
carbon for all 27 reaction yields, the differences in the
mean percent carbon at each reaction time, and the number of
replicates at each time in order to determine if the mean
yield for each reaction time was statistically different

40
from those at the other reaction times. In the comparison
of the 24 hour reactions to the 36 hour reactions, tca-]c was
0.984; for the 24 hour reaction time versus the 48 hour
reaction time, tca-|c was 0.148 and for the 36 hour reaction
time versus the 48 hour reaction time, tcalc was 0.835.
Since all of the calculated t values are less than the
critical t value (tcrit) at the 80% confidence level
(tcrit,80%= 1*34 for 16 degrees of freedom), there is no
significant statistical difference in the reaction yields
for reaction times of 24, 36 and 48 hours at the 80%
confidence level (Peters et al., 1974). Therefore,
reactions were carried out for 24 hours (as also recommended
by Kinkel and Unger (1984)) unless otherwise noted.
Once the reaction time was complete, the product was
washed in order to remove excess reagents. Each bonded
phase product was washed three times with each solvent using
the rinse sequence methylene chloride, methanol, 50/50 (v/v)
methanol/water, methanol and diethyl ether. After the ether
was allowed to evaporate from the product, the derivatized
silica was dried in a vacuum oven at 125 C for 16-24 hours.
(Caution: It is imperative that the ether be completely
evaporated from the product prior to drying the product in
the vacuum oven in order to avoid a possible explosion.)
Products were analyzed by in-house elemental analysis
performed at least in duplicate for each sample.
Reliability of the elemental analysis was confirmed by
repeated submission of a standard packing material over a

41
two year period; for 66 measurements the resultant standard
deviation was + 0.20% carbon.
Syntheses Utilizing Ultrasonic Waves
Use of Ultrasound as a Reaction Catalyst
The region of frequencies above 16 kHz is beyond the
sensitivity of the human ear; it is therefore termed the
ultrasound region. The first reported use of ultrasound in
organic chemistry was in 1938, but it was not until the late
1970s that ultrasound was used to speed reactions in
nonaqueous media; indeed the use of ultrasound as a reaction
catalyst is still in its infancy (Boudjouk, 1986; Bremner,
1986). Ultrasonic radiation can be introduced to the
reaction medium either by immersion of the reaction vessel
into the liquid of a common laboratory ultrasonic cleaning
bath or by introduction of an ultrasonic generating probe
directly into the reaction medium.
Ultrasonic frequencies span the range of 20 kHz to 10
MHz, with associated acoustic wavelengths of 7.6 to 0.015
cm. Therefore sonochemistry cannot be accounted for in
terms of direct coupling of the acoustic field with chemical
species on a molecular level (Suslick, 1986). However, the
effects of ultrasound can be attributed to three different
phenomena. The variation of sonic pressure causes the rapid
movement (oscillation) of fluids, subjecting them to
compression and rarefaction. Negative pressure in the
rarefaction region gives rise to cavitation, the formation
and collapse of microbubbles. The violent implosion of

42
these microbubbles generates powerful shock waves with a
considerable energy output (Boudjouk, 1986; Bremner, 1986;
Suslick, 1986). Pressures in the kilobar range and
temperatures of 2000-3000 C have been estimated in the
region of the collapsing bubble for time periods in the
nanosecond range (Sehgal et al., 1980). The third
contributing phenomenon is microstreaming, where a large
amount of vibrational energy is put into small volumes with
little heating (Bremner, 1986). The extremes of temperature
and pressure generated by ultrasonic waves cause the
generation of free radicals and ions, the dispersion of
chemical layers and the promotion of intimate contact
between reactants. Emulsification of immiscible liquids and
enhanced mass transfer at solid-liquid interfaces are
secondary effects of u11ras on ification. All of these
effects can contribute to the promotion of chemical
reactions (Bremner, 1986; Suslick, 1986).
Effect of Ultrasound on the Bonding Reaction
In order to define the surface coverage of the bonded
silica in an unambiguous and pertinent manner, the surface
coverage should be expressed as the number of silane
molecules attached to the surface, usually as micromoles of
bonded silane molecules per square meter of silica surface,
taking into account the increase in weight of the silica
after the bonding reaction. These surface coverages are
calculated from the percentage of carbon as obtained from
elemental analysis of the bonded phase (Berendsen and de

43
Galan, 1978b). This calculation is quite straightforward
for monoreactive silanes and for monoch1 oros i 1anes (the most
commonly used monoreactive reagents) can be expressed by
a = t (%C) (IQ6) (1)
(12.011) (nc) (S) (100-L(%C/(12.011)(nc)](M-36.5) )
where a is the surface coverage (pmoles/m^); %z is grams
carbon per 100 grams bonded silica, as obtained from
elemental analysis; nc is the number of carbon atoms per
mole silane; M is the molecular weight of the silane; and S
is the surface area of the native silica in m^/g. Although
the typical value for the average surface hydroxyl
concentration of amorphous silica is 8 pmol/m^ (Cheng and
McCown, 1985), in practice octadecyl bonded phase coverages
are limited to about 3 pmol/m^ due to steric considerations
(Berendsen et al. 1980a; Berendsen and de Galan, 1978a and
1978b; Cheng and McCown, 1985; Snyder and Kirkland, 1979).
Three sets of experiments were compared in order to
determine the effect of ultrasound on the silica bonding
reaction. In all cases dimethyloctadecylchlorosilane was
the reactive silane, methylene chloride was the reaction
solvent and 2,6-lutidine the acid-acceptor catalyst; all
reaction mixtures were stirred during the reaction time
period (24 hours). In the first set of experiments, the
reaction mixture was stirred at ambient temperature (22.0
C); in the second set the reaction mixture was refluxed at
50.0 C, The third set of experiments was performed at 28.5
C, but the reaction vessels were immersed in an ultrasonic

44
cleaning bath. The refluxed stationary phases had an
average bonding density (_+ one standard deviation over three
trials) of 2.82 + 0.02 umol/m2. The room temperature
reaction resulted in a bonding density of 2.69 umol/m2 with
a range of + 0.03 ymol/m2 over two trials; the ultrasound
reaction gave a bonded phase (over two trials) with an
average bonding density of 2.71 + 0.01 ymol/m2. The small
bonding density difference between the stirred reaction at
ambient temperature and the one at reflux temperature is not
surprising as Lork et al. (1986) have shown that the bonding
density increases slightly and in a linear fashion with
increasing reaction temperature when monoch1 oros i 1 anes are
used as the silanizing reagent. These experimental results
show that ultrasound is indeed a viable method for the
bonded phase synthesis, giving results which are comparable
to those obtained using traditional reflux techniques.
Effect of Subambient Temperature on the Ultrasound
Reaction
Two sets of experiments were performed using ultrasound
in conjunction with subambient reaction temperatures. In
achieving high bonding densities one of the greatest
obstacles is increasing steric hindrance at the silica
surface as more and more bulky dimethy1octadecy1sily1 groups
are bonded to the surface. It is possible that at low
temperatures the bonding density might be enhanced due to
the increased order (decreased entropy) in a lower
temperature system. It is here that the ultrasound reactions
are most unique, as they allow the temperature of the

reaction to be controlled independently of the ultrasonic
driving force. Additionally, low reaction temperatures have
often been found to enhance reaction yields for
ultrasonically catalysed chemical reactions. One
explanation for this phenomenon is that low temperatures
cause the vapor pressures of the reactants to be decreased,
enabling increased efficiency of u11rason ica11y produced
cavitation (Boudjouk, 1986; Bremner, 1986; Suslick, 1986).
In order to overcome the slower kinetics expected at lower
temperatures, reaction times were increased beyond the usual
24 hour time period.
In the first set of experiments, two reaction vessels
were sonicated and stirred at 15.0 C for 48 hours with a
resultant average bonding density ( + the range) of 2.74 +
0.00 y m o 1 / m 2 Since this result was little different from
that at room temperature, it was decided to increase the
reaction time as well as to decrease the reaction
temperature. In this set of experiments, two reaction
flasks were sonicated and stirred at 8.5 C for 101 hours
with a resultant average bonding density of 2.84 + 0.01
y m o 1 / m 2 a slightly higher value than for those ultrasound
reactions run at higher temperatures. These preliminary
results indicated that subarnbient temperatures could indeed
enhance the ultrasonic silica bonding reaction.
The Use of 4-Dimethyl aminopyridine as the Acid-Acceptor
Catalyst
There are also advantages in the use of 4-dimethyl-
ami nopyri di ne (4-DMAP) as the acid-acceptor catalyst. The

46
presence of the dimethy lamino group should serve to make
this base better at forming a reactive intermediate with the
silane than 2,6-1utidine. In addition it has a relatively
high melting point (108-110 C) which allows it to be
oven-dried rather than necessitating distillation to remove
adsorbed water. The odor of 4-DMAP is also quite mild in
comparison to that of 2,6-1utidine.
The first set of experiments using 4-DMAP as the
acid-acceptor catalyst was performed using methylene
chloride as the solvent and dimethy1octadecy1ch1 oros i 1 ane as
the reactive silane. The reaction mixture was refluxed and
stirred at 50.0 C for 24 hours. The bonded phase product
had an average bonding density (+ the range for two trials)
of 3.44 + 0.02 umol/m^, much higher than that achieved in
our previous syntheses using 2,6-lutidine (2.82 + 0.02
nmol/m^). This bonding density is also greater than that
(3.34 ymol/m2) achieved by Kinkel and Unger (1984) under
reflux conditions using methylene chloride and 2,6-lutidine.
This is especially significant because the silane in our
experiments was used as received from a commercial source;
Kinkel and Unger synthesized and then distilled their silane
under reduced pressure in order to obtain a reactive silane
of the utmost purity. Silane purity has been shown to be a
very important factor in obtaining high bonding densities
(Kinkel and Unger, 1984).
The second set of experiments using 4-DMAP as the
acid-acceptor catalyst was run with the same reagents as

47
described above; the reaction mixture was immersed in the
ultrasonic bath and stirred at a temperature of 31.0 C for
24 hours. The average bonding density of the resultant
bonded phases ( + the range for two trials) was 3.35 + 0.05
timol/m2, again much higher than that achieved under similar
circumstances using 2,6-lutidine as the acid-acceptor
catalyst (2.71 + 0.01 ymol/m^).
A low temperature ultrasound reaction was then carried
out under the same conditions as stated above, with a
reaction temperature of 4.0 C for a duration of 97 hours.
For the two trials, an average bonding density of 3.24 +
0.01 ymol/m^ was obtained. A second set of low temperature
ultrasound reactions was performed under analogous
conditions with a reaction temperature of 3.0 C for 144
hours. The bonded phase resulting from this experiment had
a higher bonding density than achieved in any of our
previous attempts; the average + the range for the set was
3.60 + 0.01 jjmol/m^. To our knowledge, this bonding density
is higher than any previously reported in the literature
using dimethyloctadecylchlorosi1ane as the reactive silane
(Berendsen et al., 1980a; Cheng and McCown, 1985; Kinkel and
Unger, 1984) .
To ensure that the high bonding density in this second
low temperature experiment was a result of the ultrasonic
driving force as well as the lengthy reaction time, two
other reactions were carried out. In one, the reaction was
performed exactly as above (at 3.0 C for 144 hours with

stirring) except that the reaction flask was not sonicated.
In the other, the reaction was stirred for 144 hours, but
the reaction mixture was refluxed at 50.0 C rather than
sonicated. From duplicate elemental analyses of each of
these two materials, the average bonding density (+ the
range) for the silica stirred (but not sonicated) at 3.0 C
for 144 hours was 3.48 _+ 0.00 nmol/m^j that for the silica
refluxed and stirred at 50.0 C for 144 hours was 3.44 +
0.03 ymol/m^. Since the absolute error in the elemental
analysis is + 0.20% carbon, which corresponds to + 0.03
o
iimol/m11 for the octadecyl packings, the differences in
bonding density between these two materials and the silica
which was sonicated at 3.0 C for 144 hours (3.60 ymol/m^)
is both real and significant. Therefore it can be concluded
that subambient ultrasound reactions are especially
efficacious for synthesizing stationary phases with very
high alkyl bonding densities.
In order to investigate the effect of superambient
temperatures on the ultrasonic bonding reaction, two types
of experiments were performed. In the first, the reaction
was carried out by stirring with the same reagents as
previously described for a reaction time of 24 hours and
with the ultrasonic bath maintained at a temperature of
50.0 C. For two trials, the average + the range was 3.34 +
0.04 ymol/rn2, virtually identical to that achieved under
ambient ultrasonic conditions (3.35 + 0.05 umol/m^). in the
second experiment, the reagents were stirred and sonicated

49
at 31.0 C for 1 hour and then refluxed and stirred at 50.0
C for an additional 23 hours, in hopes that the preliminary
sonication of the reagents would permit greater
accessibility of the reactive silane to silanols located
deep within the silica pores. The resulting bonding density
(for two trials) of 3.42 + 0.03 pmol/m^ is comparable to
that achieved under reflux conditions alone (3.44 + 0.02
prnol/m^). These results indicate that silica bonding
reactions performed in an ultrasonic bath are not affected
by superambient temperatures; this is in contrast to those
performed at subambient temperatures, which were found to
give an increasing yield as the temperature was decreased.
Experiments were also carried out in the ultrasonic
bath at 28.0 C for 24 hours using trimethy1ch1 oros i 1 ane
(TMCS) as the reactive silane, 4-DMAP as the base and
methylene chloride as the reaction solvent. TMCS is a much
smaller molecule than the octadecyl silane and therefore
should approximate the maximum bonding density obtainable in
these reactions when steric hindrance is minimized. The
average bonding density achieved in the two trials was 3.51
+ 0.01 pmol/m2; the octadecyl bonding densities achieved in
the above reactions show that the TMCS bonding density at
ambient temperatures can be exceeded under subambient
conditions even with bulky octadecyl reagents. The results
of these 4-DMAP experiments, as summarized in Table 2-1,
demonstrate that it is indeed a superior acid-acceptor
catalyst to 2,6-lutidine for reversed phase bonding
reactions.

Table 2-1. Comparison of silica octadecyl bonding densities using
4-DMAP and 2,6-lutidine as acid-acceptor catalysts.
Reaction
Conditions
Temperature (C)
Reaction
Time (h)
Co Bonding
1 4 D M A P
Density (ymol/m
2,6-lutidine
Ref 1uxed
50.0
24
3.44
2.82
Ultrasound
28.0
24
-
2.71
Ultrasound
31.0
24
3.35
-
U1trasound
8.5
101
-
ro
oo
-F*
U1trasound
4.0
97
3.24
-
U1trasound
3.0
144
3.60
-
Stirred Only
3.0
144
3.48
-
Ref 1uxed
50.0
144
3.44

51
The use of ultrasound as a driving force for reversed
phase syntheses has been shown to be a viable synthetic
procedure. Ultrasonic syntheses performed at subambient
temperatures have proven to be especially effective for the
production of high alkyl bonding density stationary phases.
The use of 4-dimethy1 aminopyridine as the acid-acceptor
catalyst is recommended due to its ease of use and the
resulting high bonding densities. The small ranges of the
bonding density for duplicate syntheses show that reversed
phase packings with reproducible bonding densities can be
synthesized by these methods.

CHAPTER III
SYNTHESES OF CONTROLLED PORE GLASS-BASED RP STATIONARY
PHASES
Comparison of Controlled Pore Glass and Silica as Supports
for RP Stationary Phases
The most commonly used column packing materials for
reversed phase liquid chromatography (RPLC) are based on
microparticulate silica. As has been described in Chapter
II, this material is modified by chemically bonding alkyl
chains of the desired length onto the silica surface. The
use of such siliceous supports is widespread due to their
high reactivity and relatively low cost. Silica-based RP
bonded phases also exhibit good column stability within the
pH range of 2.5 to 7.5 (Melander and Horvath, 1980).
However, such materials are not without problems. The
surface of silica gel is very porous in nature and there is
a wide distribution in the size of these pores. This can
affect chromatographic selectivity by causing size exclusion
effects. This broad pore size distribution is one of the
contributors to the problem of inhomogeneous energies of
transfer between the stationary and mobile phases for
solutes in RPLC, leading to distorted peak shapes and
decreasing chromatographic efficiency. The effects of pore
size and structure have received much attention in size
exclusion chromatography, but these parameters have garnered
52

53
little attention in reversed phase systems (Sander and Wise,
1984b). At present, there is no satisfactory explanation of
the effect of pore size distribution on the properties of
hydrocarbonaceous bonded phases; however differences in the
pore structure of the support material may account for some
of the differences observed in the RPLC behavior of various
commercial bonded phases having the same alkyl chain length
but different siliceous substrates (Melander and Horvath,
1980).
Controlled pore glass (CPG>) offers an ideal medium for
investigating the effects of pore size and structure on RP
retention and selectivity. CPG, which has mainly been used
in size exclusion chromatography, consists of nearly pure
quartz glass with pores of uniform size. In CPG, the pore
diameter is the same at the surface as it is in the interior
of the particle; 80% of the pores show a deviation of less
than + 10% from the nominal pore diameter (Fluka). Chemical
modification of the surface of CPG is accomplished by
reacting surface silanol groups with the appropriate
reactive silane, as has been described for silica in Chapter
II. Although such reactions have been performed to prepare
CPG RPLC bonded phases (Dawidowicz et a 1 ., 1983 ; Dawidowicz
and Rayss, 1985 ; Rayss et al. 1983 ; Suprynowicz et al.,
1978 and 1985) commercial bonded phases based on CPG are
currently impractical due to its much greater expense
compared to that of silica.

54
The differences in the pore structures of silica and
CPG come about from the differences in their chemical
compositions and in their manufacturing processes. The
manufacture of chromatoyraphic silica is described in detail
by Unger (1979). The starting materials in the manufacture
of porous silica are soluble silicates such as sodium
silicates, silicon tetrachloride or tetraalkoxysilanes. By
adjusting the pH of an aqueous solution of the starting
material within a range of 8 to 9, silica sols are made. In
the sol, polysilicic acids are formed by polycondensation
and polymerization, growing into colloidal particles ranging
from 1 to 100 nm. The sol consists of spherically shaped,
nonporous and amorphous discrete silica particles. Unless
stabilized, the discrete particles in the sol aggregate,
mainly due to gelling. The particles become linked together
to eventually form a three dimensional packing of silica
particles that is a gelatinous mass called silica hydrogel.
The hydrogel is washed and water is then removed by heating.
This dehydration results in shrinkage from the partial
collapse of the globular hydrogel structure; the resultant
xerogel consists of hard porous grains. The silica
particles are also cemented together by dissolution-
deposition processes. The conversion of the hydrogel to the
xerogel is the origin of the porosity of chromatographic
silica. This porosity comes about from compaction of the
dispersed silica in the hydrogel; the pore space is made up
of the interparticle interstices and voids. This results in

55
a totally porous structure; moreover these pores are quite
irregularly shaped. Factors which can be varied to control
the final pore structure include changes in the sol and/or
hydrogel pH, changes in the duration of the hydrogel
ripening (via stabilization of the sol), variation of pH
during washing of the hydrogel, and substitution of an
organic liquid wash for the hydrogel rather than an aqueous
one (Unger, 1979). The chemical composition of the final
amorphous silica can be exemplified by the composition of
the Davisil silica used in our experiments; it consists of
99.60% by weight of Si02 and 0.10% Na20 with the remaining
0.30% made up of other metal oxides (Grace, 1984).
The procedure used to produce controlled pore glasses
was first reported by Wolfgang Haller in 1965 (1965a and
1965b). The starting material consists of a Vycor type
glass consisting of 7% Na20, Z3% B203 and 70% S i 0 2. The
glass is crushed, fractionated to the desired particle size
distribution by sieving, and then heated at approximately
600 C for the desired number of hours. This heating period
causes fusion to take place within the glass, resulting in
the formation of microheterogeneous regions in the
continuous silica network. This alkali borate-rich
microphase is then removed from the glass by a series of
acidic and basic leachings, resulting in a finished material
which is porous throughout its entire volume (Dawidowicz et
al., 1983). The diameter of the pores is determined by the
length and temperature of the heat treatment (Haller,

56
1965a). The chemical composition of controlled pore glass
is also different from that of amorphous silica; the
composition of the finished product is typically 96% by
weight of SO2, 3% B2O3, less than 1% Na2, and a trace
amount of other metal oxides (Electro-nucleonics Inc.,
1987 ) .
Experimental Procedure
Reagents
All of the reagents used in the preparation of the
reversed phase CP6 packings were as described in Chapter II,
with the exception of CPG being used as the support material
instead of silica. All CPG was manufactured by Electro
nucleonics Inc. (Fairfield, NJ). The CPG denoted as CPG-86
was from a single lot of CPG-10-75A (Fluka Chemical Corp,;
Hauppauge, NY) and had a mean pore diameter of 86 Angstroms
with a pore size distribution of + 9.8%. The absolute
surface area (Sg^y, as measured by BET analysis) was 153.1
m^/g; the particle size range was 37-74 urn and the nitrogen
pore volume was 0.48 cm^/g. The CPG denoted as CPG-167 was
from a single lot of PG-170-400 (Sigma Chemical Co., St.
Louis, MO) with a mean pore diameter of 167 Angstroms and a
pore size distribution of + 9.6%. The absolute surface area
was 161 m2/g, the particle size distribution was 37-74 ym
and the nitrogen pore volume was 1.0 cm^/g.
Bonded Phase Preparation
Both CPG's were acid leached, dried and reacted with
the appropriate silane reagents for derivatization as

57
described in Chapter II. The only difference in the
procedure between the CPG and silica was in the method of
agitation. CPG is more mechanically fragile than silica;
therefore direct stirring via a magnetic stirring bar is
inadvisable. Agitation was accomplished by a rotary
evaporator; a nitrogen gas line was attached to what is
normally the vacuum outlet in order to maintain a dry
atmosphere. An evacuated glass Dewar-type condenser
(fabricated in-house) was used to join the reaction flask to
the rotary evaporator; this piece of glassware was necessary
in order to prevent the escape of the reaction solvent,
especially under reflux conditions.
Reactions were performed under ambient as well as
reflux conditions; ultrasonic reactions were also carried
out. The reaction products were washed and vacuum dried as
described in Chapter II. Evaluation of the bonding
procedure was performed via in-house elemental analysis.
Scanning electron micrographs of the acid-leached CPG-86
(Figures 3-1 and 3-2) show that like the silica used in
previous reactions, the CPG also exhibits an irregular
particle shape as well as an irregular surface. This
irregularity is not surprising considering that CPG
particles of the desired size range are obtained by
mechanically crushing and sieving bulk Vycor glass (Haller,
1965a).

Figure 3
-1. Scanning electron micrograph of acid-1eached CPG-86;
820X magnification.
U~.
CO

Figure 3-2. Scanning electron micrograph of acid-leached CPG-86;
3010X magnification.
<_n

60
Comparison of Silica and CPG Bonding Densities via Reflux
and Ultrasonic Syntheses
The controlled pore glasses of both pore sizes (denoted
CPG-86 and CPG-167) were derivatized under the same types of
reaction conditions as for the Davisil silica. Two aspects
in particular were to be examined by these experiments; the
first was to determine the reactivity of CPG compared to
that of amorphous silica and the second was to determine the
effect of pore size on CPG reactivity. From pore shape
indices, it has been found that the pore shape of siliceous
pores is not usually cylindrical; therefore the cylindrical
pore model is not a good approximation for silica (Nikolov,
1986). Since CPG has a very uniform pore diameter compared
to that of silica and the CPG pores are much larger than the
molecular dimensions of the reactive silane (the average
molecular cross-sectional area for the octadecy1ch1 oro-
silane is 50.95 (Angstromsper molecule and its length is
24.72 Angstroms (Cheng and McCown, 1985)) it was expected
that the CPG's would exhibit higher reactivity and therefore
result in alkyl bonding densities higher than those achieved
with silica. In addition, it was expected that CPG-167
would be more reactive than CPG-86 due to its larger pore
size. Other workers have found that octadecyl bonding
density increases with increasing pore size for silica
substrates (Engelhardt et al., 1982; Sander and Wise, 1984b;
Sands et al., 1986; Staroverov et al., 1986).
In the first set of reactions, both CPG-86 and CPG-167
were rotated at ambient temperature (26.0 C) for 24 hours

61
using dimethyloctadecylchlorosilane as the reactive silane,
2,6-lutidine as the acid-acceptor catalyst and methylene
chloride as the reaction solvent. All bonding densities are
calculated from duplicate elemental analyses as described in
Chapter II and the mean value + the range is reported in all
cases. The bonding densities for the CPG-86 and the CPG-167
were 2.56 + 0.03 ymol/m^ and 2.07 + 0.02 ymol/m^
respectively. In the second set of experiments, the same
reagents and reaction time as above were used to react both
CPG's, but the reactions were performed under reflux
conditions at a temperature of 50.0 C. The CPG-86 bonding
density was 2.63 _+ 0.00 ymol/m2 and that for the CPG-167 was
2.28 + 0.00 pmol/m^. As expected from previous work with
silica (Chapter II), reflux temperatures resulted in a
higher bonding density than ambient temperatures.
The next three sets of experiments were run for 24
hours with rotation under ultrasound conditions as described
in Chapter II. The first set of experiments used the same
reagents as described above at a temperature of 28.0 C.
The CPG-86 had a resultant bonding density of 2.55 + 0.01
ymol/m^- that for the CPG-167 was 2.04 + 0.03 ymol/m^. As
in the case for the silica, bonding densities achieved under
ultrasonic conditions with 2,6-lutidine as the acid-acceptor
catalyst were comparable to those achieved at ambient
temperatures.
The second set of ultrasound experiments was run under
the same conditions as the first set and at a temperature of

62
28.5 C, except that 4-dimethyl aminopyridine (4-DMAP) was
used as the acid-acceptor catalyst instead of 2,6-lutidine.
The bonding densities for CPG-86 and CPG-167 were 3.30 +
0.02 and 3.04 + 0.08 umol/m2 respectively. The 4-DMAP had
again proven to be a superior acid-acceptor catalyst to the
2,6-lutidine as had been the case for silica. The third set
of ultrasound experiments was performed at 28.0 C using
4-DMAP as the acid-acceptor catalyst, methylene chloride as
the reaction solvent and trimethy1 chiorosi1ane (TMCS) as the
reactive silane. TMCS was used in order to approximate the
maximum bonding density achievable under minimum steric
hindrance conditions, as explained in Chapter II. The TMCS
bonding density was 4.19 + 0.02 umol/m^ for CPG-86 and 5.15
+ 0.10 umol/m^ for CPG-167. As expected, use of a less
bulky silane reagent resulted in a higher alkyl bonding
density, since steric hindrances are minimized.
A comparison of the bonding densities of the 147
Angstrom pore size silica, 86 Angstrom CPG and 167 Angstrom
CPG achieved under all sets of conditions is tabulated in
Table 3-1. As seen from these results, neither of our
expectations was realized. In all of the octadecyl silane
reactions, reactivity of the amorphous silica was greater
than that of either CPG. Even more puzzling, the smaller
pore CPG (CPG-86) exhibited greater reactivity than the
wider pore CPG (CPG-167). In the case where TMCS was the
reactive silane, these trends were reversed. This seems to
indicate that the reactivity trends for the silane bonding

Table 3-1.
Compa rison
silica, 86
controlled
of octadecyl bonding densities
Angstrom controlled pore glass
pore gl ass (CPG-167 ).
for 147
(CPG-86)
Angstrom
and 167
(pore size
Angstrom
Reaction
Conditions1
Temperatu re
(C) silica8
Bonding Density (jimol/m^)
CPG-86 CPG-167
Refluxed/
2,6-1utidine
50.0
2 .82
2.63
2.28
U1trasound/
2,6-1utidine
28.0
2.71
2.55
2.04
U1trasound/
4-DMAP
28.5
3.35
3.30
3.04
Ambient/
2,6-lutidine
26.0
2 .69
2.56
2.07
Ci sil ane2/
Ultrasound/
28.0
3.51
4.19
5.15
4-DMAP
1 Reaction method/acid-acceptor catalyst.
silane used instead of Cig silane in order to estimate bonding density
achievable using a less bulky silane reagent.
CT>
CO

64
reactions could be due to steric problems, since TMCS is
much smaller than dimethy1octadecy1 chi oros i 1ane, but the
lower reactivity of the CPG-167 compared to that of CPG-86
for the octadecy! reaction contradicts this theory. The
chemical composition of the CPG's may be implicated in this
problem, since CPG contains 3% B2O3 and silica contains no
more than trace amounts. Other workers have found that
heating CPG to a temperature of 700 C for 5 to 100 hours
results in migration of boron atoms to the glass surface and
consequent enrichment of boron on the surface of the porous
glass. They further found that this surface boron
enrichment resulted in higher octadecy1 bonding densities
than achieved on untreated CPG (Dawidowicz et al., 1983;
Dawidowicz et al., 1986; Dawidowicz and Rayss, 1986; Rayss
et al., 1983; Rayss and Dawidowicz, 1986; Suprynowicz et
al., 1985). At present, we are unable to explain the
anomalous octadecy! bonding behavior of the controlled pore
glasses.

CHAPTER IV
CORRELATIONS BETWEEN CHROMATOGRAPHIC RETENTION AND OCTADECYL
BONDING DENSITY
Chromatographic Determination of Thermodynamic Partition
Coefficients
Retention in any chromatographic process occurs when
the solute of interest is transferred from the mobile phase
to the stationary phase. The process of transfer of the
solute between the mobile phase and the stationary phase is
characterized by the thermodynamic distribution or partition
coefficient, K, which is the ratio of the concent ration of
the solute in the stationary and mobile phase.
Chromatographic retention is a function of the distribution
coefficient and the volumes of the respective phases, and is
most often described by the capacity factor, k', the ratio
of the number of moles of solute in the stationary phase and
in the mobile phase. The capacity factor also expresses the
ratio between the amount of time the solute spends in the
stationary phase and in the mobile phase. It is the
commonly used measure to describe retention because it
accounts for differences in column dimensions and mobile
phase flow rates; it is also easy to measure as
k' = (Vr Vm)/Vm where Vr is the solute retention volume
and Vm is the mobile phase void volume. Measurement of the
capacity factor provides valuable thermodynamic information
65

about solute retention in a particular chromatographic
system since retention is related to the thermodynamic
distribution coefficient through the volume phase ratio
Vs/vm (stationary/mobile) in that k = K(Vs/Vm). Therefore
for rigorous theoretical treatment of chromatographic
retention, the phase ratio must be accurately known in order
to determine the partition coefficient.
Measurement of the Mobile Phase Volume in RPLC
The determination of the mobile phase volume, Vm, in
liquid chromatographic systems is a problem that has
generated great interest as well as considerable
controversy. Its value is an essential component for the
calculation of the capacity factor k1 as well as for the
thermodynamic distribution coefficient K. Many workers have
addressed this dilemma, yet there is little consensus on a
generally applicable convention for measuring V m (Berendsen
et al. 1980b; Engelhardt et al., 1984; Gutnikov and Hung,
1984; Knox and Kaliszan, 1985; Le Ha et al., 1982; McCormick
and Karger, 1980; Melander et al., 1983a and 1983b; Slaats
et al., 1981; Wainwright et al., 1985; Wells and Clark,
1981). The problem is especially complex for RPLC, since
preferential sorption of mobile phase components by the
stationary phase results in the formation of a solvation
layer on this surface. The thickness and composition of the
solvation layer varies with the bulk composition of the
mobile phase and the local concentration of organic modifier
in the solvation layer may be greater than that in the bulk

67
mobile phase due to the hydrophobicity of the stationary
phase. The composition of the solvation layer also varies
with its distance from the anchored ends of the RP chains;
therefore its presence results in an ill-defined boundary
between the stationary and mobile phases (Berendsen et al.
1980b; Gutnikov and Hung, 1984; Le Ha et al., 1982, Knox and
Kaliszan, 1985).
There are three general categories of procedures used
to determine Vm: the use of unretained compounds, the
linearization of the net retention time for a homologous
series and static methods (Berendsen et al., 1980b). The
choice of an "unretained" compound for Vm measurements in
RPLC systems is a difficult one. In any case, neither its
heat of sorption nor its size should differ from those of
the mobile phase components. For this reason, mobile phase
constituents and especially their deuterated analogs are
often used (Engelhardt et al., 1984). However, this choice
is not without its problems. Since these compounds are
transparent in the UV region, sensitive detection of them
requires a refractive index detector. The use of deuterated
organic modifier as an unretained compound is only valid
when there is a large amount of organic modifier present in
the mobile phase since in organic-lean mobile phases the
marker will be slightly retained, especially in the case of
methanol/water mobile phase systems (Engelhardt et al.,
1984). The same is true for D 2 0 in organic-rich mobile
phase systems; this phenomenon is attributed to D2O

68
adsorption onto the residual silanol groups on the
stationary phase surface (McCormick and Karger, 1980).
Melander et al. (1983a, p. 213) suggest that "... the
most weakly bound solvent component is not present in the
solvation layer." and that this component should be used for
the determination of Vm. They concur with McCormick and
Karger (1980) that D2O is a useful probe for mobile phase
volume determination unless the mobile phase is water-lean.
As secondary probes of Vm, fructose and urea have been
suggested for all compositions of methanol/water mobile
phases and for acetonitrile volume fractions from 0 to 0.75
for acetonitrile/water mobile phase (Melander et al.,
1983a). Gutnikov and Hung (1984) have also proposed the use
of oxalohydroxamic acid as a UV-detectable Vm probe.
The use of UV-active inorganic salts such as nitrates
has also been recommended for determination of RPLC dead
volumes; however in unbuffered mobile phases the dead
volumes obtained increase with increasing amount of salt
injected. Nitrate is also prevented from penetrating the
stationary phase pores by the Donnan potential which comes
about from the negatively charged silicate ions present on
the stationary phase surface (Berendsen et al., 1980b;
Engelhardt et al., 1984; Wells and Clark, 1981). Knox and
Kaliszan (1985) suggest using a volume fraction-weighted
average of the retention volumes of isotopically labelled
forms of the mobile phase components. However, besides the
aforementioned problems associated with the detection of

69
these species, this method adopts the convention that Vm is
the total volume of all mobile phase components within the
column bed; the solvation layer adsorbed on the stationary
phase is thereby included in the mobile phase volume.
The linearization of a homologous series of compounds
in order to find the column dead volume has been widely used
in gas chromatography; therefore it is not surprising that
this method has also been applied for LC systems. This
method assumes that there is a linear relationship between
the logarithm of the net retention time tr and the carbon
number of a homologous series (Berendsen et al., 1980b; Laub
and Madden, 1985; Wainwright et al., 1985). The mobile
phase volume can be obtained by comparing the retention
times for two consecutive homologs, termed n and n + 1. For a
homologous series the ratio of capacity factors for
consecutive homologs is assumed to be constant and
^r,n + l = A(tr>n) (A 1)tg
By plotting trjn+1 versus tr n, the slope A can be obtained
and tg can be determined from the intercept and multiplied
by the mobile phase volume flow rate to obtain Vm. The
results obtained can be precise to within 1% if alkyl-
benzenes are the homologous series used (Berendsen et al.,
1980b); however the use of homologous aromatic alcohols
gives inconsistent data due to their interactions with
silanol groups on the stationary phase (Laub and Madden,
1985) .
The linearization method is not without criticism. It
assumes that the relationship between the logarithm of the

retention times for a homologous series is a linear function
of the number of carbons in the series and therefore that
the change in free energy of partitioning per methylene
group is constant. This implies that as the number of
methylene groups increase, the rest of the molecule has a
constant effect on stationary phase interactions. Yet in
some cases the relationship between the logarithm of
retention and carbon number is not linear, implying that
this assumption is invalid. The linearization method is
also very time consuming, since the retention time
measurements must be determined very precisely in order to
obtain precision in the Vm value (Knox and Kaliszan, 1985).
Determination of Vm by the static method is a
gravimetric procedure. A thermostatted packed LC column is
filled successively with two pure liquids with greatly
different densities and weighed. From the differences in
the column masses, w, and the density of each liquid, d, the
total mobile phase volume can be calculated, since
(Berendsen et al., 1980b; Knox and Kaliszan, 1985; McCormick
and Karger, 1980). The mobile phase volume as determined by
this method is the maximum volume within the column that is
accessible to a molecule comparable in size to those used in
the procedure (McCormick and Karger, 1980). However, this
method ignores the possibility of a solvation layer on the
stationary phase and therefore can overestimate the value of
a dynamic Vm by as much as 15% for a pure methanol mobile

71
phase (Berendsen et al., 1980b). The volume of the mobile
phase as determined by this method is useful for a reference
point, both in terms of whether or not a compound
experiences retention in a chromatographic system and in
understanding the changes in the solvation layer which occur
when the bulk mobile phase composition is changed (McCormick
and Karger, 1980). Knox and Kaliszan (1985) even argue that
in the theoretical treatment of thermodynamic aspects of
chromatography that the maximum Vm value is the pertinent
one. They argue that since the thickness of the boundary
between the bulk stationary phase and bulk mobile phase is
on the order of one nanometer that its position cannot be
sufficiently well defined to give an accurate measure of the
two volumes and that calculational methods for the volume of
the solvation layer are very arbitrary. Because the
gravimetric procedure gives a precise and reproducible value
for Vm that is unambiguous and convenient to measure, this
convention was chosen to determine the mobile phase volume
for the chromatographic thermodynamic distribution
coefficients calculated in this work.
Measurement of the Stationary Phase Volume in RPLC
Although much work has been done on measuring the
volume of the mobile phase, measurement of Vs, the volume of
the stationary phase has not been as thoroughly investigated
(Berendsen et al., 1980b; Jandera et al, 1982; McCormick and
Karger, 1980; Melander et al., 1980; Sander and Field, 1980;
Slaats et al., 1981). In determining a method for the

72
measurement of Vs, a convention for defining Vs must be
chosen, since the choice of the phase ratio must be
compatible with the definition of K that is in agreement
with the molecular mechanism of retention. Jandera et al.
(1982) have defined the stationary phase volume as that
fraction of the column volume that is not occupied by the
mobile phase. While this choice is certainly convenient and
can be readily determined, it is at best a crude measure, as
similar (or even identical) values of Vs would be obtained
for stationary phases made from the same bulk silica but
with different bonding densities of alkyl chains, or
possibly even of different chain lengths. Any determination
of stationary phase volume based solely on mobile phase
volume measurements is doomed to failure, as such a
measurement cannot be sufficiently sensitive to ascertain
bonding density or small chain length differences.
Melander and Horvath (1980) have suggested defining the
phase ratio as the ratio of the surface area of the
adsorbent (m2) divided by the column dead volume (cm^).
While this approach is an improvement in definition, it
again fails to account for certain variations in the
structure of the bonded phase and it implies that adsorption
is the sole mechanism in RPLC retention. The major drawback
to this proposed phase ratio convention, however, lies in
the accurate measurement of the two parameters involved. As
previously mentioned, chromatographers have been unable to
embrace any one of the commonly used methods for determining

73
column dead volumes as being accurate and consistent enough
for precise work (Engelhardt et al., 1984; Melander et al.,
1982; Smith et al., 1986). Melander and Horvath (1980)
state that a small relative error in the determination of
the column dead volume results in a commensurate relative
error in calculating both the capacity factor and the Gibbs
free energy of the solute transfer.
Other problems exist as well. The surface area of the
adsorbent is usually found by use of the BET analysis
method. It should be noted that the surface area of the
adsorbent must be determined after derivatization with the
alkyl ligand, as the surface area of the derivatized silica
will be significantly different from that of the
underivatized support. Although use of the BET method for
surface area determination is widespread, this method is
inappropriate in assessing that surface area of derivatized
silica packings which is chromatographical 1y significant.
The BET method measures the area of surface that is
accessible to a small molecular probe such as nitrogen. Yet
in an irregular surface such as porous silica, there may
exist many pores which are large enough to allow nitrogen
in, but which are too small to allow the passage of any
larger molecules of chromatographic interest.
Chromatographic support surface area data based on BET
analysis is usually overestimated, and the amount of
overestimation is by no means a constant, depending on the
base silica structure and the derivatization method.

74
Melander and Horvath (1980, p. 270) state that . any
estimation of "stationary phase volume" on the basis of BET
surface area of the support is likely to be inaccurate."
Due to the errors in determining both adsorbent surface area
and column dead volume, there will consequently be a large
error propagated in the subsequent calculation of the phase
ratio if Melander and Horvath's convention is used.
Sander and Field (1980) have estimated the phase ratio
by constructing physical models of the bonded phase using
manufacturer's data regarding silanol surface coverage and
percent carbon loading. This approach is quite reasonable
from a theoretical standpoint, as it accounts for variation
in bonding density and alkyl chain length. However because
it is based on models it can only be an estimate of V s; the
construction of such physical models is also time consuming.
In determining the stationary phase volume, the
pertinent volume should be the volume of the alkyl chains
bonded to the silica surface. Dill (1987a) has performed
statistical mechanical calculations based on a lattice
interphase model of RPLC stationary phases which describe
chromatographic retention in reversed phase systems. These
calculations have shown that in a well endcapped column,
chain interactions with solutes are the most important
stationary phase contribution to solute retention.
Therefore the calculation of V$ should give only the actual
volume of the alkyl chains bonded to the support surface.
The assumption here is that all of the bonded stationary

7b
phase volume is accessible to the solute.
A simple method for calculating the stationary phase
volume has been devised in our laboratory. The only
measurements necessary are the carbon load of the packing
and the actual weight of packing contained in the
chromatographic column. Wise and May (1983) proposed that
the surface density of a bonded alkyl ligand (in micromoles
of alkyl ligand per square meter of packing surface) can be
calculated by
Cs = %C (106) (4-1)
1200 nc SBET
where Cs is the bonding density (ymol/m2), %C is the carbon
loading of the packing as determined from elemental analysis
or by a gravimetric procedure (Cheng, 1985), nc is the
number of carbons in the alkyl ligand, and Sggy is the
surface area of the derivatized packing as determined by
BET analysis. But the volume of the stationary phase, V$,
can be expressed as
Vs = ( C s ) (SBET >(v)(Wp)(10"6) (4-2)
where v is the molar volume of the bonded alkyl group in
cirr/mole and Wp is the weight of the bonded packing
contained in the chromatographic column. Molar volume, v,
i s
v=M/d (4-3)
where M is the weight of the bonded phase alkyl group and d
is the density of the bonded alkyl group. Cheng (1985) has
experimentally determined the pertinent densities of
commonly used bonded silanes and reported values of 0.8607

76
g/cm^, 0.8625 g/cm^, and 0.8638 g/cm^ respectively for the
octadecy1dimethy1sily1, octy1dimethy1sily1, and
trimethylsilyl bonded groups. Substitution of equations 4-1
and 4-3 into equation 4-2 results in the volume of the
stationary phase, Vs (in cm^), as expressed by the following
formu1 a
r---.(%C)(M)(Wp)
(l00)(12.011)\nr)(d)
(4-4).
This method provides a much more accurate calculation
of Vs than has been previously possible. A principal
advantage of this method is that the surface area of the
packing is not used in determining Vs, which eliminates the
errors associated with this measurement. The stationary
phase volume that is calculated in this method is the volume
that is important in the chromatographic process, i.e. the
actual volume of the bonded alkyl chains themselves. The
precision is limited only by the carbon loading
determination (+ 0.20 %C for our departmental elemental
analysis) and by the measurement of the mass of packing in
the column ( + 0.1 mg on any analytical balance).
Calculation of the volume of the stationary phase by this
method provides the means for a more accurate and uniform
determination of the phase ratio.
Experimental Procedure
Preparation of Bonded Phases of Varied Bonding Densities
All of the reagents used in the preparation of the
silica and CPG bonded phases are described in Chapters II
and III. Silica bonded phases with octadecyl bonding

77
densities greater than or equal to 2.75 ymol/m2 were
prepared under traditional reflux conditions as well as
under ambient, subambient and superambient ultrasonic
conditions, using 2,6-lutidine or 4-DMAP as the
acid-acceptor catalyst as described in Chapter II.
Controlled pore glass bonded phases with octadecyl bonding
densities of 3.30 and 2.63 pmol/m^ were prepared from the 86
Angstrom CPG (CPG-86) under ambient ultrasonic conditions
using 4-DMAP as the acid-acceptor catalyst or under reflux
conditions using 2,6-lutidine as the acid-acceptor catalyst
respectively as described in Chapter III. Table 4-1 lists
the experimental conditions, acid-acceptor catalyst and
resultant octadecyl bonding density for each of these
stationary phases.
In order to prepare bonded phases with octadecyl
bonding densities less than 2.6 pmol/m2, the experimental
conditions of the bonding reaction must be altered so that a
less than maximal bonding density is achieved. One strategy
that can be used to accomplish this is to use a less than
stoichiometric amount of the reactive silane. Another
approach is to partially cover some of the reactive silanols
with trimethylsilane before exhaustive derivatization with
the octadecyl silane reagent (Marshall et al., 1984 and
1986). By varying the amount of trimethylsilane precoverage
and then reacting the precovered silicas with an excess of
the octadecylsilane, lower coverage octadecyl bonded phases
of varying bonding densities can be synthesized.

Table 4-1. High octadecyl bonding density reversed phase packings.
Packing
Identifier
Base
Packing
Reaction
Conditions
T emperatu re
(C)
Reaction
Time (h)
Acid-Acceptor
Cata!yst
Co BondingDensity
(umol/m2)
C18-2
silica
ref 1ux
50.0
24
2,6-1utidine
2.75
LT2
silica
u11 rasound
8.5
101
2 ,6 1 u t i d i n e
2 .84
DMAP1
silica
ultrasound
28.0
24
4-DMAP
3 .06
DMAP1/31
silica
ultrasound
28.0/4.0
24/97
4-DMAP
3.15
DMAP3
silica
u1trasound
4.0
97
4-DMAP
3.24
US/ref1
silica
ultrasound
50.0
24
4-DMAP
3.34
ref/DMAP
silica
reflux
50.0
24
4-DMAP
3.43
rederDMAP12
silica
ultrasound
28.0
24
4-DMAP
3.56
DMAP5
silica
ultrasound
3.0
144
4-DMAP
3 .60
CPG2
CPG-86
reflux
50.0
24
2,6-lutidine
2 .68
CPG4
CPG-86
ultrasound
28.5
24
4-DMAP
3.30
Packing DMAP1/3 is a 50/50 (weight/weight) mixture of packings DMAP1 and DMAP3.
Packing rederDMAPl was obtained by reacting packing DMAP1 with the octadecyl silane
under identical conditions as for DMAP1.
00

79
Based on Kinkel and Unger's (1984) estimation of a
maximum of five micromoles of reactive hydroxyl sites per
square meter of silica surface, amounts of trimethyl chioro-
silane (TMCS) correspond'ng to approximately 5%, 10%, 15%,
30% and 40% coverage of these hydroxyl sites were reacted as
described in Chapters II and III under ambient conditions
(at a temperature of 26.5 C) for 24 hours to partially
precover the silica and controlled pore glass supports. The
reaction solvent was dry methylene chloride and 2,6-lutidine
was used as the acid-acceptor catalyst. It should be noted
that it is unlikely that exactly 5%, 10%, 15%, 30% or 40% of
the surface hydroxyl groups on the supports were reacted;
the amounts of TMCS used merely represent some fraction of
the amount necessary for total coverage of the surface
(Marshall et a 1., 1984). After TMCS precoverage, the
supports were washed and dried as described in Chapters II
and III. The precovered supports were then reacted with a
twofold excess of octadecy1dimethyl chi oros i 1ane and a
fourfold excess of 2,6-lutidine with methylene chloride at a
temperature of 26.5 C for 24 hours and washed and dried as
described in Chapters II and III. The resultant C-^ and C^8
bonding densities for these precovered bonded phases are
summarized in Tables 4-2 and 4-3.
HPLC Column Packing Procedures
HPLC columns were assembled from 15 cm lengths of 1/4"
outer diameter and 4.6 mm inner diameter seamless precision
bore polished HPLC tubing (Alltech Associates, Inc.,

80
Table 4-2. Bonding densities for precovered silica reversed
phase packings.
Packing
Identifier
Ci Bonding Density
1 Umol/m?)
C-L3 Bonding Density
(ymol)
5%
0 .63
2.07
10%
0.98
2.09
15%
1 .50
1.98
30%
1.38
1.74
40%
1 .90
1 .60

81
Table 4-3. Bonding densities for precovered 85 Angstrom
controlled pore glass reversed phase packings.
Packing
Identifier
C]_ Bonding Density
(umo 1/nr )
Co Bonding Density
(pmol /wr)
5% CPG
0.55
3.21
10% CPG
1.08
2.83
15% CPG
2.25
1 .70
30% CPG
1.27
2.72
40% CPG
1 .34
2.59

82
Deerfield, IL) and from Swagelok 1/4" to 1/16" zero dead
volume reducing union chromatographic end fittings (Crawford
Fitting Company, Solon, OH) which had been fitted with 2 urn
passivated 316 stainless steel frits (Alltech Associates,
Inc., Deerfield, IL). The column tubing and end fittings
were made of 316 stainless steel and were passivated prior
to use by ultrasonication for 30 minutes in 3 M nitric acid,
followed by an aqueous and a methanol rinse.
Silica columns were packed using a Shandon high
pressure HPLC column packer with a 33 ml slurry reservoir
(Shandon Southern Instruments, Inc., Sewickley, PA).
Approximately 1.5 grams of derivatized silica were slurried
in 30 ml of chloroform and sonicated for 10 minutes. The
column was then packed at a packing pressure of 6000 psi in
the downward position using a sequence of 150 ml each of
50/50 (v/v) chioroform/methanol, methanol and 50/50
methanol/water. The column was then removed from the packer
fittings, the packing leveled with a spatula and the
remaining column end fitting installed. Controlled pore
glass columns were packed in an identical manner except for
the type of packer used. Since CPG is mechanically fragile
and brittle (Fluka), it cannot be packed using a high
pressure packer. Therefore a Beckman Model 100A HPLC
pump(Beckman Instruments Inc., San Ramon, CA) running at a
flow rate of 9.9 ml/min was used in conjunction with
continuous mechanical vibration to pack the CPG columns.
Packing in such a manner generated a packing pressure of

83
200-300 psi, preserving the integrity of the CPG packings
and resulting in a stable packing bed. All columns were
equilibrated with the desired chromatographic mobile phase
by passing approximately 125 ml of the mobile phase through
the column immediately prior to use.
Chromatographic Measurements
The liquid chromatographic system used for
chromatographic measurements consisted of a Valeo C6W
injector (Valeo Instrument Company, Inc., Houston, TX) with
a 10 microliter sample loop, a Beckman Model 100A isocratic
HPLC pump, a Beckman Model 153 fixed wavelength 254 nm UV
detector and a Fisher Recordall chart recorder (Fisher
Scientific, Fairlawn, NJ). Samples were loaded in the
injection loop via a Hamilton 705 SNR syringe (Reno, NV).
Temperature control of the chromatographic column was
accomplished by passing thermostatted water through a water
jacket fitted around the column. Superambient temperatures
were controlled using a Lauda Model MT heater/circu1ator
(Brinkmann Instruments Company, Westbury, NY). Subambient
temperatures were brought about by a Neslab Endocal 800
water bath (Neslab Instruments, Portsmouth, NH). Mobile
phases were made of HPLC grade methanol or acetonitrile
(Fisher Scientific, Fairlawn, NJ) mixed with water which had
been prepared as described in Chapter II; mobile phase
compositions are designated by volume ratios of organic
modifier to water. Mobile phases were premixed by adding
the appropriate volume of organic modifier to the

84
appropriate volume of water; these mobile phases were then
mixed well and placed in an ultrasonic bath for 15-30
minutes in order to degas them. The flow rate of the mobile
phase in all cases was 1.5 ml/min. Naphthalene (Eastman
Organic Chemicals, Rochester, NY) was chosen as a small
nonpolar test solute; standards were made up in HPLC grade
methanol for use in the retention studies. Solute retention
and column holdup volumes were measured from the chart
recorder tracings. The solvent disturbance peak was used to
determine column mobile phase volumes for the calculation of
capacity factors; since this disturbance comes about from
the methanol in which the test solute is dissolved, its
choice for Vm falls under the category of using an
unretained compound to determine Vm. This convention was
chosen in order to account for variances in the individual
column dead volumes resulting from differences in the
stationary phase packing density within the chromatographic
columns .
Measurement of Vm and V£
The gravimetric procedure previously described was used
to calculate Vm. While an ambient temperature of 25.0 C
was maintained, 150 ml of methylene chloride was passed
through a 15 cm LC column packed with either silica or CPG.
The column was then capped and weighed on an analytical
balance. The procedure was duplicated using methanol as the
mobile phase and by dividing the difference in column masses
by the difference in the solvent densities at 25.0 C (1.318

85
and 0.7866 g/cm3 for methanol and methylene chloride
respectively) the silica column dead volume was determined
to be 1.805 ml while Vm for the CPG column was 1.752 ml. It
was assumed that the mobile phase volumes measured by this
procedure will be constant (within experimental error) for
any of the silica or CPG reversed phase columns used in this
work, since in each case the packings were based on the same
starting material.
The stationary phase volume for each LC column was
calculated using Equation 4-4. For the precovered bonded
phases the total volume of both the trimethyl- and
octadecy1 si 1 y 1 alkyl groups was used for V$. Percent carbon
for each column packing was obtained from in-house elemental
analysis and the densities used for the trimethylsilyl and
octadecy1sily1 groups were 0.8638 and 0.8607 g/cm3
respectively as reported by Cheng (1985). The weight of the
packing contained in the chromatographic column was
determined by weighing an empty chromatographic column,
packing it as described previously in this chapter and
drying it at 100 C to constant weight in a gas
chromatograph with a constant helium flow through the
column. From the mass differences in the two weighings the
weight of the column packing was determined to be 1.1705 g
for the silica columns and 1.2898 for the CPG columns. The
capacity factors for the naphthalene solute were determined
by triplicate injections onto each of the chromatographic
columns of different alkyl bonding density using mobile

86
phases consisting of 55/45 methanol/water and 85/15
acetonitrile/water. Thermodynamic partition coefficients
for the transfer of the naphthalene solute from the
stationary phase to the mobile phase were calculated by
dividing the capacity factor by the volume phase ratio.
Results
Silica-based Stationary Phases
Thermodynamic partition coefficients for the
naphthalene solute as a function of silica stationary phase
octadecyl bonding density are listed in Tables 4-4, 4-5 and
4-6 respectively for the 55/45 methanol/water mobile phase
system at 20.0 C and 35.0 C and for the 85/15
acetonitrile/water mobile phase system at 35.0 C.
Graphical representations of these data are shown in Figures
4-1, 4-2 and 4-3.
In all cases the partition coefficient increased as a
linear function of bonding density until a bonding density
of 3.1 umol/m^ was reached. The best fit line for this
linear region of each plot was calculated using least
squares linear regression. For 55/45 methanol/water at
20.0 C the slope and y-intercept for the best fit line were
30.1 and 12.3 respectively with a coefficient of correlation
of 0.989. At 35.0 C for 55/45 methanol/water the slope was
21.0, the y-intercept 12.7 and the coefficient of
correlation 0.973. For the 85/15 acetonitrile/water mobile
phase at 35.0 C the slope was 1.20, the y-intercept 1.54
and the coefficient of correlation 0.982. In all cases the

87
Table 4-4. Naphthalene thermodynamic partition
coefficients at 20.0 C as a function of
silica octadecyl bonding density for
55/45 methanol/water mobile phase.
C^g Bonding Density Naphthalene Thermodynamic Partition
(ymol/rrr) Coefficient at 20.0 C
1 .60
57.6
1.74
66.7
1 .98
69 .6
2.07
78.6
2.09
75.8
2.75
94.3
2.84
96.2
3.06
104
3.15
97.2
3.24
93.4
3.34
89.6
3 .43
87.2
3.56
86.1
3.60
85.9

88
Table 4-5. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
silica octadecyl bonding density for
55/45 methanol/water mobile phase.
Co Bonding Density
(umol/nr)
Naphthalene Thermodynamic Partition
Coefficient at 35.0 C
1 .60
43.4
1 .74
50.7
1 .98
51 .3
2.07
60.5
2.16
59.5
2.75
69.8
2.84
71 .3
3.06
76.1
3 .15
70.9
3.24
69.2
3.34
66.8
3.43
65.0
3.56
63.9
3.60
63.7

89
Table 4-6. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.
Co Bonding Density
(pmol/nr )
Naphthalene Thermodynamic Partition
Coefficient at 35.0 C
1 .60
3.28
1 .74
3.69
1 .98
3.79
2.07
4.20
2.09
4.16
2.75
4.90
2 .84
4.95
3.06
5.09
3.15
4.88
3.24
4.73
3.34
4.66
3.56
4.48
3 .60
4.41

147 A Silica at 20 C
Ce Bonding Density (pmoi/ m2)
Figure 4-1. Naphthalene thermodynamic partition coefficient at 20.0 C
as a function of silica octadecyl bonding density for
55/45 methanol/water mobile phase.

147 A Silica at 35 C
C(8 Bonding Density (jjmoi/ m2)
Figure 4-2. Naphthalene thermodynamic partition coefficient at 35.0 C
as a function of silica octadecyl bonding density for
55/45 methanol/water mobile phase.

6.0-
147 & Silica at 35 C
~i i ( 1 1 1 1
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
C|8 Bonding Density (jjmoi/m*)
Figure 4-3. Naphthalene thermodynamic partition coefficient at 35.0
as a function of silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.

93
coefficient of correlation showed a definite linear
relationship between partition coefficient and bonding
density, especially in light of the uncertainties in the
measurements of bonding density, Vs, Vm and capacity factor.
The amount of error propagated in the bonding density could
be calculated from the variances in percent carbon and
support surface area; similarly, that for the stationary
phase volume (as calculated by Equation 4-4) could be
calculable from the variances in percent carbon, weight of
the packing contained in the column and density of the alkyl
group bonded to the silica surface. However, these
calculable sources of error are small when compared to the
incalculable sources of error in the stationary and mobile
phase volumes. The primary source of error in Vm
determination is the choice of convention for its
measurement, as discussed earlier in this chapter. In fact
the choice of Vm convention is the most significant source
of error in the calculation of capacity factors as well,
since k1 = (Vr-Vm)/Vm, where V r is the retention volume of
the solute of interest. The most significant error in Vs is
certainly the determination of the volume of the solvation
layer associated with the stationary phase surface. As
explained earlier in this chapter, we have developed an
equation (4-4) to calculate the stationary phase volume
which gives an accurate volume for the alkyl chains bonded
to the silica surface; however this convention does not take
the solvation layer volume into account. This omission was

94
deliberate because the solvation layer volume is very
difficult to measure experimentally, moreover it changes
substantially as the mobile phase composition is changed
(Berendsen et a 1., 1980b; McCormick and Karger, 1980). Our
calculations of Vs may therefore underestimate the true
stationary phase volume, which will encompass the solvation
layer volume as well as the bonded chain volume. Moreover,
our static Vm value is larger than those calculated by most
dynamic means since the static value includes the solvation
layer volume in the mobile phase volume. The combination of
these two effects results in an overall value for the volume
phase ratio (stationary/mobi1e) that is smaller than the
"true" value; consequently the solute thermodynamic
partition coefficient as determined chromatographically will
probably be overestimated, but the exact amount of
overestimation is incalculable at this time. Although this
possible overestimation of the partition coefficient would
cause the plots of partition coefficient versus bonding
density in Figures 4-1, 4-2 and 4-3 to be shifted
vertically, it should be noted that this would change
neither the shape of the plots nor their maxima. Error bars
based on the range of partition coefficient values resulting
from the imprecision in the elemental analysis for the
silica packings (+ 0.20% carbon) are shown for Figure 4-3.
At bonding densities greater than about 3.1 ymol/m^ the
partition coefficient begins to decrease as the bonding
density is increased. This trend is evident for both mobile

phase systems and/or temperatures. The overall behavior
under the conditions specified above was that the partition
coefficient increased linearly as a function of bonding
density until a maximum was reached at a certain "critical"
bonding density in the vicinity of 3.1 pmol/m^. Once this
critical bonding density is reached, the earlier trend is
reversed and the partition coefficient decreases with
increasing bonding density. Comparisons between these
experimental trends and Dill's (1987a and 1987b) theoretical
predictions are made later in Chapter VI. These comparisons
result in the interpretation of the experimental results,
giving a wealth of information about the fundamental
mechanisms of small solute retention in reversed phase
liquid chromatographic systems.
Controlled Pore Glass-based Stationary Phases
Naphthalene thermodynamic partition coefficients as a
function of CPG stationary phase octadecyl bonding density
for 55/45 methanol/water at 80.0 C and 35.0 C and for
85/15 acetonitrile/ water mobile phase systems at 35.0 C
are listed in Tables 4-7, 4-8 and 4-9 respectively.
Graphical representations of these data are shown in Figures
4-4, 4-5 and 4-6.
In all three cases the partition coefficient reached a
local maximum at about 2.7 ymol/m^. For the 55/45
methanol/water mobile phase, at bonding densities higher
than this value the partition coefficient decreased to a
local minimum at approximately 2.8 pmol/m^ for a temperature

96
Table 4-7. Naphthalene thermodynamic partition
coefficients at 20.0 C as a function of
CP6-86 octadecyl bonding density for
55/45 methanol/water mobile phase.
C]_g Bonding Density Naphthalene Thermodynamic Partition
( pinol /m)Coefficient at 20.0 C
1 .70
89.3
2.68
103
2.72
95.2
2.83
93.6
3.21
98.1
3.30
102

97
Table 4-8. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
CPG-86 octadecyl bonding density for
55/45 methanol/water mobile phase.
C^g Bonding Density Naphthalene Thermodynamic Partition
(umol /irr ) Coefficient at 20.0 C
1 .70
69.3
2 .68
75.1
2.72
70.0
2 .83
67.1
3.21
67.0
3.30
69.7

98
Table 4-9. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
CPG-86 octadecyl bonding density for
85/15 acetonitri 1 e/water mobile phase.
Cio Bonding Density
(nmol/nr)
Naphthalene Thermodynamic Partition
Coefficient at 35.0 C
1.70
11 .0
2.59
13.5
2.68
15.0
2.72
13.9
2 .83
11 .9
3.21
9.76
3.30
9.74

120-
MO -
100-
Partltion
Coefficient,
K 90-
80-
70-
~T~
0.5
i 1 1 1 1 r-
1.0 1.5 2.0 2.5 3.0 3.5
C(8 Bonding Density (pmol/m2)
Figure 4-4. Naphthalene thermodynamic partition coefficient at 20.0
as a function of CPG-86 octadecyl bonding density for
55/45 methanol/water mobile phase.

Cl8 Bonding Density (jmoi/mZ)
Figure 4-5. Naphthalene thermodynamic partition coefficient at 35.0 C
as a function of CPG-86 octadecyl bonding density for
55/45 methanol/water mobile phase.
100

86 l CPG at 35 t
C|8 Bonding Density (imol/m2)
Figure 4-6. Naphthalene thermodynamic partition coefficient at 35.0 C
as a function of CPG-86 octadecyl bonding density for
85/15 acetonitrile/water mobile phase.

102
of 20.0 C and in the area of 2.8 to 3.2 ymol/m2 at 35.0 C.
At bonding densities higher than the local minimum the
partition coefficient shows an increasing trend. This
increasing trend contrasts with the behavior of the silica
bonded phases, where the partition coefficient continuously
decreases for bonding densities greater than about 3.1
ymol/m2. In the 85/15 acetonitrile/water mobile phase
system the partition coefficient exhibited a decreasing
trend with increasing octadecyl bonding density once the
local maximum at 2.7 ymol/m2 is reached; this trend is
similar to that exhibited by the silica bonded phases.
It is premature at this point in time to make any
further comparisons between the behavior of the CPG bonded
phases and that of the silica bonded phases as regards the
relationship between bonding density and partition
coefficient. The fourteen silica packings evaluated span a
bonding density range from 1.60 to 3.60 ymol/m2 while the
seven CPG packings span a range of 1.70 to 3.30 ymol/m2,
with four of the seven packings in the narrow range of 2.6
to 2.8 ymol/m2. Since there is such a small number of data
points for the CPG packings, the scatter plots shown in
Figures 4-4 through 4-6 are rather speculative and therefore
can serve as only the most preliminary indication of the
partitioning behavior of the CPG packings.
The sources of error in the calculations of capacity
factor, Vs, Vm and partition coefficient have previously
been discussed for the silica bonded phases; these errors

103
are also valid for the CPG bonded phases. In addition, the
retention volumes of the naphthalene solute are difficult to
measure precisely from their resultant chromatographic
peaks. Since the CPG has a particle size range of 37 to 74
pm, the solute peaks are very broad and therefore the peak
maxima are difficult to pinpoint precisely. The solvent
disturbance peak was too indistinct and ambiguous for use in
measuring Vm; therefore Vm was determined by injection of
D2O. This should result in a fairly accurate Vm measurement
for the 55/45 methanol/water mobile phase, but this method
probably overestimates V|n for the 85/15 acetonitrile/water
mobile phase since this is a water-lean system (McCormick
and Karger, 1980; Melander, et al., 1980). Because of these
considerations, measurements of solute capacity factors for
the CPG bonded phases are even less accurate than those for
the silica bonded phases.
The calculated bonding densities for the CPG packings
are less precise than those for the silica packings due to
imprecision in their elemental analysis for percent carbon.
The analyst measuring the percent carbon in these packings
(Courtney, 1987) had expressed his concern for the elemental
analysis precision because the CPG packings were harder to
weigh accurately due to handling difficulties as well as
being susceptible to incomplete combustion during the
analysis. These concerns were reflected in the precision of
the percent carbon analysis for the CPG packings; for any
given set of elemental analyses for a single CPG sample a

104
much wider range of percent carbon values was obtained than
in an analogous situation with silica packings. In the
worst case situation, the standard deviation in the percent
carbon analysis was + 0.61% carbon. Error bars based on the
range in the partition coefficient values which would result
from this worst case standard deviation in the percent
carbon value are shown for Figure 4-6. Comparison of Figure
4-6 to Figure 4-3 demonstrates that even the errors due to
uncertainty in the elemental analysis are much more
substantial for the CPG packings than for the silica
packings. Additionally, due to the abovementioned problems
there are no doubt greater errors in the values for
octadecyl bonding density, capacity factor and Vm for CPG
packings than for silica packings. Therefore it must be
again stressed that the data depicting the behavior of the
thermodynamic partition coefficient with respect to
octadecyl bonding density for the CPG bonded phases is
tentative at best and should be regarded as only a most
preliminary prediction of retention characteristics for CPG
chromatographic systems.

CHAPTER V
CORRELATIONS BETWEEN CHROMATOGRAPHIC SELECTIVITY AND ALKYL
BONDING DENSITY
Introduction
Chromatographic selectivity (a) is the difference in
retention between two solute molecules. Chromatographic
selectivity is an important thermodynamic measurement in
studies of the solute distribution process since it is
directly related to the difference in the Gibbs free energy
of transfer from the mobile phase to the stationary phase
for two solutes:
In a = a(aG)/RT ,
where aG is the Gibbs free energy, R is the gas constant,
and T is the absolute temperature. Consequently, any two
solutes possessing different free energies of transfer will
be differentially retained (Lochmuller et a 1 ., 1985;
Melander and Horvath, 1982). Selectivity between two
solutes is measured as the ratio of their capacity factors;
aab = k a / ^ b' and 1 s defined such that a _> 1.0.
Functional group selectivity is the change in retention for
a given solute caused by the addition (or subtraction) of a
particular functional group. Evaluation of functional group
selectivity is accomplished by measuring the capacity
factors for a homologous series of compounds which differ
105

106
from each other by the functional group in question, i.e. a
homologous series of alkylbenzenes for methylene
selectivity. The natural logarithm of the capacity factor
is then plotted versus the unit number of the functional
group for each homolog; the slope of the resultant line is
the natural logarithm of the group selectivity, In a.
Figure 5-1 illustrates such a plot of methylene selectivity
on column DMAP 3 for the homologous alky1 benzene series of
toluene through penty1 benzene. The slope of the plot, which
1 s ln methylene* is 0.6773 ; the resultant amet tiy ] ene value
is then 1.969.
Antle and Snyder (1984) and Antle et al. (1985) state
that there are two different types of RP column selectivity,
namely solvophobic and chemical. Solvophobic selectivity
arises from hydrophobic interactions between the solute
molecules and the stationary phase. Chemical selectivity
comes about from strong interactions (for example, hydrogen
bonding or comp 1exation) between the solute molecules and
specific active sites such as silanol groups or trace metal
contaminants on the silica surface (Antle and Snyder, 1984;
Jandera, 1986). A third type of selectivity, shape
selectivity, can also be exhibited by chemically bonded
phases. Since these phases consist of lengthy alkyl chains
bonded to the silica surface, the conformation of the bonded
chains can play an important role in retention, especially
for large molecules. When these chains are well solvated by
the mobile phase, such as when the mobile phase has a large

In k'
Figure 5-
Methylene selectivity plot for silica column DMAP3.
The slope of the plot is In methylene'
O

108
proportion of organic modifier, the chains become more fully
extended and shape selectivity is increased (Martire and
Boehm, 1983). Wise and Sander (1985) refer to this as the
"slot model." They have postulated that when the closely
packed bonded RP chains are extended, the stationary phase
surface can be visualized as containing a number of long
narrow "slots" between these extended chains into which
solute molecules can penetrate. Since planar and/or linear
molecules can more deeply penetrate these slots and
therefore interact more strongly with the stationary phase,
they are preferent i a 1ly retained over nonplanar and/or
nonlinear molecules.
Examination of chromatographic selectivity can be very
useful in studies of retention mechanisms in LC. As
discussed in Chapter IV, the capacity factor, k1, is the
most widely studied chromatographic parameter, since it is a
normalized measure of solute retention. However, the
capacity factor is directly proportional to the volume phase
ratio (stationary/mobi1e) of the chromatographic column.
The phase ratio is dependent upon bonded group chain length,
alkyl bonding density, the pore structure of the silica
support and the homogeneity of the packing bed of the
column. Therefore, when comparing intercolumn capacity
factors there are many variables to consider, making it very
difficult to draw conclusions about intercolumn retention
behavior. Selectivity values for solutes are not affected
by the phase ratio of the chromatographic column, since they

109
are measurements of retention differences (i.e. ratios)
rather than absolute measures of retention. Therefore
intercolumn selectivity differences are not due to different
column phase ratios, but rather are due to actual
differences in the structure of the bonded alkyl chains in
the different columns. In general, selectivity values for a
particular type of bonded phase are independent of the
specific column used (Antle and Snyder, 1984; Colin et al.,
1983a and 1983b; Krstulovic et al., 1983; Melander and
Horvath, 1982), implying that very fundamental aspects of
the retention process are reflected by selectivity behavior.
Examination of methylene selectivity offers an
additional advantage for retention mechanism studies. Since
methylene selectivity is solely due to solvophobic
selectivity, it is quite insensitive to the presence of
residual silanol groups on the bonded phase surface. For
solutes which have highly polar and/or hydrogen bonding
functional groups, the presence of these silanol groups can
lead to poor chromatographic peak shape as well as anomalous
retention due to chemical selectivity. However, methylene
selectivity values for a homologous series of such compounds
will be largely unaffected by these specific interactions,
even though retention of individual members of the series
may be susceptible to these chemical interactions (Johnson,
1986).
In the present work, chromatographic selectivity was
examined as a function of stationary phase alkyl bonding

110
density. When the same mobile phase composition is utilized
in comparing different stationary phase selecti vities,
mobile phase contributions to the free energy of transfer
should be equivalent. Under such conditions, changes in
selectivity are attributable to differences in the
stationary phase structure (Lochmuller et al., 1985).
Methylene selectivity and phenyl selectivity were examined
on octadecyl silica and CPG reversed phases. Methylene
selectivity was examined using the alkylbenzenes as test
solutes; phenyl selectivity was probed with the phenyl
homologous series consisting of benzene, biphenyl and
p-terphenyl, whose structures are shown in Figure 5-2.
National Bureau of Standards (NBS) column evaluation test
mixture 1 (PAH) was also used to measure overall polycyclic
aromatic hydrocarbon (PAH) selectivity; this mixture
contains benzo[a]pyrene (BaP), 1,2 :3,4 : 5 ,6 : 7 ,8-
tetrabenzonaphthalene (TBN) and phenanthro[3 ,4-c]-
phenanthrene (PhPh), whose structures are shown in Figure
5-3.
Experimental Procedure
The liquid chromatographic system, silica and CPG
columns and solvents used for the selectivity measurements
are described in Chapter IV. Toluene (Eastman Organic
Chemicals, Rochester, NY), ethylbenzene (Fisher Scientific,
Fairlawn, NJ), propyl benzene (Alfa Products, Danvers, MA),
butylbenzene (Eastman) and penty1 benzene (Alfa) standards
were made up in HPLC grade methanol for methylene

Ill
Figure 5-
Biphenyl
Structures of phenyl selectivity test
solutes.

112
Figure 5
Benzo[a]pyrene (BaP)
Phenanthro [3,4 c| phenanth rene
(PhPh)
l,2:3,4:5,6:7,8-Tetrabenzonaphthalene (TBN)
3. Structures of National Bureau of Standards
(NBS) column evaluation test mixture number
1 (PAH) solutes.

selectivity studies. Benzene (Ma11inekrodt, Inc., Paris,
KY), biphenyl (Eastman, recrystallized three times from
ethanol) and p-terphenyl (Siyma Chemical Co., St. Louis, MO)
methanolic standards comprised the phenyl selectivity test
solutes. The NBS (Gaithersburg, MD) column evaluation test
mixture was kindly supplied by Dr. Lane Sander. Methylene
and phenyl selectivity studies were conducted at 35.0 C
with a 55/45 methanol/water mobile phase on the silica
columns; these studies were also performed with a 85/15
acetonitrile/water mobile phase at 35.0 C for both the
silica and CPG columns. The NBS test mixture was also
evaluated on the silica and CPG columns with a 85/15
acetonitrile/water mobile phase but at ambient temperature.
Results and Conclusions
Silica-Based Stationary Phases
Methylene and phenyl selectivities as a function of
octadecyl bonding density for the 55/45 methanol/water and
85/15 acetonitrile/water mobile phase systems are tabulated
in Tables 5-1 and 5-2. Since the selectivity values are
calculated from the slopes of plots of In k1 versus homolog
unit number for each stationary phase, the least squares
linear regression coefficients of correlation for each of
these plots are included to verify that linear behavior is
being followed. Colin et al. (1983a) state that a linear
relationship exists between In k and the homolog unit
number for unit numbers above three to five. This number of
units is termed the critical carbon number and it results

Table 5-1. Methylene and phenyl se1ecti vities at 35.0 C as a
function of silica octadecyl bonding density for
55/45 methanol/v/ater mobile phase.
g Bonding
ensity
mol/m2)
Methylene
Selectivity
Methylene^
Correlation
Coefficient
Phenyl
Selectivity
P heny12
Correlation
Coefficient
1 .60
1.721
0.9989
5.479
0.9998
1 .74
1 .916
0.9993
7 .272
0.9997
1 .98
1 .959
0.9997
7.053
1.0000
2.07
1.925
0.9994
7.385
0.9996
2.09
1 .967
0.9999
7 196
1.0000
2.75
1 .936
0.9993
7.606
0.9999
3.06
1 .986
0.9996
7 .830
0.9999
3.24
1 .969
0.9995
7.941
0.9999
3.34
1 .968
0.9994
8.134
0.9997
3.56
1.927
0.9994
7.965
0.9998
3.60
1 .963
0.9997
8.170
0.9997
Coefficient
of correlation for the
plot of In k1
versus carbon
number; slope of this line is ln(methylene selectivity).
Coefficient of correlation for the plot of In k' versus phenyl
number; slope of this line is ln(phenyl selectivity).

Table 5-2. Methylene and phenyl selectivities at 35.0 C as a
function of silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.
C13 Bonding
Density
(ymo1/n/)
Methylene
Selectivity
Methy1 ene
Correlation
Coefficient
Phenyl
Selectivity
Phenyl^
Correlation
Coefficient
1 .60
1 .134
0 .9971
1 .423
0.9984
1 .74
1 .291
0.9991
1.901
0.9996
1 .98
1.275
0.9993
1 .924
0.9994
2.07
1.214
0.9935
1.944
1 .0000
2.09
1.308
0.9994
1 .950
0.9999
2.75
1.530
0.9906
2.000
0.9995
2 .84
1.352
0.9983
2.042
0.9999
3.06
1.339
0.9988
2.039
0.9997
3.15
1 344
0.9974
2.011
0.9995
3.24
1.348
0.9990
2 .033
0.9992
3.34
1 .364
0.9943
2.016
0.9992
3.56
1 .350
0.9995
2 .031
0.9995
3 .60
1 .358
0.9999
2.111
0.9997
* Coefficient of correlation for the plot of In k1 versus carbon
number; slope of this line is ln(methylene selectivity).
*- Coefficient of correlation for the plot of In k versus phenyl
number; slope of this line is ln(phenyl selectivity).

116
from the fact that the effect of an additional homolog unit
should only become constant when it is sufficiently removed
from the basic functional group. Thus for homologs below
the critical carbon number, the plot of In k' versus homolog
unit number is expected to exhibit curvature. However, this
departure from linearity is generally small for RPLC
systems, causing a very limited influence on the average
slope of the plot (Colin et al., 1983a). This expected
curvature was not found for either mobile phase system, as
all of the correlation coefficients are greater than or
equal to 0.991.
Methylene selectivity versus octadecyl bonding density
is plotted in Figure 5-4 for the 55/45 methanol/water system
and in Figure 5-5 for the 85/15 acetonitrile/water system
for all of the silica stationary phases except for the
lowest bonding density phase (1.60 ymol/m^). This bonded
phase can be omitted from both of the silica methylene
selectivity plots because its selectivity in both cases can
be shown to be an outlier based on the Q-test at the 99%
confidence level for the 55/45 methanol/water mobile phase
and at the 90% level for the 85/15 aceton itri 1e/water mobile
phase data (Peters et al., 1974). The average methylene
selectivity value + one standard deviation for the remaining
ten bonded phases with the methanol/water mobile phase is
1.952 + 0.024; for the remaining twelve stationary phases in
the acetonitrile/water system this value is 1.339 + 0.074.
Using 55/45 methanol/water mobile phase systems and reversed

2.00
1.98 -
Methylene 1-96"
Selectivity
1.94 -
1.92 -
1.90 -
1.
Bonding Density (jimol / square meter)
mobile phase: 55/45 MeOH/water
H- 1- 1 h-
2.0 2.5 3.0 3.5
Figure 5-4. Plot of methylene selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 55/45
methanol/water mobile phase.

1.6
1.5 -
Methylene
Selectivity 14_.
1.3 --
1.2
1.5
mobile phase: 85/15 ACN/water
h1 1 1 1
2.0 2.5 3.0 3.5 4.0
Bonding Density (|nmol / square meter)
Figure 5-5. Plot of methylene selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 85/15
acetonitri 1 e/water mobile phase.

119
phase octadecyl columns, Colin et al. (1983a) and Karger et
al. (1976) report methylene selectivity values of 2.14 and
2.0 respectively. For octadecyl silica columns and 8b/15
acetonitrile/water mobile phases Colin et al. (1983a),
Karger et al. (1976) and Krstulovic et al. (1983) report
methylene selectivity values of 1.40, 1.3 and 1.4
respectively; therefore our reported methylene selectivity
values are comparable to literature values in both mobile
phase systems. It is not surprising that these methylene
selectivities are approximately constant in either system
since methylene selectivity is a type of solvophobic
selectivity, coming about solely from hydrophobic
interactions between the solute molecules and the stationary
phase. It was expected that such a nonspecific interaction
would be unaffected by the greater chain ordering resulting
from increasing octadecyl bonding density. The differences
in methylene selectivity values for the two mobile phase
systems is due to differences in bonded chain solvation.
Acetonitrile is able to better solvate the hydrocarbonaceous
bonded chains and therefore results in a more robust
solvation layer than methanol does. Since methylene
selectivity is a measure of the hydrophobic interactions
between a methylene group and the stationary phase, the
methylene selectivity value for the acetonitrile system is
smaller than that for the methanol system because there is
less difference in hydrophobicity between a methylene group
and the solvated stationary phase structure in the

acetonitrile system. Put another way, the acetonitrile/
bonded phase interphase is more nonpolar than the methanolic
one; therefore a methylene group will experience less
hydrophobic interactions in the acetonitrile system,
resulting in a lower methylene selectivity value (Karger et
al 1976 ) .
Examination of the relationship between phenyl
selectivity and bonding density is facilitated by inspection
of Figures 5-6 and 5-7 for the 55/45 methanol/water and
85/15 acetonitrile/water mobile phase systems respectively.
The plots show that phenyl selectivity increases with
increasing octadecyl bonding density in an approximately
linear fashion with least squares linear regression slopes
of 0.547 and 0.0835 and coefficients of correlation of 0.956
and 0.917 for the methanol/water and acetonitrile/water
systems respectively. This correlation between phenyl
selectivity and octadecyl bonding density can be attributed
to shape selectivity. As previously mentioned, other
workers have noted that chromatographic selectivity is
affected by the shape of the solute molecules (Lochmuller et
al., 1985; Martire and Boehm, 1983; Tanaka et al., 1982).
They have predicted that solute selectivity should decrease
as a function of solute shape in the order rodlike > planar
> chainlike. It has also been suggested that selectivity of
rodlike or rigid solutes increases with increasing bonded
chain surface coverage (Engelhardt et al., 1982;
Hemetsberger et al., 1979; Wise et al., 1981). This effect

8.2
8.0
7.8
Phenyl
Selectivity 76
7.4
7.2
7.0
1.5 2.0 2.5 3.0 3.5 4.0
Bonding Density (jimol / square meter)
Figure 5-6. Plot of phenyl selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 55/45
methanol/water mobile phase.

2.2
Phenyl
Selectivity
2.1 --
2.0 -
1.9
1.5 2.0 2.5 3.0 3.5 4.0
Bonding Density (|imol / square meter)
mobile phase: 85/15 ACN/water
r = 0=917
Figure 5-7. Plot of phenyl selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 85/15
acetonitrile/water mobile phase.
ro

has been explained in terms of increased ordering of the
bonded RP chains (Krstulovic et a 1 1983; Lochmuller et
al-, 1985; Marti re and Boehm, 1983).
Sander and Wise (1984a and 1984b; Wise and Sander,
1985) and Wise and May (1983) have extensively examined the
effect of alkyl bonding density on retention and selectivity
for polycyclic aromatic hydrocarbons (PAH). They have
studied the PAH selectivity of monomeric and polymeric
octadecyl phases with bonding density ranges of 1.8 to 3.2
ymol/rn^ and 2.7 to 7.3 ymol/m^ respectively. Their studies
indicate that the polymeric phases exhibit much greater PAH
selectivity than the monomeric ones and that the polymeric
phase PAH selectivity increases with increasing bonded phase
surface coverage. They initially attributed this behavior
to some fundamental difference in the structures of the
monomeric and polymeric bonded phases. However, Verzele and
Mussche (1983) concluded that there is no true difference in
the nature of polymeric and monomeric bonded phases, and
that their differences in chromatographic behavior are
attributable to differences in surface coverage. In later
comparisons of monomeric and polymeric bonded phases of
varying bonded alkyl chain length, Sander and Wise (1987)
concluded that the changes in PAH selectivity that they had
earlier observed were not necessarily due to fundamental
differences in the two phases but rather could be attributed
to changes in the overall stationary phase thickness.

124
Wise and Sander's (1985) "slot model" was postulated
based on their PAH selectivity studies. They found that for
polymeric phases with high bonding densities (greater than
about 5.1 pmo 1/m^) nonplanar solutes eluted before planar
ones and that nonlinear solutes eluted before linear ones,
even if the solutes compared had similar molecular weight,
overall shape and molecular dimensions. Additionally, they
found that selectivity between p1 anar/nonp1 anar and
linear/nonlinear PAHs increases with the degree of
nonplanarity and nonlinearity. Their "slot model"
postulates that nonplanar solutes have a greater
"thickness", hindering penetration of the solute into the
narrow slots between the bonded alkyl chains. If both wide
and narrow slots exist in the stationary phase structure due
to inhomogeneous distribution of the bonded chains on the
surface, retention will be greatest for long narrow solutes,
since they would fit into more available slots than thicker
"square" shaped molecules. The situation is analogous for
linear molecules, which would show greater retention than
nonlinear ones. This also corresponds with Marti re and
Boehm's "unified theory of retention and selectivity in
liquid chromatography" (1983) which predicts that shape
selectivity is greater for rigid rod solutes than for
globular solutes, especially when the stationary phase
chains are fully extended or more rigid. Sander and Wise
(1985) argue that higher alkyl density polymeric phases are
more extended and rigid than low density polymeric or
monomeric ones.

125
Sander and Wise (1984a) have devised a simple empirical
LC test to gauge the relative monomeric or polymeric nature
of a bonded phase. They found that the elution order of a
three component PAH test mixture of phenanthro[3,4-c]-
phenanthrene (PhPh), 1 ,2 :3 ,4 : 5 ,6 :7 ,8-tetrabuty1naphtha 1 ene
(TBN) and benzo[a]pyrene (BaP) at ambient temperature with a
85/15 acetonitrile/water mobile phase is dependent on the
type of phase and the surface coverage. For monomeric
phases (bonding densities up to about 3.2 ymol/rn2) the
elution order is BaP <_ PhPh < TBN; for oligomeric phases
(bonding densities of 3.3 to about 4.2 pmol/m2) the elution
order is PhPh < BaP < TBN. Polymeric phases (bonding
density > 4.3 ymol/m^) give the elution order PhPh < TBN <
BaP. Monomeric phases were synthesized with the
monoch1 oros i 1 ane; oligomeric and polymeric phases were
prepared using the trichlorosi1ane reagent. Each type of
phase also results in a different narrow range of values for
TBN/BaP selectivity. By examining the elution order of the
compounds in the test mixture, PAH selectivity of any RP
column can be quickly predicted.
The retention behavior of these compounds can be
attributed to their shapes. Phenanthro[3,4-c]phenanthrene
and TBN are nonplanar, due to steric hindrance of
neighboring aromatic rings. Of the two, PhPh is the more
nonplanar, exhibiting a helical shape, while TBN is
described as saddle shaped. Although TBN and PhPh are six
ring PAHs, BaP is a five ring PAH and is completely planar;

therefore as just discussed it exhibits greater retention as
the polymeric character of the bonded phase (and its bonding
density) is increased (Sander and Wise, 1984a).
In light of what has just been discussed, the trend of
greater phenyl selectivity with increasing octadecyl bonding
density shown by our work is not surprising. These results
correlate well with Sander and Wise's (1985) "slot model",
since with increasing bonding density the "slots" between
the extended octadecyl chains become increasingly long and
narrow. Therefore selectivity is expected to increase for
planar, linear solutes such as the biphenyl and p-terphenyl
used to measure phenyl selectivity in this work. However, a
more rigorous explanation for the correlation between phenyl
selectivity and bonding density can be offered in light of
the Dill (1987a) interphase stationary phase model discussed
in Chapter I. As alkyl surface densities increase, the
corresponding configurational constraints are also
increased, creating a more rigid and ordered chain packing
structure. In this model, the driving force for retention
is the creation of a solute-sized cavity in the stationary
phase chain packing structure. As bonding density and
consequently chain ordering are increased the energy
required for cavity formation also increases. Creation of a
narrow linear cavity for solute retention will require less
energy than that required to create a wider cavity such as
is needed for nonlinear or nonplanar solute retention.
Therefore selectivity for these linear and planar molecules

127
will increase with alkyl bonding density, as predicted by
this theory and as borne out by our experimental results.
It is interesting to compare the slope of the 85/15
acetonitrile/water phenyl selectivity plot (0.0835) to that
for the 55/45 methanol/water plot (0.547). This disparity
can probably be attributed to the different structures of
the solvation layers on the bonded phase surfaces in the two
very different mobile phase systems. The 85/15
acetonitrile/water solvation layer is relatively robust; at
any of the bonded phase alkyl densities the stationary phase
surface will be well solvated and the chains well extended.
This means that relative retention will only be affected to
a small extent by changes in bonding density; chain ordering
will increase very little with increased packing constraints
because the chains are already well extended and relatively
ordered. In 55/45 methanol/water the chains are not well
solvated and are in a relatively collapsed configuration;
they are rather disordered. As the bonding density
increases the chains become increasingly ordered as well as
being much more extended. Thus shape selectivity will be
affected by bonding density to a much greater extent in the
methanol/water system; this is exhibited by the larger slope
of the phenyl selectivity plot.
The selectivity behavior of Sander and Wise's PAH test
mixture on these silica columns, compiled in Table 5-3,
further confirms that shape selectivity increases with
increasing alkyl bonding density. For bonding densities of

128
Table 5-3. Tetrabuty1 naphtha 1 ene(TBN)/benzo[a]pyrene(BaP)
selectivity as a function of silica octadecyl
bonding density for 85/15 acetonitri 1 e/v/ater
mobile phase.
Cig Bonding
Density
(umol/)
TBN/BaP1
Selectivity
0
Stationary
Phase
Behavior
T emperature
(c)
1 .60
1 .60
monomeric
25.0
1.74
1.63
monomeric
24.0
1.98
1 .68
monomeric
26.0
2.07
1.72
monomeric
26.0
2.09
1.70
monomeric
26.0
2.75
1.73
monomeric
25.5
2 .84
1.72
monomeric
25.5
3.06
1.75
monomeric
25.0
3.15
1 .73
monomeric
25.0
3.24
1.72
monomeric
27.0
3.34
1.70
monomeric
29.0
3.56
1.69
monomeric
26.0
3 .60
1 .56
o 1 i g o m e r i c
26.0
y Ratio of k j p to k'Bap.
Stationary phase characterization based on classification
system of Sander and Wise (1984a). If solute elution
order is B aP_

be monomeric; elution order of PhPh to be oligomeric.

1.74 to 3.56 pmol/m2 the TBN/BaP selectivity is about 1.7
and the elution order is BaP = PhPh < TBN. At 3.60 u mol/in2,
the elution order changes to PhPh < BaP < TBN and the
TBN/BaP selectivity is 1.56. The planar BaP molecule is now
retained longer than the helical PhPh. Although this is
classified as "oligomeric" type behavior by Sander and Wise
(1984a), this stationary phase was prepared using the
monochlorosilane as opposed to the trichlorosilane reagent
used by Sander and Wise to prepare the oligomeric bonded
phases. The oligomeric bonded phases are actually polymeric
type phases whose bonding density (or "thickness") has been
controlled by sequential polymerization. The fact that our
monomeric phase exhibits the same PAH selectivity as Sander
and Wise's polymeric phases indicates that PAH selectivity
and shape selectivity are probably not a function of the
degree of stationary phase polymerization but rather are a
function of alkyl bonding density. The correlation of
phenyl selectivity with alkyl bonding density further
supports this conclusion.
Controlled Pore Glass-Based Stationary Phases
Table 5-4 is a compilation of methylene and phenyl
selectivities of the controlled pore glass (CPG) octadecyl
bonded phases versus their octadecyl bonding density for the
85/15 acetonitrile/water mobile phase; this data is also
plotted in Figures 5-8 and 5-9. Although very scattered,
the methylene selectivity seems to be approximately
constant, as in the case of the silica stationary phases.

130
The average methylene selectivity + one standard deviation
is 1.210 + 0.031. The argument for constant methylene
selectivity for the silica bonded phases can similarly be
applied to these CPG phases.
Phenyl selectivity for these CPG phases does not show
the linear trend exhibited by the silica bonded phases. The
phenyl selecti vities are quite scattered and do not seem to
correlate with bonding density in any manner. Therefore, no
conclusions can be drawn about the effect of bonding density
on phenyl selectivity for the CPG phases. Selectivity
results from the NBS PAH test mixture are listed in Table
5-5. These phases exhibit monomeric behavior at all of the
bonding densities examined; the TBN/BaP selectivity values
range from 1.66 to 1.80 and are more scattered than those
for the silica. In summary, our CPG selectivity data is
inconclusive, especially when compared to the silica data.
Further experiments on this support are warranted if its
selectivity trends are to be determined.

Table 5-4. Methylene and phenyl selectivities at 35.0 C as a
function of controlled pore glass octadecyl bonding
density for 85/15 acetonitrile/water mobile phase.
Co Bonding Methylene Methylene^ Phenyl Phenyl^
Density Selectivity Correlation Selectivity Correlation
( Mino! /mz) Coef f i ci ent Coef f i ci en t
1 .70
1 .174
0.9874
1.489
0.9902
2.59
1.199
0.9928
1.636
0.9961
2 .68
1.252
0.9980
1 .719
0.9975
2.72
1.222
0.9995
1.557
0.9983
2.83
1 .210
0.9937
1 .678
0.9971
3.21
1.240
0.9976
1.748
0.9966
3.30
1.171
0.9879
1.577
0.9980
* Coefficient of correlation
number; slope of this line
Coefficient of correlation
number; slope of this line
for the plot of In k1 versus carbon
is In(methyl ene selectivity),
for the plot of In k1 versus phenyl
is ln(phenyl selectivity).

1.26
1.24 -
Methylene 1-22"'
Selectivity
1.20 -
1.18 -
1.16
1.6
mobile phase: 85/15 ACN/water
-i 1 1 1 1 1 1 t-
1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2
Bonding Density (pmol / square meter)
3.4
Figure 5-8. Plot of methylene selectivity versus octadecyl bonding
density for CPG-86-based columns at 35.0 C for 85/15
acetonitrile/water mobile phase.
132

Phenyl
Selectivity
1.8
1.7
1.6
1.5
1.4
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
Bonding Density (pmol / square meter)
B
a
a
mobile phase: 85/15 ACN/water
, 1 1 1 1 1
Figure 5-9.
Plot of phenyl selectivity versus octadecyl bonding
density for CPG-86-based columns at 35.0 C for 85/15
acetonitrile/water mobile phase.

134
Table 5-5. Tetrabutylnaphthalene(TBN)/benzo[a]pyrene(BaP)
selectivity as a function of controlled pore
glass octadecyl bonding density for 85/15
acetonitrile/water mobile phase.
Co Bonding
Density
(ymol/m)
TBN/B aP1
Selectivity
Stationary^
Phase
Behavior
T emperatu re
(C)
1 .70
1 .66
monomeric
24.5
2.59
1.79
monomeric
25.0
2.68
1 .74
monomeric
26.0
2.72
1.71
monomeric
25.0
2.83
1 .79
monome ric
25.0
3.21
1 .80
monomeric
25.5
3.30
1.72
monomeric
25.5
y Ratio of k 1 ydn to k'Bap.
Stationary phase characterization based on classification
system of Sander and Wise (1984a). If solute elution
order is BaP<_PhP h be monomeric; elution order of PhPh to be oligomeric.

CHAPTER VI
CONCLUSIONS
Syntheses of RP Stationary Phases
The results presented in Chapter II show that the use
of ultrasound as a driving force for the synthesis of
reversed phase (RP) stationary phases is a viable
alternative to traditional reflux methods. This especially
has advantages on an industrial-sized scale; the use of
ultrasonification at ambient temperatures results in
stationary phases of comparable bonding density (3.35
umol/m^) to those produced under refluxed conditions (3.44
ymol/m2), with considerably less power consumed and
therefore at a reduced cost. Another advantage of
ultrasonification over reflux in a manufacturing situation
is with respect to Occupational Safety and Health
Administration (OSHA) guidelines; the use of reflux requires
adherence to very stringent OSHA fire codes, resulting in
additional manufacturing expenses. Ultrasound reactions at
subambient temperature (i.e. 3.0 C) produce stationary
phases with very high alkyl bonding densities (3.60
ymol/m^). Although our reason for pursuing the synthesis of
high density reversed phases was to examine their retention
and selectivity characteristics, there are practical
advantages to these phases as well. In order to separate
135

136
two compounds with very similar free energies of retention
(and therefore very similar retention times) their
chromatographic resolution, Rs, must be maximized. Since
Rs = ((N1/2)/4) ((a -1) / a) (k'/U'+l)),
where N is the number of theoretical plates (column
efficiency), a is the selectivity factor for the two
compounds and k' is the capacity factor of the longer
retained compound, an increase in a will increase Rs,
resulting in an improved separation of the two compounds.
Increasing selectivity is the optimum method for increasing
chromatographic resolution without increasing the analysis
time. As we have shown in our experimental results and
discussions in Chapter V, one method of increasing a is to
increase the alkyl bonding density. Therefore, these high
density phases offer a practical solution for the resolution
of difficult to separate compounds.
High alkyl density stationary phases are also more
amenable to alkaline pH conditions than are low density
phases. Silica-based bonded phases are usually only
practical in the pH range of 2.5 to 7.5 since at pH lower
than 2.5 the Si-C bond is cleaved and at pH higher than 7.5
appreciable dissolution of the siliceous support occurs
(Melander and Horvath, 1980). High density bonded phases
can offer a better "protection" of the siliceous support
from the mobile phase since by virtue of the closely spaced
alkyl groups less of the mobile phase is able to reach the
silica surface. Preliminary longevity studies of an

137
ultrasound synthesized stationary phase with a bonding
density of 3.35 ymol/m^ have shown impressive ruggedness--
chromatographi c performance did not appreciably degrade in
terms of efficiency or selectivity until 6000 ml of 30/30/40
methanol/acetonitri1 e/pH 11 buffer had been passed through
the column (Novak, 1987). It can be concluded that these
high density phases show great promise for extending the
viable working pH range in reversed phase systems.
Buszewski et al. (1986) have prepared monomeric octadecyl
stationary phases of various bonding densities; they found
that the high density phases resulted in better resolution
of purine compounds than the lower density phases because
the higher alkyl surface coverage shields residual silanol
groups on the silica surface, resulting in a marked decrease
in peak tailing. High alkyl density phases should then be
well suited for the improved separation of other types of
basic compounds. In summary, high alkyl density reversed
phase packings offer many practical advantages over lower
density phases.
For both silica and controlled pore glass (CPG) bonded
phase syntheses, 4-dimethyl aminopyridine (4-DMAP) proved to
be a much more effective acid-acceptor catalyst than
2,6-1utidine. As with silica, ultrasound CPG reactions
resulted in bonding densities comparable to those achieved
under traditional refluxed conditions. However, at any of
the octadecyl reaction conditions, the silica substrate had
consistently higher reactivity than the CPG, resulting in

138
higher silica bonding densities. Contrary to our
expectations, the smaller pore diameter CPG (86 Angstroms)
was more reactive in the octadecyl reaction than the 167
Angstrom CPG. Both of these trends were reversed in the
trimethyl si 1ane bonding reaction. Except for the higher
B 2 0 3 content of the CPG material, we have been unable to
postulate a reason for the seemingly anomalous CPG results.
Validity of Chromatographic Partition Coefficient
Measurements
Experimental
In order to discuss the implications of the behavior of
chromatographic partition coefficients as a function of
octadecyl bonding density, the validity of the
chromatographic partition coefficient (K) measurement should
first be established. Recall that K = k 1 / (V s / V rn).
Measurement of k1, the solute capacity factor is defined as
(Vr-Vm)/Vm where Vr is the solute retention volume and Vm is
the mobile phase void volume. The problems involved in the
measurement of Vm and V$ have been discussed in Chapter IV,
as has our rationale for our choices of conventions for
their measurement. In order to test the validity of the
chromatographic partition coefficient measurement, we
decided to perform an independent dynamic measurement of the
partition coefficient. A known mass of a chromatographic
test solute (benzene or naphthalene) was placed in a stirred
volumetric flask containing a known mass of stationary phase
DMAP5 (3.60 pmol/m^) and a known mass of mobile phase (55/45
methanol/water or 85/15 acetonitrile/water). The stationary

139
phase volume (Vs) was calculated using equation 4-4; Vm was
calculated by dividing the mobile phase mass by its density,
which had been measured at ambient temperature. The
volumetric flask was sealed and stirred continuously over a
48 hour period. Ten microliter samples of the mobile phase
portion of the mixture were taken in quadruplicate while
stirring continued at 16, 20, 32 and 48 hour intervals from
when the sample was added to the system. These samples were
injected into the HPLC system (described in Chapter IV) at
35.0 C consisting of column DMAP5 and the appropriate
methanol or acetonitrile mobile phase system and the sample
responses (peak heights) were measured. The amount of test
solute in the sample was determined by comparison of the
sample response to a calibration plot prepared for standard
solutions of the solute under the same chromatographic
conditions. For benzene in 55/45 methanol/water, the total
range of the calibration plot was from 1297 to 0.1297 p g
benzene; the log-log calibration plot is shown in Figure
6-1. For naphthalene in 85/15 acetonitrile/water, the total
range of the calibration plot was from 100.0 to 0.01000 pg
and its corresponding log-log plot is shown in Figure 6-2.
Once the sample response of the unknown was measured, the
calibration standards bracketing the unknown were diluted
and their responses measured in order to prepare a more
precisely determined calibration plot in the concentration
range of the unknown. The benzene and naphthalene
calibration plots are shown respectively in Figures 6-3 and

In Response
F igure 6-1
In (jag Benzene)
Log-log calibration plot for benzene on column DMAP5 at
35.0 C for 55/45 methanol/water mobile phase.
140

12
10
8
In Response
6
4
2
-6 -4 -2 0 2 4 6
In (jug Naphthalene)
I I i 8 fr
Figure 6-2. Log-log calibration plot for naphthalene on column DMAP5
at 35.0 C for 85/15 acetonitrile/water mobile phase.

Response
Figure 6-3. Calibration plot for benzene on column DMAP5 at
35.0 C for 55/45 methanol/water mobile phase.

Response
(X 1000)
jig Naphthalene
Figure 6-4.
Calibration plot for naphthalene on column DMAP5
at 35.0 C for 85/15 aceton itri 1e/water mobile phase.

144
6-4. All calibration standards were injected at least in
quadruplicate.
Precautions were taken in order to ensure an accurate
dynamic measurement of the partition coefficient. The
initial calibration plots were prepared over a large
concentration range in order to find the solute
concentration at the saturation point of the stationary
phase at which the column is overloaded, resulting in
nonlinear response. The amount of solute that was added to
the volumetric flask was calculated so that upon dilution
the solute concentration would be within the linear range of
the calibration plot. The solute was added to and sampled
from the volumetric flask under continuous stirring so as to
lessen the probability of solute adsorption on the flask
walls and on the stationary phase surface. The detector
responses for replicate injections at each calibration level
were all used in preparing the calibration plot rather than
using the average response at each level; this ensures that
an outlier resonse will have a negligible effect on the
overall slope and intercept of the plot. The volumetric
flask contents were sampled at four different time intervals
to ensure that complete equilibration of the solute between
the mobile and stationary phases had occurred.
Results and Conclusions
The individual responses of the 16 solute samples taken
from the volumetric flasks at different sampling times were
each within one standard deviation of the average response

of the 16 injections; therefore equilibrium distribution of
the sample between the stationary and mobile phases was
complete at the first sampling interval (16 hours). For the
benzene solute in 55/45 methanol/water the least squares
linear regression equation of the calibration plot was:
Response = (181.96 response u n i t s / p g benzene) (p g benzene) -
6.90 with a coefficient of correlation of 0.9991. The
average response ( + one standard deviation) for the dynamic
benzene sample was 140.09 _+ 5.49. This average response
corresponded to 0.8078 pg benzene in the equilibrium mobile
phase samples injected; the benzene amount range (calculated
from the average response plus and minus one standard
deviation) was from 0.8380 to 0.7777 pg benzene. The amount
of benzene that partitioned into the stationary phase was
calculated as the difference between the original amount of
benzene added to the flask (corrected for mobile phase
dilution) and the equilibrium amount in the mobile phase.
The calculation of the dynamic partition coefficient is:
K = equilibrium pg benzene in stationary phase/Vg
equilibrium pg benzene in mobile phase/Vm
where Vs and Vm are the appropriate volumes for the
volumetric flask contents. The average dynamic partition
coefficient for benzene between the 55/45 methanol/water
mobile phase and the DMAP5 stationary phase was 8.52; when
the benzene concentration range for the measurement was
considered, the dynamic equilibrium partition coefficient
ranged from 5.97 to 11.25.

146
For the naphthalene solute in 85/15 acetonitrile/water
the least squares linear regression calibration plot
equation was:
Response = (4035.65 response u n i t s / y g naphthalene) x
(y g naphthalene) 3.29 with a coefficient of correlation of
0.9999. The average response of the dynamic naphthalene
sample + one standard deviation was 430.87 + 13.89. This
response correlated to an equilibrium amount of naphthalene
in the mobile phase of 0.1076 yg with a range of 0.1110 yg
to 0.1041 yg when + one standard deviation was considered.
The average dynamic partition coefficient for naphthalene
between 85/15 acetonitrile/water mobile phase and DMAP5
stationary phase was 0.041; the high end of the naphthalene
range could not be used since the amount of naphthalene it
corresponded to (0.1110 yg) was larger than the original
amount put into the volumetric flask, once dilution was
accounted for (0.1077 yg). When the low end of the
naphthalene concentration range was considered the dynamic
partition coefficient was 1.51.
When chromatographic partition coefficients are
compared to those obtained in the dynamic equilibrium
experiment, the chromatographic K is consistently higher.
For the benzene system, Kchromatographic = 17.98 and
^dynamic = 8.51; for the acetonitrile system
^chromatographic 4*41 and ^dynemiC = 0.041. There are a
number of reasons for these discrepancies. When the
dilution factor for the amount of solute added to the

147
volumetric flask was determined, it was based solely on the
volume of mobile phase in the flask; the volume of the
stationary phase was not considered since the density of the
derivatized silica was unknown. When the contents of the
stirred flask were sampled, it was impossible to only draw
up mobile phase for sampling; even with great care some
silica was always pulled up into the syringe as well.
Therefore the sample response was that for the solute in 10
ul of mobile phase and stationary phase and not just for the
mobile phase. This will cause an overestimation of the
amount of solute in the mobile phase. It is quite likely
that all of the solute was not available for partitioning
into the bonded alkyl chains of the stationary phase since
some of it could have been entrapped in the silica pores or
adsorbed onto the interior of the volumetric flask. The
phase ratios for the dynamic equilibrium systems were
0.01609 and 0.02297 for the benzene and naphthalene systems
respectively; in the chromatographic column the phase ratio
was 0.1897. The phase ratio in the dynamic system of
necessity had to be much smaller (larger Vm) in order to be
able to stir the mixture. Therefore there was ten times
more mobile phase in the dynamic system than in the
chromatographic one, and this will lead to a larger amount
of solute partitioning into mobile phase in the dynamic
system than in the chromatographic system, causing a lower
value for the partition coefficient.

148
Finally, it was predicted in Chapter IV that the
chromatographic partition coefficient was probably
overestimated since the solvation layer volume could not be
accurately estimated. This would result in too small a
value for Vs and too large a value of Vm, resulting overall
in an overestimation of Kc|nromat0grap|11- c. The comparison of
the results for the benzene system are encouraging in this
respect; there is about a factor of two difference in the
chromatographic and dynamic partition coefficients and this
is quite good when all sources of error are considered. The
acetonitrile system showed a much larger discrepancy, but
this is not unexpected. The k values in the 85/15
acetonitri 1 e/water system have much more error associated in
their measurement than in the methanolic mobile system since
the retention times in the acetonitrile system were shorter
for the solute (less than 1.5 minutes) as was the system
"dead time" due to a thicker solvation layer in acetonitrile
systems than in methanolic ones (McCormick and Karger,
1980). Additionally, the molar amount of naphthalene in the
acetonitrile dynamic system was eight times less (and the
mass amount 13 times less) than the amount of benzene put
into the methanolic system; this was necessary because of
the large differences in their molar absorptivi ties at 254
nm. If the absolute amount of error is constant in both
systems, the relative amount of error in the naphthalene
system will be much larger than that in the benzene system
due to the much smaller amount of solute involved. In

149
conclusion, the results of the dynamic equilibrium
experiments show that the chromatographic method of determining
partition coefficients may hold great promise in systems with
moderate to long (> 5 minutes) retention times, especially if
the solvation layer volume can be accounted for accurately.
The Effect of Octadecyl Bonding Density on the
Chromatographic Partition Coefficient
Examination of Tables 4-4, 4-5 and 4-6 and their
corresponding figures (4-1, 4-2 and 4-3) shows that for a
small nonpolar solute like naphthalene in a methanol/water
or acetonitrile/water mobile phase system, solute retention
(as measured by its partition coefficient) increases
linearly until a bonding density of about 3.1 umol/m2 is
reached. At bonding densities higher than this, the
partition coefficient begins to decrease as the bonding
density increases. Although errors in the absolute value of
the partition coefficient (due to ignoring the solvation
layer volume) have been discussed in the previous section as
well as in Chapter IV, these unidirectional errors will
result in an overestimation of the partition coefficient by
a relatively constant amount. As mentioned in Chapter IV,
this will cause the curves in Figures 4-1 through 4-3 to be
shifted vertically by a constant amount, but the shape of
the plot and the trends exhibited by it as well as its
maximum will be unchanged.
The retention trends in these plots are best examined
in light of Dill's (1987a and 1987b) theory of RPLC
retention, which was discussed in Chapter I. Our

150
experimental plots of partition coefficient versus bonding
density correlate very well with Dill's predictions. The
low density region which was predicted to be from about 0 to
2.7 ymo 1/m2 (Dill, 1987b) occurs from 0 to about 3.1 ymol/m^
in our plots. In this region, solute retention increases
linearly with increasing alkyl surface density because at
low alkyl densities configurational constraints are very
small and therefore chain packing has no effect on solute
retention. Nonpolar solute retention, as exemplified by the
naphthalene solute, increases as the chain volume increases
since there is more alkyl chain volume for the solute to
partition into. Dill predicted that the y-intercept of this
linear region, where octadecyl bonding density was zero,
would also occur at zero (no solute retention), yet all of
our plots gave nonzero intercepts. There are two theories
to explain this effect--either the naphthalene solute
exhibited a small amount of adsorption behavior on the
silica in addition to its partitioning behavior into the
alkyl chains, or the nonzero intercept was a result of
additional solute retention on the bonded trimethylsilyl
groups used to deactivate the low density bonded phases. In
order to decide which theory was correct, naphthalene
retention on a bare silica column and a trimethylsilyl (TMS)
column was examined. In both mobile phase systems at both
temperatures, the naphthalene solute was completely
unretained on the bare silica column. In contrast, using
the TMS column with both mobile phase systems at both

151
temperatures resulted in naphthalene being slightly retained
(k1 = 1.15 for 55/45 methanol/water at 35.0 C and 1.42 at
20.0 C; k' = 0.148 for 85/15 acetonitrile/water at 35.0
C). The corresponding chromatographic partition
coefficients for 55/45 methanol/water at 35.0 C and 20.0 C
and for 85/15 acetonitri1e/water at 35.0 C are 21.9, 26.8
and 2.79; the respective intercept values for the linear
regions of the plots are 12.7, 12.3 and 1.54. It is not
surprising that the partition coefficients for naphthalene
on the TMS column are much larger than the plot intercepts
since the bonding densities for the low density bonded
phases ranged from 0.63 to 1.90 ymol/rn^ and that for the TMS
column was 3.16 ymol/m2; it would be expected that partition
coefficient would increase with bonding density for the
bonded TMS groups since chain ordering could not occur.
Therefore the nonzero intercept behavior is due to the
presence of the trimethy1sily1 groups in the low density
columns. Also note that the acetonitrile mobile phase
system results in a much smaller slope in the linear region
than the methanolic system. This can be explained (as in
Chapter V with selectivity plots) by the more robust nature
of the acetonitrile solvation layer; changes in bonding
density will affect retention to a much smaller extent than
in the methanolic systems, where the mobile phase solvation
is not so extensive.
In the high bonding density region (greater than 3.1
y mo 1/m^) where bonding densities have surpassed the

152
projected critical bonding density predicted by Dill (1987a)
the partition coefficient definitively decreases with
increasing bonding density, just as Dill's model had
predicted. At these high bonding densities packing
constraints are severe, and as the densities get higher more
and more energy is required to create a solute sized cavity
in the interphase structure. Since in the Dill model
(1987a) the driving force for solute retention is the
creation of this interphase solute cavity, the
chromatographic partition coefficient for the solute (and
hence its retention) decreases due to the increasing amount
of energy necessary for cavity formation. If we were able
to synthesize extremely high density phases (about 8
ymol/m^), Dill (1987a) predicts that solute retention would
no longer occur since the solute would be energetically
unable to penetrate the packing structure.
The exhibited close correlation between Dill's
predictions (1987a and 1987b) and our experimental data
leaves little doubt that partitioning is the dominant mode
of RPLC retention. Were Melander and Horvath's (1980)
"solvophobic" model true, retention would be unaffected by
the surface chain density; in contrast partitioning is
strongly affected by this parameter. The curious reader is
probably wondering why this retention behavior has never
been noticed prior to now. Recall that the most widely used
parameter for the examination of retention behavior is the
capacity factor, k1, which is the product of the partition

153
coefficient and the volume phase ratio (stat ionary/mobi1e).
Examination of the partition coefficients in Tables 4-4, 4-5
and 4-6 shows that they decrease in very small increments
for bonding densities ranging from 3.15 to 3.60 pmol/m^.
However, at these same bonding densities, Vs has increased
from 0.3094 cm^ to 0.3424 cm^ in this range, while Vm has
remained essentially constant. Therefore the cumulative
effect of these two changing parameters on k1 was for k1 to
increase very slightly; this increase was so slight that k'
essentially remains constant over this bonding density
range. To our knowledge, this is the first systematic study
wherein the effects of bonding density on retention was
examined; our approach is also unique and fundamental
because we have deconvoluted the effects of changing phase
ratio from the measured chromatographic quantity (k1) by
examining the thermodynamic partition coefficient, which is
the most fundamental parameter involved in solute retention.
One significance of this work is that it gives insight
into the RPLC retention process at the molecular level.
Such fundamental information is of great importance, since
once RPLC retention is fully understood prediction of RPLC
retention for new solutes will be facilitated, perhaps
eventually leading to a predictive RPLC retention index
system. One additional strength of this theory is that it
is predictive without the use of adjustable parameters.
In addition to the insight that this work gives on the
importance of chain ordering and density on RPLC retention,

154
this work is also relevant to partitioning behavior in
micelles, membranes, vesicles and other organized
assemblies. These organized systems are of great
physiological importance, especially in terms of studying
drug structure and pharmacological activity. It is possible
that octanol-water partition coefficients may be better
correlated with chromatographic partition coefficients than
with log k'. Since the structure of RPLC stationary phases
is much more rnembrane-1 i ke than that of the octanol-water
system, RPLC retention data has also been used to try to
characterize the lipophilic nature of solutes and therefore
it has been used as a parameter in the quantitative
structure activity relationships that relate drug structures
with activity (Braumann, 1986; Carney, 1985; Kaliszan, 1986;
Miller et a!., 1985). Since in RPLC alkyl densities can be
varied, the RPLC systems have an advantage over organized
assemblies for these studies. This work may therefore have
far reaching significance in understanding the behavior of
other organized assemblies as well as in the understanding
of RPLC retention.
Suggestions for Future Work
Improved Syntheses of High Alkyl Density Bonded Phases
In light of the high bonding densities that we have
been able to achieve at subambient temperatures using
ultrasound as a driving force for the synthesis of alkyl
derivatized stationary phases, future studies of this
technique are warranted. Even lower subambient reaction

155
temperatures such as -10 or -20 C should be investigated
since lower temperatures will result in still more ordered
systems as well as further reducing the reaction solvent
vapor pressure, promoting even more efficient ultrasonic
cavitation (Suslick, 1986). Along this same line of
reasoning, the use of a reaction solvent with a lower vapor
pressure than methylene chloride may improve ultrasonic
efficiency. Suslick (1986) states that the greater the
solvent vapor pressure within the cavitation bubble prior to
collapse, the less effective the collapse. He also
recommends solvents with low chemical reactivity in order to
minimize the solvent concentration in the vapor phase of the
cavitation event. Therefore, these ultrasound reactions
should be attempted using toluene as the reaction solvent,
since it is chemically inert, has a high vapor pressure and
is a good solvent in terms of silane solubility.
According to Suslick (1986), sonochemical reactivity is
also greatly affected by the ambient gas atmosphere. It is
desirable to maximize the temperature reached during
microbubble collapse since this is one of the driving forces
behind sonocatalysis. The maximum temperature attained
during cavitation is strongly dependent on the polytropic
ratio (Cp/Cv) of the ambient gas, since this defines the
amount of heat released during the adiabatic compression of
the gas. Suslick (1986) states that cavitation in the
presence of xenon (Cp/Cv = 1.67) versus freon (Cp/Cv = 1.1)
would result in a sevenfold ratio of maximum cavitation

156
temperatures. For argon or helium, Cp/Cv = 1.67 whereas for
nitrogen, Cp/Cv = 1.40, so an argon or helium atmosphere
will result in a higher cavitational temperature than that
achieved with nitrogen. He also mentions that nitrogen
undergoes redox and radical reactions in the presence of
ultrasound. For these reasons, Suslick (1986) advocates the
use of helium or argon atmospheres for sonochemical
reactions, and the effect of these gases on the ultrasound
RP syntheses should certainly be examined.
Although dimethy1octadecy1ch1 oros i 1ane of high purity
is commercially available, there are other silane reagents
which can be synthesized that could give even higher bonding
densities than those achieved using the chiorosi1ane. Szabo
et al. (1984) have reported that octadecyl bonded silicas
with bonding densities of 4.18 umol/m^ have resulted from
reaction of silica with di rnethy 1 octadecy 1 ( di methy 1 ami no) -
silane at 125 C for 120 hours; they also give the reaction
scheme for this reagent's synthesis. It is postulated that
this silane results in very high bonding densities because
the dimethyl ami no moiety is a better leaving group than
chloride (Szabo et al., 1984). Golding et al. (1987) have
also synthesized a new silane which results in higher
bonding densities. They have synthesized octadecy1dihydro-
chlorosi1ane; in this silane two hydrogen atoms have
replaced the two methyl groups found in the commercially
available silane reagent. It is thought that the
dihydros i 1ane leads to higher surface coverages because of

157
its reduced size; replacing the two bulky methyl groups with
hydrogens should greatly reduce steric hindrance at the
silica surface. Golding et al ( 1987 ) were able to achieve
bonding densities of about 4.6 pmol/m^ using this reagent.
It is suggested that one or both of these novel silane
reagents be tried in the ultrasound synthesis.
Our optimization of the reaction variables
(temperature, reaction time, amounts of reagents used, etc.)
has thus far followed the "educated trial and error" method,
whereby the optimum conditions cited in the literature were
used as a starting point and variables were altered in a
unilateral fashion. In order to find the best conditions
for ultrasound syntheses, the reaction variables should be
optimized systematically using a statistically sound method
such as experimental design or simplex optimization. By
application of Plackett-Burman matrix statistics, Jones
(1987a and 1987b) was able to reduce the number of
experiments necessary to optimize the bonding reaction to
twenty-four, even though twenty-one variables were involved.
Such a study should now be done on the ultrasound synthesis.
Besides the variables mentioned above, the effect of higher
acoustic power of the ultrasound source should also be
examined.
Finally, bonding reactions run at high pressures should
be attempted. Synthetic groups at the University of Florida
are able to run reactions at pressures of 10,000 atmospheres
or more. Bonding reactions run at these high pressures may

158
result in reversed phases with very high alkyl bonding
densities.
Bonded Phase Efficiency Studies
Although the bonding densities for reversed phases
synthesized by refluxed and ultrasound methods have been
compared, their chromatographic efficiencies have not.
Chromatographic efficiency is expressed as the number of
theoretical plates, N, or as the reduced plate height, h.
Column efficiency is evaluated by measuring the plate height
for a solute at a number of mobile phase flow rates; plate
height is plotted versus mobile phase linear velocity to
obtain a van Deemter plot (Snyder and Kirkland, 1979). It
would be interesting to compare column efficiencies for two
bonded phases (one prepared by ultrasonic methods and the
other by reflux) of comparable octadecyl bonding density.
We have postulated that the bonded alkyl groups of
ultrasonically synthesized stationary phases may be
distributed more homogeneously than those of refluxed
phases; this could result from increased pore penetration by
the silane reagent under ultrasonic conditions. If this is
the case, a more homogeneous distribution of bonded alkyl
groups should effect a more homogeneous energy of transfer
of the solute between the mobile and stationary phases. If
this occurs, the corresponding chromatographic peak should
exhibit a narrower and more Gaussian distribution, resulting
in a lower (improved) reduced plate height. These
efficiency comparisons between the ultrasound and refluxed

159
phases have practical significance, as enhanced column
efficiencies bring about improved resolution between
chromatographic peaks, an important consideration in
difficult separations. Efficiency comparisons between
ultrasonic phases of average (around 2.8 pmol/m^) and high
(around 3.6 ymol/m^) bonding densities should also be
carried out, in order to see if the higher bonding density
phases have a correspondingly higher resistance to mass
transfer and thus a lower chromatographic efficiency.
Sander and Wise (1987) have noted that column efficiency
degrades with increased bonding density for polymeric
stationary phases; it is important that monomeric phases be
evaluated in the same manner.
As explained in Chapter IV, stationary phases of lower
octadecyl bonding density were synthesized by "pre-
endcapping" the silica with a less than stoichiometric
amount of trimethylchlorosilane (TMCS) prior to exhaustive
octadecy1 ation. Marshall et al. (1984 and 1986 ) have
reported that the "pre-endcapping" treatment results in more
efficient stationary phases as well as significant
reductions in peak tailing. This is explained in terms of
increased homogeneity of the bonded octadecyl groups. The
TMCS is postulated to bond at the most reactive silanol
sites, deactivating them and creating a more homogeneous
silanol distribution for reaction with the octadecyl silane
(Marshall et al., 1984 and 1986). Since we have synthesized
a number of these "pre-endcapped" phases, it would be

illuminating to see if we obtain the same results as
Marshall et al. as well as to study the efficiency of these
phases as a function of degree of "pre-endcapping. Such
studies can supply additional information on the prevalence
of reactive silanols on the silica surface.
Further Retention and Selectivity Studies
Once bonded phases of even higher alkyl densities
(> 3.6 pmol/m^) can be synthesized, they must be examined in
terms of both chromatographic retention and selectivity.
The partition coefficients of small solutes on these very
high density phases should be evaluated in order to further
test Dill's (1987a) predictions. These partition
coefficient experiments (as desribed in Chapter IV) should
also be carried out with different types of solutes: large
molecules (such as PAHs) as well as those with different
types of shapes such as rodlike, chainlike, planar and
nonplanar molecules. This will illuminate whether the
relationship between bonding density and partition
coefficient predicted by Dill (1987a) and demonstrated in
our experiments with small molecules will also hold true for
larger molecules of different shapes. These experiments
should provide additional fundamental information about the
nature and organization of the bonded interphase region.
Since a change in mobile phase composition will affect chain
ordering by changing the stationary phase solvation layer,
partition coefficient experiments (as described above)
performed under different mobile phase conditions should

161
give additional information about bonded chain organization
under these conditions.
The use of 1^C enriched methanol and acetonitrile and
D 2 0 mobile phases in conjunction with ^C and ^ H FT-NMR
experiments could also be utilized in order to quantitate
the stationary phase solvation layer. This would give
information about the solvation structure as well as lead to
a more accurate determination of the column phase ratio,
since the solvation layer volume could now be added to the
bonded chain volume (as calculated in Chapter IV) to get the
total stationary phase volume. The solvation layer volume
could also be subtracted from the maximum mobile phase
volume (as determined in Chapter IV) to obtain a more
accurate Vm value; the combination of these two effects
would be a very accurate determination of the phase ratio
and capacity factor and therefore of chromatographic
partition coefficients. Carbon-13 FT-NMR spin lattice
relaxation time (T^) experiments to study chain mobility on
bonded phases have been carried out by other workers (Bayer
et a 1 1986; Gangoda and Gilpin, 1983; Gilpin and Gangoda,
1984; Shah et al., 1987) but the effect of bonding density
on chain mobility should also be explored. Additionally,
temperature effects on mobility and retention should be
studied over a wide temperature range using solution state
1 O
FT-NMR and liquid chromatographic measurements. It
would be particularly interesting to see if there is a
certain "critical" temperature at which the retention or

152
mobility behavior exhibits an abrupt change due to a
conformational change in the interphase structure. Such
changes in reversed phase behavior have been noted in gas
phase experiments (Claudy et al.,1985; Gilpin, 1984; Gonnet,
et al., 1985) but have not been reported under true liquid
chromatographic conditions.
Selectivity experiments (such as described in Chapter
V) should also be carried out with large molecules such as
PAHs as well as with solutes of different shapes: rodlike,
chainlike, planar and nonplanar. Although such experiments
have been performed (Lochmuller et al., 1985; Tanaka et al.,
1982; Wise and Sander, 1985) they too have not been carried
out on monomeric stationary phases of varying octadecyl
density; therefore the effects of chain ordering on
selectivity has not been fully explored for these different
types of molecules. Again, selectivities for these
compounds using mobile phases of different compositions
should provide information on stationary phase structure
under different mobile phase conditions. As mentioned in
Chapter V selectivity experiments actually provide more
information about stationary phase structure than retention
experiments; since they are measures of retention
differences of solutes they are unaffected by the column
phase ratio (Antle and Snyder, 1984; Colin et al., 1983a and
1983b). Selectivity behavior of these solutes at different
temperatures should be examined as well. In particular,
selectivity behavior at subambient temperatures should be

163
evaluated since the separation factor a for two solutes
increases with decreasing temperature (Snyder, 1979). It is
likely that this effect is due to a more ordered bonded
phase structure at lower temperatures. These selectivity
studies would have great practical significance as well,
because the most effective way to increase the resolution of
difficult to separate compounds is to increase
chromatographic selectivity.

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Slaats, E.H.; Markovski, W.; Fekete, J.; Poppe, H.
"Distribution Equilibria of Solvent Components in
Reversed-Phase Liquid Chromatographic Columns and
Relationship with the Mobile Phase Volume," J. Chromatoqr.
1981 207 299-323 .
Smith, R. J.; Nieass, C. S.; Wainwright, W. S. "A Review of
Methods for the Determination of Hold-Up Volume in Modern
Liquid Chromatography," J. Liq. Chromatoqr. 1986, 9,
1387-1430.
Snyder, L. R. "Temperature-Induced Selectivity in
Separations by Reversed-Phase Liquid Chromatography," J.
Chromatoqr. 1979, 179, 167-172.
Snyder, L. R.; Kirkland, J. J. Introduction to Liquid
Chromatography 2nd ed.; Wi 1 ey-I ntersci ence: "New York,
1979, Chapter 7.
Spacek, P.; Kubin, M.; Vozka, S.; Porsch, B. "Influence of
the Amount of Bonded Non-Polar Phase and the Length of
Attached Alkyl Chains on Retention Character!stics of
Silica-Based Sorbents for Reversed-Phase High Performance
Liquid Chromatography," J. Liq. Chromatoqr. 1980, 3,
1465-1480.
Staroverov, S. M.; Serdan, A. A.; Lisichkin, G. V.
"Structure of the Bonded Layer and Selectivity of Chemically
Modified Stationary Phases for Chromatography," J.
Chromatogr. 1986, 364, 377-388.
Suprynowicz, Z.; Rayss, J.; Dawidowicz, A. L.; Lodkowski, R.
"The Influence of the Concentration of Surface Boron Atoms
on the Properties of Column Packings with Bonded C^g Groups,
Prepared from Control 1ed-Porosity Glasses. II. Liquid
Chromatography," Chromatographi a 1985 2_0 677 -680.
Suprynowicz, Z.; Waksmundzki, A.; Buszewski, B.; Gawdzik, J.
"The Preparation and Chromatographic Properties of
Chemically Bonded Stationary Phase ODS-Type on Porous Glass
for Use in HPLC," Chemia Analit.yczna 1978, 2_3 325-332.

173
Sus lick, K. S. "Synthetic Applications of Ultrasound," Mod.
Synth. Methods 1986, 4, 1-60.
Szabo, K.; Le Ha, N.; Schneider, P .; Zeltner, P.; Kovats, E.
"Monofunctional (Dimethyl a ini no) si 1 an e as Silylating Agent,"
Helv. Chim. Acta 1984, 67, 2128-2142.
Tanaka, N.; Tokuda, Y.; Iwaguchi, K.; Araki, M. "Effect of
Stationary Phase Structure on Retention and Selectivity in
Reversed-Phase Liquid Chromatography," J. Chromatogr. 1982,
239, 761-772.
Unger, K. K. Porous Silica, Elsevier: New York, 1979,
Chapters 1-3.
Verzele, M.; Mussche, P. "Monomeric and Polymeric
Deri vat ization in Reversed-Phase High-Performanee Liquid
Chromatographic Materials," J. Chromatoqr. 1983, 254,
117-122.
Wainwright, M. S.; Nieass, C. S.; Haken, J. K.; Chaplin, R.
P. "Use of Retention Plots of n-Alkyl Benzenes for
Determining Dead Times in Liquid and Gas Chromatography,"
J. Chromatogr. 1985, 321, 287-293.
Wells, M. J. M.; Clark, C. R. "Liquid Chromatographic
Elution Characteristics of Some Solutes Used to Measure
Column Void Volume on C1S Bonded Phases," Anal. Chem. 1981,
53, 1341-1345.
Wise, S. A.; Bonnett, W. J.; Guenther, F. R.; May, W. E. "A
Relationship between Reversed-Phase C-¡_g Liquid
Chromatographic Retention and the Shape of Polycyclic
Aromatic Hydrocarbons," J. Chromatogr. Sci. 1981, 19 ,
457-465.
Wise, S. A.; May, W. E. "Effect of Co Surface Coverage on
Selectivity in Reversed-Phase Liquid Chromatography of
Polycyclic Aromatic Hydrocarbons," Anal. Chem. 1983 55 ,
1479-1485.
Wise, S. A.; Sander, L. C. "Factors Affecting the
Reversed-Phase Liquid Chromatographic Separation of
Polycyclic Aromatic Hydrocarbon Isomers," J. High Resolut.
Chromatogr. Chromatogr. Commun. 1985, 8, 248-255.

BIOGRAPHICAL SKETCH
Karen Belinda Sentell was born in Charleston, South
Carolina, on January 28, 1957. She attended Brentwood
Elementary School and Gordon H. Garrett High School (both in
Charleston Heights, South Carolina), graduating from Gordon
H. Garrett High School in June, 1974. She lived and worked
in Charleston, South Carolina, until September, 1979, when
she moved to Columbia, South Carolina. She entered the
University of South Carolina (Columbia, South Carolina) in
January, 1980, receiving her B.S. in chemistry (Magna Cum
Laude) in December, 1982. She entered graduate school at
the University of Florida in January, 1983, where she was
awarded a University of Florida Women's Fellowship. She was
awarded two American Chemical Society Analytical Division
Graduate Fe11owships--a summer fellowship for 1985 and a
full-year fellowship for 1986-1987. Upon completion of the
requirements for the degree of Doctor of Philosophy
(December, 1987) she served as a Leopold Schepp Foundation
Postdoctoral Fellow at the University of Florida under Dr.
John G. Dorsey.
174

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Johri GL Dorsey, Chairman
Associate Professor of Chenrb
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
ft- Tj
Anna F. Brajter-Toth
Assistant Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
/ /
\
WTff iam Weltner, Jr
Professor of Chemistry
\
XJ

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Food Science and
Human Nutrition
This dissertation was submitted to the Graduate Faculty of
the Department of Chemistry in the College of Liberal Arts
and Sciences and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
December, 1987
Dean, Graduate School



7b
phase volume is accessible to the solute.
A simple method for calculating the stationary phase
volume has been devised in our laboratory. The only
measurements necessary are the carbon load of the packing
and the actual weight of packing contained in the
chromatographic column. Wise and May (1983) proposed that
the surface density of a bonded alkyl ligand (in micromoles
of alkyl ligand per square meter of packing surface) can be
calculated by
Cs = %C (106) (4-1)
1200 nc SBET
where Cs is the bonding density (ymol/m2), %C is the carbon
loading of the packing as determined from elemental analysis
or by a gravimetric procedure (Cheng, 1985), nc is the
number of carbons in the alkyl ligand, and Sggy is the
surface area of the derivatized packing as determined by
BET analysis. But the volume of the stationary phase, V$,
can be expressed as
Vs = ( C s ) (SBET >(v)(Wp)(10"6) (4-2)
where v is the molar volume of the bonded alkyl group in
cirr/mole and Wp is the weight of the bonded packing
contained in the chromatographic column. Molar volume, v,
i s
v=M/d (4-3)
where M is the weight of the bonded phase alkyl group and d
is the density of the bonded alkyl group. Cheng (1985) has
experimentally determined the pertinent densities of
commonly used bonded silanes and reported values of 0.8607


136
two compounds with very similar free energies of retention
(and therefore very similar retention times) their
chromatographic resolution, Rs, must be maximized. Since
Rs = ((N1/2)/4) ((a -1) / a) (k'/U'+l)),
where N is the number of theoretical plates (column
efficiency), a is the selectivity factor for the two
compounds and k' is the capacity factor of the longer
retained compound, an increase in a will increase Rs,
resulting in an improved separation of the two compounds.
Increasing selectivity is the optimum method for increasing
chromatographic resolution without increasing the analysis
time. As we have shown in our experimental results and
discussions in Chapter V, one method of increasing a is to
increase the alkyl bonding density. Therefore, these high
density phases offer a practical solution for the resolution
of difficult to separate compounds.
High alkyl density stationary phases are also more
amenable to alkaline pH conditions than are low density
phases. Silica-based bonded phases are usually only
practical in the pH range of 2.5 to 7.5 since at pH lower
than 2.5 the Si-C bond is cleaved and at pH higher than 7.5
appreciable dissolution of the siliceous support occurs
(Melander and Horvath, 1980). High density bonded phases
can offer a better "protection" of the siliceous support
from the mobile phase since by virtue of the closely spaced
alkyl groups less of the mobile phase is able to reach the
silica surface. Preliminary longevity studies of an


26
Some researchers have examined solute retention as a
function of increasing alkyl chain length of the bonded
ligands. Colin et al. (1983b) and Jinno and Kawasaki
(1984c) found that log k' increased with increasing bonded
chain length, while Melander and Horvath (1980) have stated
that more or less contradictory results have appeared in the
literature on the influence of chain length. Spacek et al.
(1980) and Berendsen and de Galan (1978b) have also noted a
general trend of increased retention with increasing chain
length of the bonded ligand, but their results were less
conclusive than those of Jinno and Kawasaki (1984c). It is
not clear from any of these studies whether this trend is
due to the actual increased partitioning of the solute into
the longer alkyl chains or whether this trend is an artifact
of the retention parameter measured. This is because in all
of these studies, it is the capacity factor, k' that is
used to quantitate retention. But k = K(Vs/Vm) where K is
the chromatograpnic partition coefficient and Vs/Vm is the
volume phase ratio of the stationary and mobile phases. It
is obvious that as the chain length of the bonded alkyl
ligand is increased, a corresponding increase in V $ will
occur, as pointed out by Colin et al. (1983b). Therefore it
is unclear as to whether solute retention increased because
of increasing partition coefficient or merely because of the
phase ratio increase. In order to reliably determine the
cause of changes in actual solute retention as stationary
phase parameters are changed, the changes in the solute
partition coefficient must be examined.


53
little attention in reversed phase systems (Sander and Wise,
1984b). At present, there is no satisfactory explanation of
the effect of pore size distribution on the properties of
hydrocarbonaceous bonded phases; however differences in the
pore structure of the support material may account for some
of the differences observed in the RPLC behavior of various
commercial bonded phases having the same alkyl chain length
but different siliceous substrates (Melander and Horvath,
1980).
Controlled pore glass (CPG>) offers an ideal medium for
investigating the effects of pore size and structure on RP
retention and selectivity. CPG, which has mainly been used
in size exclusion chromatography, consists of nearly pure
quartz glass with pores of uniform size. In CPG, the pore
diameter is the same at the surface as it is in the interior
of the particle; 80% of the pores show a deviation of less
than + 10% from the nominal pore diameter (Fluka). Chemical
modification of the surface of CPG is accomplished by
reacting surface silanol groups with the appropriate
reactive silane, as has been described for silica in Chapter
II. Although such reactions have been performed to prepare
CPG RPLC bonded phases (Dawidowicz et a 1 ., 1983 ; Dawidowicz
and Rayss, 1985 ; Rayss et al. 1983 ; Suprynowicz et al.,
1978 and 1985) commercial bonded phases based on CPG are
currently impractical due to its much greater expense
compared to that of silica.


169
Karger, B. L.; Gant, J. R.; Hartkopf, A.; Weiner, P. H.
"Hydrophobic Effects in Reversed-Phase Liquid
Chromatoy raphy," J Chrornatogr. 1976 128 66-78 .
Kinkel, J. N ; Unger, K. K. "Role of Solvent and Base in
the Silanization Reaction of Silicas for Reversed-Phase
High-Performance Liquid Chromatography," J. Chromatoqr.
1984, 316, 193-200.
Knox, J. H.; Kaliszan, R. "Theory of Solvent Disturbance
Peaks and Determination of Thermodynamic Dead-Volume in
Column Liquid Chrornatoqraphy," J. Chromatoqr. 1985, 349 ,
211-234.
Kohler, J.; Chase, D. B.; Farlee, R. D.; Vega, A. J.;
Kirkland, J. J. "Comprehensive Characterization of Some
Silica-Based Stationary Phases for High-Performance Liquid
Chromatography," J. Chromatoqr. 1986 352 275-305 .
Kohler, 0.; Kirkland, J. J. "Improved Silica-Based Column
Packings for High-Performance Liquid Chromatography," J.
Chromatogr. 1987 385 125-150.
Krstulovic, A. M.; Colin, H. ; Tchapla, A.; Guiochon, G.
"Effects of the Bonded Alkyl Chain Length on Methylene
Selectivity in Reversed-Phase Liquid Chromatography, "
Chromatographia 1983, 17, 228-230.
Laub, R. J.; Madden, S. J. "Solute Retention in Column
Liquid Chromatography. V. The Column Dead Volume," J. Liq.
Chromatogr. 1985, 8, 173-186.
Le Ha, N.; Ungvaral, J.; Kovats, E. "Adsorption Isotherm at
the Liquid-Solid Interface and the Interpretation of
Chromatographi c Data," Anal Chem. 1982 5_4, 2410-2421.
Lehtonen, P. "Use of Molecular Connectivity Indices to
Predict LC Retention of Dansylamides in Six Different Eluent
Systems," Chromatographi a 1984 JL_9_, 316-321.
Lochmuller, C. H.; Hunnicutt, M. L.; Mullaney, J. F.
"Effect of Bonded-Chain Rigidity on Selectivity in
Reversed-Phase Liquid Chromatography." J P h.y s Chem. 1985 ,
89, 5770-5772.
Lochmuller, C. H.; Wilder, D. R. "The Sorption Behavior of
Alkyl Bonded Phases in Reversed Phase High Performance
Liquid Chromatography," J. Chromatoqr. Sci. 19 79 17 ,
574-579.
Lork, K. D.; Unger, K. K.; Kinkel, J. N. "Role of the
Functional Group in n-0ctyldimethyl si 1anes in the Synthesis
of Co Reversed-Phase Silica Packings for High-Performance
Liquid Chromatography," J. Chromatoqr. 1986 352 199-211 .


147
volumetric flask was determined, it was based solely on the
volume of mobile phase in the flask; the volume of the
stationary phase was not considered since the density of the
derivatized silica was unknown. When the contents of the
stirred flask were sampled, it was impossible to only draw
up mobile phase for sampling; even with great care some
silica was always pulled up into the syringe as well.
Therefore the sample response was that for the solute in 10
ul of mobile phase and stationary phase and not just for the
mobile phase. This will cause an overestimation of the
amount of solute in the mobile phase. It is quite likely
that all of the solute was not available for partitioning
into the bonded alkyl chains of the stationary phase since
some of it could have been entrapped in the silica pores or
adsorbed onto the interior of the volumetric flask. The
phase ratios for the dynamic equilibrium systems were
0.01609 and 0.02297 for the benzene and naphthalene systems
respectively; in the chromatographic column the phase ratio
was 0.1897. The phase ratio in the dynamic system of
necessity had to be much smaller (larger Vm) in order to be
able to stir the mixture. Therefore there was ten times
more mobile phase in the dynamic system than in the
chromatographic one, and this will lead to a larger amount
of solute partitioning into mobile phase in the dynamic
system than in the chromatographic system, causing a lower
value for the partition coefficient.


55/45 methanol/water and 85/15 acetonitrile/water mobile
phase systems.
For the silica packings in both mobile phase systems,
the partition coefficients linearly increased until a
critical bonding density of about 3.1 pmol/m^ was reached;
after this point the partition coefficients began decreasing
with increasing bonding density. This behavior supports a
partitioning retention mechanism for RPLC. In this model,
the driving force for retention is the creation of a solute
sized cavity in the stationary phase interphase structure.
Beyond the critical density, increased alkyl bonding density
results in enhanced interphase chain packing constraints
which increase the energy necessary for solute cavity
formation, resulting in decreased chromatographic partition
coefficients.
Methylene and phenyl selectivity were also examined as
a function of octadecyl bonding density. Methylene
selectivity was approximately constant, but phenyl
selectivity increased linearly with bonding density. This
further supports the partitioning theory; methylene
selectivity is not expected to be affected by chain ordering
but phenyl selectivity for the linear solutes used should
increase as the interphase packing structure becomes more
ordered. Identical selectivity and retention studies on the
CPG bonded phases garnered inconclusive results, as no
obvious trends were discernable in either study.
This work supports a partitioning mechanism for RPLC
retention and as such gives insight into the retention
v i i i


In Response
F igure 6-1
In (jag Benzene)
Log-log calibration plot for benzene on column DMAP5 at
35.0 C for 55/45 methanol/water mobile phase.
140


Figure 3
-1. Scanning electron micrograph of acid-1eached CPG-86;
820X magnification.
U~.
CO


Seh gal, C.; Sutherland, R. G.; Verral, R. E. "Sono-
luminescence of NO- and NOo-Saturated Water as a Probe of
Acoustic Cavitation," J Ph.ys. Chem. 1980, 84-, 396-401.
Shah, P.; Rogers, L. B.; Fetzer, J. C. "Differences in
Carbon 13 Nuclear Magnetic Resonance Spectra for Monomeric
and Polymeric Octadecyl Derivatized Silica Column Packings
for Liquid Chromatography," J. Chromatoqr. 1987, 388,
411-419.
Sinanoglu, 0. in Molecular Associations in Biology. B.
Pullman, Ed.; Academic Press: New York, 1968, 427-445.
Slaats, E.H.; Markovski, W.; Fekete, J.; Poppe, H.
"Distribution Equilibria of Solvent Components in
Reversed-Phase Liquid Chromatographic Columns and
Relationship with the Mobile Phase Volume," J. Chromatoqr.
1981 207 299-323 .
Smith, R. J.; Nieass, C. S.; Wainwright, W. S. "A Review of
Methods for the Determination of Hold-Up Volume in Modern
Liquid Chromatography," J. Liq. Chromatoqr. 1986, 9,
1387-1430.
Snyder, L. R. "Temperature-Induced Selectivity in
Separations by Reversed-Phase Liquid Chromatography," J.
Chromatoqr. 1979, 179, 167-172.
Snyder, L. R.; Kirkland, J. J. Introduction to Liquid
Chromatography 2nd ed.; Wi 1 ey-I ntersci ence: "New York,
1979, Chapter 7.
Spacek, P.; Kubin, M.; Vozka, S.; Porsch, B. "Influence of
the Amount of Bonded Non-Polar Phase and the Length of
Attached Alkyl Chains on Retention Character!stics of
Silica-Based Sorbents for Reversed-Phase High Performance
Liquid Chromatography," J. Liq. Chromatoqr. 1980, 3,
1465-1480.
Staroverov, S. M.; Serdan, A. A.; Lisichkin, G. V.
"Structure of the Bonded Layer and Selectivity of Chemically
Modified Stationary Phases for Chromatography," J.
Chromatogr. 1986, 364, 377-388.
Suprynowicz, Z.; Rayss, J.; Dawidowicz, A. L.; Lodkowski, R.
"The Influence of the Concentration of Surface Boron Atoms
on the Properties of Column Packings with Bonded C^g Groups,
Prepared from Control 1ed-Porosity Glasses. II. Liquid
Chromatography," Chromatographi a 1985 2_0 677 -680.
Suprynowicz, Z.; Waksmundzki, A.; Buszewski, B.; Gawdzik, J.
"The Preparation and Chromatographic Properties of
Chemically Bonded Stationary Phase ODS-Type on Porous Glass
for Use in HPLC," Chemia Analit.yczna 1978, 2_3 325-332.


22
and distance from the silica surface is termed a "disorder
gradient"; the bonded chains have much greater orientational
order at the anchored ends and this order decreases with the
distance from the attached end. This is in contrast to bulk
matter phases in which by definition properties are
invariant with spatial position (Dill, 1987a).
In Dill's (1987a) retention theory, the nature of the
retention process is dependent upon the nature of the
molecular organization within the interphase. There are
three factors which determine this organization: the first
is those constraints imposed by the surface density and
chain lengths of the alkyl groups bonded to the surface and
by the surface's geometry; the second requirement is that in
highly aqueous mobile phases the interphase region must
largely exclude the solvent due to hydrophobic effects; the
interphase volume will be filled by chain segments and
solute molecules. The final requirement is that the chains
adopt as much disorder as is consistent with the other two
constraints in order to conform to the second law of
thermodynamics. This approach allows the consideration of
any possible geometry of the silica surface onto which the
chains are bonded. Dill (1987a) assumes that the silica
surface is approximately planar; in terms of molecular
dimensions this should be a good approximation for
chromatographic silicas, which commonly have pore diameters
of 60 to 100 Angstroms or more; the effects of curvature
will be small unless the radii of curvature are a few
molecular chain lengths or less (Marqusee and Dill, 1986).


68
adsorption onto the residual silanol groups on the
stationary phase surface (McCormick and Karger, 1980).
Melander et al. (1983a, p. 213) suggest that "... the
most weakly bound solvent component is not present in the
solvation layer." and that this component should be used for
the determination of Vm. They concur with McCormick and
Karger (1980) that D2O is a useful probe for mobile phase
volume determination unless the mobile phase is water-lean.
As secondary probes of Vm, fructose and urea have been
suggested for all compositions of methanol/water mobile
phases and for acetonitrile volume fractions from 0 to 0.75
for acetonitrile/water mobile phase (Melander et al.,
1983a). Gutnikov and Hung (1984) have also proposed the use
of oxalohydroxamic acid as a UV-detectable Vm probe.
The use of UV-active inorganic salts such as nitrates
has also been recommended for determination of RPLC dead
volumes; however in unbuffered mobile phases the dead
volumes obtained increase with increasing amount of salt
injected. Nitrate is also prevented from penetrating the
stationary phase pores by the Donnan potential which comes
about from the negatively charged silicate ions present on
the stationary phase surface (Berendsen et al., 1980b;
Engelhardt et al., 1984; Wells and Clark, 1981). Knox and
Kaliszan (1985) suggest using a volume fraction-weighted
average of the retention volumes of isotopically labelled
forms of the mobile phase components. However, besides the
aforementioned problems associated with the detection of


56
1965a). The chemical composition of controlled pore glass
is also different from that of amorphous silica; the
composition of the finished product is typically 96% by
weight of SO2, 3% B2O3, less than 1% Na2, and a trace
amount of other metal oxides (Electro-nucleonics Inc.,
1987 ) .
Experimental Procedure
Reagents
All of the reagents used in the preparation of the
reversed phase CP6 packings were as described in Chapter II,
with the exception of CPG being used as the support material
instead of silica. All CPG was manufactured by Electro
nucleonics Inc. (Fairfield, NJ). The CPG denoted as CPG-86
was from a single lot of CPG-10-75A (Fluka Chemical Corp,;
Hauppauge, NY) and had a mean pore diameter of 86 Angstroms
with a pore size distribution of + 9.8%. The absolute
surface area (Sg^y, as measured by BET analysis) was 153.1
m^/g; the particle size range was 37-74 urn and the nitrogen
pore volume was 0.48 cm^/g. The CPG denoted as CPG-167 was
from a single lot of PG-170-400 (Sigma Chemical Co., St.
Louis, MO) with a mean pore diameter of 167 Angstroms and a
pore size distribution of + 9.6%. The absolute surface area
was 161 m2/g, the particle size distribution was 37-74 ym
and the nitrogen pore volume was 1.0 cm^/g.
Bonded Phase Preparation
Both CPG's were acid leached, dried and reacted with
the appropriate silane reagents for derivatization as


16
Snyder's polarity index) to determine the value of Ix for
"standard" solutes with different functional groups, and
then found the average value of these Ix's for a given
solute in many different mobile and stationary phases. They
then assumed that Ix will be constant for compounds which
undergo the same type of interactions with the mobile phase
as the "standard" solute does. Therefore if Vx for a similar
compound is known, its retention can be predicted from
equation 1, since A, B and I are already known from the
standard data (Jandera et al., 1982).
Although this model is useful in a practical sense
since it is based on empirical considerations, it cannot be
completely justified in a theoretical sense. Although the
solute experiences stronger interactions with the mobile
phase than with the stationary phase, Jandera et al. (1982)
do not prove that stationary phase interactions can be
completely ignored. They also state that the standard
compounds must be chosen "correctly" or else the predictive
power of the model fails; "correct" solutes will be those
with little or no specific interactions (such as hydrogen
bonding) between the solutes and mobile phase components.
This severely limits the types of compounds whose retention
can be predicted from this model. Finally, at present the
model cannot give very accurate (about 5 to 20% accuracy)
retention predictions (Jandera et al., 1982).


23
Dill (1987a) considers partition and adsorption
separately as alternative RPLC retention mechanisms. In
both cases a lattice interphase model is used for the bonded
phase surface and statistical thermodynamic calculations are
used to predict solute retention in the system (Dill, 1987a;
Marqusee and Dill, 1986; Martire and Boehm, 1983). Dill
(1987a) predicts the equilibrium partition coefficient for a
solute from the mobile phase to the stationary phase from
the chemical potentials of the solute in the mobile phase
system and in the bonded chain interphase. These
calculations include the entropy of mixing of the solute and
solvent in the mobile phase, the decrease in configurational
entropy of the bonded chains when the solute is inserted
within the interphase and the total contact free energy of
the system, which will be due to intermolecular interactions
of the molecules with their neighbors. After careful
consideration of both the partitioning and adsorption
retention mechanisms in conjunction with his interphase
model and available experimental evidence, Dill (1987a)
concludes that the principal retention mechanism in RPLC is
partitioning due to two lines of evidence. Partitioning
will be affected by the surface density of the bonded alkyl
chains; adsorption will not. Therefore if partitioning is
dominant, after a certain critical bonding density, solute
retention should decrease with increasing alkyl chain
surface density. It has been observed that there is less
solute retention in bonded alkyl phase stationary phase


79
Based on Kinkel and Unger's (1984) estimation of a
maximum of five micromoles of reactive hydroxyl sites per
square meter of silica surface, amounts of trimethyl chioro-
silane (TMCS) correspond'ng to approximately 5%, 10%, 15%,
30% and 40% coverage of these hydroxyl sites were reacted as
described in Chapters II and III under ambient conditions
(at a temperature of 26.5 C) for 24 hours to partially
precover the silica and controlled pore glass supports. The
reaction solvent was dry methylene chloride and 2,6-lutidine
was used as the acid-acceptor catalyst. It should be noted
that it is unlikely that exactly 5%, 10%, 15%, 30% or 40% of
the surface hydroxyl groups on the supports were reacted;
the amounts of TMCS used merely represent some fraction of
the amount necessary for total coverage of the surface
(Marshall et a 1., 1984). After TMCS precoverage, the
supports were washed and dried as described in Chapters II
and III. The precovered supports were then reacted with a
twofold excess of octadecy1dimethyl chi oros i 1ane and a
fourfold excess of 2,6-lutidine with methylene chloride at a
temperature of 26.5 C for 24 hours and washed and dried as
described in Chapters II and III. The resultant C-^ and C^8
bonding densities for these precovered bonded phases are
summarized in Tables 4-2 and 4-3.
HPLC Column Packing Procedures
HPLC columns were assembled from 15 cm lengths of 1/4"
outer diameter and 4.6 mm inner diameter seamless precision
bore polished HPLC tubing (Alltech Associates, Inc.,


CHs
i
Si-O-H + CI-SMCI-WirCHs
i
ch3
Figure 2-1. Bonding reaction for monomeric
reversed phase packings.
CHs
Si-0-Si-(CH2)i7 CH3 + HCI
CH3
octadecy1
CO
IX}


42
these microbubbles generates powerful shock waves with a
considerable energy output (Boudjouk, 1986; Bremner, 1986;
Suslick, 1986). Pressures in the kilobar range and
temperatures of 2000-3000 C have been estimated in the
region of the collapsing bubble for time periods in the
nanosecond range (Sehgal et al., 1980). The third
contributing phenomenon is microstreaming, where a large
amount of vibrational energy is put into small volumes with
little heating (Bremner, 1986). The extremes of temperature
and pressure generated by ultrasonic waves cause the
generation of free radicals and ions, the dispersion of
chemical layers and the promotion of intimate contact
between reactants. Emulsification of immiscible liquids and
enhanced mass transfer at solid-liquid interfaces are
secondary effects of u11ras on ification. All of these
effects can contribute to the promotion of chemical
reactions (Bremner, 1986; Suslick, 1986).
Effect of Ultrasound on the Bonding Reaction
In order to define the surface coverage of the bonded
silica in an unambiguous and pertinent manner, the surface
coverage should be expressed as the number of silane
molecules attached to the surface, usually as micromoles of
bonded silane molecules per square meter of silica surface,
taking into account the increase in weight of the silica
after the bonding reaction. These surface coverages are
calculated from the percentage of carbon as obtained from
elemental analysis of the bonded phase (Berendsen and de


120-
MO -
100-
Partltion
Coefficient,
K 90-
80-
70-
~T~
0.5
i 1 1 1 1 r-
1.0 1.5 2.0 2.5 3.0 3.5
C(8 Bonding Density (pmol/m2)
Figure 4-4. Naphthalene thermodynamic partition coefficient at 20.0
as a function of CPG-86 octadecyl bonding density for
55/45 methanol/water mobile phase.


40
from those at the other reaction times. In the comparison
of the 24 hour reactions to the 36 hour reactions, tca-]c was
0.984; for the 24 hour reaction time versus the 48 hour
reaction time, tca-|c was 0.148 and for the 36 hour reaction
time versus the 48 hour reaction time, tcalc was 0.835.
Since all of the calculated t values are less than the
critical t value (tcrit) at the 80% confidence level
(tcrit,80%= 1*34 for 16 degrees of freedom), there is no
significant statistical difference in the reaction yields
for reaction times of 24, 36 and 48 hours at the 80%
confidence level (Peters et al., 1974). Therefore,
reactions were carried out for 24 hours (as also recommended
by Kinkel and Unger (1984)) unless otherwise noted.
Once the reaction time was complete, the product was
washed in order to remove excess reagents. Each bonded
phase product was washed three times with each solvent using
the rinse sequence methylene chloride, methanol, 50/50 (v/v)
methanol/water, methanol and diethyl ether. After the ether
was allowed to evaporate from the product, the derivatized
silica was dried in a vacuum oven at 125 C for 16-24 hours.
(Caution: It is imperative that the ether be completely
evaporated from the product prior to drying the product in
the vacuum oven in order to avoid a possible explosion.)
Products were analyzed by in-house elemental analysis
performed at least in duplicate for each sample.
Reliability of the elemental analysis was confirmed by
repeated submission of a standard packing material over a


112
Figure 5
Benzo[a]pyrene (BaP)
Phenanthro [3,4 c| phenanth rene
(PhPh)
l,2:3,4:5,6:7,8-Tetrabenzonaphthalene (TBN)
3. Structures of National Bureau of Standards
(NBS) column evaluation test mixture number
1 (PAH) solutes.


165
Bremner, D. "Chemical Ultrasonics," Chem. Br. 1986, 22,
633-638.
Buszewski, B.; Sebekova, K.; Bosek, P.; Berek, D.
"Dependence of the Separation of Some Biological Substances
on the Carbon Content of C18 Chemically Bonded Phases,"
Chromatoqr. 1986, 367, 171-180.
Carney, C.F. "Estimation of Molecular Parameters by HPLC,"
J. Liq. Chromatoqr. 1985, 8, 2781-2804.
Cheng, W. "Differential Density Method for Determination of
Carbon Load on Chromatographic Packings," Anal. Chem.
1985, 57, 2409-2412.
Cheng, W.; McCown, M. "Effect of Alkyl Chain Length on
Surface Silanization of Silica," J. Chromatoqr. 1985, 318,
173-185.
Claudy, P.; Letoffe, J. M.; Gaget, C.; Morel, D.; Serpinet,
J. "Long Chain Alkyl Grafts and Mixed Alkyl-Alkane Layers
at the Surface of Macroporous Silicas. Their Gas
Chromatographic Properties below and above the Phase
Transition," J. Chromatoqr. 1985 329 331-349 .
Clough, S.; Goldman, E.; Williams, S.; George, B. "Starting
Recalcitrant Grignard Reactions," J. Chem. Ed. 1986, 63,
176.
Colin, H.; Guiochon, G.; Yun, Z.; Diez-Masa, J. C.; Jandera,
J. "Selectivity for Homologous Series in Reversed-phase LC:
III. Investigation of Non-specific Selectivity," J.
Chromatoqr. Sci. 1983a, 21, 179-184.
Colin, H.; Krstulovic, A.; Guiochon, G.; Yun, Z.
"Stationary Phase Effects in Reversed-Phase Liquid
Chromatography," J. Chromatoqr. 1983b, 255 295-309 .
Cooke, N. H. C.; Olsen, K. "Some Modern Concepts in
Reversed-Phase Liquid Chromatography on Chemically Bonded
Alkyl Stationary Phases," J. Chromatoqr. Sci. 1980, 18,
512-524.
Corriu, R. J. P.; Guerin, C. "Nucleophilic Displacement at
Silicon; Stereochemistry and Mechanistic Implications," J.
Organomet. Chem. 1980, 198, 231-320.
Courtney, M. Personal Communication, 1987.
D'Amboise, M.; Bertrand, M. J. "General Index of Molecular
Complexity and Chromatographic Retention Data," J.
Chromatogr. 1986, 361, 13-24.


116
from the fact that the effect of an additional homolog unit
should only become constant when it is sufficiently removed
from the basic functional group. Thus for homologs below
the critical carbon number, the plot of In k' versus homolog
unit number is expected to exhibit curvature. However, this
departure from linearity is generally small for RPLC
systems, causing a very limited influence on the average
slope of the plot (Colin et al., 1983a). This expected
curvature was not found for either mobile phase system, as
all of the correlation coefficients are greater than or
equal to 0.991.
Methylene selectivity versus octadecyl bonding density
is plotted in Figure 5-4 for the 55/45 methanol/water system
and in Figure 5-5 for the 85/15 acetonitrile/water system
for all of the silica stationary phases except for the
lowest bonding density phase (1.60 ymol/m^). This bonded
phase can be omitted from both of the silica methylene
selectivity plots because its selectivity in both cases can
be shown to be an outlier based on the Q-test at the 99%
confidence level for the 55/45 methanol/water mobile phase
and at the 90% level for the 85/15 aceton itri 1e/water mobile
phase data (Peters et al., 1974). The average methylene
selectivity value + one standard deviation for the remaining
ten bonded phases with the methanol/water mobile phase is
1.952 + 0.024; for the remaining twelve stationary phases in
the acetonitrile/water system this value is 1.339 + 0.074.
Using 55/45 methanol/water mobile phase systems and reversed


25
densities, chain configurational constraints are very small
and interphase chain packing will have no effect on solute
retention; solute retention will increase as the volume of
chains increases since there will be more alkyl phase for
the solutes to partition into. At a chain density of zero
(bare silica) the nonpolar solute will be unretained (Dill,
1987b).
Once a critical bonding density (predicted to be about
2.7 umol/rn^) is reached, the bonding density is high enough
for severe configurational constraints to result. In the
Dill (1987a) retention model, the free energy involved in
retention is determined by the differences in the free
energy between the creation of a solute sized cavity in the
interphase region and the destruction of a solute sized
cavity in the mobile phase. As alkyl bonding density is
increased past the critical bonding density, the chain
packing constraints become more and more severe, requiring
larger amounts of energy to create a solute cavity in the
interphase structure. Thus in the high density region
solute partitioning is predicted to decrease with increasing
alkyl bonding density due to the increasingly prohibitive
expenditure necessary for interphase cavity creation to
accomodate the solute. Dill (1987a) predicts that at 8.1
umol/m^, the maximum achievable bonding density if every
surface hydroxyl group on the silica is derivatized with an
alkyl ligand, solute retention would be zero and the solute
would be completely excluded from the interphase chain
packing structure.


64
reactions could be due to steric problems, since TMCS is
much smaller than dimethy1octadecy1 chi oros i 1ane, but the
lower reactivity of the CPG-167 compared to that of CPG-86
for the octadecy! reaction contradicts this theory. The
chemical composition of the CPG's may be implicated in this
problem, since CPG contains 3% B2O3 and silica contains no
more than trace amounts. Other workers have found that
heating CPG to a temperature of 700 C for 5 to 100 hours
results in migration of boron atoms to the glass surface and
consequent enrichment of boron on the surface of the porous
glass. They further found that this surface boron
enrichment resulted in higher octadecy1 bonding densities
than achieved on untreated CPG (Dawidowicz et al., 1983;
Dawidowicz et al., 1986; Dawidowicz and Rayss, 1986; Rayss
et al., 1983; Rayss and Dawidowicz, 1986; Suprynowicz et
al., 1985). At present, we are unable to explain the
anomalous octadecy! bonding behavior of the controlled pore
glasses.


Phenyl
Selectivity
1.8
1.7
1.6
1.5
1.4
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
Bonding Density (pmol / square meter)
B
a
a
mobile phase: 85/15 ACN/water
, 1 1 1 1 1
Figure 5-9.
Plot of phenyl selectivity versus octadecyl bonding
density for CPG-86-based columns at 35.0 C for 85/15
acetonitrile/water mobile phase.


88
Table 4-5. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
silica octadecyl bonding density for
55/45 methanol/water mobile phase.
Co Bonding Density
(umol/nr)
Naphthalene Thermodynamic Partition
Coefficient at 35.0 C
1 .60
43.4
1 .74
50.7
1 .98
51 .3
2.07
60.5
2.16
59.5
2.75
69.8
2.84
71 .3
3.06
76.1
3 .15
70.9
3.24
69.2
3.34
66.8
3.43
65.0
3.56
63.9
3.60
63.7


CHAPTER I
INTRODUCTION
Models of Reversed Phase Liquid Chromatographic Retention
Retention Indices Based on Solute Descriptors
Reversed phase liquid chromatography (RPLC) is one of
the most popular and powerful analytical separation methods.
In RPLC, the stationary phase support consists of silica
particles which are typically 3 to 10 pm in diameter; alkyl
chains, usually 8 or 18 carbons in length, are attached to
oxygen atoms on the silica surface via covalent bonds,
resulting in a nonpolar surface. The mobile phase consists
of water and an organic modifier such as methanol,
acetonitrile or tetrahydrofuran. Thus the mobile phase is
much more polar than the stationary phase; mobile phase
polarity is adjusted by varying the volume ratios of water
and organic modifier. It has been estimated that 80-90% of
the high performance liquid chromatography (HPLC) systems
currently in use are reversed phase systems (Melander and
Horvath, 1980). Yet many practitioners of RPLC view this
technique as "black magic" because its retention mechanism
is not well understood, especially at the molecular level.
This makes the prediction of retention for new compounds of
interest extremely difficult. A basic understanding of RPLC
retention at the molecular level is a necessity for the
1


23
was verified by actual chromatographic behavior. These
experiments were carried out using both silica and
controlled pore glass substrates in order to compare the two
materials as stationary phase supports.


12
10
8
In Response
6
4
2
-6 -4 -2 0 2 4 6
In (jug Naphthalene)
I I i 8 fr
Figure 6-2. Log-log calibration plot for naphthalene on column DMAP5
at 35.0 C for 85/15 acetonitrile/water mobile phase.


171
Miller, M. M. ; Wasik, S. P ; Huang, G.-L.; Shiu, W. -Y. ;
Mackay, D. "Relationships between Octanol-Water Partition
Coefficients and Aqueous Solubility," Environ. Sci. Technol.
1985 19 522-529 .
Nikolov, R. N. "Theoretical Aspects of the Pore
Distribution and its Determination by Size-Exclusion
Chromatography," J Chromatogr. 1986 364 163-182 .
Novak, J. W. Personal Communication. Aluminum Corporation
of America; Alcoa Center, PA; 1987.
Peters, D. G.; Hayes, J. M.; Hieftje, G. M. Chemical
Separations and Measurements: Theory and Practice of
Analytical Chemistry", 1st ed.~; w7 B. Saunders Company:
Philadelphia, Pa., 1974, Chapter 2.
Petrovic, S. M.; Lomic, S.; Sefer, I. "Utilization of the
Functional Group Contribution Concept in Liquid
Chromatography of Chemically Bonded Reversed Phases," J.
Chromatogr. 1985, 348, 49-65.
Rayss, J.; Dawidowicz, A. "The Distribution of
N-Octadecanol on the Boron Enriched Controlled-Porosity
Glass Surface," Z. P hys C hemie 1986 267 113-119 .
Rayss, J.; Dawidowicz, A.; Suprynowicz, Z.; Buszewski, B.
"A Study of the Properties of Octadecyl Phases Bonded to
Controlled-Porosity Glasses. II. Application in Liquid
Chromatography," Chromatographia 1983, 17, 437-440.
Sander, L. C.; Field, L. R. "Effect of Eluent Composition
on Thermodynamic Properties in High-Performance Liquid
Chromatography," Anal Chem. 1980 5_2 2009-2013 .
Sander, L. C.; Wise, S. A. "Synthesis and Characterization
of Polymeric C^g Stationary Phases for Liquid
Chromatography, Anal Chem. 1984a, 5j5 504-510.
Sander, L. C.; Wise, S. A. "Influence of Substrate
Parameters on Column Selectivity with Alkyl Bonded-Phase
Sorbents," J. Chromatogr. 1984b, 316 163-181.
Sander, L. C.; Wise, S. A. "Effect of Phase Length on
Column Selectivity for the Separation of Polycyclic Aromatic
Hydrocarbons by Reversed-Phase Liquid Chromatography," Anal.
Chem. 1987, 59, 2309-2313.
Sands, B. W.; Kim, Y. S.; Bass, J. L. "Characterization of
Bonded-Phase Silica Gels With Different Pore Diameters," J.
Chromatogr. 1986, 360, 353-369.


173
Sus lick, K. S. "Synthetic Applications of Ultrasound," Mod.
Synth. Methods 1986, 4, 1-60.
Szabo, K.; Le Ha, N.; Schneider, P .; Zeltner, P.; Kovats, E.
"Monofunctional (Dimethyl a ini no) si 1 an e as Silylating Agent,"
Helv. Chim. Acta 1984, 67, 2128-2142.
Tanaka, N.; Tokuda, Y.; Iwaguchi, K.; Araki, M. "Effect of
Stationary Phase Structure on Retention and Selectivity in
Reversed-Phase Liquid Chromatography," J. Chromatogr. 1982,
239, 761-772.
Unger, K. K. Porous Silica, Elsevier: New York, 1979,
Chapters 1-3.
Verzele, M.; Mussche, P. "Monomeric and Polymeric
Deri vat ization in Reversed-Phase High-Performanee Liquid
Chromatographic Materials," J. Chromatoqr. 1983, 254,
117-122.
Wainwright, M. S.; Nieass, C. S.; Haken, J. K.; Chaplin, R.
P. "Use of Retention Plots of n-Alkyl Benzenes for
Determining Dead Times in Liquid and Gas Chromatography,"
J. Chromatogr. 1985, 321, 287-293.
Wells, M. J. M.; Clark, C. R. "Liquid Chromatographic
Elution Characteristics of Some Solutes Used to Measure
Column Void Volume on C1S Bonded Phases," Anal. Chem. 1981,
53, 1341-1345.
Wise, S. A.; Bonnett, W. J.; Guenther, F. R.; May, W. E. "A
Relationship between Reversed-Phase C-¡_g Liquid
Chromatographic Retention and the Shape of Polycyclic
Aromatic Hydrocarbons," J. Chromatogr. Sci. 1981, 19 ,
457-465.
Wise, S. A.; May, W. E. "Effect of Co Surface Coverage on
Selectivity in Reversed-Phase Liquid Chromatography of
Polycyclic Aromatic Hydrocarbons," Anal. Chem. 1983 55 ,
1479-1485.
Wise, S. A.; Sander, L. C. "Factors Affecting the
Reversed-Phase Liquid Chromatographic Separation of
Polycyclic Aromatic Hydrocarbon Isomers," J. High Resolut.
Chromatogr. Chromatogr. Commun. 1985, 8, 248-255.


Table 4-1. High octadecyl bonding density reversed phase packings.
Packing
Identifier
Base
Packing
Reaction
Conditions
T emperatu re
(C)
Reaction
Time (h)
Acid-Acceptor
Cata!yst
Co BondingDensity
(umol/m2)
C18-2
silica
ref 1ux
50.0
24
2,6-1utidine
2.75
LT2
silica
u11 rasound
8.5
101
2 ,6 1 u t i d i n e
2 .84
DMAP1
silica
ultrasound
28.0
24
4-DMAP
3 .06
DMAP1/31
silica
ultrasound
28.0/4.0
24/97
4-DMAP
3.15
DMAP3
silica
u1trasound
4.0
97
4-DMAP
3.24
US/ref1
silica
ultrasound
50.0
24
4-DMAP
3.34
ref/DMAP
silica
reflux
50.0
24
4-DMAP
3.43
rederDMAP12
silica
ultrasound
28.0
24
4-DMAP
3.56
DMAP5
silica
ultrasound
3.0
144
4-DMAP
3 .60
CPG2
CPG-86
reflux
50.0
24
2,6-lutidine
2 .68
CPG4
CPG-86
ultrasound
28.5
24
4-DMAP
3.30
Packing DMAP1/3 is a 50/50 (weight/weight) mixture of packings DMAP1 and DMAP3.
Packing rederDMAPl was obtained by reacting packing DMAP1 with the octadecyl silane
under identical conditions as for DMAP1.
00


144
6-4. All calibration standards were injected at least in
quadruplicate.
Precautions were taken in order to ensure an accurate
dynamic measurement of the partition coefficient. The
initial calibration plots were prepared over a large
concentration range in order to find the solute
concentration at the saturation point of the stationary
phase at which the column is overloaded, resulting in
nonlinear response. The amount of solute that was added to
the volumetric flask was calculated so that upon dilution
the solute concentration would be within the linear range of
the calibration plot. The solute was added to and sampled
from the volumetric flask under continuous stirring so as to
lessen the probability of solute adsorption on the flask
walls and on the stationary phase surface. The detector
responses for replicate injections at each calibration level
were all used in preparing the calibration plot rather than
using the average response at each level; this ensures that
an outlier resonse will have a negligible effect on the
overall slope and intercept of the plot. The volumetric
flask contents were sampled at four different time intervals
to ensure that complete equilibration of the solute between
the mobile and stationary phases had occurred.
Results and Conclusions
The individual responses of the 16 solute samples taken
from the volumetric flasks at different sampling times were
each within one standard deviation of the average response


163
evaluated since the separation factor a for two solutes
increases with decreasing temperature (Snyder, 1979). It is
likely that this effect is due to a more ordered bonded
phase structure at lower temperatures. These selectivity
studies would have great practical significance as well,
because the most effective way to increase the resolution of
difficult to separate compounds is to increase
chromatographic selectivity.


119
phase octadecyl columns, Colin et al. (1983a) and Karger et
al. (1976) report methylene selectivity values of 2.14 and
2.0 respectively. For octadecyl silica columns and 8b/15
acetonitrile/water mobile phases Colin et al. (1983a),
Karger et al. (1976) and Krstulovic et al. (1983) report
methylene selectivity values of 1.40, 1.3 and 1.4
respectively; therefore our reported methylene selectivity
values are comparable to literature values in both mobile
phase systems. It is not surprising that these methylene
selectivities are approximately constant in either system
since methylene selectivity is a type of solvophobic
selectivity, coming about solely from hydrophobic
interactions between the solute molecules and the stationary
phase. It was expected that such a nonspecific interaction
would be unaffected by the greater chain ordering resulting
from increasing octadecyl bonding density. The differences
in methylene selectivity values for the two mobile phase
systems is due to differences in bonded chain solvation.
Acetonitrile is able to better solvate the hydrocarbonaceous
bonded chains and therefore results in a more robust
solvation layer than methanol does. Since methylene
selectivity is a measure of the hydrophobic interactions
between a methylene group and the stationary phase, the
methylene selectivity value for the acetonitrile system is
smaller than that for the methanol system because there is
less difference in hydrophobicity between a methylene group
and the solvated stationary phase structure in the


Topological descriptors such as molecular connectivity
indices are used to correlate chromatographic retention with
3
molecular structure. These indices are numerical values
which quantitatively describe carbonaceous adjacency
relationships in the molecular structure of a solute
(Lehtonen, 1984). Molecular connectivity indices have been
shown to be proportional to the cavity surface area of a
molecule. When a nonpolar hydrocarbon solute is introduced
into an aqueous or hydroorganic environment, a large
negative entropy of solution results. It has been suggested
that this negative entropy is a result of structural
ordering around the hydrocarbon molecule (Karger et al.,
1976). This ordering comes about from the formation of a
cavity of water molecules around the hydrocarbon molecule.
To overcome the entropy loss, nonpolar molecule segments
will try to remove themselves from the aqueous medium and/or
they will group together. The term "hydrophobic effects" is
used to decribe these two phenomena of cavity formation and
nonpolar clustering. The calculated surface area of the
water cavity is significant because it can be related to the
solubility of hydrocarbons in water (Karger et al., 1976).
Likewise molecular connectivity, since it is also
proportional to the cavity surface area, has also been
correlated to non-electrolyte water solubility; however in
contrast to cavity surface area, simple first order
molecular connectivity indices are quite easy to calculate.
Since in some cases the logarithm of the aqueous solubility


8
retention is controlled by the thermodynamic equilibrium of
the solute between the mobile phase and stationary phase,
retention could theoretically be predicted from standard
Gibbs free energies since aG = -RT In K, where AG is the
standard Gibbs free energy, R is the gas constant, T the
absolute temperature and K the equilibrium distribution
constant for the solute between the stationary and mobile
phases. Since k' = K(Vs/Vm) where V$ and Vm are the
stationary and mobile phase volumes, the capacity factor for
a solute (and thus its normalized retention) could easily be
calculated were aG, Vs and Vm known. Since experimental
aG values for these systems are unavailable, they are often
estimated using liquid mixture models with readily available
physical parameters, such as Hildebrand solubility
parameters and group contribution concepts. But Hildebrand
solubility parameters are only useful for qualitative
descriptions of chromatographic behavior; therefore the
group contribution concept is often used, since it was
developed to predict activity coefficients in nonelectrolyte
liquid mixtures (Petrovic et a 1 ., 1985 ). This concept is
the basis for the UNIFAC model for chromatographic
retention, which combines a model based on extension of
quasi-chemical theory of liquid mixtures (UNIQUAC) with the
concept of functional group solubility. In this method
solute activity coefficients in the mobile and stationary
phases are calculated via structural and binary parameters
characterizing the mutual interaction energy of the


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i i i
ABSTRACT. vi i
CHAPTERS
I INTRODUCTION 1
Models of Reversed Phase Liquid Chrornatographi c
Retenti on 1
Theories of Retention in RPLC 17
II SYNTHESES OF SILICA-BASED RP STATIONARY PHASES 29
Experimental Considerations in the Synthesis
of RP Stationary Phases 29
Experimental Procedure 34
Syntheses Utilizing Ultrasonic Waves 41
Effect of Subambient Temperature on the
Ultrasound Reaction 44
The Use of 4-Dimethylamino pyridine as the
Aci d-Acceptor Catalyst 45
III SYNTHESES OF CONTROLLED PORE GLASS-BASED RP
STATIONARY PHASES 52
Comparison of Controlled Pore Glass and Silica
as Supports for RP Stationary Phases 52
Experimental Procedure 56
Comparison of Silica and CPG Bonding Densities
via Reflux and Ultrasonic Syntheses 60
IV CORRELATIONS BETWEEN CHROMATOGRAPHIC RETENTION
AND OCTADECYL BONDING DENSITY 65
Chromatographic Determination of Thermodynamic
Partition Coefficients 65
Experimental Procedure 76
Resul ts . 86
v


72
measurement of Vs, a convention for defining Vs must be
chosen, since the choice of the phase ratio must be
compatible with the definition of K that is in agreement
with the molecular mechanism of retention. Jandera et al.
(1982) have defined the stationary phase volume as that
fraction of the column volume that is not occupied by the
mobile phase. While this choice is certainly convenient and
can be readily determined, it is at best a crude measure, as
similar (or even identical) values of Vs would be obtained
for stationary phases made from the same bulk silica but
with different bonding densities of alkyl chains, or
possibly even of different chain lengths. Any determination
of stationary phase volume based solely on mobile phase
volume measurements is doomed to failure, as such a
measurement cannot be sufficiently sensitive to ascertain
bonding density or small chain length differences.
Melander and Horvath (1980) have suggested defining the
phase ratio as the ratio of the surface area of the
adsorbent (m2) divided by the column dead volume (cm^).
While this approach is an improvement in definition, it
again fails to account for certain variations in the
structure of the bonded phase and it implies that adsorption
is the sole mechanism in RPLC retention. The major drawback
to this proposed phase ratio convention, however, lies in
the accurate measurement of the two parameters involved. As
previously mentioned, chromatographers have been unable to
embrace any one of the commonly used methods for determining


might be facilitated under ultrasonification. The use of
ultrasound has two distinct advantages over traditional
reflux methods. The ultrasonic waves serve as a driving
force which is controlled independently of temperature,
allowing reaction temperatures to be varied over any desired
range. Secondly, the power of the ultrasonic driving force
can be varied by using a variable power ultrasonic probe.
We have investigated the effect of ultrasound on the
silane bonding reaction, including the effects of subambient
and superambient temperatures on the ultrasonic reaction.
In addition to these investigations, a novel base,
4-dimethyl aminopyridine, was utilized as the acid-acceptor
catalyst, in hopes that it might prove superior to
2,6-lutidine.
Experimental Procedure
Reagents
All of the organic solvents used were supplied by
Fisher Scientific (Fair!awn, NJ). Water was deionized,
passed through a Barnstead Nanopure (Boston, MA)
purification system, irradiated in a Photronix Model 816
HPLC reservoir with a UV source (Photronix Corp., Medway,
MA) for at least 48 hours, and filtered through a 0.45 pm
Nylon 66 membrane (Rainin, Woburn, MA). The methanol used
was HPLC grade; the chloroform, methylene chloride, and
diethyl ether were reagent grade. Methylene chloride was
dried by stirring over phosphorus pentoxide (Fisher
Scientific) for 24 hours, followed by distillation under a
dry nitrogen atmosphere.


93
coefficient of correlation showed a definite linear
relationship between partition coefficient and bonding
density, especially in light of the uncertainties in the
measurements of bonding density, Vs, Vm and capacity factor.
The amount of error propagated in the bonding density could
be calculated from the variances in percent carbon and
support surface area; similarly, that for the stationary
phase volume (as calculated by Equation 4-4) could be
calculable from the variances in percent carbon, weight of
the packing contained in the column and density of the alkyl
group bonded to the silica surface. However, these
calculable sources of error are small when compared to the
incalculable sources of error in the stationary and mobile
phase volumes. The primary source of error in Vm
determination is the choice of convention for its
measurement, as discussed earlier in this chapter. In fact
the choice of Vm convention is the most significant source
of error in the calculation of capacity factors as well,
since k1 = (Vr-Vm)/Vm, where V r is the retention volume of
the solute of interest. The most significant error in Vs is
certainly the determination of the volume of the solvation
layer associated with the stationary phase surface. As
explained earlier in this chapter, we have developed an
equation (4-4) to calculate the stationary phase volume
which gives an accurate volume for the alkyl chains bonded
to the silica surface; however this convention does not take
the solvation layer volume into account. This omission was


154
this work is also relevant to partitioning behavior in
micelles, membranes, vesicles and other organized
assemblies. These organized systems are of great
physiological importance, especially in terms of studying
drug structure and pharmacological activity. It is possible
that octanol-water partition coefficients may be better
correlated with chromatographic partition coefficients than
with log k'. Since the structure of RPLC stationary phases
is much more rnembrane-1 i ke than that of the octanol-water
system, RPLC retention data has also been used to try to
characterize the lipophilic nature of solutes and therefore
it has been used as a parameter in the quantitative
structure activity relationships that relate drug structures
with activity (Braumann, 1986; Carney, 1985; Kaliszan, 1986;
Miller et a!., 1985). Since in RPLC alkyl densities can be
varied, the RPLC systems have an advantage over organized
assemblies for these studies. This work may therefore have
far reaching significance in understanding the behavior of
other organized assemblies as well as in the understanding
of RPLC retention.
Suggestions for Future Work
Improved Syntheses of High Alkyl Density Bonded Phases
In light of the high bonding densities that we have
been able to achieve at subambient temperatures using
ultrasound as a driving force for the synthesis of alkyl
derivatized stationary phases, future studies of this
technique are warranted. Even lower subambient reaction


14
estimated P values do not in general correlate as well with
retention as measured ones (Braumann, 1986; Funasaki et al.,
1986). This approach is very much like that of Jinno and
Kawasaki (1984a and 1984c) previously discussed except that
Jinno and Kawasaki examined substituent group hydrophobicity
(pi parameters) in relation to retention whereas Funasaki et
al ( 1986 ) examined its relation with overall molecular
hydrophobicity (log P).
Funasaki et al. (1986) also examined the effect of
temperature and mobile phase composition on the degree of
correlation between log k' and log P. Not s u rpri s i ng 1 y ,
they found that they were better correlated when both were
measured at the same temperature than when they were
measured at different temperatures. They also found that
the log k' value estimated from extrapolation to zero
percent methanol (i.e. totally aqueous) mobile phase gave a
better correlation with log P than those obtained with
hydroorganic mobile phases. As in all other cases
previously discussed, the extent of correlation will be
dependent upon the test solutes, the chromatographic column
and the experimental conditions used (Funasaki et al.,
1986 ).
In summary, most of the retention indices based on
solute descriptors tend to accurately predict retention for
certain small sets of similar compounds; they are by no
means universal. Additionally, some of them require
extensive calculations and/or experimental data in order to


1.26
1.24 -
Methylene 1-22"'
Selectivity
1.20 -
1.18 -
1.16
1.6
mobile phase: 85/15 ACN/water
-i 1 1 1 1 1 1 t-
1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2
Bonding Density (pmol / square meter)
3.4
Figure 5-8. Plot of methylene selectivity versus octadecyl bonding
density for CPG-86-based columns at 35.0 C for 85/15
acetonitrile/water mobile phase.
132


67
mobile phase due to the hydrophobicity of the stationary
phase. The composition of the solvation layer also varies
with its distance from the anchored ends of the RP chains;
therefore its presence results in an ill-defined boundary
between the stationary and mobile phases (Berendsen et al.
1980b; Gutnikov and Hung, 1984; Le Ha et al., 1982, Knox and
Kaliszan, 1985).
There are three general categories of procedures used
to determine Vm: the use of unretained compounds, the
linearization of the net retention time for a homologous
series and static methods (Berendsen et al., 1980b). The
choice of an "unretained" compound for Vm measurements in
RPLC systems is a difficult one. In any case, neither its
heat of sorption nor its size should differ from those of
the mobile phase components. For this reason, mobile phase
constituents and especially their deuterated analogs are
often used (Engelhardt et al., 1984). However, this choice
is not without its problems. Since these compounds are
transparent in the UV region, sensitive detection of them
requires a refractive index detector. The use of deuterated
organic modifier as an unretained compound is only valid
when there is a large amount of organic modifier present in
the mobile phase since in organic-lean mobile phases the
marker will be slightly retained, especially in the case of
methanol/water mobile phase systems (Engelhardt et al.,
1984). The same is true for D 2 0 in organic-rich mobile
phase systems; this phenomenon is attributed to D2O


2.2
Phenyl
Selectivity
2.1 --
2.0 -
1.9
1.5 2.0 2.5 3.0 3.5 4.0
Bonding Density (|imol / square meter)
mobile phase: 85/15 ACN/water
r = 0=917
Figure 5-7. Plot of phenyl selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 85/15
acetonitrile/water mobile phase.
ro


60
Comparison of Silica and CPG Bonding Densities via Reflux
and Ultrasonic Syntheses
The controlled pore glasses of both pore sizes (denoted
CPG-86 and CPG-167) were derivatized under the same types of
reaction conditions as for the Davisil silica. Two aspects
in particular were to be examined by these experiments; the
first was to determine the reactivity of CPG compared to
that of amorphous silica and the second was to determine the
effect of pore size on CPG reactivity. From pore shape
indices, it has been found that the pore shape of siliceous
pores is not usually cylindrical; therefore the cylindrical
pore model is not a good approximation for silica (Nikolov,
1986). Since CPG has a very uniform pore diameter compared
to that of silica and the CPG pores are much larger than the
molecular dimensions of the reactive silane (the average
molecular cross-sectional area for the octadecy1ch1 oro-
silane is 50.95 (Angstromsper molecule and its length is
24.72 Angstroms (Cheng and McCown, 1985)) it was expected
that the CPG's would exhibit higher reactivity and therefore
result in alkyl bonding densities higher than those achieved
with silica. In addition, it was expected that CPG-167
would be more reactive than CPG-86 due to its larger pore
size. Other workers have found that octadecyl bonding
density increases with increasing pore size for silica
substrates (Engelhardt et al., 1982; Sander and Wise, 1984b;
Sands et al., 1986; Staroverov et al., 1986).
In the first set of reactions, both CPG-86 and CPG-167
were rotated at ambient temperature (26.0 C) for 24 hours


152
projected critical bonding density predicted by Dill (1987a)
the partition coefficient definitively decreases with
increasing bonding density, just as Dill's model had
predicted. At these high bonding densities packing
constraints are severe, and as the densities get higher more
and more energy is required to create a solute sized cavity
in the interphase structure. Since in the Dill model
(1987a) the driving force for solute retention is the
creation of this interphase solute cavity, the
chromatographic partition coefficient for the solute (and
hence its retention) decreases due to the increasing amount
of energy necessary for cavity formation. If we were able
to synthesize extremely high density phases (about 8
ymol/m^), Dill (1987a) predicts that solute retention would
no longer occur since the solute would be energetically
unable to penetrate the packing structure.
The exhibited close correlation between Dill's
predictions (1987a and 1987b) and our experimental data
leaves little doubt that partitioning is the dominant mode
of RPLC retention. Were Melander and Horvath's (1980)
"solvophobic" model true, retention would be unaffected by
the surface chain density; in contrast partitioning is
strongly affected by this parameter. The curious reader is
probably wondering why this retention behavior has never
been noticed prior to now. Recall that the most widely used
parameter for the examination of retention behavior is the
capacity factor, k1, which is the product of the partition


Figure 2-2. Scanning electron micrograph of acid-leached Davisil
silica; 500X magnification.
CO
Ch


35
Dimethyloctadecylchlorosilane, n-octyldimethylchloro-
silane and trimethy1ch1 oros i 1 ane (99.9%) were used as
received from Petrarch Systems (Bristol, PA). The
2,6-lutidine (Sigma Chemical Co., St. Louis, MO) was stirred
for 24 hours over barium oxide (Fisher Scientific) prior to
distillation under dry nitrogen atmosphere; 4-dimethy1 ami no
pyridine (4-DMAP; Nepera Inc., Harriman, NY) was oven dried
at 80 C for 24 hours before use.
The chromatographic silica was from a single lot of
Davisil (W. R. Grace, Baltimore, MD) synthetic amorphous
silica, grade 641LC0X1823. The silica had an average pore
diameter of 147 Angstroms, an absolute surface area (SB£j,
as measured by BET analysis) of 300 m^/g, a particle size
range of 20-30 pm with an 80% distribution of 23 + 10 pm and
a nitrogen pore volume of 1.10 cm3/g (Grace, 1984). As
recommended by Snyder and Kirkland (1979) the silica was
acid leached in 0.1 M nitric acid at 90 C for 24 hours in
order to fully hydroxylate the surface and to remove any
metal contaminants remaining from the manufacturing process.
The silica was then washed thoroughly with water until all
traces of the nitric acid had been removed and dried under
vacuum at 240 C for 24 hours prior to use in order to
remove physically adsorbed water from the surface (Unger,
1979). Scanning electron micrographs of the acid-leached
silica (Figures 2-2 and 2-3) show that this type of
chromatographic silica exhibits an irregular shape as well
as an irregular surface. This surface irregularity is
reflected in the silica's high surface area.


77
densities greater than or equal to 2.75 ymol/m2 were
prepared under traditional reflux conditions as well as
under ambient, subambient and superambient ultrasonic
conditions, using 2,6-lutidine or 4-DMAP as the
acid-acceptor catalyst as described in Chapter II.
Controlled pore glass bonded phases with octadecyl bonding
densities of 3.30 and 2.63 pmol/m^ were prepared from the 86
Angstrom CPG (CPG-86) under ambient ultrasonic conditions
using 4-DMAP as the acid-acceptor catalyst or under reflux
conditions using 2,6-lutidine as the acid-acceptor catalyst
respectively as described in Chapter III. Table 4-1 lists
the experimental conditions, acid-acceptor catalyst and
resultant octadecyl bonding density for each of these
stationary phases.
In order to prepare bonded phases with octadecyl
bonding densities less than 2.6 pmol/m2, the experimental
conditions of the bonding reaction must be altered so that a
less than maximal bonding density is achieved. One strategy
that can be used to accomplish this is to use a less than
stoichiometric amount of the reactive silane. Another
approach is to partially cover some of the reactive silanols
with trimethylsilane before exhaustive derivatization with
the octadecyl silane reagent (Marshall et al., 1984 and
1986). By varying the amount of trimethylsilane precoverage
and then reacting the precovered silicas with an excess of
the octadecylsilane, lower coverage octadecyl bonded phases
of varying bonding densities can be synthesized.


81
Table 4-3. Bonding densities for precovered 85 Angstrom
controlled pore glass reversed phase packings.
Packing
Identifier
C]_ Bonding Density
(umo 1/nr )
Co Bonding Density
(pmol /wr)
5% CPG
0.55
3.21
10% CPG
1.08
2.83
15% CPG
2.25
1 .70
30% CPG
1.27
2.72
40% CPG
1 .34
2.59


158
result in reversed phases with very high alkyl bonding
densities.
Bonded Phase Efficiency Studies
Although the bonding densities for reversed phases
synthesized by refluxed and ultrasound methods have been
compared, their chromatographic efficiencies have not.
Chromatographic efficiency is expressed as the number of
theoretical plates, N, or as the reduced plate height, h.
Column efficiency is evaluated by measuring the plate height
for a solute at a number of mobile phase flow rates; plate
height is plotted versus mobile phase linear velocity to
obtain a van Deemter plot (Snyder and Kirkland, 1979). It
would be interesting to compare column efficiencies for two
bonded phases (one prepared by ultrasonic methods and the
other by reflux) of comparable octadecyl bonding density.
We have postulated that the bonded alkyl groups of
ultrasonically synthesized stationary phases may be
distributed more homogeneously than those of refluxed
phases; this could result from increased pore penetration by
the silane reagent under ultrasonic conditions. If this is
the case, a more homogeneous distribution of bonded alkyl
groups should effect a more homogeneous energy of transfer
of the solute between the mobile and stationary phases. If
this occurs, the corresponding chromatographic peak should
exhibit a narrower and more Gaussian distribution, resulting
in a lower (improved) reduced plate height. These
efficiency comparisons between the ultrasound and refluxed


Ill
Figure 5-
Biphenyl
Structures of phenyl selectivity test
solutes.


Cl8 Bonding Density (jmoi/mZ)
Figure 4-5. Naphthalene thermodynamic partition coefficient at 35.0 C
as a function of CPG-86 octadecyl bonding density for
55/45 methanol/water mobile phase.
100


61
using dimethyloctadecylchlorosilane as the reactive silane,
2,6-lutidine as the acid-acceptor catalyst and methylene
chloride as the reaction solvent. All bonding densities are
calculated from duplicate elemental analyses as described in
Chapter II and the mean value + the range is reported in all
cases. The bonding densities for the CPG-86 and the CPG-167
were 2.56 + 0.03 ymol/m^ and 2.07 + 0.02 ymol/m^
respectively. In the second set of experiments, the same
reagents and reaction time as above were used to react both
CPG's, but the reactions were performed under reflux
conditions at a temperature of 50.0 C. The CPG-86 bonding
density was 2.63 _+ 0.00 ymol/m2 and that for the CPG-167 was
2.28 + 0.00 pmol/m^. As expected from previous work with
silica (Chapter II), reflux temperatures resulted in a
higher bonding density than ambient temperatures.
The next three sets of experiments were run for 24
hours with rotation under ultrasound conditions as described
in Chapter II. The first set of experiments used the same
reagents as described above at a temperature of 28.0 C.
The CPG-86 had a resultant bonding density of 2.55 + 0.01
ymol/m^- that for the CPG-167 was 2.04 + 0.03 ymol/m^. As
in the case for the silica, bonding densities achieved under
ultrasonic conditions with 2,6-lutidine as the acid-acceptor
catalyst were comparable to those achieved at ambient
temperatures.
The second set of ultrasound experiments was run under
the same conditions as the first set and at a temperature of


In k'
Figure 5-
Methylene selectivity plot for silica column DMAP3.
The slope of the plot is In methylene'
O


80
Table 4-2. Bonding densities for precovered silica reversed
phase packings.
Packing
Identifier
Ci Bonding Density
1 Umol/m?)
C-L3 Bonding Density
(ymol)
5%
0 .63
2.07
10%
0.98
2.09
15%
1 .50
1.98
30%
1.38
1.74
40%
1 .90
1 .60


CHAPTER VI
CONCLUSIONS
Syntheses of RP Stationary Phases
The results presented in Chapter II show that the use
of ultrasound as a driving force for the synthesis of
reversed phase (RP) stationary phases is a viable
alternative to traditional reflux methods. This especially
has advantages on an industrial-sized scale; the use of
ultrasonification at ambient temperatures results in
stationary phases of comparable bonding density (3.35
umol/m^) to those produced under refluxed conditions (3.44
ymol/m2), with considerably less power consumed and
therefore at a reduced cost. Another advantage of
ultrasonification over reflux in a manufacturing situation
is with respect to Occupational Safety and Health
Administration (OSHA) guidelines; the use of reflux requires
adherence to very stringent OSHA fire codes, resulting in
additional manufacturing expenses. Ultrasound reactions at
subambient temperature (i.e. 3.0 C) produce stationary
phases with very high alkyl bonding densities (3.60
ymol/m^). Although our reason for pursuing the synthesis of
high density reversed phases was to examine their retention
and selectivity characteristics, there are practical
advantages to these phases as well. In order to separate
135


33
considerable effect on the strength of the bond between the
silicon and oxygen atoms. Solvents which have both a
pronounced Lewis acid and Lewis base character cause the
Si-0 bond strength to be weakened and facilitate the bonding
reaction. The solvent can also activate the silicon atom of
the organohalosilane by forming a pentacoordinated
intermediate through nucleophilic attack. The resultant
bond lengthening causes nucleophilic activation to occur,
favoring attack by a second nucleophile (such as the base).
The solvent may influence the base as well, as it is known
that in aprotic polar solvents the nucleophilic character of
reactants is more pronounced. All of these considerations
may have a synergistic relationship as well. Based on their
experimental work with organoha1 os i 1anes, Kinkel and Unger
(1984) found that methylene chloride and N,N-dimethyl-
formamide were the most effective solvents for the bonding
reaction.
Many organic reactions have been shown to be enhanced
by ultrasound (Boudjouk, 1986; Brernner, 1986; Clough et al.,
1986; Han and Boudjouk, 1982 and 1983; Suslick, 1986).
Boudjouk and Han (1981) have shown that in the presence of
ultrasonic waves both alkyl and aryl ch1 oros i 1anes could be
coupled over lithium wire; without ultrasonification, this
reaction occurred to no appreciable extent. Reactions at
solid-liquid interfaces are also particularly enhanced by
ultrasound (Brernner, 1986; Suslick, 1986). It is then
reasonable to assume that reversed phase bonding reactions


41
two year period; for 66 measurements the resultant standard
deviation was + 0.20% carbon.
Syntheses Utilizing Ultrasonic Waves
Use of Ultrasound as a Reaction Catalyst
The region of frequencies above 16 kHz is beyond the
sensitivity of the human ear; it is therefore termed the
ultrasound region. The first reported use of ultrasound in
organic chemistry was in 1938, but it was not until the late
1970s that ultrasound was used to speed reactions in
nonaqueous media; indeed the use of ultrasound as a reaction
catalyst is still in its infancy (Boudjouk, 1986; Bremner,
1986). Ultrasonic radiation can be introduced to the
reaction medium either by immersion of the reaction vessel
into the liquid of a common laboratory ultrasonic cleaning
bath or by introduction of an ultrasonic generating probe
directly into the reaction medium.
Ultrasonic frequencies span the range of 20 kHz to 10
MHz, with associated acoustic wavelengths of 7.6 to 0.015
cm. Therefore sonochemistry cannot be accounted for in
terms of direct coupling of the acoustic field with chemical
species on a molecular level (Suslick, 1986). However, the
effects of ultrasound can be attributed to three different
phenomena. The variation of sonic pressure causes the rapid
movement (oscillation) of fluids, subjecting them to
compression and rarefaction. Negative pressure in the
rarefaction region gives rise to cavitation, the formation
and collapse of microbubbles. The violent implosion of


49
at 31.0 C for 1 hour and then refluxed and stirred at 50.0
C for an additional 23 hours, in hopes that the preliminary
sonication of the reagents would permit greater
accessibility of the reactive silane to silanols located
deep within the silica pores. The resulting bonding density
(for two trials) of 3.42 + 0.03 pmol/m^ is comparable to
that achieved under reflux conditions alone (3.44 + 0.02
prnol/m^). These results indicate that silica bonding
reactions performed in an ultrasonic bath are not affected
by superambient temperatures; this is in contrast to those
performed at subambient temperatures, which were found to
give an increasing yield as the temperature was decreased.
Experiments were also carried out in the ultrasonic
bath at 28.0 C for 24 hours using trimethy1ch1 oros i 1 ane
(TMCS) as the reactive silane, 4-DMAP as the base and
methylene chloride as the reaction solvent. TMCS is a much
smaller molecule than the octadecyl silane and therefore
should approximate the maximum bonding density obtainable in
these reactions when steric hindrance is minimized. The
average bonding density achieved in the two trials was 3.51
+ 0.01 pmol/m2; the octadecyl bonding densities achieved in
the above reactions show that the TMCS bonding density at
ambient temperatures can be exceeded under subambient
conditions even with bulky octadecyl reagents. The results
of these 4-DMAP experiments, as summarized in Table 2-1,
demonstrate that it is indeed a superior acid-acceptor
catalyst to 2,6-lutidine for reversed phase bonding
reactions.


110
density. When the same mobile phase composition is utilized
in comparing different stationary phase selecti vities,
mobile phase contributions to the free energy of transfer
should be equivalent. Under such conditions, changes in
selectivity are attributable to differences in the
stationary phase structure (Lochmuller et al., 1985).
Methylene selectivity and phenyl selectivity were examined
on octadecyl silica and CPG reversed phases. Methylene
selectivity was examined using the alkylbenzenes as test
solutes; phenyl selectivity was probed with the phenyl
homologous series consisting of benzene, biphenyl and
p-terphenyl, whose structures are shown in Figure 5-2.
National Bureau of Standards (NBS) column evaluation test
mixture 1 (PAH) was also used to measure overall polycyclic
aromatic hydrocarbon (PAH) selectivity; this mixture
contains benzo[a]pyrene (BaP), 1,2 :3,4 : 5 ,6 : 7 ,8-
tetrabenzonaphthalene (TBN) and phenanthro[3 ,4-c]-
phenanthrene (PhPh), whose structures are shown in Figure
5-3.
Experimental Procedure
The liquid chromatographic system, silica and CPG
columns and solvents used for the selectivity measurements
are described in Chapter IV. Toluene (Eastman Organic
Chemicals, Rochester, NY), ethylbenzene (Fisher Scientific,
Fairlawn, NJ), propyl benzene (Alfa Products, Danvers, MA),
butylbenzene (Eastman) and penty1 benzene (Alfa) standards
were made up in HPLC grade methanol for methylene


96
Table 4-7. Naphthalene thermodynamic partition
coefficients at 20.0 C as a function of
CP6-86 octadecyl bonding density for
55/45 methanol/water mobile phase.
C]_g Bonding Density Naphthalene Thermodynamic Partition
( pinol /m)Coefficient at 20.0 C
1 .70
89.3
2.68
103
2.72
95.2
2.83
93.6
3.21
98.1
3.30
102


43
Galan, 1978b). This calculation is quite straightforward
for monoreactive silanes and for monoch1 oros i 1anes (the most
commonly used monoreactive reagents) can be expressed by
a = t (%C) (IQ6) (1)
(12.011) (nc) (S) (100-L(%C/(12.011)(nc)](M-36.5) )
where a is the surface coverage (pmoles/m^); %z is grams
carbon per 100 grams bonded silica, as obtained from
elemental analysis; nc is the number of carbon atoms per
mole silane; M is the molecular weight of the silane; and S
is the surface area of the native silica in m^/g. Although
the typical value for the average surface hydroxyl
concentration of amorphous silica is 8 pmol/m^ (Cheng and
McCown, 1985), in practice octadecyl bonded phase coverages
are limited to about 3 pmol/m^ due to steric considerations
(Berendsen et al. 1980a; Berendsen and de Galan, 1978a and
1978b; Cheng and McCown, 1985; Snyder and Kirkland, 1979).
Three sets of experiments were compared in order to
determine the effect of ultrasound on the silica bonding
reaction. In all cases dimethyloctadecylchlorosilane was
the reactive silane, methylene chloride was the reaction
solvent and 2,6-lutidine the acid-acceptor catalyst; all
reaction mixtures were stirred during the reaction time
period (24 hours). In the first set of experiments, the
reaction mixture was stirred at ambient temperature (22.0
C); in the second set the reaction mixture was refluxed at
50.0 C, The third set of experiments was performed at 28.5
C, but the reaction vessels were immersed in an ultrasonic


54
The differences in the pore structures of silica and
CPG come about from the differences in their chemical
compositions and in their manufacturing processes. The
manufacture of chromatoyraphic silica is described in detail
by Unger (1979). The starting materials in the manufacture
of porous silica are soluble silicates such as sodium
silicates, silicon tetrachloride or tetraalkoxysilanes. By
adjusting the pH of an aqueous solution of the starting
material within a range of 8 to 9, silica sols are made. In
the sol, polysilicic acids are formed by polycondensation
and polymerization, growing into colloidal particles ranging
from 1 to 100 nm. The sol consists of spherically shaped,
nonporous and amorphous discrete silica particles. Unless
stabilized, the discrete particles in the sol aggregate,
mainly due to gelling. The particles become linked together
to eventually form a three dimensional packing of silica
particles that is a gelatinous mass called silica hydrogel.
The hydrogel is washed and water is then removed by heating.
This dehydration results in shrinkage from the partial
collapse of the globular hydrogel structure; the resultant
xerogel consists of hard porous grains. The silica
particles are also cemented together by dissolution-
deposition processes. The conversion of the hydrogel to the
xerogel is the origin of the porosity of chromatographic
silica. This porosity comes about from compaction of the
dispersed silica in the hydrogel; the pore space is made up
of the interparticle interstices and voids. This results in


Response
(X 1000)
jig Naphthalene
Figure 6-4.
Calibration plot for naphthalene on column DMAP5
at 35.0 C for 85/15 aceton itri 1e/water mobile phase.


85
and 0.7866 g/cm3 for methanol and methylene chloride
respectively) the silica column dead volume was determined
to be 1.805 ml while Vm for the CPG column was 1.752 ml. It
was assumed that the mobile phase volumes measured by this
procedure will be constant (within experimental error) for
any of the silica or CPG reversed phase columns used in this
work, since in each case the packings were based on the same
starting material.
The stationary phase volume for each LC column was
calculated using Equation 4-4. For the precovered bonded
phases the total volume of both the trimethyl- and
octadecy1 si 1 y 1 alkyl groups was used for V$. Percent carbon
for each column packing was obtained from in-house elemental
analysis and the densities used for the trimethylsilyl and
octadecy1sily1 groups were 0.8638 and 0.8607 g/cm3
respectively as reported by Cheng (1985). The weight of the
packing contained in the chromatographic column was
determined by weighing an empty chromatographic column,
packing it as described previously in this chapter and
drying it at 100 C to constant weight in a gas
chromatograph with a constant helium flow through the
column. From the mass differences in the two weighings the
weight of the column packing was determined to be 1.1705 g
for the silica columns and 1.2898 for the CPG columns. The
capacity factors for the naphthalene solute were determined
by triplicate injections onto each of the chromatographic
columns of different alkyl bonding density using mobile


98
Table 4-9. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
CPG-86 octadecyl bonding density for
85/15 acetonitri 1 e/water mobile phase.
Cio Bonding Density
(nmol/nr)
Naphthalene Thermodynamic Partition
Coefficient at 35.0 C
1.70
11 .0
2.59
13.5
2.68
15.0
2.72
13.9
2 .83
11 .9
3.21
9.76
3.30
9.74


acetonitrile system. Put another way, the acetonitrile/
bonded phase interphase is more nonpolar than the methanolic
one; therefore a methylene group will experience less
hydrophobic interactions in the acetonitrile system,
resulting in a lower methylene selectivity value (Karger et
al 1976 ) .
Examination of the relationship between phenyl
selectivity and bonding density is facilitated by inspection
of Figures 5-6 and 5-7 for the 55/45 methanol/water and
85/15 acetonitrile/water mobile phase systems respectively.
The plots show that phenyl selectivity increases with
increasing octadecyl bonding density in an approximately
linear fashion with least squares linear regression slopes
of 0.547 and 0.0835 and coefficients of correlation of 0.956
and 0.917 for the methanol/water and acetonitrile/water
systems respectively. This correlation between phenyl
selectivity and octadecyl bonding density can be attributed
to shape selectivity. As previously mentioned, other
workers have noted that chromatographic selectivity is
affected by the shape of the solute molecules (Lochmuller et
al., 1985; Martire and Boehm, 1983; Tanaka et al., 1982).
They have predicted that solute selectivity should decrease
as a function of solute shape in the order rodlike > planar
> chainlike. It has also been suggested that selectivity of
rodlike or rigid solutes increases with increasing bonded
chain surface coverage (Engelhardt et al., 1982;
Hemetsberger et al., 1979; Wise et al., 1981). This effect


108
proportion of organic modifier, the chains become more fully
extended and shape selectivity is increased (Martire and
Boehm, 1983). Wise and Sander (1985) refer to this as the
"slot model." They have postulated that when the closely
packed bonded RP chains are extended, the stationary phase
surface can be visualized as containing a number of long
narrow "slots" between these extended chains into which
solute molecules can penetrate. Since planar and/or linear
molecules can more deeply penetrate these slots and
therefore interact more strongly with the stationary phase,
they are preferent i a 1ly retained over nonplanar and/or
nonlinear molecules.
Examination of chromatographic selectivity can be very
useful in studies of retention mechanisms in LC. As
discussed in Chapter IV, the capacity factor, k1, is the
most widely studied chromatographic parameter, since it is a
normalized measure of solute retention. However, the
capacity factor is directly proportional to the volume phase
ratio (stationary/mobi1e) of the chromatographic column.
The phase ratio is dependent upon bonded group chain length,
alkyl bonding density, the pore structure of the silica
support and the homogeneity of the packing bed of the
column. Therefore, when comparing intercolumn capacity
factors there are many variables to consider, making it very
difficult to draw conclusions about intercolumn retention
behavior. Selectivity values for solutes are not affected
by the phase ratio of the chromatographic column, since they


62
28.5 C, except that 4-dimethyl aminopyridine (4-DMAP) was
used as the acid-acceptor catalyst instead of 2,6-lutidine.
The bonding densities for CPG-86 and CPG-167 were 3.30 +
0.02 and 3.04 + 0.08 umol/m2 respectively. The 4-DMAP had
again proven to be a superior acid-acceptor catalyst to the
2,6-lutidine as had been the case for silica. The third set
of ultrasound experiments was performed at 28.0 C using
4-DMAP as the acid-acceptor catalyst, methylene chloride as
the reaction solvent and trimethy1 chiorosi1ane (TMCS) as the
reactive silane. TMCS was used in order to approximate the
maximum bonding density achievable under minimum steric
hindrance conditions, as explained in Chapter II. The TMCS
bonding density was 4.19 + 0.02 umol/m^ for CPG-86 and 5.15
+ 0.10 umol/m^ for CPG-167. As expected, use of a less
bulky silane reagent resulted in a higher alkyl bonding
density, since steric hindrances are minimized.
A comparison of the bonding densities of the 147
Angstrom pore size silica, 86 Angstrom CPG and 167 Angstrom
CPG achieved under all sets of conditions is tabulated in
Table 3-1. As seen from these results, neither of our
expectations was realized. In all of the octadecyl silane
reactions, reactivity of the amorphous silica was greater
than that of either CPG. Even more puzzling, the smaller
pore CPG (CPG-86) exhibited greater reactivity than the
wider pore CPG (CPG-167). In the case where TMCS was the
reactive silane, these trends were reversed. This seems to
indicate that the reactivity trends for the silane bonding


57
described in Chapter II. The only difference in the
procedure between the CPG and silica was in the method of
agitation. CPG is more mechanically fragile than silica;
therefore direct stirring via a magnetic stirring bar is
inadvisable. Agitation was accomplished by a rotary
evaporator; a nitrogen gas line was attached to what is
normally the vacuum outlet in order to maintain a dry
atmosphere. An evacuated glass Dewar-type condenser
(fabricated in-house) was used to join the reaction flask to
the rotary evaporator; this piece of glassware was necessary
in order to prevent the escape of the reaction solvent,
especially under reflux conditions.
Reactions were performed under ambient as well as
reflux conditions; ultrasonic reactions were also carried
out. The reaction products were washed and vacuum dried as
described in Chapter II. Evaluation of the bonding
procedure was performed via in-house elemental analysis.
Scanning electron micrographs of the acid-leached CPG-86
(Figures 3-1 and 3-2) show that like the silica used in
previous reactions, the CPG also exhibits an irregular
particle shape as well as an irregular surface. This
irregularity is not surprising considering that CPG
particles of the desired size range are obtained by
mechanically crushing and sieving bulk Vycor glass (Haller,
1965a).


1.6
1.5 -
Methylene
Selectivity 14_.
1.3 --
1.2
1.5
mobile phase: 85/15 ACN/water
h1 1 1 1
2.0 2.5 3.0 3.5 4.0
Bonding Density (|nmol / square meter)
Figure 5-5. Plot of methylene selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 85/15
acetonitri 1 e/water mobile phase.


Jandera, P .; Colin, H.; Guiochon, G. "Interaction Indexes
for Prediction of Retention in Reversed-Phase Liquid
Chromatography," Anal Chem. 1982 5_4, 435-441.
Jandera, P.; Spacek, M. "Method for Characterization of
Selectivity in Reversed-Phase Liquid Chromatography III.
Retention Behavior in Gradient-Elution Chromatography:
Application to the Chromatography of Pesticide Compounds,"
J. Chromatogr. 1986 366 107-126.
Jinno, K. "Effect of Alkyl Chain Length of the Bonded
Stationary Phase on Solute Retention in Reversed-Phase
High-Performance Liquid Chromatography," Chromatographia
1982, 15, 667-668.
Jinno, K.; Kawasaki, K. "Retention Prediction of
Substituted Benzenes in Reversed-Phase HPLC,"
Chromatograph i a 1984a, 1_8, 90-95.
Jinno, K.j Kawasaki, K. "The Correlation Between Molecular
Polarizability of PAHs and Their Retention Data on Various
Stationary Phases in Reversed-Phase HPLC," Chromatographia
1984b, 18, 103-105.
Jinno, K.; Kawasaki, K. "Effect of the Chain Length of
Chemically Bonded Phases on Retention of Substituted Benzene
Derivatives in Reversed-Phase Liquid Chromatography,"
Chromatographi a 1984c, _18_, 499-502.
Jinno, K.; Okamoto, M. "Effect of Stationary Phase
Properties and Solute Molecular Size on Retention of PAHs in
Reversed-Phase Liquid Chromatography," Chromatographia
1984, 18, 677-679.
Johnson, B. P. Ph.D. Dissertation, University of Florida,
1986 .
Jones, K. "Optimisation Procedure for the Silanisation of
Silicas for Reversed-Phase High-Performance Liquid
Chromatography I. Elimination of Non-Significant
Variables, J Chromatogr. 1987a, 392 1-10 .
Jones, K. "Optimisation Procedure for the Silanisation of
Silicas for Reversed-Phase High-Performance Liquid
Chromatography II. Detailed Examination of Significant
Variables," J. Chromatogr. 1987b, 392 11- 16 .
Kaliszan, R. "Quantitative Relationships between Molecular
Structure and Chromatographic Retention. Implications in
Physical, Analytical, and Medicinal Chemistry," CRC Crit.
Rev. Anal Chem. 1986, 1_6 323-383.


104
much wider range of percent carbon values was obtained than
in an analogous situation with silica packings. In the
worst case situation, the standard deviation in the percent
carbon analysis was + 0.61% carbon. Error bars based on the
range in the partition coefficient values which would result
from this worst case standard deviation in the percent
carbon value are shown for Figure 4-6. Comparison of Figure
4-6 to Figure 4-3 demonstrates that even the errors due to
uncertainty in the elemental analysis are much more
substantial for the CPG packings than for the silica
packings. Additionally, due to the abovementioned problems
there are no doubt greater errors in the values for
octadecyl bonding density, capacity factor and Vm for CPG
packings than for silica packings. Therefore it must be
again stressed that the data depicting the behavior of the
thermodynamic partition coefficient with respect to
octadecyl bonding density for the CPG bonded phases is
tentative at best and should be regarded as only a most
preliminary prediction of retention characteristics for CPG
chromatographic systems.


150
experimental plots of partition coefficient versus bonding
density correlate very well with Dill's predictions. The
low density region which was predicted to be from about 0 to
2.7 ymo 1/m2 (Dill, 1987b) occurs from 0 to about 3.1 ymol/m^
in our plots. In this region, solute retention increases
linearly with increasing alkyl surface density because at
low alkyl densities configurational constraints are very
small and therefore chain packing has no effect on solute
retention. Nonpolar solute retention, as exemplified by the
naphthalene solute, increases as the chain volume increases
since there is more alkyl chain volume for the solute to
partition into. Dill predicted that the y-intercept of this
linear region, where octadecyl bonding density was zero,
would also occur at zero (no solute retention), yet all of
our plots gave nonzero intercepts. There are two theories
to explain this effect--either the naphthalene solute
exhibited a small amount of adsorption behavior on the
silica in addition to its partitioning behavior into the
alkyl chains, or the nonzero intercept was a result of
additional solute retention on the bonded trimethylsilyl
groups used to deactivate the low density bonded phases. In
order to decide which theory was correct, naphthalene
retention on a bare silica column and a trimethylsilyl (TMS)
column was examined. In both mobile phase systems at both
temperatures, the naphthalene solute was completely
unretained on the bare silica column. In contrast, using
the TMS column with both mobile phase systems at both


ACKNOWLEDGEMENTS
There are many people whom I wish to acknowledge for
their assistance with this work. Thanks are extended to Mel
Courtney for performing the numerous elemental analyses of
my packings and to the technical staff in the departmental
machine shop and glassblowing shop for their courteous
assistance and advice. I am grateful to Dr. John Gerdes for
suggesting the use of 4-dimethyl aminopyridine (4-DMAP) in
the bonded phase syntheses, to Nepera, Inc. for providing
the 4-DMAP, to Dr. John Novak of the Aluminum Corporation of
America for providing scanning electron micrographs of the
silica and controlled pore glass supports and to Dr. Lane
Sander of the National Bureau of Standards (NBS) for
providing the NBS column evaluation test mixture.
I would like to express my gratitude to the Society for
Analytical Chemists of Pittsburgh for funding my summer
American Chemical Society (ACS) Analytical Division Graduate
Fellowship and to Procter and Gamble for funding my full-
year ACS Analytical Division Graduate Fellowship.
Thanks are also due to my fellow Dorsey group members
(both present and former) for their friendship, advice and
support. The camaraderie within our group will be one of my
warmest memories of graduate school. I look forward to
i i i


7
Geometric descriptors are in general fairly easy to
calculate. Van der Waals volume and surface area are both
calculated from the van der Waals radii of the atoms from
which the molecule is composed (Jinno and Kawasaki, 1984a).
Length to breadth ratio (L/B) is a shape parameter based on
the rectangle with minimum area which could envelop a
molecule (Wise et al., 1981). However, all of these
descriptors considered alone or in combination were found to
have poor direct correlation with RPLC retention for
substituted benzene derivatives. Jinno and Kawasaki (1984a)
concluded that this indicates that molecular size and shape
were not the dominant forces controlling retention for these
molecules. However, they noted that size and shape are
important contributors to retention for a 1ky1benzenes and
polycyclic aromatic hydrocarbons (PAHs) (Jinno and Okamoto,
1984). Wise et al. (1981) have also found that L/B is
useful for predicting PAH elution order; however this
parameter is useless for establishing general PAH retention
indices since these indices vary according to the type of
octadecyl bonded phase column used, necessitating the
determination of a retention index equation for each
different octadecyl column.
Physical property descriptors have been the most
successful for predicting RPLC solute retention. The
physical properties on which these descriptors are based
come about from the solute's solution behavior in the mobile
and stationary phases (Karger et al., 1976). Since


retention times for a homologous series is a linear function
of the number of carbons in the series and therefore that
the change in free energy of partitioning per methylene
group is constant. This implies that as the number of
methylene groups increase, the rest of the molecule has a
constant effect on stationary phase interactions. Yet in
some cases the relationship between the logarithm of
retention and carbon number is not linear, implying that
this assumption is invalid. The linearization method is
also very time consuming, since the retention time
measurements must be determined very precisely in order to
obtain precision in the Vm value (Knox and Kaliszan, 1985).
Determination of Vm by the static method is a
gravimetric procedure. A thermostatted packed LC column is
filled successively with two pure liquids with greatly
different densities and weighed. From the differences in
the column masses, w, and the density of each liquid, d, the
total mobile phase volume can be calculated, since
(Berendsen et al., 1980b; Knox and Kaliszan, 1985; McCormick
and Karger, 1980). The mobile phase volume as determined by
this method is the maximum volume within the column that is
accessible to a molecule comparable in size to those used in
the procedure (McCormick and Karger, 1980). However, this
method ignores the possibility of a solvation layer on the
stationary phase and therefore can overestimate the value of
a dynamic Vm by as much as 15% for a pure methanol mobile


134
Table 5-5. Tetrabutylnaphthalene(TBN)/benzo[a]pyrene(BaP)
selectivity as a function of controlled pore
glass octadecyl bonding density for 85/15
acetonitrile/water mobile phase.
Co Bonding
Density
(ymol/m)
TBN/B aP1
Selectivity
Stationary^
Phase
Behavior
T emperatu re
(C)
1 .70
1 .66
monomeric
24.5
2.59
1.79
monomeric
25.0
2.68
1 .74
monomeric
26.0
2.72
1.71
monomeric
25.0
2.83
1 .79
monome ric
25.0
3.21
1 .80
monomeric
25.5
3.30
1.72
monomeric
25.5
y Ratio of k 1 ydn to k'Bap.
Stationary phase characterization based on classification
system of Sander and Wise (1984a). If solute elution
order is BaP<_PhP h be monomeric; elution order of PhPh to be oligomeric.


103
are also valid for the CPG bonded phases. In addition, the
retention volumes of the naphthalene solute are difficult to
measure precisely from their resultant chromatographic
peaks. Since the CPG has a particle size range of 37 to 74
pm, the solute peaks are very broad and therefore the peak
maxima are difficult to pinpoint precisely. The solvent
disturbance peak was too indistinct and ambiguous for use in
measuring Vm; therefore Vm was determined by injection of
D2O. This should result in a fairly accurate Vm measurement
for the 55/45 methanol/water mobile phase, but this method
probably overestimates V|n for the 85/15 acetonitrile/water
mobile phase since this is a water-lean system (McCormick
and Karger, 1980; Melander, et al., 1980). Because of these
considerations, measurements of solute capacity factors for
the CPG bonded phases are even less accurate than those for
the silica bonded phases.
The calculated bonding densities for the CPG packings
are less precise than those for the silica packings due to
imprecision in their elemental analysis for percent carbon.
The analyst measuring the percent carbon in these packings
(Courtney, 1987) had expressed his concern for the elemental
analysis precision because the CPG packings were harder to
weigh accurately due to handling difficulties as well as
being susceptible to incomplete combustion during the
analysis. These concerns were reflected in the precision of
the percent carbon analysis for the CPG packings; for any
given set of elemental analyses for a single CPG sample a


process on a molecular level. This theory is predictive
without adjustable parameters and is relevant to
partitioning behavior in organized assemblies,
micelles, membranes and vesicles.
including


102
of 20.0 C and in the area of 2.8 to 3.2 ymol/m2 at 35.0 C.
At bonding densities higher than the local minimum the
partition coefficient shows an increasing trend. This
increasing trend contrasts with the behavior of the silica
bonded phases, where the partition coefficient continuously
decreases for bonding densities greater than about 3.1
ymol/m2. In the 85/15 acetonitrile/water mobile phase
system the partition coefficient exhibited a decreasing
trend with increasing octadecyl bonding density once the
local maximum at 2.7 ymol/m2 is reached; this trend is
similar to that exhibited by the silica bonded phases.
It is premature at this point in time to make any
further comparisons between the behavior of the CPG bonded
phases and that of the silica bonded phases as regards the
relationship between bonding density and partition
coefficient. The fourteen silica packings evaluated span a
bonding density range from 1.60 to 3.60 ymol/m2 while the
seven CPG packings span a range of 1.70 to 3.30 ymol/m2,
with four of the seven packings in the narrow range of 2.6
to 2.8 ymol/m2. Since there is such a small number of data
points for the CPG packings, the scatter plots shown in
Figures 4-4 through 4-6 are rather speculative and therefore
can serve as only the most preliminary indication of the
partitioning behavior of the CPG packings.
The sources of error in the calculations of capacity
factor, Vs, Vm and partition coefficient have previously
been discussed for the silica bonded phases; these errors


CHAPTER V
CORRELATIONS BETWEEN CHROMATOGRAPHIC SELECTIVITY AND ALKYL
BONDING DENSITY
Introduction
Chromatographic selectivity (a) is the difference in
retention between two solute molecules. Chromatographic
selectivity is an important thermodynamic measurement in
studies of the solute distribution process since it is
directly related to the difference in the Gibbs free energy
of transfer from the mobile phase to the stationary phase
for two solutes:
In a = a(aG)/RT ,
where aG is the Gibbs free energy, R is the gas constant,
and T is the absolute temperature. Consequently, any two
solutes possessing different free energies of transfer will
be differentially retained (Lochmuller et a 1 ., 1985;
Melander and Horvath, 1982). Selectivity between two
solutes is measured as the ratio of their capacity factors;
aab = k a / ^ b' and 1 s defined such that a _> 1.0.
Functional group selectivity is the change in retention for
a given solute caused by the addition (or subtraction) of a
particular functional group. Evaluation of functional group
selectivity is accomplished by measuring the capacity
factors for a homologous series of compounds which differ
105


Silane Bonding Reaction
It is essential that the silane bonding reaction be
carried out under scrupulously dry conditions in order to
prevent the water-initiated dimerization of the silane
reagent. Glassware used in the derivatization reaction was
presilanized by etching the surface with a 10% (v/v)
hydrofluoric acid (Fisher Scientific) solution, drying, and
then soaking the glassware for an hour in a 5% (v/v)
trimethylchlorosilane in chloroform solution. Immediately
prior to use, the glassware was oven dried at 125 C for at
least 4 hours in order to remove trace moisture and allowed
to cool in a dry box under nitrogen atmosphere. The reagents
were mixed together in the dry box and the reaction flasks
kept under dry nitrogen atmosphere at all times. Based on
Kinkel and Unger's (1984) estimation of a maximum of five
micromoles of reactive hydroxyl sites per square meter of
silica surface, a twofold excess of the silane reagent was
added to achieve exhaustive derivatization of the silica
surface. A fourfold excess of the base (2 ,6-1utidine or
4-DMAP) was added both to serve as an acid-acceptor catalyst
for the HC1 produced in the reaction and to act as a
reactive intermediate at the silica-solution interface. Dry
methylene chloride was used as the reaction solvent, using a
ratio of 10 ml of methylene chloride per gram of base
silica.
The reaction flasks were sonicated by immersion to the
flask neck in an ultrasonic cleaning bath (Bransonic model


2
formulation of a predictive retention index system. Such a
retention index system would allow accurate interlaboratory
comparison of RPLC retention data.
Chromatographic retention is most often quantified by
the capacity factor, k'. Thermodynamically, the capacity
factor for a chromatographic solute is the ratio of the
number of moles of solute in the stationary and mobile
phases. The capacity factor is also a measure of solute
retention which normalizes for the mobile phase flow rate
and the physical dimensions of the chromatographic column,
since k' = (Vr Vm)/Vm, where Vr is the solute retention
volume and Vm is the retention volume of an unretained
solute, often called the dead volume. Many different
investigators have attempted to correlate RPLC retention
data with topological, geometric and/or calculated physical
property descriptors of chromatographic solutes in order to
predict RP retention. Topological descriptors include
molecular connectivity, molecular complexity and correlation
factor; van der Waals volume, molecular surface area and
length/breadth parameters are geometric decriptors.
Physical property descriptors include hydrophobic
substituent constants, UNIFAC models of activity
coefficients, and octanol/water partition coefficients
(D'Amboise and Bertrand, 1986; Funasaki et al., 1986; Jinno
and Kawasaki, 1984a, 1984b and 1984c; Petrovic et al.,
1985 ) .


161
give additional information about bonded chain organization
under these conditions.
The use of 1^C enriched methanol and acetonitrile and
D 2 0 mobile phases in conjunction with ^C and ^ H FT-NMR
experiments could also be utilized in order to quantitate
the stationary phase solvation layer. This would give
information about the solvation structure as well as lead to
a more accurate determination of the column phase ratio,
since the solvation layer volume could now be added to the
bonded chain volume (as calculated in Chapter IV) to get the
total stationary phase volume. The solvation layer volume
could also be subtracted from the maximum mobile phase
volume (as determined in Chapter IV) to obtain a more
accurate Vm value; the combination of these two effects
would be a very accurate determination of the phase ratio
and capacity factor and therefore of chromatographic
partition coefficients. Carbon-13 FT-NMR spin lattice
relaxation time (T^) experiments to study chain mobility on
bonded phases have been carried out by other workers (Bayer
et a 1 1986; Gangoda and Gilpin, 1983; Gilpin and Gangoda,
1984; Shah et al., 1987) but the effect of bonding density
on chain mobility should also be explored. Additionally,
temperature effects on mobility and retention should be
studied over a wide temperature range using solution state
1 O
FT-NMR and liquid chromatographic measurements. It
would be particularly interesting to see if there is a
certain "critical" temperature at which the retention or


139
phase volume (Vs) was calculated using equation 4-4; Vm was
calculated by dividing the mobile phase mass by its density,
which had been measured at ambient temperature. The
volumetric flask was sealed and stirred continuously over a
48 hour period. Ten microliter samples of the mobile phase
portion of the mixture were taken in quadruplicate while
stirring continued at 16, 20, 32 and 48 hour intervals from
when the sample was added to the system. These samples were
injected into the HPLC system (described in Chapter IV) at
35.0 C consisting of column DMAP5 and the appropriate
methanol or acetonitrile mobile phase system and the sample
responses (peak heights) were measured. The amount of test
solute in the sample was determined by comparison of the
sample response to a calibration plot prepared for standard
solutions of the solute under the same chromatographic
conditions. For benzene in 55/45 methanol/water, the total
range of the calibration plot was from 1297 to 0.1297 p g
benzene; the log-log calibration plot is shown in Figure
6-1. For naphthalene in 85/15 acetonitrile/water, the total
range of the calibration plot was from 100.0 to 0.01000 pg
and its corresponding log-log plot is shown in Figure 6-2.
Once the sample response of the unknown was measured, the
calibration standards bracketing the unknown were diluted
and their responses measured in order to prepare a more
precisely determined calibration plot in the concentration
range of the unknown. The benzene and naphthalene
calibration plots are shown respectively in Figures 6-3 and


REFERENCES
Antle, P. E.; Goldberg, A. P.; Snyder, L. R.
"Characterization of Silica-Based Reversed-Phase Columns
with Respect to Retention Selectivity. Solvophobic
Effects," J. Chromatogr. 1985, 321, 1-32.
Antle, P. E.; Snyder, L. R. "Selecting Columns for
Reversed-Phase HPLC. Part I. Column Selectivity," L_C
1984, 2, 840-846.
Bayer, E.; Paulus, A.; Peters, B.; Laupp, G.; Reiners, J.;
Albert, K. "Conformational Behavior of Alkyl Chains of
Reversed Phases in High-Performance Liquid Chromatography,"
J. Chromatogr. 1986 364 25-37 .
Berendsen, G. E.; de Galan, L. "A Geometrical Model for
Chemically Bonded TMS and PDS Phases," J. Liq. Chromatogr.
1978a, 1, 403-426.
Berendsen, G. E.; de Galan, L. "Preparation and
Chromatographic Properties of Some Chemically Bonded Phases
for Reversed-Phase Liquid Chromatography," J. Liq.
Chromatogr. 1978b, 561-586.
Berendsen, G. E.; Pikaart, K. A.; de Galan, L. "Preparation
of Various Bonded Phases for HPLC Using Monochlorosilanes,"
J Liq. C hromatogr. 1980a, 3, 1437-1464.
Berendsen, G. E.; Shoenmakers, P. J.; de Galan, L.; Vigh,
G.; Varga-Puchony, Z.; Inczedy, J. "On the Determination of
the Hold-Up Time in Reversed Phase Liquid Chromatography,"
J. Liq. Chromatogr. 1980b, 3, 1669-1686 .
Boudjouk, P. "Synthesis With Ultrasonic Waves," J. Chem.
Ed. 1986, 63, 427-429.
Boudjouk, P.; Han, B.-H. "Organic Sonochemistry.
Ultrasound Promoted Coupling of Chiorosi1anes in the
Presence of Lithium Wire," Tetrahedron Lett. 1981, 22,
3813-3814.
Braumann, T. "Determination of Hydrophobic Parameters by
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373 119 1-225 .
164


4
of a solute is proportional to the logarithm of its capacity
factor k1, it was expected that molecular connectivity would
also be proportional to k1, allowing prediction of retention
to be made from these molecular connectivity calculations
(Karger et al., 1976 ).
In their comparisons of experimental capacity factors
to those predicted via simple molecular connectivity
calculations, Karger et al. (1976) found very good agreement
for para-substituted phenols and primary alcohols. However,
the predicted k' values were uniformly high for secondary
alcohols. This is because the experimental log k' values
for two of the primary alcohols were used to determine the
slope and intercept in the presumed linear relationship
between log k' and molecular connectivity index for both
types of alcohols. The high predicted k1 values for the
secondary alcohols reflect that their steric environment is
different from that of the primary alcohols, showing that
simple molecular connectivity indices can only be used to
predict relative retention for compounds with the same
functional group as those used for standards (Karger et al.,
1976 ).
Lehtonen (1984) used molecular connectivity indices of
different orders, which correct for complex branching as
well as for the nature of atoms other than carbon which make
up the solute framework, to predict retention behavior for
16 dansy1 ami des. Predicted and experimental k' values could
be correlated very well by combinations of different order


127
will increase with alkyl bonding density, as predicted by
this theory and as borne out by our experimental results.
It is interesting to compare the slope of the 85/15
acetonitrile/water phenyl selectivity plot (0.0835) to that
for the 55/45 methanol/water plot (0.547). This disparity
can probably be attributed to the different structures of
the solvation layers on the bonded phase surfaces in the two
very different mobile phase systems. The 85/15
acetonitrile/water solvation layer is relatively robust; at
any of the bonded phase alkyl densities the stationary phase
surface will be well solvated and the chains well extended.
This means that relative retention will only be affected to
a small extent by changes in bonding density; chain ordering
will increase very little with increased packing constraints
because the chains are already well extended and relatively
ordered. In 55/45 methanol/water the chains are not well
solvated and are in a relatively collapsed configuration;
they are rather disordered. As the bonding density
increases the chains become increasingly ordered as well as
being much more extended. Thus shape selectivity will be
affected by bonding density to a much greater extent in the
methanol/water system; this is exhibited by the larger slope
of the phenyl selectivity plot.
The selectivity behavior of Sander and Wise's PAH test
mixture on these silica columns, compiled in Table 5-3,
further confirms that shape selectivity increases with
increasing alkyl bonding density. For bonding densities of


therefore as just discussed it exhibits greater retention as
the polymeric character of the bonded phase (and its bonding
density) is increased (Sander and Wise, 1984a).
In light of what has just been discussed, the trend of
greater phenyl selectivity with increasing octadecyl bonding
density shown by our work is not surprising. These results
correlate well with Sander and Wise's (1985) "slot model",
since with increasing bonding density the "slots" between
the extended octadecyl chains become increasingly long and
narrow. Therefore selectivity is expected to increase for
planar, linear solutes such as the biphenyl and p-terphenyl
used to measure phenyl selectivity in this work. However, a
more rigorous explanation for the correlation between phenyl
selectivity and bonding density can be offered in light of
the Dill (1987a) interphase stationary phase model discussed
in Chapter I. As alkyl surface densities increase, the
corresponding configurational constraints are also
increased, creating a more rigid and ordered chain packing
structure. In this model, the driving force for retention
is the creation of a solute-sized cavity in the stationary
phase chain packing structure. As bonding density and
consequently chain ordering are increased the energy
required for cavity formation also increases. Creation of a
narrow linear cavity for solute retention will require less
energy than that required to create a wider cavity such as
is needed for nonlinear or nonplanar solute retention.
Therefore selectivity for these linear and planar molecules


CHAPTER III
SYNTHESES OF CONTROLLED PORE GLASS-BASED RP STATIONARY
PHASES
Comparison of Controlled Pore Glass and Silica as Supports
for RP Stationary Phases
The most commonly used column packing materials for
reversed phase liquid chromatography (RPLC) are based on
microparticulate silica. As has been described in Chapter
II, this material is modified by chemically bonding alkyl
chains of the desired length onto the silica surface. The
use of such siliceous supports is widespread due to their
high reactivity and relatively low cost. Silica-based RP
bonded phases also exhibit good column stability within the
pH range of 2.5 to 7.5 (Melander and Horvath, 1980).
However, such materials are not without problems. The
surface of silica gel is very porous in nature and there is
a wide distribution in the size of these pores. This can
affect chromatographic selectivity by causing size exclusion
effects. This broad pore size distribution is one of the
contributors to the problem of inhomogeneous energies of
transfer between the stationary and mobile phases for
solutes in RPLC, leading to distorted peak shapes and
decreasing chromatographic efficiency. The effects of pore
size and structure have received much attention in size
exclusion chromatography, but these parameters have garnered
52


71
phase (Berendsen et al., 1980b). The volume of the mobile
phase as determined by this method is useful for a reference
point, both in terms of whether or not a compound
experiences retention in a chromatographic system and in
understanding the changes in the solvation layer which occur
when the bulk mobile phase composition is changed (McCormick
and Karger, 1980). Knox and Kaliszan (1985) even argue that
in the theoretical treatment of thermodynamic aspects of
chromatography that the maximum Vm value is the pertinent
one. They argue that since the thickness of the boundary
between the bulk stationary phase and bulk mobile phase is
on the order of one nanometer that its position cannot be
sufficiently well defined to give an accurate measure of the
two volumes and that calculational methods for the volume of
the solvation layer are very arbitrary. Because the
gravimetric procedure gives a precise and reproducible value
for Vm that is unambiguous and convenient to measure, this
convention was chosen to determine the mobile phase volume
for the chromatographic thermodynamic distribution
coefficients calculated in this work.
Measurement of the Stationary Phase Volume in RPLC
Although much work has been done on measuring the
volume of the mobile phase, measurement of Vs, the volume of
the stationary phase has not been as thoroughly investigated
(Berendsen et al., 1980b; Jandera et al, 1982; McCormick and
Karger, 1980; Melander et al., 1980; Sander and Field, 1980;
Slaats et al., 1981). In determining a method for the


166
Dawidowicz, A. L.; Rayss, J. "The Influence of the
Concentration of Surface Boron Atoms on the Properties of
Column Packings with Bonded Cig Groups Prepared from
Contro11ed-Porosity Glasses: I. Gas Chromatography,"
Chromatographi a 1985 2_0, 555-558 .
Dawidowicz, A.; Rayss, J.; Suprynowicz, Z. "A Study of the
Properties of Octadecyl Phases Bonded to Controlled Porosity
Glasses. Investigations by "Inverse" Gas Chromatography,"
Chromatographi a 1983 17_, 157 159.
Dawidowicz, A. L.; Rayss, J.; Surowiec, K. "The Influence
of Boron Atoms Introduced on the Silica Gel Surface upon the
Properties of n-Octadecanol Films," Z. Phys. Chemie 1986,
267 401-41 1 .
Dill, K. A. "The Mechanism of Solute Retention in
Reversed-Phase Liquid Chromatography," J. Phys. Chem.
1987a, 91, 1980-1988.
Dill, K. A. Personal Communication. 1987b.
Electro-nucleonics, Inc. Personal Communication.
F a irfi eld NJ; 1987 .
Engelhardt, H.; Dreyer, B.; Schmidt, H. "Properties and
Diversity of C18 Bonded Phases," Chromatographia 1982, 16,
11-17 .
Engelhardt, H.; Muller, H.; Dreyer, B. "Is There a "True"
Dead Volume for HPLC C o 1 u m n s it C h romatograph i a 1984 19 ,
240-245.
Fluka Chemical Corp. "CPG Controlled Pore Glass,"
manufacturer's literature, Hauppage, NY.
Funasaki, N.; Hada, S.; Neya, S. "Prediction of Retention
Times in Reversed-Phase High-Performance Liquid
Chromatography from the Chemical Structure," J. Chrornatogr.
1986, 361, 33-45.
Gangoda, M. E.; Gilpin, R. K. "NMR Investigations of ^ C
Labeled Alkyl Modified Silica," J. Magn. Reson. 1983 53 ,
140-143.
Gilpin, R. K. "The Bonded Phase: Structure and Dynamics,"
J. Chrornatogr. Sci 1984, 2_2, 371-377.
Gilpin, R. K.; Gangoda, M. E. "Nuclear Magnetic Resonance
Spectrometry of Alkyl Ligands Immobilized on Reversed-Phase
Liquid Chromatographic Surfaces," Anal. Chem. 1984, 56,
1470-1473.


CHAPTER II
SYNTHESES OF SILICA-BASED RP STATIONARY PHASES
Experimental Considerations in the Synthesis of RP
Stationary Phases
Reversed phase bonded silicas are the most popular
packings used in high performance liquid chromatography
(HPLC). Although the role of the mobile phase in
chromatographic retention and selectivity has been
extensively studied, that of the stationary phase has only
come under intense scrutiny recently and as a result the
effects of the stationary phase on these chromatographic
properties is not yet fully understood. One reason for this
dearth of knowledge is the lack of precise and reliable
methods for determining bonded phase characteristics such as
the density, homogeneity and topographical distribution of
the bonded alkyl ligands and the residual hydroxyl groups on
the support surface. These properties are a direct
consequence of the bulk silica medium and the reagent and
reaction conditions for the silanization process (Kinkel and
Unger, 1984). In order to obtain reversed phase packings
with reproducible surface characteristics, the silanization
reaction conditions must be painstakingly controlled.
In the preparation of reversed phase packings, one
objective is the modification of as many surface hydroxyl
29


INTERPHASE SOLUBILITY AND
CHROMATOGRAPHIC RETENTION
By
KAREN BELINDA SENTELL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987


21
In their adsorption model, Melander and Horvath (1980)
use very simple descriptions of the stationary phase surface
which contain two gross oversimplifications. They imply
first of all that the bonded chains are rigid rods
containing no internal degrees of freedom. But at the
temperatures commonly used in RPLC, the bonded alkyl chains
are quite disordered (Dill, 1987a). Melander and Horvath
(1980) also use stationary phase models wherein the bonded
alkyl chains are fully exposed to the mobile phase.
However, in a hydroorganic mobile phase system the chains
cannot be fully exposed to such a highly aqueous
environment; such a configuration would be prohibitively
expensive in free energy terms (Dill, 1987a).
Dill (1987a) has proposed an alternative model of the
RPLC stationary phase surface which regards the grafted
phase as an organized "interphase" similar to those found in
surfactant aggregates such as monolayers, bilayers, micelles
and microemulsions (Marqusee and Dill, 1986). Interphases
are composed of alkyl chains that have one end anchored at
an interface; their thickness is on the order of a few
molecular dimensions. The anchored chain density (i.e. the
number of chains anchored per unit silica surface area) is
sufficiently high as to cause severe configurational
constraints. Two important properties distinguish this
system as an interphase; its surface area/volume ratio is
high and its properties vary with the distance from the
anchored end. The relationship between orientational order


156
temperatures. For argon or helium, Cp/Cv = 1.67 whereas for
nitrogen, Cp/Cv = 1.40, so an argon or helium atmosphere
will result in a higher cavitational temperature than that
achieved with nitrogen. He also mentions that nitrogen
undergoes redox and radical reactions in the presence of
ultrasound. For these reasons, Suslick (1986) advocates the
use of helium or argon atmospheres for sonochemical
reactions, and the effect of these gases on the ultrasound
RP syntheses should certainly be examined.
Although dimethy1octadecy1ch1 oros i 1ane of high purity
is commercially available, there are other silane reagents
which can be synthesized that could give even higher bonding
densities than those achieved using the chiorosi1ane. Szabo
et al. (1984) have reported that octadecyl bonded silicas
with bonding densities of 4.18 umol/m^ have resulted from
reaction of silica with di rnethy 1 octadecy 1 ( di methy 1 ami no) -
silane at 125 C for 120 hours; they also give the reaction
scheme for this reagent's synthesis. It is postulated that
this silane results in very high bonding densities because
the dimethyl ami no moiety is a better leaving group than
chloride (Szabo et al., 1984). Golding et al. (1987) have
also synthesized a new silane which results in higher
bonding densities. They have synthesized octadecy1dihydro-
chlorosi1ane; in this silane two hydrogen atoms have
replaced the two methyl groups found in the commercially
available silane reagent. It is thought that the
dihydros i 1ane leads to higher surface coverages because of


55
a totally porous structure; moreover these pores are quite
irregularly shaped. Factors which can be varied to control
the final pore structure include changes in the sol and/or
hydrogel pH, changes in the duration of the hydrogel
ripening (via stabilization of the sol), variation of pH
during washing of the hydrogel, and substitution of an
organic liquid wash for the hydrogel rather than an aqueous
one (Unger, 1979). The chemical composition of the final
amorphous silica can be exemplified by the composition of
the Davisil silica used in our experiments; it consists of
99.60% by weight of Si02 and 0.10% Na20 with the remaining
0.30% made up of other metal oxides (Grace, 1984).
The procedure used to produce controlled pore glasses
was first reported by Wolfgang Haller in 1965 (1965a and
1965b). The starting material consists of a Vycor type
glass consisting of 7% Na20, Z3% B203 and 70% S i 0 2. The
glass is crushed, fractionated to the desired particle size
distribution by sieving, and then heated at approximately
600 C for the desired number of hours. This heating period
causes fusion to take place within the glass, resulting in
the formation of microheterogeneous regions in the
continuous silica network. This alkali borate-rich
microphase is then removed from the glass by a series of
acidic and basic leachings, resulting in a finished material
which is porous throughout its entire volume (Dawidowicz et
al., 1983). The diameter of the pores is determined by the
length and temperature of the heat treatment (Haller,


86 l CPG at 35 t
C|8 Bonding Density (imol/m2)
Figure 4-6. Naphthalene thermodynamic partition coefficient at 35.0 C
as a function of CPG-86 octadecyl bonding density for
85/15 acetonitrile/water mobile phase.


94
deliberate because the solvation layer volume is very
difficult to measure experimentally, moreover it changes
substantially as the mobile phase composition is changed
(Berendsen et a 1., 1980b; McCormick and Karger, 1980). Our
calculations of Vs may therefore underestimate the true
stationary phase volume, which will encompass the solvation
layer volume as well as the bonded chain volume. Moreover,
our static Vm value is larger than those calculated by most
dynamic means since the static value includes the solvation
layer volume in the mobile phase volume. The combination of
these two effects results in an overall value for the volume
phase ratio (stationary/mobi1e) that is smaller than the
"true" value; consequently the solute thermodynamic
partition coefficient as determined chromatographically will
probably be overestimated, but the exact amount of
overestimation is incalculable at this time. Although this
possible overestimation of the partition coefficient would
cause the plots of partition coefficient versus bonding
density in Figures 4-1, 4-2 and 4-3 to be shifted
vertically, it should be noted that this would change
neither the shape of the plots nor their maxima. Error bars
based on the range of partition coefficient values resulting
from the imprecision in the elemental analysis for the
silica packings (+ 0.20% carbon) are shown for Figure 4-3.
At bonding densities greater than about 3.1 ymol/m^ the
partition coefficient begins to decrease as the bonding
density is increased. This trend is evident for both mobile


86
phases consisting of 55/45 methanol/water and 85/15
acetonitrile/water. Thermodynamic partition coefficients
for the transfer of the naphthalene solute from the
stationary phase to the mobile phase were calculated by
dividing the capacity factor by the volume phase ratio.
Results
Silica-based Stationary Phases
Thermodynamic partition coefficients for the
naphthalene solute as a function of silica stationary phase
octadecyl bonding density are listed in Tables 4-4, 4-5 and
4-6 respectively for the 55/45 methanol/water mobile phase
system at 20.0 C and 35.0 C and for the 85/15
acetonitrile/water mobile phase system at 35.0 C.
Graphical representations of these data are shown in Figures
4-1, 4-2 and 4-3.
In all cases the partition coefficient increased as a
linear function of bonding density until a bonding density
of 3.1 umol/m^ was reached. The best fit line for this
linear region of each plot was calculated using least
squares linear regression. For 55/45 methanol/water at
20.0 C the slope and y-intercept for the best fit line were
30.1 and 12.3 respectively with a coefficient of correlation
of 0.989. At 35.0 C for 55/45 methanol/water the slope was
21.0, the y-intercept 12.7 and the coefficient of
correlation 0.973. For the 85/15 acetonitrile/water mobile
phase at 35.0 C the slope was 1.20, the y-intercept 1.54
and the coefficient of correlation 0.982. In all cases the


82
Deerfield, IL) and from Swagelok 1/4" to 1/16" zero dead
volume reducing union chromatographic end fittings (Crawford
Fitting Company, Solon, OH) which had been fitted with 2 urn
passivated 316 stainless steel frits (Alltech Associates,
Inc., Deerfield, IL). The column tubing and end fittings
were made of 316 stainless steel and were passivated prior
to use by ultrasonication for 30 minutes in 3 M nitric acid,
followed by an aqueous and a methanol rinse.
Silica columns were packed using a Shandon high
pressure HPLC column packer with a 33 ml slurry reservoir
(Shandon Southern Instruments, Inc., Sewickley, PA).
Approximately 1.5 grams of derivatized silica were slurried
in 30 ml of chloroform and sonicated for 10 minutes. The
column was then packed at a packing pressure of 6000 psi in
the downward position using a sequence of 150 ml each of
50/50 (v/v) chioroform/methanol, methanol and 50/50
methanol/water. The column was then removed from the packer
fittings, the packing leveled with a spatula and the
remaining column end fitting installed. Controlled pore
glass columns were packed in an identical manner except for
the type of packer used. Since CPG is mechanically fragile
and brittle (Fluka), it cannot be packed using a high
pressure packer. Therefore a Beckman Model 100A HPLC
pump(Beckman Instruments Inc., San Ramon, CA) running at a
flow rate of 9.9 ml/min was used in conjunction with
continuous mechanical vibration to pack the CPG columns.
Packing in such a manner generated a packing pressure of


153
coefficient and the volume phase ratio (stat ionary/mobi1e).
Examination of the partition coefficients in Tables 4-4, 4-5
and 4-6 shows that they decrease in very small increments
for bonding densities ranging from 3.15 to 3.60 pmol/m^.
However, at these same bonding densities, Vs has increased
from 0.3094 cm^ to 0.3424 cm^ in this range, while Vm has
remained essentially constant. Therefore the cumulative
effect of these two changing parameters on k1 was for k1 to
increase very slightly; this increase was so slight that k'
essentially remains constant over this bonding density
range. To our knowledge, this is the first systematic study
wherein the effects of bonding density on retention was
examined; our approach is also unique and fundamental
because we have deconvoluted the effects of changing phase
ratio from the measured chromatographic quantity (k1) by
examining the thermodynamic partition coefficient, which is
the most fundamental parameter involved in solute retention.
One significance of this work is that it gives insight
into the RPLC retention process at the molecular level.
Such fundamental information is of great importance, since
once RPLC retention is fully understood prediction of RPLC
retention for new solutes will be facilitated, perhaps
eventually leading to a predictive RPLC retention index
system. One additional strength of this theory is that it
is predictive without the use of adjustable parameters.
In addition to the insight that this work gives on the
importance of chain ordering and density on RPLC retention,


10
In f.js was zero (since stationary phase interactions were
assumed to be very weak and nonselective). They found that
the predicted values of k1 were at best a rough estimate of
actual chromatographic retention and concluded that
therefore the solute interaction with the stationary phase
could not be ignored in the prediction of RP retention.
However they found the UNIFAC method to be useful for
predicting changes in relative solute retention with varying
mobile phase composition, since the solute activity
coefficients in the mobile phase could be accurately
calculated using UNIFAC parameters (Petrovic et al., 1985).
Hydrophobicity is the physical descriptor which has
most accurately been used to predict RPLC retention.
Hydrophobic effects between solutes and hydroorganic mobile
phases were described earlier in this chapter. Solute
hydrophobicity is usually described in terms of the pi scale
developed by Hansch and Leo (1979). By evaluation of solute
partition coefficients between n-octanol and water (P) they
were able to establish substituent hydrophobicity
parameters. The logarithm of the partition coefficient is
determined for both a compound containing the substituent
group and the parent compound; the difference in these two
log P values is pi, the hydrophobicity parameter for the
substituent (Melander and Horvath, 1980). Jinno (1982) and
Jinno and Kawasaki (1984a and 1984c) use the descriptors pi,
HA and HD, where HA is the number of electron acceptor
groups and HD is the number of electron donor groups, to


phase systems and/or temperatures. The overall behavior
under the conditions specified above was that the partition
coefficient increased linearly as a function of bonding
density until a maximum was reached at a certain "critical"
bonding density in the vicinity of 3.1 pmol/m^. Once this
critical bonding density is reached, the earlier trend is
reversed and the partition coefficient decreases with
increasing bonding density. Comparisons between these
experimental trends and Dill's (1987a and 1987b) theoretical
predictions are made later in Chapter VI. These comparisons
result in the interpretation of the experimental results,
giving a wealth of information about the fundamental
mechanisms of small solute retention in reversed phase
liquid chromatographic systems.
Controlled Pore Glass-based Stationary Phases
Naphthalene thermodynamic partition coefficients as a
function of CPG stationary phase octadecyl bonding density
for 55/45 methanol/water at 80.0 C and 35.0 C and for
85/15 acetonitrile/ water mobile phase systems at 35.0 C
are listed in Tables 4-7, 4-8 and 4-9 respectively.
Graphical representations of these data are shown in Figures
4-4, 4-5 and 4-6.
In all three cases the partition coefficient reached a
local maximum at about 2.7 ymol/m^. For the 55/45
methanol/water mobile phase, at bonding densities higher
than this value the partition coefficient decreased to a
local minimum at approximately 2.8 pmol/m^ for a temperature


47
described above; the reaction mixture was immersed in the
ultrasonic bath and stirred at a temperature of 31.0 C for
24 hours. The average bonding density of the resultant
bonded phases ( + the range for two trials) was 3.35 + 0.05
timol/m2, again much higher than that achieved under similar
circumstances using 2,6-lutidine as the acid-acceptor
catalyst (2.71 + 0.01 ymol/m^).
A low temperature ultrasound reaction was then carried
out under the same conditions as stated above, with a
reaction temperature of 4.0 C for a duration of 97 hours.
For the two trials, an average bonding density of 3.24 +
0.01 ymol/m^ was obtained. A second set of low temperature
ultrasound reactions was performed under analogous
conditions with a reaction temperature of 3.0 C for 144
hours. The bonded phase resulting from this experiment had
a higher bonding density than achieved in any of our
previous attempts; the average + the range for the set was
3.60 + 0.01 jjmol/m^. To our knowledge, this bonding density
is higher than any previously reported in the literature
using dimethyloctadecylchlorosi1ane as the reactive silane
(Berendsen et al., 1980a; Cheng and McCown, 1985; Kinkel and
Unger, 1984) .
To ensure that the high bonding density in this second
low temperature experiment was a result of the ultrasonic
driving force as well as the lengthy reaction time, two
other reactions were carried out. In one, the reaction was
performed exactly as above (at 3.0 C for 144 hours with


19
polarizability and ionization potentials of the solute and
solvent species. Electrostatic interactions consider both
dipole and ionic effects; dipole effects are calculated from
the solute dipole moment, polarizability and molecular
radius as well as from the dielectric constant of the
solvent. Ionic effects are calculated from conventional
electrostatic theories such as Debye-Huckel treatments. The
entropic term is a measure of solute "free volume," which is
the volume that the molecule encounters before colliding
with another molecule. The "free volume" is assumed to be
proportional to the solute molar volume (Melander and
Horvath, 1980).
Although the solvophobic theory outlined above is
pertinent to a 1 iquid-1 iquid system, the bonded stationary
phase has not been considered in this process. Melander and
Horvath (1980) regard the change in free energy for
retention to be a combination of the mobile phase effects
just described and a small contribution from the adsorption
of the solute onto the stationary phase surface. This
adsorption is viewed as a reversible reaction between the
solute and stationary phase to form an associated complex.
The free energy of adsorption is quantified as the van der
Waals interaction energy between the solute and stationary
phase in the absence of solvent molecules. Although
Melander and Horvath (1980) mention that an entropic term
should be introduced to account for the restricted
translational freedom of the bonded chains at the silica


about solute retention in a particular chromatographic
system since retention is related to the thermodynamic
distribution coefficient through the volume phase ratio
Vs/vm (stationary/mobile) in that k = K(Vs/Vm). Therefore
for rigorous theoretical treatment of chromatographic
retention, the phase ratio must be accurately known in order
to determine the partition coefficient.
Measurement of the Mobile Phase Volume in RPLC
The determination of the mobile phase volume, Vm, in
liquid chromatographic systems is a problem that has
generated great interest as well as considerable
controversy. Its value is an essential component for the
calculation of the capacity factor k1 as well as for the
thermodynamic distribution coefficient K. Many workers have
addressed this dilemma, yet there is little consensus on a
generally applicable convention for measuring V m (Berendsen
et al. 1980b; Engelhardt et al., 1984; Gutnikov and Hung,
1984; Knox and Kaliszan, 1985; Le Ha et al., 1982; McCormick
and Karger, 1980; Melander et al., 1983a and 1983b; Slaats
et al., 1981; Wainwright et al., 1985; Wells and Clark,
1981). The problem is especially complex for RPLC, since
preferential sorption of mobile phase components by the
stationary phase results in the formation of a solvation
layer on this surface. The thickness and composition of the
solvation layer varies with the bulk composition of the
mobile phase and the local concentration of organic modifier
in the solvation layer may be greater than that in the bulk


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INTERPHASE SOLUBILITY AND
CHROMATOGRAPHIC RETENTION
By
KAREN BELINDA SENTELL
December, 1987
Chairman: John G. Dorsey
Major Department: Chemistry
The retention and selectivity behavior of small solutes
on silica and controlled pore glass (CPG) reversed phase
liquid chromatographic (RPLC) stationary phases was studied
as a function of stationary phase alkyl bonding density.
These monomeric octadecyl phases were synthesized by both
reflux and ultrasound methods; high alkyl bonding densities
(3.60 pmol/m^) were obtained via low temperature ultrasound
reactions using 4-dimethylamino pyridine as the acid-acceptor
catalyst. Using an improved method for the calculation of
the stationary phase volume, the chromatographic capacity
factors for the solutes were divided by the volume phase
ratio (stationary/mobile) to obtain the thermodynamic
partition coefficients; their behavior as a function of
stationary phase octadecyl bonding density was examined in
vi i


20
surface, they ignore this effect because they feel its
contribution is negligible. In summary, in the solvophobic
theory of RPLC retention, retention is mainly dependent upon
the free energy of creation of a solute sized cavity in the
mobile phase; stationary phase effects are considered to be
weak and therefore rather negligible.
Partitioning Theory
One of the most severe drawbacks to Melander and
Horvath's (1980) solvophobic theory is that it is based on a
one phase model--that of the mobile phase. But RPLC
involves two phases, the stationary and mobile phases;
therefore a one phase model is not completely applicable for
such a system. Melander and Horvath (1980) view the
retention process as if there is no true transfer of the
solute from the mobile phase to the stationary phase; the
solute is merely associated with the stationary phase
through weak adsorptive effects. Melander and Horvath
(1980) also account for the stationary and mobile phase
interactions by viewing them as bulk phases with homogeneous
properties throughout. In reality, the stationary
phase/mobile phase boundary is a highly heterogeneous area
consisting of the core silica particles, the alkyl chains
bonded to the silica surface, residual silanol groups
remaining on the silica surface and the mobile phase
solvating these silanols and the bonded chains. In such a
heterogeneous system, it is highly unlikely that bulk phase
thermodynamic considerations based on ideal solution
behavior are applicable (Marqusee and Dill, 1986).


Table 2-1. Comparison of silica octadecyl bonding densities using
4-DMAP and 2,6-lutidine as acid-acceptor catalysts.
Reaction
Conditions
Temperature (C)
Reaction
Time (h)
Co Bonding
1 4 D M A P
Density (ymol/m
2,6-lutidine
Ref 1uxed
50.0
24
3.44
2.82
Ultrasound
28.0
24
-
2.71
Ultrasound
31.0
24
3.35
-
U1trasound
8.5
101
-
ro
oo
-F*
U1trasound
4.0
97
3.24
-
U1trasound
3.0
144
3.60
-
Stirred Only
3.0
144
3.48
-
Ref 1uxed
50.0
144
3.44


6
whose applicability is general and which does not require
the use of experimental or empirical data (D'Amboise and
Bertrand, 1986). The GIMC is derived from combinations of
graph theory and statistical information theory. It is so
named because it considers all of the features which make a
molecule more or less complex such as size, symmetry,
branching, ring structures, multiple bonds and atomic
heterogeneity. Molecules are represented by their skeletal
molecular graph whose complexity is determined from a
statistical information theory derived formula. Since any
observable behavior related to a molecule's complexity is a
function of the GIMC, chromatographic retention should also
correlate with the GIMC. D'Amboise and Bertrand (1986)
point out that GIMC is able to make retention predictions
for solutes such as alcohols or fatty acids, which are not
well correlated with hyrophobicity. They point out that
GIMC is a structure sensitive parameter representing the
various reactive attributes of a molecule; therefore it
should be related to the interaction mechanism in retention.
However, plots of log k' versus GIMC for alcohols show
distinct curvature, especially for alcohols with five or
less carbons. Other difficulties exist as well. The index
does not seem to be well applicable to molecules with
different heteroatoms that are similarly bonded or for
molecules belonging to nonhomologous series. Correlation of
data between different stationary phases has also proven to
be a problem (D'Amboise and Bertrand, 1986).


reaction to be controlled independently of the ultrasonic
driving force. Additionally, low reaction temperatures have
often been found to enhance reaction yields for
ultrasonically catalysed chemical reactions. One
explanation for this phenomenon is that low temperatures
cause the vapor pressures of the reactants to be decreased,
enabling increased efficiency of u11rason ica11y produced
cavitation (Boudjouk, 1986; Bremner, 1986; Suslick, 1986).
In order to overcome the slower kinetics expected at lower
temperatures, reaction times were increased beyond the usual
24 hour time period.
In the first set of experiments, two reaction vessels
were sonicated and stirred at 15.0 C for 48 hours with a
resultant average bonding density ( + the range) of 2.74 +
0.00 y m o 1 / m 2 Since this result was little different from
that at room temperature, it was decided to increase the
reaction time as well as to decrease the reaction
temperature. In this set of experiments, two reaction
flasks were sonicated and stirred at 8.5 C for 101 hours
with a resultant average bonding density of 2.84 + 0.01
y m o 1 / m 2 a slightly higher value than for those ultrasound
reactions run at higher temperatures. These preliminary
results indicated that subarnbient temperatures could indeed
enhance the ultrasonic silica bonding reaction.
The Use of 4-Dimethyl aminopyridine as the Acid-Acceptor
Catalyst
There are also advantages in the use of 4-dimethyl-
ami nopyri di ne (4-DMAP) as the acid-acceptor catalyst. The


Table 5-4. Methylene and phenyl selectivities at 35.0 C as a
function of controlled pore glass octadecyl bonding
density for 85/15 acetonitrile/water mobile phase.
Co Bonding Methylene Methylene^ Phenyl Phenyl^
Density Selectivity Correlation Selectivity Correlation
( Mino! /mz) Coef f i ci ent Coef f i ci en t
1 .70
1 .174
0.9874
1.489
0.9902
2.59
1.199
0.9928
1.636
0.9961
2 .68
1.252
0.9980
1 .719
0.9975
2.72
1.222
0.9995
1.557
0.9983
2.83
1 .210
0.9937
1 .678
0.9971
3.21
1.240
0.9976
1.748
0.9966
3.30
1.171
0.9879
1.577
0.9980
* Coefficient of correlation
number; slope of this line
Coefficient of correlation
number; slope of this line
for the plot of In k1 versus carbon
is In(methyl ene selectivity),
for the plot of In k1 versus phenyl
is ln(phenyl selectivity).


147 A Silica at 35 C
C(8 Bonding Density (jjmoi/ m2)
Figure 4-2. Naphthalene thermodynamic partition coefficient at 35.0 C
as a function of silica octadecyl bonding density for
55/45 methanol/water mobile phase.


128
Table 5-3. Tetrabuty1 naphtha 1 ene(TBN)/benzo[a]pyrene(BaP)
selectivity as a function of silica octadecyl
bonding density for 85/15 acetonitri 1 e/v/ater
mobile phase.
Cig Bonding
Density
(umol/)
TBN/BaP1
Selectivity
0
Stationary
Phase
Behavior
T emperature
(c)
1 .60
1 .60
monomeric
25.0
1.74
1.63
monomeric
24.0
1.98
1 .68
monomeric
26.0
2.07
1.72
monomeric
26.0
2.09
1.70
monomeric
26.0
2.75
1.73
monomeric
25.5
2 .84
1.72
monomeric
25.5
3.06
1.75
monomeric
25.0
3.15
1 .73
monomeric
25.0
3.24
1.72
monomeric
27.0
3.34
1.70
monomeric
29.0
3.56
1.69
monomeric
26.0
3 .60
1 .56
o 1 i g o m e r i c
26.0
y Ratio of k j p to k'Bap.
Stationary phase characterization based on classification
system of Sander and Wise (1984a). If solute elution
order is B aP_

be monomeric; elution order of PhPh to be oligomeric.


Table 3-1.
Compa rison
silica, 86
controlled
of octadecyl bonding densities
Angstrom controlled pore glass
pore gl ass (CPG-167 ).
for 147
(CPG-86)
Angstrom
and 167
(pore size
Angstrom
Reaction
Conditions1
Temperatu re
(C) silica8
Bonding Density (jimol/m^)
CPG-86 CPG-167
Refluxed/
2,6-1utidine
50.0
2 .82
2.63
2.28
U1trasound/
2,6-1utidine
28.0
2.71
2.55
2.04
U1trasound/
4-DMAP
28.5
3.35
3.30
3.04
Ambient/
2,6-lutidine
26.0
2 .69
2.56
2.07
Ci sil ane2/
Ultrasound/
28.0
3.51
4.19
5.15
4-DMAP
1 Reaction method/acid-acceptor catalyst.
silane used instead of Cig silane in order to estimate bonding density
achievable using a less bulky silane reagent.
CT>
CO


continued association with them during my postdoctoral
tenure.
The love and moral support from my parents, Bobby and
Ruth Sentell, have helped to sustain me throughout my
education. They are responsible for instilling in me a love
of reading, a respect for education and an unquenchable
thirst for knowledge. I am grateful to them and to my
sister, Michelle, for their encouragement during the
toughest times.
My deepest gratitude is extended to my graduate
research advisor, Dr. John G. Dorsey, for his advice and
guidance. He is the epitome of what a research advisor
should aspire to be and has served as an inspiration to me
both as a research scientist and as a teacher. I have
greatly enjoyed our conversations and I look forward to our
continued professional interaction over the next year of my
postdoctoral appointment. I also thank him for encouraging
my oenophilic tendencies; after all, everyone needs to
develop a new vice now and then.
Lastly, I want to thank my fiance, Daniel Coffman. His
love, patience and support have sustained me even when I was
discouraged and disheartened; without his help I could never
have completed this work. In addition to his moral support,
I would also like to thank him for his expert drafting and
technical assistance as well as for accompanying me on my
numerous midnight sorties to check on my reactions. He
deserves my heartfelt gratitude now and forever for always
being there when I need him.


87
Table 4-4. Naphthalene thermodynamic partition
coefficients at 20.0 C as a function of
silica octadecyl bonding density for
55/45 methanol/water mobile phase.
C^g Bonding Density Naphthalene Thermodynamic Partition
(ymol/rrr) Coefficient at 20.0 C
1 .60
57.6
1.74
66.7
1 .98
69 .6
2.07
78.6
2.09
75.8
2.75
94.3
2.84
96.2
3.06
104
3.15
97.2
3.24
93.4
3.34
89.6
3 .43
87.2
3.56
86.1
3.60
85.9


5
connectivity indices. However, a computer program had to be
used in order to find the best combination of indices to
obtain good correlation and these combinations were often
nonlinear, involving combinations of connectivity indices
raised to powers ranging from -2 to +2. The choice of
indices also varied according to which organic modifier was
used in the mobile phase as well as its percent composition.
Although Lehtonen obtained good correlations between
predicted and experimental retention, his method requires
extensive computer calculations as no general connectivity
index combination was applicable even within the same class
of solutes (dansy1 ami des). These two types of examples
point out the shortcomings in the use of molecular
connectivity indices to predict retention: the capacity
factors for at least two members of the same class of
compounds must be determined in order to find the
proportionality constant between capacity factor and
molecular connectivity, these predictions are only valid for
compounds of the same functional group as the standards, for
complex molecules a computer program must be used in order
to find the best combination of indices to predict retention
and this combination for a particular type of molecule may
change if the mobile phase composition is altered. This
method is also unable to distinguish between geometric
isomers (Funasaki et al., 1986).
Molecular complexity is a topological descriptor and
the general index of molecular complexity (GIMC) is an index


151
temperatures resulted in naphthalene being slightly retained
(k1 = 1.15 for 55/45 methanol/water at 35.0 C and 1.42 at
20.0 C; k' = 0.148 for 85/15 acetonitrile/water at 35.0
C). The corresponding chromatographic partition
coefficients for 55/45 methanol/water at 35.0 C and 20.0 C
and for 85/15 acetonitri1e/water at 35.0 C are 21.9, 26.8
and 2.79; the respective intercept values for the linear
regions of the plots are 12.7, 12.3 and 1.54. It is not
surprising that the partition coefficients for naphthalene
on the TMS column are much larger than the plot intercepts
since the bonding densities for the low density bonded
phases ranged from 0.63 to 1.90 ymol/rn^ and that for the TMS
column was 3.16 ymol/m2; it would be expected that partition
coefficient would increase with bonding density for the
bonded TMS groups since chain ordering could not occur.
Therefore the nonzero intercept behavior is due to the
presence of the trimethy1sily1 groups in the low density
columns. Also note that the acetonitrile mobile phase
system results in a much smaller slope in the linear region
than the methanolic system. This can be explained (as in
Chapter V with selectivity plots) by the more robust nature
of the acetonitrile solvation layer; changes in bonding
density will affect retention to a much smaller extent than
in the methanolic systems, where the mobile phase solvation
is not so extensive.
In the high bonding density region (greater than 3.1
y mo 1/m^) where bonding densities have surpassed the


has been explained in terms of increased ordering of the
bonded RP chains (Krstulovic et a 1 1983; Lochmuller et
al-, 1985; Marti re and Boehm, 1983).
Sander and Wise (1984a and 1984b; Wise and Sander,
1985) and Wise and May (1983) have extensively examined the
effect of alkyl bonding density on retention and selectivity
for polycyclic aromatic hydrocarbons (PAH). They have
studied the PAH selectivity of monomeric and polymeric
octadecyl phases with bonding density ranges of 1.8 to 3.2
ymol/rn^ and 2.7 to 7.3 ymol/m^ respectively. Their studies
indicate that the polymeric phases exhibit much greater PAH
selectivity than the monomeric ones and that the polymeric
phase PAH selectivity increases with increasing bonded phase
surface coverage. They initially attributed this behavior
to some fundamental difference in the structures of the
monomeric and polymeric bonded phases. However, Verzele and
Mussche (1983) concluded that there is no true difference in
the nature of polymeric and monomeric bonded phases, and
that their differences in chromatographic behavior are
attributable to differences in surface coverage. In later
comparisons of monomeric and polymeric bonded phases of
varying bonded alkyl chain length, Sander and Wise (1987)
concluded that the changes in PAH selectivity that they had
earlier observed were not necessarily due to fundamental
differences in the two phases but rather could be attributed
to changes in the overall stationary phase thickness.


17
Theories of Retention in RPLC
Solvophobic Theory
In order to truly understand the retention process in
RPLC and thereby be able to predict solute retention, the
retention process must be examined at the molecular level.
At present there are two main schools of thought on the
retention mechanism of RPLC at the molecular level. The
solvophobic theory espoused by Melander and Horvath (1980)
states that RP retention comes about from solute binding
onto the stationary phase from the mobile phase and is
mainly due to hydrophobic interactions between the solute
and the mobile phase. Other workers have utilized
statistical mechanical analysis based on mean field lattice
theory to show that RPLC solute retention is due to solute
partitioning from the mobile phase into the bonded
stationary phase chains (Dill, 1987a and 1987b; Marqusee and
Dill, 1986; Martire and Boehm, 1983). The main tenets of
both of these proposed theories will be outlined below.
Melander and Horvath's (1980) solvophobic theory
assumes that the mobile phase plays the dominant role in the
RPLC retention process. This is because the stationary
phase is nonpolar; therefore the only attractive forces
occurring between the stationary phase and a nonpolar solute
will be van der Waals forces, which are weak and
nonspecific. They attribute the interactions between the
solute and the mobile phase to a type of hydrophobic effect,
which was discussed earlier in this chapter. In the


137
ultrasound synthesized stationary phase with a bonding
density of 3.35 ymol/m^ have shown impressive ruggedness--
chromatographi c performance did not appreciably degrade in
terms of efficiency or selectivity until 6000 ml of 30/30/40
methanol/acetonitri1 e/pH 11 buffer had been passed through
the column (Novak, 1987). It can be concluded that these
high density phases show great promise for extending the
viable working pH range in reversed phase systems.
Buszewski et al. (1986) have prepared monomeric octadecyl
stationary phases of various bonding densities; they found
that the high density phases resulted in better resolution
of purine compounds than the lower density phases because
the higher alkyl surface coverage shields residual silanol
groups on the silica surface, resulting in a marked decrease
in peak tailing. High alkyl density phases should then be
well suited for the improved separation of other types of
basic compounds. In summary, high alkyl density reversed
phase packings offer many practical advantages over lower
density phases.
For both silica and controlled pore glass (CPG) bonded
phase syntheses, 4-dimethyl aminopyridine (4-DMAP) proved to
be a much more effective acid-acceptor catalyst than
2,6-1utidine. As with silica, ultrasound CPG reactions
resulted in bonding densities comparable to those achieved
under traditional refluxed conditions. However, at any of
the octadecyl reaction conditions, the silica substrate had
consistently higher reactivity than the CPG, resulting in


18
specialized RPLC environment, they have adopted a variation
of this effect, termed the "solvophobic" theory, since the
hydrophobic theory assumes a totally aqueous environment and
RPLC mobile phases are generally a mixture of aqueous and
organic components. Solvophobic theory is based on a theory
of solvent effects on chemical equilibria developed by
Sinanoglu (1968). The theory states that chromatographic
retention is based on the free energy change as the solute
is transferred from a hypothetical gas phase at atmospheric
pressure to the mobile phase. The energy involved in this
process is calculated in two steps. In the first, a cavity
of the proper shape and size for the solute molecule is
formed in the solvent. In the second, the solute enters the
cavity and interacts with the surrounding solvent molecules
via van der Waals and electrostatic interactions (Melander
and Horvath, 1980) .
The free energy change accompanying the mobile phase
cavity formation comes about from the fact that the solvent
surface area will increase by the molecular surface area of
the solute (Melander and Horvath, 1980). Therefore the
mobile phase free energy will increase by an amount
proportional to the solvent surface tension and the increase
in area. The change in free energy due to the interaction
of the solute with the surrounding solvent molecules will be
due to chemical and entropic effects. The chemical effects
are van der Waals interactions and electrostatic effects.
The van der Waals interaction energies are a function of the


15
predict retention. At present, none of these solute
descriptor index systems is adequate for reliable prediction
of RPLC retention.
Empirical Prediction of Retention
Jandera and coworkers (Colin et al., 1983a; Jandera,
1986; Jandera et al., 1982; Jandera and Spacek, 1986) have
developed an empirical model to predict absolute or relative
retention. They assumed that the stationary phase
contribution to retention is very small compared to that of
the mobile phase and that nonpolar interactions between the
solute, stationary phase and mobile phase cancel each other.
If this is true, the energy of transfer of the solute from
the mobile to the stationary phase will depend on the
interaction energy (energy of cohesion) between mobile phase
molecules and the interaction energy between the mobile
phase and solute. They defined an interaction index,
determined from retention data in hydroorganic mobile phase
systems, which describes polar interactions between solute
molecules and the mobile phase components.
The interaction index for a solute (Ix) can be
determined if the volume of interaction (Vx) for the solute
and the column phase ratio (Vs/Vm) are known, since in this
model ,
log (kx'/Vx) log ((Vs/Vm)/Vx) = A BIx (1)
where A and B are constants which depend on the stationary
and mobile phases used. Jandera et al. (1982) plotted
(log kx log (Vs/Vm))/Vx versus solute polarity (based on


73
column dead volumes as being accurate and consistent enough
for precise work (Engelhardt et al., 1984; Melander et al.,
1982; Smith et al., 1986). Melander and Horvath (1980)
state that a small relative error in the determination of
the column dead volume results in a commensurate relative
error in calculating both the capacity factor and the Gibbs
free energy of the solute transfer.
Other problems exist as well. The surface area of the
adsorbent is usually found by use of the BET analysis
method. It should be noted that the surface area of the
adsorbent must be determined after derivatization with the
alkyl ligand, as the surface area of the derivatized silica
will be significantly different from that of the
underivatized support. Although use of the BET method for
surface area determination is widespread, this method is
inappropriate in assessing that surface area of derivatized
silica packings which is chromatographical 1y significant.
The BET method measures the area of surface that is
accessible to a small molecular probe such as nitrogen. Yet
in an irregular surface such as porous silica, there may
exist many pores which are large enough to allow nitrogen
in, but which are too small to allow the passage of any
larger molecules of chromatographic interest.
Chromatographic support surface area data based on BET
analysis is usually overestimated, and the amount of
overestimation is by no means a constant, depending on the
base silica structure and the derivatization method.


152
mobility behavior exhibits an abrupt change due to a
conformational change in the interphase structure. Such
changes in reversed phase behavior have been noted in gas
phase experiments (Claudy et al.,1985; Gilpin, 1984; Gonnet,
et al., 1985) but have not been reported under true liquid
chromatographic conditions.
Selectivity experiments (such as described in Chapter
V) should also be carried out with large molecules such as
PAHs as well as with solutes of different shapes: rodlike,
chainlike, planar and nonplanar. Although such experiments
have been performed (Lochmuller et al., 1985; Tanaka et al.,
1982; Wise and Sander, 1985) they too have not been carried
out on monomeric stationary phases of varying octadecyl
density; therefore the effects of chain ordering on
selectivity has not been fully explored for these different
types of molecules. Again, selectivities for these
compounds using mobile phases of different compositions
should provide information on stationary phase structure
under different mobile phase conditions. As mentioned in
Chapter V selectivity experiments actually provide more
information about stationary phase structure than retention
experiments; since they are measures of retention
differences of solutes they are unaffected by the column
phase ratio (Antle and Snyder, 1984; Colin et al., 1983a and
1983b). Selectivity behavior of these solutes at different
temperatures should be examined as well. In particular,
selectivity behavior at subambient temperatures should be


9
functional groups in the system. The solute activity
coefficient is the product of a combinatorial and residual
contribution. The combinatorial contribution is dependent
on the size and shape of the molecules in the system; the
residual depends on the interaction energy of functional
group pairs, as well as the fraction of the surfaces on
these groups which are available for mutual interactions
(Petrovic et al. 1985 ) .
Assuming infinite dilution, the relationship between
the capacity factor of a solute i and its activity
coefficient (f-j) in the mobile and stationary phases can be
written as
In ki1 = In i + In (Vs/Vm)
therefore In k ^ 1 = In f-¡m In f-¡ s + In (Vs/Vm). Petrovic
et al. ( 1985 ) assume that f^s and Vs/Vm are constants;
therefore In k^1 and In f-¡m are linearly related with a
slope of one. If the activity coefficients of the solute i
in the chromatographic system and Vs/Vm are known, retention
can then be predicted. Petrovic et al. (1985) calculated
infinite dilution activity coefficients of solutes in the
mobile phase from experimental gas-liquid chromatographic
data and then correlated them with their RP retention values
to see how well the UNIFAC method could predict
chromatographic behavior. They assumed that Vs/Vm would be
the same for any octadecyl RP column at any methanol/water
mobile phase composition (an assumption that will be
thoroughly disputed in Chapter IV of this tome) and that


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Food Science and
Human Nutrition
This dissertation was submitted to the Graduate Faculty of
the Department of Chemistry in the College of Liberal Arts
and Sciences and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
December, 1987
Dean, Graduate School


27
In this study, we examined the effects of octadecyl
alkyl chain bonding density on both retention and selectvity
of small nonpolar solutes. We were particularly interested
in experimental verification of the molecular mechanism of
RPLC retention proposed by Dill (1987a and 1987b). Although
Sander and Wise (1984a and 1984b; Wise and Sander, 1985) and
Wise and May (1983) have extensively examined the effect of
alkyl bonding density on retention and selectivity for PAHs,
they have mainly examined polymeric stationary phases, which
are not as well structurally characterized as the monomeric
stationary phases used in our study. Another problem with
their work is that they examined capacity factor (k1)
behavior in their retention studies, which fails to account
for phase ratio changes. Additionally, PAHs are not ideal
solutes for such a study, since their large sizes and
unusual shapes are not typical of most chromatographic
solutes.
In order to determine the effect of interphase chain
packing on solute partitioning, the behavior of the
chromatographic partition coefficient was examined as a
function of octadecyl bonding density. Chromatographic
selectivity was also studied as a function of bonding
density for solutes of different sizes and shapes. Novel
synthetic methods utilizing ultrasound as a reaction driving
force were devised to obtain stationary phases with high
bonding densities. In this manner, we were able to see if
Dill's (1987a and 1987b) proposed RPLC retention mechanism


of the 16 injections; therefore equilibrium distribution of
the sample between the stationary and mobile phases was
complete at the first sampling interval (16 hours). For the
benzene solute in 55/45 methanol/water the least squares
linear regression equation of the calibration plot was:
Response = (181.96 response u n i t s / p g benzene) (p g benzene) -
6.90 with a coefficient of correlation of 0.9991. The
average response ( + one standard deviation) for the dynamic
benzene sample was 140.09 _+ 5.49. This average response
corresponded to 0.8078 pg benzene in the equilibrium mobile
phase samples injected; the benzene amount range (calculated
from the average response plus and minus one standard
deviation) was from 0.8380 to 0.7777 pg benzene. The amount
of benzene that partitioned into the stationary phase was
calculated as the difference between the original amount of
benzene added to the flask (corrected for mobile phase
dilution) and the equilibrium amount in the mobile phase.
The calculation of the dynamic partition coefficient is:
K = equilibrium pg benzene in stationary phase/Vg
equilibrium pg benzene in mobile phase/Vm
where Vs and Vm are the appropriate volumes for the
volumetric flask contents. The average dynamic partition
coefficient for benzene between the 55/45 methanol/water
mobile phase and the DMAP5 stationary phase was 8.52; when
the benzene concentration range for the measurement was
considered, the dynamic equilibrium partition coefficient
ranged from 5.97 to 11.25.


157
its reduced size; replacing the two bulky methyl groups with
hydrogens should greatly reduce steric hindrance at the
silica surface. Golding et al ( 1987 ) were able to achieve
bonding densities of about 4.6 pmol/m^ using this reagent.
It is suggested that one or both of these novel silane
reagents be tried in the ultrasound synthesis.
Our optimization of the reaction variables
(temperature, reaction time, amounts of reagents used, etc.)
has thus far followed the "educated trial and error" method,
whereby the optimum conditions cited in the literature were
used as a starting point and variables were altered in a
unilateral fashion. In order to find the best conditions
for ultrasound syntheses, the reaction variables should be
optimized systematically using a statistically sound method
such as experimental design or simplex optimization. By
application of Plackett-Burman matrix statistics, Jones
(1987a and 1987b) was able to reduce the number of
experiments necessary to optimize the bonding reaction to
twenty-four, even though twenty-one variables were involved.
Such a study should now be done on the ultrasound synthesis.
Besides the variables mentioned above, the effect of higher
acoustic power of the ultrasound source should also be
examined.
Finally, bonding reactions run at high pressures should
be attempted. Synthetic groups at the University of Florida
are able to run reactions at pressures of 10,000 atmospheres
or more. Bonding reactions run at these high pressures may


2.00
1.98 -
Methylene 1-96"
Selectivity
1.94 -
1.92 -
1.90 -
1.
Bonding Density (jimol / square meter)
mobile phase: 55/45 MeOH/water
H- 1- 1 h-
2.0 2.5 3.0 3.5
Figure 5-4. Plot of methylene selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 55/45
methanol/water mobile phase.


147 A Silica at 20 C
Ce Bonding Density (pmoi/ m2)
Figure 4-1. Naphthalene thermodynamic partition coefficient at 20.0 C
as a function of silica octadecyl bonding density for
55/45 methanol/water mobile phase.


130
The average methylene selectivity + one standard deviation
is 1.210 + 0.031. The argument for constant methylene
selectivity for the silica bonded phases can similarly be
applied to these CPG phases.
Phenyl selectivity for these CPG phases does not show
the linear trend exhibited by the silica bonded phases. The
phenyl selecti vities are quite scattered and do not seem to
correlate with bonding density in any manner. Therefore, no
conclusions can be drawn about the effect of bonding density
on phenyl selectivity for the CPG phases. Selectivity
results from the NBS PAH test mixture are listed in Table
5-5. These phases exhibit monomeric behavior at all of the
bonding densities examined; the TBN/BaP selectivity values
range from 1.66 to 1.80 and are more scattered than those
for the silica. In summary, our CPG selectivity data is
inconclusive, especially when compared to the silica data.
Further experiments on this support are warranted if its
selectivity trends are to be determined.


stirring) except that the reaction flask was not sonicated.
In the other, the reaction was stirred for 144 hours, but
the reaction mixture was refluxed at 50.0 C rather than
sonicated. From duplicate elemental analyses of each of
these two materials, the average bonding density (+ the
range) for the silica stirred (but not sonicated) at 3.0 C
for 144 hours was 3.48 _+ 0.00 nmol/m^j that for the silica
refluxed and stirred at 50.0 C for 144 hours was 3.44 +
0.03 ymol/m^. Since the absolute error in the elemental
analysis is + 0.20% carbon, which corresponds to + 0.03
o
iimol/m11 for the octadecyl packings, the differences in
bonding density between these two materials and the silica
which was sonicated at 3.0 C for 144 hours (3.60 ymol/m^)
is both real and significant. Therefore it can be concluded
that subambient ultrasound reactions are especially
efficacious for synthesizing stationary phases with very
high alkyl bonding densities.
In order to investigate the effect of superambient
temperatures on the ultrasonic bonding reaction, two types
of experiments were performed. In the first, the reaction
was carried out by stirring with the same reagents as
previously described for a reaction time of 24 hours and
with the ultrasonic bath maintained at a temperature of
50.0 C. For two trials, the average + the range was 3.34 +
0.04 ymol/rn2, virtually identical to that achieved under
ambient ultrasonic conditions (3.35 + 0.05 umol/m^). in the
second experiment, the reagents were stirred and sonicated


39
B-2200R-1, Branson Cleaning Equipment Co., Shelton, CT) with
a power rating of 100 W and a frequency of 55 kHz. Stirring
of the reagents within the flasks was accomplished by
rotating a magnetic bar submerged in the bath adjacent to
the reaction flask, resulting in the corresponding rotation
of a magnetic stirring bar within the flask. Temperature
control of the ultrasonic bath was accomplished by passing a
thermostatted solution of ethylene glycol and water through
coiled copper tubing lining the inner perimeter of the bath.
The solution was thermostatted by an Endocal or Exacal water
bath (Neslab Instruments, Portsmouth, NH). Refluxed
reactions were carried out at 50 C using an oil bath and
magnetic stirrer. Control reactions were carried out by
stirring the reaction mixture at room temperature.
Refluxed reactions utilizing n-octyldimethylchloro-
silane as the reactive silane, 2,6-lutidine as the acid-
acceptor catalyst and methylene chloride as the reaction
solvent were carried out for reaction times of 24, 36 and 48
hours in order to ascertain whether the differences in
reaction time would make a statistically significant
difference in the reaction yield. Nine replicate reactions
were performed for each reaction time. Student's t (tca]c)
was calculated from the pooled standard deviation of percent
carbon for all 27 reaction yields, the differences in the
mean percent carbon at each reaction time, and the number of
replicates at each time in order to determine if the mean
yield for each reaction time was statistically different


167
Golding, R. D.; Barry, A. J.; Burke, M. F. "Synthesis of
Three A1ky1dihydroch1 o ros i 1anes and Their Application in
Studies of Steric Factors in' the Surface Deactivation of
Porous Silica," J. Chromatogr. 1987 384 105-1 16 .
Gonnet, C.; Morel; D.; Ramamonji nirina, E.; Serpinet, J.;
Claudy, P.; Letoffe, J. M. "Insertion of Various Long Alkyl
Chain Molecules in Brush-Type Grafted Monolayers.
Chromatographic Study of the Resulting Materials," J.
Chromatogr. 1985 330 227-241 .
Grace, W. R. manufacturer's literature, Baltimore, MD;
1984.
Gutnikov, G.; Hung, L.-B. "Convenient Estimation of the
Mobile Phase Volume for Water-Rich Eluents in Reversed-Phase
Liquid Chromatography," Chromatographia 1984, JJ5, 260-265.
Haller, W. "Rearrangement Kinetics of the Liquid-Liquid
Immiscible Microphases in Alkali Borosilicate Melts," J.
Chem. Ph.ys. 1965a, 42., 686-693.
Haller, W. "Chromatography on Glass of Controlled Pore
Size," Nature 1965b, 106, 693-696.
Han, B.-H.; Boudjouk, P. "Organic Sonochemistry.
Ultrasound-Promoted Reaction of Zinc With
a,a-Dibromo-o-Xy1 ene. Evidence for Facile Generation of
o-Xylylene," J. Org. Chem. 1982, 47., 751-752.
Han, B.-H.; Boudjouk, P. "Organic Sonoche in istry.
Ultrasonic Acceleration of the Hydros i 1 ation Reaction,"
Organometal1ics 1983, 2 769-771.
Hansch, C.; Leo, A. Substituent Constants for Correlation
Analysis in Chemistry and Biology. John Wiley and Sons:
New York, 1979, 13-17.
Hemetsberger, H.; Behrensmeyer, P.; Henning, J.; Ricken, H.
"Reversed Phase, High-Performance Liquid Chromatography:
Effect of the Structure of the Chemically Bonded Hydrocarbon
Ligand on Retention and Selectivity," Chromatographia 1979,
12, 71-76.
Hurtubise, R. J.; Allen, T. W.; Silver, H. F. "Comparison
of Molecular Connectivity and a Chromatographic Correlation
Factor in Reversed-Phase High-Performance Liquid
Chromatography for Polycyclic Aromatic Hydrocarbons," J.
Chromatogr. 1982, 231, 517-522.
Jandera, P. "Method for Characterization of Selectivity in
Reversed-Phase Liquid Chromatography I. Derivation of the
Method and Verification of the Assumptions," J. Chromatogr.
1986, 352, 91-110.


Table 5-2. Methylene and phenyl selectivities at 35.0 C as a
function of silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.
C13 Bonding
Density
(ymo1/n/)
Methylene
Selectivity
Methy1 ene
Correlation
Coefficient
Phenyl
Selectivity
Phenyl^
Correlation
Coefficient
1 .60
1 .134
0 .9971
1 .423
0.9984
1 .74
1 .291
0.9991
1.901
0.9996
1 .98
1.275
0.9993
1 .924
0.9994
2.07
1.214
0.9935
1.944
1 .0000
2.09
1.308
0.9994
1 .950
0.9999
2.75
1.530
0.9906
2.000
0.9995
2 .84
1.352
0.9983
2.042
0.9999
3.06
1.339
0.9988
2.039
0.9997
3.15
1 344
0.9974
2.011
0.9995
3.24
1.348
0.9990
2 .033
0.9992
3.34
1 .364
0.9943
2.016
0.9992
3.56
1 .350
0.9995
2 .031
0.9995
3 .60
1 .358
0.9999
2.111
0.9997
* Coefficient of correlation for the plot of In k1 versus carbon
number; slope of this line is ln(methylene selectivity).
*- Coefficient of correlation for the plot of In k versus phenyl
number; slope of this line is ln(phenyl selectivity).


Response
Figure 6-3. Calibration plot for benzene on column DMAP5 at
35.0 C for 55/45 methanol/water mobile phase.


44
cleaning bath. The refluxed stationary phases had an
average bonding density (_+ one standard deviation over three
trials) of 2.82 + 0.02 umol/m2. The room temperature
reaction resulted in a bonding density of 2.69 umol/m2 with
a range of + 0.03 ymol/m2 over two trials; the ultrasound
reaction gave a bonded phase (over two trials) with an
average bonding density of 2.71 + 0.01 ymol/m2. The small
bonding density difference between the stirred reaction at
ambient temperature and the one at reflux temperature is not
surprising as Lork et al. (1986) have shown that the bonding
density increases slightly and in a linear fashion with
increasing reaction temperature when monoch1 oros i 1 anes are
used as the silanizing reagent. These experimental results
show that ultrasound is indeed a viable method for the
bonded phase synthesis, giving results which are comparable
to those obtained using traditional reflux techniques.
Effect of Subambient Temperature on the Ultrasound
Reaction
Two sets of experiments were performed using ultrasound
in conjunction with subambient reaction temperatures. In
achieving high bonding densities one of the greatest
obstacles is increasing steric hindrance at the silica
surface as more and more bulky dimethy1octadecy1sily1 groups
are bonded to the surface. It is possible that at low
temperatures the bonding density might be enhanced due to
the increased order (decreased entropy) in a lower
temperature system. It is here that the ultrasound reactions
are most unique, as they allow the temperature of the


89
Table 4-6. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.
Co Bonding Density
(pmol/nr )
Naphthalene Thermodynamic Partition
Coefficient at 35.0 C
1 .60
3.28
1 .74
3.69
1 .98
3.79
2.07
4.20
2.09
4.16
2.75
4.90
2 .84
4.95
3.06
5.09
3.15
4.88
3.24
4.73
3.34
4.66
3.56
4.48
3 .60
4.41


BIOGRAPHICAL SKETCH
Karen Belinda Sentell was born in Charleston, South
Carolina, on January 28, 1957. She attended Brentwood
Elementary School and Gordon H. Garrett High School (both in
Charleston Heights, South Carolina), graduating from Gordon
H. Garrett High School in June, 1974. She lived and worked
in Charleston, South Carolina, until September, 1979, when
she moved to Columbia, South Carolina. She entered the
University of South Carolina (Columbia, South Carolina) in
January, 1980, receiving her B.S. in chemistry (Magna Cum
Laude) in December, 1982. She entered graduate school at
the University of Florida in January, 1983, where she was
awarded a University of Florida Women's Fellowship. She was
awarded two American Chemical Society Analytical Division
Graduate Fe11owships--a summer fellowship for 1985 and a
full-year fellowship for 1986-1987. Upon completion of the
requirements for the degree of Doctor of Philosophy
(December, 1987) she served as a Leopold Schepp Foundation
Postdoctoral Fellow at the University of Florida under Dr.
John G. Dorsey.
174


selectivity studies. Benzene (Ma11inekrodt, Inc., Paris,
KY), biphenyl (Eastman, recrystallized three times from
ethanol) and p-terphenyl (Siyma Chemical Co., St. Louis, MO)
methanolic standards comprised the phenyl selectivity test
solutes. The NBS (Gaithersburg, MD) column evaluation test
mixture was kindly supplied by Dr. Lane Sander. Methylene
and phenyl selectivity studies were conducted at 35.0 C
with a 55/45 methanol/water mobile phase on the silica
columns; these studies were also performed with a 85/15
acetonitrile/water mobile phase at 35.0 C for both the
silica and CPG columns. The NBS test mixture was also
evaluated on the silica and CPG columns with a 85/15
acetonitrile/water mobile phase but at ambient temperature.
Results and Conclusions
Silica-Based Stationary Phases
Methylene and phenyl selectivities as a function of
octadecyl bonding density for the 55/45 methanol/water and
85/15 acetonitrile/water mobile phase systems are tabulated
in Tables 5-1 and 5-2. Since the selectivity values are
calculated from the slopes of plots of In k1 versus homolog
unit number for each stationary phase, the least squares
linear regression coefficients of correlation for each of
these plots are included to verify that linear behavior is
being followed. Colin et al. (1983a) state that a linear
relationship exists between In k and the homolog unit
number for unit numbers above three to five. This number of
units is termed the critical carbon number and it results


124
Wise and Sander's (1985) "slot model" was postulated
based on their PAH selectivity studies. They found that for
polymeric phases with high bonding densities (greater than
about 5.1 pmo 1/m^) nonplanar solutes eluted before planar
ones and that nonlinear solutes eluted before linear ones,
even if the solutes compared had similar molecular weight,
overall shape and molecular dimensions. Additionally, they
found that selectivity between p1 anar/nonp1 anar and
linear/nonlinear PAHs increases with the degree of
nonplanarity and nonlinearity. Their "slot model"
postulates that nonplanar solutes have a greater
"thickness", hindering penetration of the solute into the
narrow slots between the bonded alkyl chains. If both wide
and narrow slots exist in the stationary phase structure due
to inhomogeneous distribution of the bonded chains on the
surface, retention will be greatest for long narrow solutes,
since they would fit into more available slots than thicker
"square" shaped molecules. The situation is analogous for
linear molecules, which would show greater retention than
nonlinear ones. This also corresponds with Marti re and
Boehm's "unified theory of retention and selectivity in
liquid chromatography" (1983) which predicts that shape
selectivity is greater for rigid rod solutes than for
globular solutes, especially when the stationary phase
chains are fully extended or more rigid. Sander and Wise
(1985) argue that higher alkyl density polymeric phases are
more extended and rigid than low density polymeric or
monomeric ones.


faster solute mass transfer kinetics (Cooke and Olsen,
1980). For octadecy1 dimethy1ch1 oros i 1ane, the most commonly
used monoreactive silane, the resulting bonding reaction is
depicted in Figure 2-1.
Kinkel and Unger (1984) have studied the roles of the
solvent and the base in these monofunctional bonding
reactions and have found their choice to be crucial. When
al ky 1 halosi 1anes are reacted with silica, a base is added to
serve as the acid-acceptor catalyst, binding the haloacid
formed during the reaction and driving the equilibrium to
the product side. In addition, the base favorably affects
the kinetics of the silanization reaction. Mechanistic
studies of these types of reactions (Corriu and Guerin,
1980) have shown that two molecules of base attack one
molecule of silane, activating the Si-X bond such that a
reactive intermediate and a hydrohalide are formed.
Formation of this reactive intermediate greatly increases
the kinetics of the bonding reaction; indeed, the addition
of the acid-acceptor catalyst results in approximately 90%
of the total conversion taking place within the first hour
of the reaction. In their study, Kinkel and Unger (1984)
found that the two most effective acid-acceptor catalysts
for organohalosilanes were imidazole and 2,6-lutidine.
The reaction solvent must also be carefully chosen.
The solvent can interact specifically with the silane, the
base and the surface silanol groups on the silica. When the
solvent interacts with a silanol group, there is a


84
appropriate volume of water; these mobile phases were then
mixed well and placed in an ultrasonic bath for 15-30
minutes in order to degas them. The flow rate of the mobile
phase in all cases was 1.5 ml/min. Naphthalene (Eastman
Organic Chemicals, Rochester, NY) was chosen as a small
nonpolar test solute; standards were made up in HPLC grade
methanol for use in the retention studies. Solute retention
and column holdup volumes were measured from the chart
recorder tracings. The solvent disturbance peak was used to
determine column mobile phase volumes for the calculation of
capacity factors; since this disturbance comes about from
the methanol in which the test solute is dissolved, its
choice for Vm falls under the category of using an
unretained compound to determine Vm. This convention was
chosen in order to account for variances in the individual
column dead volumes resulting from differences in the
stationary phase packing density within the chromatographic
columns .
Measurement of Vm and V£
The gravimetric procedure previously described was used
to calculate Vm. While an ambient temperature of 25.0 C
was maintained, 150 ml of methylene chloride was passed
through a 15 cm LC column packed with either silica or CPG.
The column was then capped and weighed on an analytical
balance. The procedure was duplicated using methanol as the
mobile phase and by dividing the difference in column masses
by the difference in the solvent densities at 25.0 C (1.318


146
For the naphthalene solute in 85/15 acetonitrile/water
the least squares linear regression calibration plot
equation was:
Response = (4035.65 response u n i t s / y g naphthalene) x
(y g naphthalene) 3.29 with a coefficient of correlation of
0.9999. The average response of the dynamic naphthalene
sample + one standard deviation was 430.87 + 13.89. This
response correlated to an equilibrium amount of naphthalene
in the mobile phase of 0.1076 yg with a range of 0.1110 yg
to 0.1041 yg when + one standard deviation was considered.
The average dynamic partition coefficient for naphthalene
between 85/15 acetonitrile/water mobile phase and DMAP5
stationary phase was 0.041; the high end of the naphthalene
range could not be used since the amount of naphthalene it
corresponded to (0.1110 yg) was larger than the original
amount put into the volumetric flask, once dilution was
accounted for (0.1077 yg). When the low end of the
naphthalene concentration range was considered the dynamic
partition coefficient was 1.51.
When chromatographic partition coefficients are
compared to those obtained in the dynamic equilibrium
experiment, the chromatographic K is consistently higher.
For the benzene system, Kchromatographic = 17.98 and
^dynamic = 8.51; for the acetonitrile system
^chromatographic 4*41 and ^dynemiC = 0.041. There are a
number of reasons for these discrepancies. When the
dilution factor for the amount of solute added to the


12
phase composition a least squares fitting for a large data
set must take place in order to determine these
proportionality constants, requiring a tremendous amount of
data both in terms of pi (and possibly sigma) values for
each solute and in terms of retention values for these
solutes at each mobile phase composition. Jinno and
Kawasaki (1984a and 1984c) also have not shown that this
model is applicable for larger more complex molecules.
Funasaki et a 1 (1986) examined retention behavior for
alcohols and ethers with positional and geometric isomers
and examined the correlations between the solute's log k1
and molecular cavity surface area (S), the logarithm of the
aqueous solubility (log Cw) and the logarithm of the
octanol-water partition coefficient (log P). As previously
mentioned in this chapter, it was found that S and k' were
very well correlated, even in the case of conformational
isomers. This is because S for a molecule in water is
defined as the area of the surface traced out by the center
of a water molecule rolling over the van der Waals surface
of the solute molecule (Funasaki et a 1 ., 1986 ). However S
is very difficult to accurately calculate, requiring the
construction of solute molecular models for rigorous work,
as well as detailed knowledge of the molecular conformation
of the molecule of interest. If S is calculated from group
surface areas, an easier but less rigorous approach, the
molecular surface area is usually overestimated for very
crowded molecules. Using S to predict retention does have


76
g/cm^, 0.8625 g/cm^, and 0.8638 g/cm^ respectively for the
octadecy1dimethy1sily1, octy1dimethy1sily1, and
trimethylsilyl bonded groups. Substitution of equations 4-1
and 4-3 into equation 4-2 results in the volume of the
stationary phase, Vs (in cm^), as expressed by the following
formu1 a
r---.(%C)(M)(Wp)
(l00)(12.011)\nr)(d)
(4-4).
This method provides a much more accurate calculation
of Vs than has been previously possible. A principal
advantage of this method is that the surface area of the
packing is not used in determining Vs, which eliminates the
errors associated with this measurement. The stationary
phase volume that is calculated in this method is the volume
that is important in the chromatographic process, i.e. the
actual volume of the bonded alkyl chains themselves. The
precision is limited only by the carbon loading
determination (+ 0.20 %C for our departmental elemental
analysis) and by the measurement of the mass of packing in
the column ( + 0.1 mg on any analytical balance).
Calculation of the volume of the stationary phase by this
method provides the means for a more accurate and uniform
determination of the phase ratio.
Experimental Procedure
Preparation of Bonded Phases of Varied Bonding Densities
All of the reagents used in the preparation of the
silica and CPG bonded phases are described in Chapters II
and III. Silica bonded phases with octadecyl bonding


13
one very strong merit--since the slope of a log k1 versus S
plot is related to interfacial tension, the dependence of
log k on the organic modifier content of the mobile phase
can be predicted (Funasaki et a!., 1986).
Funasaki et al. (1986) found the correlation between
log k' and the logarithm of solute aqueous solubility, Cw,
to be rough at best. The main drawbacks to this method are
that the extent of correlation will depend on whether Cw was
measured for the compound in the gas, liquid or solid state,
that some compounds are infinitely water soluble and that
the correlation is particularly poor for branched solutes.
In contrast, they found that the correlation between the
logarithm of the solute octanol-water partition coefficient
(log P) and log k' was particularly good for the solutes
examined (alcohols and ethers) in methanol/water mobile
phases. Braumann (1986) reports that other workers have
found good correlations between log k' and log P for a
variety of compounds including PAHs, alkylbenzenes,
substituted benzenes, pesticides, phenols and barbiturates;
again correlations were much better with methanolic mobile
phases than with those containing acetonitrile, due to
methanol's hydrogen bonding properties. One drawback to
this method is that octanol-water partition coefficients are
measured via shake-flask methods, which are very time
consuming. However, P values are tabulated for many
compounds and additivity rules using Hansch's pi parameters
may be used to estimate P for other compounds although


51
The use of ultrasound as a driving force for reversed
phase syntheses has been shown to be a viable synthetic
procedure. Ultrasonic syntheses performed at subambient
temperatures have proven to be especially effective for the
production of high alkyl bonding density stationary phases.
The use of 4-dimethy1 aminopyridine as the acid-acceptor
catalyst is recommended due to its ease of use and the
resulting high bonding densities. The small ranges of the
bonding density for duplicate syntheses show that reversed
phase packings with reproducible bonding densities can be
synthesized by these methods.


Figure 2-3. Scanning electron micrograph of acid-leached Davisil
silica; 3000X magnification.
CO


97
Table 4-8. Naphthalene thermodynamic partition
coefficients at 35.0 C as a function of
CPG-86 octadecyl bonding density for
55/45 methanol/water mobile phase.
C^g Bonding Density Naphthalene Thermodynamic Partition
(umol /irr ) Coefficient at 20.0 C
1 .70
69.3
2 .68
75.1
2.72
70.0
2 .83
67.1
3.21
67.0
3.30
69.7


11
correlate with RPLC retention for substituted benzenes
(excluding phenol). They found a very good correlation
between In k and pi, and poor correlations between In k1
and molecular connectivity, correlation factor (number of
double bonds plus number of primary and secondary carbons
minus 0.5 for nonaromatic rings) and van der Waals volume
and surface area. They interpret this to mean that the size
and shape of these molecules were not dominant for
controlling their retention. However, when the size and
shape of the solute molecules are a dominant retention force
such as for PAHs and hydroaromatics, the correlation factor
(F) has been found to correlate quite well with log k1
(Hurtubise et a 1., 1982). If a linear combination of HA, HD
and pi descriptors was used, they found an even better
correlation between predicted and experimental k1 values.
For phenol solutes, hydrophobicity alone was not an adequate
retention descriptor so they added Hammett's acidity
parameters (sigma) to account for the strong hydrogen
bonding ability of the phenols (Jinno and Kawasaki, 1984a
and 1984c). Linear combinations of pi and sigma for the
phenol solutes resulted in excellent correlations with
retention behavior. However, for both sets of solutes the
parameters had to be multiplied by certain proportionality
constants in order to correctly predict retention and these
constants were different not only for different organic
modifiers in the mobile phase but also for each volume
composition. This requires that at every different mobile


V CORRELATIONS BETWEEEN CHROMATOGRAPHIC SELECTIVITY
AND OCTADECYL BONDING DENSITY 105
I ntroducti on 105
Experimental Procedure ...110
Results and Conclusions 113
VI CONCLUSIONS 135
Syntheses of RP Stationary Phases 135
Validity of Chromatographic Partition
Coefficient Measurements 138
The Effect of Octadecyl Bonding Density on the
the Chromatographic Partition Coefficient 149
Suggestions for Future Work 154
REFERENCES 164
BIOGRAPHICAL SKETCH
174


6.0-
147 & Silica at 35 C
~i i ( 1 1 1 1
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
C|8 Bonding Density (jjmoi/m*)
Figure 4-3. Naphthalene thermodynamic partition coefficient at 35.0
as a function of silica octadecyl bonding density for
85/15 acetonitrile/water mobile phase.


155
temperatures such as -10 or -20 C should be investigated
since lower temperatures will result in still more ordered
systems as well as further reducing the reaction solvent
vapor pressure, promoting even more efficient ultrasonic
cavitation (Suslick, 1986). Along this same line of
reasoning, the use of a reaction solvent with a lower vapor
pressure than methylene chloride may improve ultrasonic
efficiency. Suslick (1986) states that the greater the
solvent vapor pressure within the cavitation bubble prior to
collapse, the less effective the collapse. He also
recommends solvents with low chemical reactivity in order to
minimize the solvent concentration in the vapor phase of the
cavitation event. Therefore, these ultrasound reactions
should be attempted using toluene as the reaction solvent,
since it is chemically inert, has a high vapor pressure and
is a good solvent in terms of silane solubility.
According to Suslick (1986), sonochemical reactivity is
also greatly affected by the ambient gas atmosphere. It is
desirable to maximize the temperature reached during
microbubble collapse since this is one of the driving forces
behind sonocatalysis. The maximum temperature attained
during cavitation is strongly dependent on the polytropic
ratio (Cp/Cv) of the ambient gas, since this defines the
amount of heat released during the adiabatic compression of
the gas. Suslick (1986) states that cavitation in the
presence of xenon (Cp/Cv = 1.67) versus freon (Cp/Cv = 1.1)
would result in a sevenfold ratio of maximum cavitation


1.74 to 3.56 pmol/m2 the TBN/BaP selectivity is about 1.7
and the elution order is BaP = PhPh < TBN. At 3.60 u mol/in2,
the elution order changes to PhPh < BaP < TBN and the
TBN/BaP selectivity is 1.56. The planar BaP molecule is now
retained longer than the helical PhPh. Although this is
classified as "oligomeric" type behavior by Sander and Wise
(1984a), this stationary phase was prepared using the
monochlorosilane as opposed to the trichlorosilane reagent
used by Sander and Wise to prepare the oligomeric bonded
phases. The oligomeric bonded phases are actually polymeric
type phases whose bonding density (or "thickness") has been
controlled by sequential polymerization. The fact that our
monomeric phase exhibits the same PAH selectivity as Sander
and Wise's polymeric phases indicates that PAH selectivity
and shape selectivity are probably not a function of the
degree of stationary phase polymerization but rather are a
function of alkyl bonding density. The correlation of
phenyl selectivity with alkyl bonding density further
supports this conclusion.
Controlled Pore Glass-Based Stationary Phases
Table 5-4 is a compilation of methylene and phenyl
selectivities of the controlled pore glass (CPG) octadecyl
bonded phases versus their octadecyl bonding density for the
85/15 acetonitrile/water mobile phase; this data is also
plotted in Figures 5-8 and 5-9. Although very scattered,
the methylene selectivity seems to be approximately
constant, as in the case of the silica stationary phases.


109
are measurements of retention differences (i.e. ratios)
rather than absolute measures of retention. Therefore
intercolumn selectivity differences are not due to different
column phase ratios, but rather are due to actual
differences in the structure of the bonded alkyl chains in
the different columns. In general, selectivity values for a
particular type of bonded phase are independent of the
specific column used (Antle and Snyder, 1984; Colin et al.,
1983a and 1983b; Krstulovic et al., 1983; Melander and
Horvath, 1982), implying that very fundamental aspects of
the retention process are reflected by selectivity behavior.
Examination of methylene selectivity offers an
additional advantage for retention mechanism studies. Since
methylene selectivity is solely due to solvophobic
selectivity, it is quite insensitive to the presence of
residual silanol groups on the bonded phase surface. For
solutes which have highly polar and/or hydrogen bonding
functional groups, the presence of these silanol groups can
lead to poor chromatographic peak shape as well as anomalous
retention due to chemical selectivity. However, methylene
selectivity values for a homologous series of such compounds
will be largely unaffected by these specific interactions,
even though retention of individual members of the series
may be susceptible to these chemical interactions (Johnson,
1986).
In the present work, chromatographic selectivity was
examined as a function of stationary phase alkyl bonding


Figure 3-2. Scanning electron micrograph of acid-leached CPG-86;
3010X magnification.
<_n


159
phases have practical significance, as enhanced column
efficiencies bring about improved resolution between
chromatographic peaks, an important consideration in
difficult separations. Efficiency comparisons between
ultrasonic phases of average (around 2.8 pmol/m^) and high
(around 3.6 ymol/m^) bonding densities should also be
carried out, in order to see if the higher bonding density
phases have a correspondingly higher resistance to mass
transfer and thus a lower chromatographic efficiency.
Sander and Wise (1987) have noted that column efficiency
degrades with increased bonding density for polymeric
stationary phases; it is important that monomeric phases be
evaluated in the same manner.
As explained in Chapter IV, stationary phases of lower
octadecyl bonding density were synthesized by "pre-
endcapping" the silica with a less than stoichiometric
amount of trimethylchlorosilane (TMCS) prior to exhaustive
octadecy1 ation. Marshall et al. (1984 and 1986 ) have
reported that the "pre-endcapping" treatment results in more
efficient stationary phases as well as significant
reductions in peak tailing. This is explained in terms of
increased homogeneity of the bonded octadecyl groups. The
TMCS is postulated to bond at the most reactive silanol
sites, deactivating them and creating a more homogeneous
silanol distribution for reaction with the octadecyl silane
(Marshall et al., 1984 and 1986). Since we have synthesized
a number of these "pre-endcapped" phases, it would be


illuminating to see if we obtain the same results as
Marshall et al. as well as to study the efficiency of these
phases as a function of degree of "pre-endcapping. Such
studies can supply additional information on the prevalence
of reactive silanols on the silica surface.
Further Retention and Selectivity Studies
Once bonded phases of even higher alkyl densities
(> 3.6 pmol/m^) can be synthesized, they must be examined in
terms of both chromatographic retention and selectivity.
The partition coefficients of small solutes on these very
high density phases should be evaluated in order to further
test Dill's (1987a) predictions. These partition
coefficient experiments (as desribed in Chapter IV) should
also be carried out with different types of solutes: large
molecules (such as PAHs) as well as those with different
types of shapes such as rodlike, chainlike, planar and
nonplanar molecules. This will illuminate whether the
relationship between bonding density and partition
coefficient predicted by Dill (1987a) and demonstrated in
our experiments with small molecules will also hold true for
larger molecules of different shapes. These experiments
should provide additional fundamental information about the
nature and organization of the bonded interphase region.
Since a change in mobile phase composition will affect chain
ordering by changing the stationary phase solvation layer,
partition coefficient experiments (as described above)
performed under different mobile phase conditions should


106
from each other by the functional group in question, i.e. a
homologous series of alkylbenzenes for methylene
selectivity. The natural logarithm of the capacity factor
is then plotted versus the unit number of the functional
group for each homolog; the slope of the resultant line is
the natural logarithm of the group selectivity, In a.
Figure 5-1 illustrates such a plot of methylene selectivity
on column DMAP 3 for the homologous alky1 benzene series of
toluene through penty1 benzene. The slope of the plot, which
1 s ln methylene* is 0.6773 ; the resultant amet tiy ] ene value
is then 1.969.
Antle and Snyder (1984) and Antle et al. (1985) state
that there are two different types of RP column selectivity,
namely solvophobic and chemical. Solvophobic selectivity
arises from hydrophobic interactions between the solute
molecules and the stationary phase. Chemical selectivity
comes about from strong interactions (for example, hydrogen
bonding or comp 1exation) between the solute molecules and
specific active sites such as silanol groups or trace metal
contaminants on the silica surface (Antle and Snyder, 1984;
Jandera, 1986). A third type of selectivity, shape
selectivity, can also be exhibited by chemically bonded
phases. Since these phases consist of lengthy alkyl chains
bonded to the silica surface, the conformation of the bonded
chains can play an important role in retention, especially
for large molecules. When these chains are well solvated by
the mobile phase, such as when the mobile phase has a large


149
conclusion, the results of the dynamic equilibrium
experiments show that the chromatographic method of determining
partition coefficients may hold great promise in systems with
moderate to long (> 5 minutes) retention times, especially if
the solvation layer volume can be accounted for accurately.
The Effect of Octadecyl Bonding Density on the
Chromatographic Partition Coefficient
Examination of Tables 4-4, 4-5 and 4-6 and their
corresponding figures (4-1, 4-2 and 4-3) shows that for a
small nonpolar solute like naphthalene in a methanol/water
or acetonitrile/water mobile phase system, solute retention
(as measured by its partition coefficient) increases
linearly until a bonding density of about 3.1 umol/m2 is
reached. At bonding densities higher than this, the
partition coefficient begins to decrease as the bonding
density increases. Although errors in the absolute value of
the partition coefficient (due to ignoring the solvation
layer volume) have been discussed in the previous section as
well as in Chapter IV, these unidirectional errors will
result in an overestimation of the partition coefficient by
a relatively constant amount. As mentioned in Chapter IV,
this will cause the curves in Figures 4-1 through 4-3 to be
shifted vertically by a constant amount, but the shape of
the plot and the trends exhibited by it as well as its
maximum will be unchanged.
The retention trends in these plots are best examined
in light of Dill's (1987a and 1987b) theory of RPLC
retention, which was discussed in Chapter I. Our


74
Melander and Horvath (1980, p. 270) state that . any
estimation of "stationary phase volume" on the basis of BET
surface area of the support is likely to be inaccurate."
Due to the errors in determining both adsorbent surface area
and column dead volume, there will consequently be a large
error propagated in the subsequent calculation of the phase
ratio if Melander and Horvath's convention is used.
Sander and Field (1980) have estimated the phase ratio
by constructing physical models of the bonded phase using
manufacturer's data regarding silanol surface coverage and
percent carbon loading. This approach is quite reasonable
from a theoretical standpoint, as it accounts for variation
in bonding density and alkyl chain length. However because
it is based on models it can only be an estimate of V s; the
construction of such physical models is also time consuming.
In determining the stationary phase volume, the
pertinent volume should be the volume of the alkyl chains
bonded to the silica surface. Dill (1987a) has performed
statistical mechanical calculations based on a lattice
interphase model of RPLC stationary phases which describe
chromatographic retention in reversed phase systems. These
calculations have shown that in a well endcapped column,
chain interactions with solutes are the most important
stationary phase contribution to solute retention.
Therefore the calculation of V$ should give only the actual
volume of the alkyl chains bonded to the support surface.
The assumption here is that all of the bonded stationary


30
groups on the silica as possible, especially the highly
acidic isolated si lands. These residual isolated silanol
groups have been shown to be the main cause of tailing of
chromatographic peaks for basic compounds, of mechanical
instability of the packing, and of low sample capacity of
the column (Kohler et al., 1986; Kohler and Kirkland, 1987).
Di- or trireactive alkylsilanes had previously found favor
over monoreactive silanes because of their greater
reactivity and the possibility of reacting simultaneously
with two or three hydroxyl groups. However, any unreacted
sites on the bonded functional groups will be hydrolyzed
upon contact with water (i.e. from the mobile phase),
forming additional undesirable silanol groups (Berendsen and
de Galan, 1978b; Snyder and Kirkland, 1979). Di- and
trireactive silane reagents also often result in
nonreproducible stationary phases since the degree of
polymerization is highly dependent on the residual water
content of the silica and the reagents used in the bonding
reaction (Snyder and Kirkland, 1979). Another drawback of
polymeric stationary phases is their lower chromatographic
efficiency, which results from poor solute mass transfer in
these relatively thick stationary phases. Therefore many
investigators now advocate the use of monofunctional silanes
for the silica derivatization reaction, since this results
in a reproducible and well defined chemically bonded phase.
Additionally, monomeric stationary phases generally exhibit
superior column performance to polymeric phases due to their


Table 5-1. Methylene and phenyl se1ecti vities at 35.0 C as a
function of silica octadecyl bonding density for
55/45 methanol/v/ater mobile phase.
g Bonding
ensity
mol/m2)
Methylene
Selectivity
Methylene^
Correlation
Coefficient
Phenyl
Selectivity
P heny12
Correlation
Coefficient
1 .60
1.721
0.9989
5.479
0.9998
1 .74
1 .916
0.9993
7 .272
0.9997
1 .98
1 .959
0.9997
7.053
1.0000
2.07
1.925
0.9994
7.385
0.9996
2.09
1 .967
0.9999
7 196
1.0000
2.75
1 .936
0.9993
7.606
0.9999
3.06
1 .986
0.9996
7 .830
0.9999
3.24
1 .969
0.9995
7.941
0.9999
3.34
1 .968
0.9994
8.134
0.9997
3.56
1.927
0.9994
7.965
0.9998
3.60
1 .963
0.9997
8.170
0.9997
Coefficient
of correlation for the
plot of In k1
versus carbon
number; slope of this line is ln(methylene selectivity).
Coefficient of correlation for the plot of In k' versus phenyl
number; slope of this line is ln(phenyl selectivity).


138
higher silica bonding densities. Contrary to our
expectations, the smaller pore diameter CPG (86 Angstroms)
was more reactive in the octadecyl reaction than the 167
Angstrom CPG. Both of these trends were reversed in the
trimethyl si 1ane bonding reaction. Except for the higher
B 2 0 3 content of the CPG material, we have been unable to
postulate a reason for the seemingly anomalous CPG results.
Validity of Chromatographic Partition Coefficient
Measurements
Experimental
In order to discuss the implications of the behavior of
chromatographic partition coefficients as a function of
octadecyl bonding density, the validity of the
chromatographic partition coefficient (K) measurement should
first be established. Recall that K = k 1 / (V s / V rn).
Measurement of k1, the solute capacity factor is defined as
(Vr-Vm)/Vm where Vr is the solute retention volume and Vm is
the mobile phase void volume. The problems involved in the
measurement of Vm and V$ have been discussed in Chapter IV,
as has our rationale for our choices of conventions for
their measurement. In order to test the validity of the
chromatographic partition coefficient measurement, we
decided to perform an independent dynamic measurement of the
partition coefficient. A known mass of a chromatographic
test solute (benzene or naphthalene) was placed in a stirred
volumetric flask containing a known mass of stationary phase
DMAP5 (3.60 pmol/m^) and a known mass of mobile phase (55/45
methanol/water or 85/15 acetonitrile/water). The stationary


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Johri GL Dorsey, Chairman
Associate Professor of Chenrb
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
ft- Tj
Anna F. Brajter-Toth
Assistant Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
/ /
\
WTff iam Weltner, Jr
Professor of Chemistry
\
XJ


125
Sander and Wise (1984a) have devised a simple empirical
LC test to gauge the relative monomeric or polymeric nature
of a bonded phase. They found that the elution order of a
three component PAH test mixture of phenanthro[3,4-c]-
phenanthrene (PhPh), 1 ,2 :3 ,4 : 5 ,6 :7 ,8-tetrabuty1naphtha 1 ene
(TBN) and benzo[a]pyrene (BaP) at ambient temperature with a
85/15 acetonitrile/water mobile phase is dependent on the
type of phase and the surface coverage. For monomeric
phases (bonding densities up to about 3.2 ymol/rn2) the
elution order is BaP <_ PhPh < TBN; for oligomeric phases
(bonding densities of 3.3 to about 4.2 pmol/m2) the elution
order is PhPh < BaP < TBN. Polymeric phases (bonding
density > 4.3 ymol/m^) give the elution order PhPh < TBN <
BaP. Monomeric phases were synthesized with the
monoch1 oros i 1 ane; oligomeric and polymeric phases were
prepared using the trichlorosi1ane reagent. Each type of
phase also results in a different narrow range of values for
TBN/BaP selectivity. By examining the elution order of the
compounds in the test mixture, PAH selectivity of any RP
column can be quickly predicted.
The retention behavior of these compounds can be
attributed to their shapes. Phenanthro[3,4-c]phenanthrene
and TBN are nonplanar, due to steric hindrance of
neighboring aromatic rings. Of the two, PhPh is the more
nonplanar, exhibiting a helical shape, while TBN is
described as saddle shaped. Although TBN and PhPh are six
ring PAHs, BaP is a five ring PAH and is completely planar;


69
these species, this method adopts the convention that Vm is
the total volume of all mobile phase components within the
column bed; the solvation layer adsorbed on the stationary
phase is thereby included in the mobile phase volume.
The linearization of a homologous series of compounds
in order to find the column dead volume has been widely used
in gas chromatography; therefore it is not surprising that
this method has also been applied for LC systems. This
method assumes that there is a linear relationship between
the logarithm of the net retention time tr and the carbon
number of a homologous series (Berendsen et al., 1980b; Laub
and Madden, 1985; Wainwright et al., 1985). The mobile
phase volume can be obtained by comparing the retention
times for two consecutive homologs, termed n and n + 1. For a
homologous series the ratio of capacity factors for
consecutive homologs is assumed to be constant and
^r,n + l = A(tr>n) (A 1)tg
By plotting trjn+1 versus tr n, the slope A can be obtained
and tg can be determined from the intercept and multiplied
by the mobile phase volume flow rate to obtain Vm. The
results obtained can be precise to within 1% if alkyl-
benzenes are the homologous series used (Berendsen et al.,
1980b); however the use of homologous aromatic alcohols
gives inconsistent data due to their interactions with
silanol groups on the stationary phase (Laub and Madden,
1985) .
The linearization method is not without criticism. It
assumes that the relationship between the logarithm of the


83
200-300 psi, preserving the integrity of the CPG packings
and resulting in a stable packing bed. All columns were
equilibrated with the desired chromatographic mobile phase
by passing approximately 125 ml of the mobile phase through
the column immediately prior to use.
Chromatographic Measurements
The liquid chromatographic system used for
chromatographic measurements consisted of a Valeo C6W
injector (Valeo Instrument Company, Inc., Houston, TX) with
a 10 microliter sample loop, a Beckman Model 100A isocratic
HPLC pump, a Beckman Model 153 fixed wavelength 254 nm UV
detector and a Fisher Recordall chart recorder (Fisher
Scientific, Fairlawn, NJ). Samples were loaded in the
injection loop via a Hamilton 705 SNR syringe (Reno, NV).
Temperature control of the chromatographic column was
accomplished by passing thermostatted water through a water
jacket fitted around the column. Superambient temperatures
were controlled using a Lauda Model MT heater/circu1ator
(Brinkmann Instruments Company, Westbury, NY). Subambient
temperatures were brought about by a Neslab Endocal 800
water bath (Neslab Instruments, Portsmouth, NH). Mobile
phases were made of HPLC grade methanol or acetonitrile
(Fisher Scientific, Fairlawn, NJ) mixed with water which had
been prepared as described in Chapter II; mobile phase
compositions are designated by volume ratios of organic
modifier to water. Mobile phases were premixed by adding
the appropriate volume of organic modifier to the


8.2
8.0
7.8
Phenyl
Selectivity 76
7.4
7.2
7.0
1.5 2.0 2.5 3.0 3.5 4.0
Bonding Density (jimol / square meter)
Figure 5-6. Plot of phenyl selectivity versus octadecyl bonding
density for silica-based columns at 35.0 C for 55/45
methanol/water mobile phase.


24
systems than in the corresponding bulk alkane systems (Colin
et a 1 1983b). Although Melander and Horvath (1980) have
interpreted this as favoring an adsorption mechanism, Dill
(1987a) interprets this in terms of partial chain ordering
in the stationary phase, leading to less retention than in
the completely disordered bulk alkane. This is supported by
the work of Lochrnuller and Wilder ( 1979 ) since solute
methylene selectivities should be unaffected by the
molecular organization of the interphase (Dill, 1987a).
The other line of evidence is that In k1 for congeneric
sets of molecules can be linearly correlated with In P
(octanol-water partition coefficient) with a slope of one.
A slope of one is expected for the partitioning mechanism,
since all of the solute surface area would be available for
partitioning within the interphase. The slope for the
adsorption mechanism is expected to be considerably less
(about 1/6) because only a small fraction of the solute
surface area would contact the hydrocarbon chains, giving a
smaller driving force for retention (Dill, 1987a).
Experimental evidence has resulted in linear plots of In k1
versus In P with a slope of one (Melander and Horvath,
1980).
Based on the partitioning mechanism of retention, Dill
(1987b) predicts that at low surface densities (less than
2.7 micromoles of bonded alkyl chains per square meter of
silica surface) nonpolar solute retention will increase
linearly with increasing surface density. At these low