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Reverse micellar mobile phases for normal phase chromatography

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Title:
Reverse micellar mobile phases for normal phase chromatography
Creator:
Hernández Torres, Maria A., 1958-
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English
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iv, 35 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Chromatography ( jstor )
Hexanes ( jstor )
Liquids ( jstor )
Micelles ( jstor )
Moisture content ( jstor )
Molecules ( jstor )
Phenols ( jstor )
Solutes ( jstor )
Solvents ( jstor )
Surfactants ( jstor )
Chemistry thesis M.S
Dissertations, Academic -- Chemistry -- UF
Liquid chromatography ( lcsh )
Micelles ( lcsh )
Surface active agents ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (M.S.)--University of Florida, 1983.
Bibliography:
Bibliography: leaves 33-34.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Maria A. Hernández Torres.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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REVERSE MICELLAR MOBILE PHASES FOR NORMAL PHASE CHROMATOGRAPHY


By

MARIA A. HERNANDEZ TORRES























A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


1983




REVERSE MICELLAR MOBILE PHASES FOR NORMAL PHASE CHROMATOGRAPHY
By
MARIA A. HERNANDEZ TORRES
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
1983


ACKNOWLEDGEMENTS
I would like to express my gratitude and appreciation to my
research director, Dr. John G. Dorsey, for his invaluable assis
tance, guidance and encouragement throughout the course of this
project.
Also, my special thanks are extended to Mr. John S. Landy, for
his help in the initial setting of the instrument and his continuous
assistance.
I would like to thank my colleagues in Dr. Dorsey's research
group for their friendship and daily encouragement.
I would also like to thank my family and friends for their pa
tience and continuous support.
Finally, I would like to mention Patty Hickerson for her prompt
ness and accuracy in typing the final draft of this thesis.
11


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
ABSTRACT i v
CHAPTER
I INTRODUCTION 1
II THEORY 9
III EXPERIMENTAL 12
Instrumentation 12
Reagents 12
Procedure and Calculations 13
IV RESULTS AND DISCUSSION 15
Retention Changes with Surfactant Concentration 15
Efficiency 23
Influence of Water Content on Retention 26
V CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK 31
REFERENCES 33
BIOGRAPHICAL SKETCH 35


Abstract of Thesis Presented to the
Graduate School of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Master of Science
REVERSE MICELLAR MOBILE PHASES FOR
NORMAL PHASE CHROMATOGRAPHY
By
Maria A. Hernandez Torres
December 1983
Chairman: John G. Dorsey
Department: Chemistry
Reverse micelles, formed in hexane, can be used in place of polar
modifier to control the strength and selectivity of normal phase mo
bile phases for liquid chromatography. The effect of surfactant con
centration (Aerosol OT) on retention of various solutes in a normal
phase liquid chromatographic system was investigated. Surfactant con
centration above the critical micelle concentration range was used to
observe the change in capacity factor, k, as the water content in the
mobile phase was varied. Small changes in k1 values were obtained at
low water content in the micellar mobile phase. Finally, the column
_2
efficiencies for dry hexane, 5/95 isopropanol/hexane and 5 x 10 M
AOT in hexane mobile phases were compared for Ultrasil-NH^ and Ultra
sphere-Si columns.
TV


CHAPTER I
INTRODUCTION
A great deal of research has been put into the improvement of
both the stationary and mobile phases of high performance liquid
1-3
chromatography (HPLC) to achieve better separations.
This study is limited to normal phase HPLC, for which the reten
tion of the normal phase separation increases with increasing solute
polarity and decreases with increasing mobile phase polarity. The
solute retention mechanism is dominated by interaction of polar sta
tionary phase sites with polar solutes.
The advantages of normal phase chromatography are summarized by
4
Abbot as follows:
1) ability to separate highly hydrophilic species which cannot
be retained on reverse phase
2) use of predominantly organic solvent-based mobile phases,
avoiding silica dissolution problems experienced in reversed
phase
3) ability to differentiate solutes based on differences in hy
drophilic structure rather than hydrophobic structure
4) compatibility of organic phase with molecules having either
low stability or aggregation problems in aqueous phases
5) lower viscosity of organic mobile phases, resulting in lower
operating pressures
1


6) mobile phase volatility, allowing simpler, more efficient
concentration and transfer steps in off-line fraction collection
and structural characterization
7) ability to separate isomers which is of great significance
in natural product and pharmaceutical chemistry
8) ability to obtain class separations which cannot be obtained
in reversed phase
9) greater compatibility of organic mobile phase with on-line
coupling
10) availability of wide range of organic solvents to capital
ize on special solvent effects
11) availability of a wide range of stationary phase selectivi-
ties, which can be utilized to obtain high separation performance
in multi-stage chromatography.
Normal phase chromatography typically encompasses adsorption
chromatography on silica and partition chromatography on cyano and
amino bonded phases.
Adsorption may be defined as the concentration of solute mole
cules at the interface of two immiscible phases. In liquid-solid ad
sorption chromatography, the mobile phase is a liquid, while the sta
tionary phase is a finely divided, usually porous, solid. The atoms
in the bulk of the solid are subjected to equal forces in all direc
tions, whereas the surface atoms experience unbalanced forces which
can attract molecules from the surrounding solution to restore the
balance.


J
In a multicomponent system, selective adsorption occurs due to
competition between the solutes and the mobile phase for the surface.
It is governed by the differences in the strengths of the adsorption
forces between the adsorbent and the adsorbates. In general, polar
compounds are more strongly adsorbed by polar solids than are non
polar compounds. Also, adsorption of a polar compound is enhanced in
a nonpolar medium, but reduced in a polar medium due to increased com
petition of the mobile phase for the surface.
The partition process of a solute occurs between a liquid mobile
phase and a liquid absorbed on, or chemically bonded onto, a porous
support. Classical liquid-liquid chromatography involves the parti
tion of the sample components between a liquid stationary phase and
the liquid mobile phase. The separation occurs due to differences in
the solubilities of the sample components in the two liquid phases.
Since liquids which are practically immiscible will usually still have
at least ppm solubility in each other, the mobile phase must be pre
saturated with the polar phase to prevent gradual column stripping and
loss of separating power. As a result, column packings have been
developed in which the stationary phase is permanently bonded to the
support by chemical bonding. Bonded phase packings are prepared by
the reaction of the surface Si-OH groups of the support particles
with various reagents (Figure 1). These siliceous supports are pre
pared with a variety of functional groups, ranging from very polar to
nonpolar, resulting in widely diverse selectivities. The aminoi-NH^)
and cyano(-CN) functional groups are polar in nature, and they are
commonly used with low polarity mobile phases for normal phase sepa
rations.


(a) SILICATE ESTERS
-Si-OH + HOR Si-OR
I
(6) SILICA-CARBON ARP SILICA-NITROGEN
i-NHCHjCHjNHj
(c) SILOXANES
1 CISiRj i
Si-OH + or Si-O-SiRj
J ROSiRj
5
Figure 1. Reactions for preparing bonded-phase packing (from ).


It has been recognized that retention on active stationary phases
such as silica and alumina with nonpolar and moderately polar eluents
is strongly influenced by the water content of the eluent.^ ^ The
surface of the silica gel is covered with Si-OH and Si-0-Si groups
which can interact with the sample molecules. There are two kinds of
hydroxyl groups covering the silica gel surface: free (OH) and reac
tive (HOH) hydroxyl groups. The reactive hydroxyl groups are very
strong bonding agents which may permanently adsorb polar sample and
water molecules onto the gel, giving nonreproducible results. Since
the water content of the absorbent markedly affects relative capacity
ratio (k1) values and band migration rates, water content must be held
constant for repeatable separation.
Surfactants are amphiphatic molecules which have distinct hydro-
phobic and hydrophilic regions. Over a narrow concentration range,
defined as the critical micelle concentration (CMC), surfactants dy
namically associate to form large molecular aggregates. In nonpolar
solvents, these have been termed reversed or inverted micelles, since
their polar groups are concentrated in the interior of the aggregate
while their hydrophobic groups extend into and are surrounded by the
bulk apolar solvent (Figure 2). In aqueous solution, the micelle is
a compact roughly spherical body with a liquid-like hydrocarbon core.
The polar head groups are at the surface (Figure 3). Micellar sys
tems have been introduced in reverse phase HPLC, and an improvement
2 3 10 11
in the separations has been reported. When using micellar
mobile phases, solutes do not partition to the bulk solvent but rather
to the discrete aggregates creating a unique separation mechanism:


b
Figure 2. Reverse micelle representation. The ionic head group (0)
points toward the center of the micelle, and the hydro
carbon chain (W) surrounds the central ionic core
(from 12).


Figure 3. Di 11-FIory1s representation of a normal micelle. The
ionic head groups are indicated by the circles, and the
hydrocarbon chains are pointing toward the center of
the micelle (froml2).


pseudo phase liquid chromatography. Some of the advantages of the
13
micelle system in RPHPLC are:
1) many hydrophobic, amphiphilic, and even hydrophilic molecules
interact differently with the micelles, allowing for a wide range
of applications
2) low cost
3) nontoxic
4) non-UV absorbing
5) simultaneous separations of hydrophobic and hydrophilic
solutes are accomplished without aqueous organic gradients
6) the addition of solutes to the mobile phase to control ionic
strength, pH, and buffer capacity will not produce solubility
problems
7) a relatively small change in the amount of surfactant will
essentially give a different mobile phase.
Our studies are directed mainly to observe the behavior of sol
utes in reverse micellar systems. Solute's retentions as a function
of surfactant concentration are examined. Also, this study attempts
to find whether the water in the chromatographic system will be en
trapped so that the retention of the solutes in the normal phase HPLC
will not be changed. The best column efficiency is sought among all
the conditions studied.


CHAPTER II
THEORY
Due to the new findings and applications of reversed or inverted
micelles in the course of the last few years, the scientific interest
with regard to nonpolar detergent solutions has extensively in-
14
creased.
The obvious difference between micelles in polar and nonpolar
surfactant solutions is their mutual structural reversion. In non
polar solvents, the inverted micelle is visualized to be built up by
a polar core covered by hydrocarbon tails (vide supra). Inverted
micelles have moderate aggregation numbers which contrast the large
micellar aggregates in aqueous surfactant solutions, e.g., for 4.9 x
-4
10 M of Aerosol 0T in pentane at 25C the mean aggregation number
(n) is 15, while for 8.2 x 10 ^ M of sodium n-dodecylsulfate (SDS)
in aqueous solution at 25C n is 64.^"^
In the formation of a micelle in apolar media, there seems to be
a general agreement with regard to the existence of premicellar ag
gregates. The monomer ? n-mer type association is unlikely to rep
resent the behavior of surfactants.^ The most universally used
treatment, the multiple equilibrium model, assumes the stepwise forma-
^1
tion of aggregates in an indefinite association: monomer dimer
k2 k3 kn
trimer n-mer, where the distribution of the
different aggregates depends on the stoichiometric surfactant
9


10
concentration. The higher the concentration of the surfactant, the
larger the aggregates. Under this treatment, the CMC can be defined
as the concentration of aggregates within a narrow distribution cen
tering around the average micellar size. On the other hand, it can
be assumed that there is no unique concentration at which the exist
ence of micelles becomes detectable; therefore, it is unjustified to
speak of one concentration, or even a narrow range of concentration,
describing the CMC of surfactants in hydrocarbon solutions.^7
The driving force behind the process of aggregation and micelli-
zation of surfactants in hydrocarbon media is essentially due to
dipole-dipole interactions between polar heads of the amphiphilic
molecules.
One important property of the micellar system is the detergency
or the ability of surfactant molecules to take up (solubilize) polar
material, i.e., water in the polar core of the inverted micelles.
There are a large number of parameters which influence and even
determine the extent of the solubilization of the polar material.
These parameters include the structure of the surfactant, the physico
chemical property of the sol ubi 1izate, temperature, pressure, electric
fields and cosurfactants.
Considerable amounts of water can be solubilized by reverse mi
celles. This surfactant solubilized water is often called waterpool.
Initial water molecules are bound to the polar head groups, and their
motion is restricted. Additional water molecules occupy the core of
the micelle, and their properties resemble bulk water. A rapid ex
change takes place between these two kinds of water molecules.


The effective polarity, acidity and microscopic viscosity of the
water pools are expected to be substantially different from those in
bulk water.
Polar substrates are expected to be localized in water pools;
therefore, controlled amounts of surfactant-entrapped water in non
polar solvents provide a unique medium for interactions of polar sub
strate.


CHAPTER III
EXPERIMENTAL
Instrumentation
A Spectra-Physics 8700 (Santa Clara, California) liquid chromato
graph equipped with a model 7125 Rheodyne sample injection valve with
a 10 microliter loop, a model 153 Beckman UV detector (254 nm) with
an 8 microliter flow cell, and a model 1210 Linear strip chart re
corder were used. The columns used were: an Altex (Berkeley, CA)
Ultrasphere-Si (15 cm x 4.6 mm ID) 5 ym particle size and an Altex
Ultrasil-NH^ (25 cm x 4.6 mm ID) 10 ym particle size. Both columns
were thermostatted at 30C 0.2 using a water jacket and a Haake D1
water circulator.
Reagents
HPLC grade n-hexane (Fisher Scientific) was used as the solvent;
it was dried using Linde Molecular Sieves (Union Carbide) type 3A.
Reagent grade 2-propanol (Fisher Scientific) was used as organic
modifier and to wash the columns.
Reagent grade Aerosol 0T (dioctyl sodium sulfosuccinate, Fisher
Scientific) was used without further purification; it was dissolved in
hexane and then filtered with a Rainin solvent filtration apparatus
through a 0.45 ym membrane filter.
Water used in the mobile phase was purified using a Barnstead
Nanopure System (Symbron Corporation).
12


The solutes were used without further purification, and all the
solute solutions were prepared in n-hexane. The solutes were: 2,4-
dinitrotoluene, naphthol (Matheson Coleman & Bell) and phenol
(Mal 1inckrodt).
Reagent grade n-pentene (J.T. Baker) was used as an unretained
compound for the column's void volume calculation.
Procedure and Calculations
The appropriate weight of surfactant (Aerosol OT) was weighed
-4
and dissolved in hexane. The concentration range was from 1 x 10 M
to 1 x 10 1 M with alternate increments of 5x and 2x, respectively.
The columns were thermostated at 30C. A silica gel precolumn was
used before the injector to prevent the silica in the column from
being dissolved by the surfactant. The columns were washed with 2-
propanol, and rinsed and stored in n-hexane.
A stock surfactant solution was prepared of 0.50M, and aliquots
_2
of 50 ml were taken and diluted with n-hexane to 5.0 x 10 M for the
study of the influence of mobile phase water content on the retention
_2
time of the solutes. In each of the six 5.0 x 10 M Aerosol 0T solu
tions, the volume percentage of water was varied (0%, 0.01%, 0.02%,
0.05%, 0.10%, 0.25%).
The capacity factor, k1, was used to compare the retention data
of the solutes and was calculated using the following equation:
o
where Vr = the retention volume of a given solute and is calculated
as the product of flow rate times retention time (ml), and VQ = the


column's void volume (ml). n-Pentene, assumed to be an unretained
solute, was used to calculate the void volume of the column.
The efficiencies of the columns were compared using the reduced
plate height, h, given by:
h = N^dp ^
where L = the column length, dp = the particle diameter, and N = the
18
plate number. The plate number, N, was calculated as follows:
N
41 7
w0.1
B/A + 1.25
(3)
where t^ = the retention time of the compound (cm), Wq ^ = the peak
width at 10% of the peak height (cm), and B/A = the asymmetry factor.


CHAPTER IV
RESULTS AND DISCUSSION
Retention Changes with Surfactant Concentration
Figures 4 and 5 show the behavior of phenol and naphthol in
hexane/Aerosol OT mobile phases. These graphs show two linear compo
nents, one above and one below the CMC. Since the formation of mi
celles in nonpolar solvents occurs in sequential steps, it is diffi
cult to assign the CMC to one point. The intersection of the two
_3
linear components was found to be 3.5 x 10 MAOT in hexane. There-
-3 -3
fore, the concentration range from 1.0 x 10 M to 5.0 x 10 M A0T
in hexane was assigned as the CMC, in agreement with the formation
of reverse micelles as the multiple equilibrium model postulates. In
both columns, Ultrasphere-Si and Ultrasil-NH^, the first large change
in k was observed in the same range of concentration, the CMC range.
In other words, the same value of CMC range for A0T in hexane was ob
tained for the two different columns and two different solutes. Table
I shows mean aggregation numbers and critical micelle concentrations
of Aerosol 0T in different hydrocarbon solvents.
At concentrations above the CMC range, the slope of the four
curves are slightly negative; with the Ultrasphere-Si column having a
greater negative slope. This can be attributed to the fact that the
silica surface has silanol groups that interact with polar compounds
15


A w
Figure 4. Effect of surfactant concentration on retention
using Ultrasphere-Si column. (0) naphthol, (I)
phenol.


A /
Figure 5. Effect of surfactant concentration on retention using
Ultrasil-NH0 column. (!) naphthol, (0) phenol.


lo
Table I
Mean Aggregation Numbers (n) and CMCs of
Aerosol OT in Different Hydrocarbon Solvents (from i4)
Solvent
Temperature
(C)
Technique3
CMC
(M)
n
Cyclohexane
37
VPO
3.9
x 10"4
17
28 4
LS
1.3
x 10'3
56
Benzene
37
VPO
3.5
x 10"4
13
RT
PA
2.2
x 10"3
-
28 4
LS
2.7
CO
1
o
f-H
X
23.6
20
TCNQ
2.0
x 10-3
-
cci4
25
VPO
1.6
1
o
r-H
X
17
37
VPO
4.0
x 10"4
17
20
TCNQ
6.0
X
I*
o
1
-
Pentane
25
LS
4.9
<-
1
o
It
X
15
a VPO: vapor pressure osmometry
LS: light scattering
PA: positron annihilation
TCNQ: solubilization of 7,7,8,8-tetracyanoquinodimethane


13
in a very strong way. Also, the polar heads of the surfactant can
interact with these active sites, and retention of polar compounds
will decrease as the number of active sites available is reduced by
the presence of free surfactant. In the Ultrasil-NH2 column, the
interaction of the polar surfactant head groups with the amino sta
tionary phase groups is less strong; therefore, the concentration of
free surfactant in the mobile phase for this column affects the re
tention of polar compounds to a lesser extent. Recently, Deming and
Tang proposed that addition of surfactant to the eluent reduces the
interfacial tension and thus decreases the retention time of the in-
19
jected samples.
Looking at Table II, one can observe a big change in k' value
-4
going from dry hexane to 5 x 10 M AOT in hexane mobile phases. Two
possible reasons for this change are (1) before the CMC range, the
surfactant is in a monomeric form, and the polar heads compete for
active sites in the stationary phase, and (2) the formation of small
aggregates at very low concentration of surfactant that can interact
with solutes. For both points, there is a decrease in the number of
active sites available at the stationary phase; therefore, a decrease
in the retention time of solutes is observed.
Above the CMC range, the k of naphthol and phenol decrease very
rapidly as the formation of aggregates increases. The reason for this
behavior is that in a reverse micellar system a polar solute will par
tition to the polar core of the micelle spending less time interacting
with the stationary phase. This separation mechanism with reverse mi-
20
celles is similar to the one discussed by Armstrong and Nome for


U.\J
Table II
k' Value for Phenol and Naphthol in Dry Hexane and
5x10"^M AOT in Hexane for Ultrasphere-Si and Ultrasil-NH^ Columns
Column
Mobile Phase
k phenol
k' naphthol
Ultrasphere-Si
dry hexane
72.53
47.50
5 x 10'4 M AOT in
hexane
27.89
20.30
Ultrasil-NH^
dry hexane
47.52
53.75
5 x 10-4 M AOT in
hexane
29.44
40.87


C. 1
micellar systems in reversed phase HPLC, or pseudo phase liquid
chromatography. In normal phase chromatography, the solvent strength
is varied in order to optimize k'. Increasing the strength of the
mobile phase by increasing the solvent polarity, a decrease in sample
k* value is observed. Preferred solvents used in normal phase to
increase the strength of the mobile phase are acetonitrile, ethanol,
ethylacetate, CHCl^, tetrahydrofuran and isopropyl ether.
Comparing micellar and organic mobile phases for normal phase
HPLC, one can say that (1) Organic mobile phases have somewhat lower
viscosity than micellar mobile phases, resulting in lower operating
pressures. For example, at a flow rate of 1 ml/min., 30C, the pres
sure for 5/95 isopropanol/hexane mobile phase was 302 psig vs. 350
-4
psig for 5 x 10 M AOT in hexane mobile phase for Ultrasphere-Si
column. Table III shows the viscosity values for most common solvents
used in normal phase HPLC. (2) Surfactants are cheaper than the HPLC
grade organic solvents; an approximate cost per gallon of organic
solvent is thirty dollars vs. twenty dollars for 500 g of Aerosol 0T
(Table III). (3) Surfactants are non-toxic vs. some of the most
commonly used organic modifiers, for example tetrahydrofuran. (4)
Micellar mobile phases offer a different selectivity for separations
of compounds, and (5) For both mobile phases, a UV detector can be
used. The molar absorptivity coefficient for Aerosol 0T was calcu
lated to be 0.9986 L/mole cm at a wavelength equal to 254 nanometers
(Table III).


Lm i
Table III
Viscosity, UV Cutoff and Price per Gallon for
Most Commonly Used Solvents in Normal Phase HPLC Frorri 5
UV
Solvent
Cutoff
(nm)
Viscosity
(cP, 25C)
Price per
Gallon
Hexane
195
0.30
28.65
i-Propyl ether
220
0.38
27.85
Tetrahydrofuran
212
0.46
47.15
i-Propanol
205
1.9
24.80
Chloroform
245
0.53
29.85
Acetonitrile
190
0.34
55.85
Ethanol
210
1.08
26.75
Ethyl acetate
256
0.43
26.05


i
Efficiency
In looking for an optimum separation, one which gives adequate
sample resolution with a minimum of time and effort, one of the para
meters to be considered is column efficiency. The number of theo
retical plates, N, was calculated in order to compare the performance
of the system under the different experimental conditions studied.
Foley and Dorsey's equation was used to calculate the number of theo-
18
retical plates. (vide supra) This equation was preferred over the
most commonly used,
N = 5.54 (tr/w0>5)2 (4)
because the former corrects for the asymmetry of skewed peaks giving
more accurate values for N. The reduced plate height was calculated
by
h = H/dp (5)
where H is the plate height, and dp is the particle diameter.
Table IV shows the theoretical plate numbers, plate heights, re-
_2
duced plate height and asymmetry ratios for phenol in 5 x 10 M AOT
in hexane, 5/95 isopropanol/hexane and dry hexane mobile phases for
Ultrasphere-Si and Ultrasil-NH^ columns.
Better efficiencies were observed for 5/95 isopropanol/hexane
mobile phase for both columns. The reason for smaller values of N in
_2
5 x 10 M AOT is the higher viscosity of the micellar solution, hav
ing smaller diffusion coefficients, therefore, slower mass transfer.
This slow mass transfer results from the slower rate of movement of a
polar sample between the stationary phase and the interior of the


4
Table IV
Theoretical Plate Numbers (N), Plate Heights (H, mm), Reduced
Plate Heights (h) and Asymmetry Ratios (B/A) for Phenol in
5 x 10~2 M AOT in Hexane, 5/95 Isopropanol/Hexane and Dry
Hexane Mobile Phases for Ultrasphere-Si and Ultrasil-NH^ Columns
Ultrasphere
-Si
5
x 10"2 M AOT
5/95
in Hexane
Isopropanol/Hexane
Dry Hexane
N
3301
8174
3987
H (mm)
0.0454
0.0184
0.0376
h
9.08
3.68
7.40
B/A
1.50
1.14
2.02
Ultrasil
-NH
2
N
902
2409
-
H (mm)
0.277
0.104
-
h
27.7
10.4
-
B/A
2.18
1.22
-


micelle. Also, higher viscosity in the interior of the micelle in
comparison of the bulk solution contributes to slow the adsorption-
desorption interchange.
Greater values for asymmetry ratio in micellar system were ob
tained in accordance with the N values. Optimization of the micellar
system has to be done in order to be a feasible medium for separa
tion.
For Ultrasphere-Si columns, higher values of N were calculated
for the three systems studied. This can be explained in terms of a
smaller particle diameter, 5 pm vs. 10 pm for Ultrasil-NH^. It seems
that the fact of smaller particle diameter dominates over the longer
column for Ultrasil-NH^ and stronger active sites in Ultrasphere-Si.
Lower efficiency was observed for phenol in Ultrasphere-Si in
dry hexane mobile phase. This was expected since it is generally
accepted that with a dry mobile phase, at low water contents, much
lower efficiencies will be obtained because of the strong active
sites of the adsorbent surface, leading to slow adsorption-desorp
tion kinetics.^ The theoretical plate number for phenol in Ultrasil-
NH^ could not be calculated because of the long retention (more than
three hours).
In Szepezy, Combellas, Claude and Rosset's paper,^ very low and
rapidly changing values of efficiency at low water contents were
pointed out, indicating the necessity for selection and control of
the water content in the mobile phase. For this reason, the next part
of this experiment was performed.


Influence of Water Content on Retention
One of the disadvantages in using normal phase chromatography
is the changes in the chromatographic characteristics (capacity
ratio, selectivities, efficiencies) with increasing water content
of the mobile phase.
In order to observe the influence of the water content on reten-
_2
tion, plots of k1 vs. volume percent of H^O in 5 x 10 M AOT/hexane
were drawn for both columns. The surfactant concentration in the
mobile phase was chosen above the CMC range to assure the existence
of micelles.
Figures 6 and 7 show the behavior of phenol, naphthol, and 2,4-
dinitrotoluene in Ultrasil-NH^ and Ultrasphere-Si columns. A smooth
change in k1 was observed at low water concentrations. For the amino
column, only a slight change was noticed; this can be attributed to
the fact that the silica surface is more active, (vide supra)
Retention values are quite sensitive to small changes in mobile
phase with low water concentration. In actual practice, it is diffi
cult to avoid small changes in mobile phase water content, for several
23
reasons;
-The water contents of the starting (supposedly dry) solvents
that comprise the mobile phase vary somewhat.
-The water is readily picked up or lost to the atmosphere, de
pending on its humidity and water content of the solvent.
-Further changes in mobile phase water content can occur as a
result of contact with the walls of intermediate containers, the
solvent reservoir, and so on.


8
7
6
5
4
3
2
I
i 1 i i i i i i i i i
0.05 0.1 0.2 0.3 0.4 0.5 0.6 07 08 0.9 100
%H20 in 5XIO'2 Maot'n hexane
7. Effect of water content variation on retention using an Ultrasil-NH?
column. (0) naphthol, (A) phenol and (X) 2,4-dinitrotoluene


0 000.01 0.02
0.05
01
0.20
Figure 6
% H20 in 5 X lO"2 Maot in hexane
Effect of water content variation on retention using an Ultrasphere-Si col
(0) 2,4-dinitrotoluene, (A) phenol and (X) naphthol


u -/
The importance of controlling the water content at low water
concentration in order to obtain reproducibility of k' values has
r 7 po
been pointed out by many authors. 5 Szepezy, Combellas, Claude
and Rosset demonstrated in their paper^ that for silica packing, a
small increase in water content corresponds to a large decrease in
k1 values. Their k' values change up to one order of magnitude for
dibutylphthalate varying from 100 to 600 ppm of water.
The solubility of water in hexane at 20C, 1 atm, is 0.0111 g
24
water per 100 g of hexane, or 0.0073 percent V/V.
Using reversed micellar system, a maximum change of 38 percent
in k1 was observed at low water compositions (Table V). This small
change in k' values as the water content is increased can be ex
plained by the fact that reverse micelles tend to solubilize a large
amount of water in their interior. The first molecules of water are
attached to the counterion (sodium) which connects, via two hydrogen
bridges to two sulfonate molecules, forming a trimer. Additional
water molecules occupy the core of the micelle, and polar substrates
are expected to be localized in this water pool. Small changes in
the amount of water do not affect the retention, since almost all the
water in the system is restricted to the interior of the micelle.
Instead, it has been concluded that small amounts of water are a pre
requisite to the formation of closed, concentration independent sur-
25
factant aggregates. Large amounts of water cause the micelle to
swell, increase the mean aggregation number and to assume a different
shape; therefore, it will be interesting to find out the amount of
water that the micelle can hold without drastic changes in the k1
value.


3U
Table V
Percent of
Varies from
and from 0.0
in 5 x
k' Values Changes as the Water Content
0.0 to 0.25 Percent for Ultrasphere-Si
to 1.0 Percent for Ultrasil-Nl^ Columns
102 M A0T in Hexane Mobile Phase
% Change
l Change
% Change
Column
Phenol
Naphthol
2,4-Dinitrotoluene
Ultrasphere-Si
11.28
10.14
32.53
Ultrasil-NH^
24.03
19.81
38.27


CHAPTER V
CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK
Reverse micellar systems are a promising mobile phase for normal
phase HPLC, as normal micellar systems have been shown to be in re
versed phase HPLC. As the concentration of surfactant in the mobile
phase is increased, the retention times of polar compounds decrease.
Drastic reductions in k1 values for surfactant concentrations above
the critical micellar concentration range were observed. Only small
changes in capacity ratio values for polar solutes were obtained at
low water contents in the mobile phase. Due to the fact that the
amount of water in the mobile phase is critical in order to obtain
reproducible retention values in normal phase chromatography, an
addition of a small amount of water in micellar mobile phases may be
a feasible means to obtain reproducible k' values. Further studies
should be done in this area. It will be interesting to look for the
maximum amount of water than can be held by the micellar mobile phase
without an abrupt change in k' value.
Lower efficiencies were obtained for micellar mobile phases be
cause of the higher viscosity of these mobile phases, leading to
slower mass transfer. Raising the temperature or adding small amounts
3
of polar organic modifiers may be a good approach to improving effi
ciencies for reverse micellar mobile phases, and should be investi
gated.
31


The selectivity (a), or relative retention of solutes, has a
considerable effect on resolution. The value of a should be as large
as possible, other factors being equal. The use of a micellar mobile
phase and changing temperature are two options that can be studied in
the future in order to increase a, thereby improving resolution.
A similar study can be performed using a cyano column and more
polar solutes in order to clarify the retention mechanism of polar
compounds in reverse micellar systems.
Aerosol OT is an anionic surfactant. A study of different anionic
surfactants, cationic surfactants and nonionic surfactants will give
different types of aggregation systems from which the separation mecha
nism can be observed.


REFERENCES
1. R.E. Majors. Recent advances in HPLC packing and columns. J.
Chromatoqr. Sci. 18, 488-511 (1980).
2. D.W. Armstrong and S.J. Henry. Use of an aqueous micellar mo
bile phase for separation of phenols and polynuclear aromatic
hydrocarbons via HPLC. J. Liq. Chromatoqr. 3, 657-662 (1980).
3. J.G. Dorsey, M.T. DeEchegaray and J.S. Landy. Efficiency en
hancement in micellar liquid chromatography. Anal. Chem. 55,
924-928 (1983).
4. S.R. Abbot. Practical aspects of normal-phase chromatography.
J. Chromatoqr. Sci. 18, 540-550 (1980).
5. L.R. Snyder and J.J. Kirkland. "Introduction to Modern Liquid
Chromatography," 2nd Ed., John Wiley and Sons, New York, 1979,
pp. 272-277.
6. L. Szepezy, C. Combellas, M. Claude and R. Rosset. Influence
of water content of the mobile phase on chromatographic perform
ance in adsorption chromatography. J. Chromatoqr, 237, 65-78
(1982).
7. L.R. Snyder. "Principles of Adsorption Chromatography," Marcel
Dekker, Inc., New York, 1968, pp. 157-159.
8. J.J. Kirkland. "Modern Practice of Liquid Chromatography,"
John Wiley and Sons, New York, 1971, pp. 215-218.
9. R.W. Yost, L.S. Ettre and R.D. Conlon. "Practical Liquid Chromo-
tography," Perkin Elmer Corporation, New York, 1980, pp. 60-62.
10. D.W. Armstrong. Pseudophase liquid chromatography: applications
to TLC. J. Liq. Chromatogr. 3, 895-900 (1980).
11. D.W. Armstrong. Application of pseudophase liquid chromato
graphy (PLC). Amer. Lab. 13, 14-20 (1981).
12. K.A. Dill and P.J. Flory. Proc. Natl. Acad. Sci. USA 78, 676-
680 (1981).
13. M.T. DeEchegaray. Effect on temperature on the elution of non
ionic solutes in micelles containing mobile phases for reverse
phase HPLC, Master's Thesis, University of Florida, Gainesville,
1982, p. 8.
33


*/T
14. H.F. Eicke. Surfactants in nonpolar solvents aggregation and
mice!1ization. Topics in Current Chemistry 87, 1-83 (1980).
15. B. Lindman and H. Wennerstrom. Micelles amphiphilic aggrega
tion in aqueous solution. Topics in Current Chemistry 87, 85-
145 (1980).
16. J.H. Fendler. Interaction and reactions in reversed micellar
system. Acc. Chem. Res. 9, 153-161 (1976).
17. K.L. Mittal. "Micellization, Solubilization and Microemul
sions," _1, Plenum, New York, 1977, pp. 429-454.
18. J.P. Foley and J.6. Dorsey. Equations for calculation of
chromatographic figures of merit for ideal and skewed peaks.
Anal. Chem. 55, 730-737 (1983).
19. M. Tang and S.N. Deming. Interfacial tension effects of non
ionic surfactants in reversed-phase liquid chromatography.
Anal. Chem. 55, 425-428 (1983).
20. D.W. Armstrong and F. Nome. Partitioning behavior of solutes
eluted with micellar mobile phase in liquid chromatography.
Anal. Chem. 53, 1662-1666 (1981).
21. L.R. Snyder and J.J. Kirland. "Introduction to Modern Liquid
Chromatography," 2nd Ed., John Wiley and Sons, New York, 1979,
pp. 248-250.
22. Rainin Catalog, 1983, pp. 124-125.
23. L.R. Snyder and J.J. Kirland, "Introduction to Modern Liquid
Chromatography," 2nd Ed., John Wiley and Sons, New York, 1979,
pp. 374-376.
24. C. Black, G.G. Joris and H.S. Taylor. The solubility of water
in hydrocarbons. J. Chem. Phys. 16, 53 (1948).
25.H.F. Eicke and H. Chisten. Is water critical to the formation
of micelles in apolar media? Helv. Chim. Acta. 61, 2258-2263
(1978).


BIOGRAPHICAL SKETCH
Maria A. Hernandez Torres was born in Mayaguez, Puerto Rico,
on May 29th, 1958. She received her elementary and high school edu
cation at the Colegio de La Milagrosa, Mayagez, Puerto Rico. In
1980, she completed her Bachelor of Science degree in chemistry at
the University of Puerto Rico Mayaguez Campus. She is now pursuing
a Master of Science degree in analytical chemistry at the University
of Florida.
35


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 thesis for the degree
of Master of Science.
John')6. Dorsey, Chairman
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 thesis for the degree
of Master of Science.
CUU
7 p,
. Winefori^rier
lames D.
Graduate Research Professor
of Chemistry
This thesis was submitted to the Graduate Faculty of the Depart
ment of Chemistry in the College of Liberal Arts and Sciences and to
the Graduate School, and was accepted for partial fulfillment of the
requirements of the degree of Master of Science.
December 1983
Dean for Graduate Studies and
Research


Full Text
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REVERSE MICELLAR MOBILE PHASES FOR NORMAL PHASE CHROMATOGRAPHY
By
MARIA A. HERNANDEZ TORRES
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
1983

ACKNOWLEDGEMENTS
I would like to express my gratitude and appreciation to my
research director, Dr. John G. Dorsey, for his invaluable assis¬
tance, guidance and encouragement throughout the course of this
project.
Also, my special thanks are extended to Mr. John S. Landy, for
his help in the initial setting of the instrument and his continuous
assistance.
I would like to thank my colleagues in Dr. Dorsey's research
group for their friendship and daily encouragement.
I would also like to thank my family and friends for their pa¬
tience and continuous support.
Finally, I would like to mention Patty Hickerson for her prompt¬
ness and accuracy in typing the final draft of this thesis.
11

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS Ü
ABSTRACT i v
CHAPTER
I INTRODUCTION 1
II THEORY 9
III EXPERIMENTAL 12
Instrumentation 12
Reagents 12
Procedure and Calculations 13
IV RESULTS AND DISCUSSION 15
Retention Changes with Surfactant Concentration 15
Efficiency 23
Influence of Water Content on Retention 26
V CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK 31
REFERENCES 33
BIOGRAPHICAL SKETCH 35

Abstract of Thesis Presented to the
Graduate School of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Master of Science
REVERSE MICELLAR MOBILE PHASES FOR
NORMAL PHASE CHROMATOGRAPHY
By
Maria A. Hernandez Torres
December 1983
Chairman: John G. Dorsey
Department: Chemistry
Reverse micelles, formed in hexane, can be used in place of polar
modifier to control the strength and selectivity of normal phase mo¬
bile phases for liquid chromatography. The effect of surfactant con¬
centration (Aerosol OT) on retention of various solutes in a normal
phase liquid chromatographic system was investigated. Surfactant con¬
centration above the critical micelle concentration range was used to
observe the change in capacity factor, k‘, as the water content in the
mobile phase was varied. Small changes in k1 values were obtained at
low water content in the micellar mobile phase. Finally, the column
_2
efficiencies for dry hexane, 5/95 isopropanol/hexane and 5 x 10 M
AOT in hexane mobile phases were compared for Ultrasil-NH^ and Ultra¬
sphere-Si columns.
TV

CHAPTER I
INTRODUCTION
A great deal of research has been put into the improvement of
both the stationary and mobile phases of high performance liquid
1-3
chromatography (HPLC) to achieve better separations.
This study is limited to normal phase HPLC, for which the reten¬
tion of the normal phase separation increases with increasing solute
polarity and decreases with increasing mobile phase polarity. The
solute retention mechanism is dominated by interaction of polar sta¬
tionary phase sites with polar solutes.
The advantages of normal phase chromatography are summarized by
4
Abbot as follows:
1) ability to separate highly hydrophilic species which cannot
be retained on reverse phase
2) use of predominantly organic solvent-based mobile phases,
avoiding silica dissolution problems experienced in reversed
phase
3) ability to differentiate solutes based on differences in hy¬
drophilic structure rather than hydrophobic structure
4) compatibility of organic phase with molecules having either
low stability or aggregation problems in aqueous phases
5) lower viscosity of organic mobile phases, resulting in lower
operating pressures
1

6) mobile phase volatility, allowing simpler, more efficient
concentration and transfer steps in off-line fraction collection
and structural characterization
7) ability to separate isomers which is of great significance
in natural product and pharmaceutical chemistry
8) ability to obtain class separations which cannot be obtained
in reversed phase
9) greater compatibility of organic mobile phase with on-line
coupling
10) availability of wide range of organic solvents to capital¬
ize on special solvent effects
11) availability of a wide range of stationary phase selectivi-
ties, which can be utilized to obtain high separation performance
in multi-stage chromatography.
Normal phase chromatography typically encompasses adsorption
chromatography on silica and partition chromatography on cyano and
amino bonded phases.
Adsorption may be defined as the concentration of solute mole¬
cules at the interface of two immiscible phases. In liquid-solid ad¬
sorption chromatography, the mobile phase is a liquid, while the sta¬
tionary phase is a finely divided, usually porous, solid. The atoms
in the bulk of the solid are subjected to equal forces in all direc¬
tions, whereas the surface atoms experience unbalanced forces which
can attract molecules from the surrounding solution to restore the
balance.

J
In a multicomponent system, selective adsorption occurs due to
competition between the solutes and the mobile phase for the surface.
It is governed by the differences in the strengths of the adsorption
forces between the adsorbent and the adsorbates. In general, polar
compounds are more strongly adsorbed by polar solids than are non¬
polar compounds. Also, adsorption of a polar compound is enhanced in
a nonpolar medium, but reduced in a polar medium due to increased com¬
petition of the mobile phase for the surface.
The partition process of a solute occurs between a liquid mobile
phase and a liquid absorbed on, or chemically bonded onto, a porous
support. Classical liquid-liquid chromatography involves the parti¬
tion of the sample components between a liquid stationary phase and
the liquid mobile phase. The separation occurs due to differences in
the solubilities of the sample components in the two liquid phases.
Since liquids which are practically immiscible will usually still have
at least ppm solubility in each other, the mobile phase must be pre¬
saturated with the polar phase to prevent gradual column stripping and
loss of separating power. As a result, column packings have been
developed in which the stationary phase is permanently bonded to the
support by chemical bonding. Bonded phase packings are prepared by
the reaction of the surface Si-OH groups of the support particles
with various reagents (Figure 1). These siliceous supports are pre¬
pared with a variety of functional groups, ranging from very polar to
nonpolar, resulting in widely diverse selectivities. The aminoi-NH^)
and cyano(-CN) functional groups are polar in nature, and they are
commonly used with low polarity mobile phases for normal phase sepa¬
rations.

(a) SILICATE ESTERS
-Si-OH + HOR Si-OR
I Í
(b) SILICA-CARBON ARP SILICA-NITROGEN
i-NH—CHjCHjNHj
(c) SILOXANES
1 CISiRj i
Si-OH + or »• Si-O-SiRj
J ROSiRj
5
Figure 1. Reactions for preparing bonded-phase packing (from ).

It has been recognized that retention on active stationary phases
such as silica and alumina with nonpolar and moderately polar eluents
is strongly influenced by the water content of the eluent.^ ^ The
surface of the silica gel is covered with Si-OH and Si-0-Si groups
which can interact with the sample molecules. There are two kinds of
hydroxyl groups covering the silica gel surface: free (OH) and reac¬
tive (HOH) hydroxyl groups. The reactive hydroxyl groups are very
strong bonding agents which may permanently adsorb polar sample and
water molecules onto the gel, giving nonreproducible results. Since
the water content of the absorbent markedly affects relative capacity
ratio (k1) values and band migration rates, water content must be held
constant for repeatable separation.
Surfactants are amphiphatic molecules which have distinct hydro-
phobic and hydrophilic regions. Over a narrow concentration range,
defined as the critical micelle concentration (CMC), surfactants dy¬
namically associate to form large molecular aggregates. In nonpolar
solvents, these have been termed reversed or inverted micelles, since
their polar groups are concentrated in the interior of the aggregate
while their hydrophobic groups extend into and are surrounded by the
bulk apolar solvent (Figure 2). In aqueous solution, the micelle is
a compact roughly spherical body with a liquid-like hydrocarbon core.
The polar head groups are at the surface (Figure 3). Micellar sys¬
tems have been introduced in reverse phase HPLC, and an improvement
2 3 10 11
in the separations has been reported. ’ ’ ’ When using micellar
mobile phases, solutes do not partition to the bulk solvent but rather
to the discrete aggregates creating a unique separation mechanism:

b
Figure 2. Reverse micelle representation. The ionic head group (0)
points toward the center of the micelle, and the hydro¬
carbon chain (v^) surrounds the central ionic core
(from 12).

Figure 3. Dill-Flory's representation of a normal micelle. The
ionic head groups are indicated by the circles, and the
hydrocarbon chains are pointing toward the center of
the micelle (froml2).

pseudo phase liquid chromatography. Some of the advantages of the
13
micelle system in RPHPLC are:
1) many hydrophobic, amphiphilic, and even hydrophilic molecules
interact differently with the micelles, allowing for a wide range
of applications
2) low cost
3) nontoxic
4) non-UV absorbing
5) simultaneous separations of hydrophobic and hydrophilic
solutes are accomplished without aqueous organic gradients
6) the addition of solutes to the mobile phase to control ionic
strength, pH, and buffer capacity will not produce solubility
problems
7) a relatively small change in the amount of surfactant will
essentially give a different mobile phase.
Our studies are directed mainly to observe the behavior of sol¬
utes in reverse micellar systems. Solute's retentions as a function
of surfactant concentration are examined. Also, this study attempts
to find whether the water in the chromatographic system will be en¬
trapped so that the retention of the solutes in the normal phase HPLC
will not be changed. The best column efficiency is sought among all
the conditions studied.

CHAPTER II
THEORY
Due to the new findings and applications of reversed or inverted
micelles in the course of the last few years, the scientific interest
with regard to nonpolar detergent solutions has extensively in-
14
creased.
The obvious difference between micelles in polar and nonpolar
surfactant solutions is their mutual structural reversion. In non¬
polar solvents, the inverted micelle is visualized to be built up by
a polar core covered by hydrocarbon tails (vide supra). Inverted
micelles have moderate aggregation numbers which contrast the large
micellar aggregates in aqueous surfactant solutions, e.g., for 4.9 x
-4
10 M of Aerosol 0T in pentane at 25°C the mean aggregation number
(n) is 15, while for 8.2 x 10 ^ M of sodium n-dodecylsulfate (SDS)
in aqueous solution at 25°C n is 64.*
In the formation of a micelle in apolar media, there seems to be
a general agreement with regard to the existence of premicellar ag¬
gregates. The monomer ? n-mer type association is unlikely to rep¬
resent the behavior of surfactants.The most universally used
treatment, the multiple equilibrium model, assumes the stepwise forma-
^1
tion of aggregates in an indefinite association: monomer â–  dimer
k2 k3 kn
trimer ■ ••• ■■■ - ■ n-mer, where the distribution of the
different aggregates depends on the stoichiometric surfactant
9

10
concentration. The higher the concentration of the surfactant, the
larger the aggregates. Under this treatment, the CMC can be defined
as the concentration of aggregates within a narrow distribution cen¬
tering around the average micellar size. On the other hand, it can
be assumed that there is no unique concentration at which the exist¬
ence of micelles becomes detectable; therefore, it is unjustified to
speak of one concentration, or even a narrow range of concentration,
describing the CMC of surfactants in hydrocarbon solutions.^7
The driving force behind the process of aggregation and micelli-
zation of surfactants in hydrocarbon media is essentially due to
dipole-dipole interactions between polar heads of the amphiphilic
molecules.
One important property of the micellar system is the detergency
or the ability of surfactant molecules to take up (solubilize) polar
material, i.e., water in the polar core of the inverted micelles.
There are a large number of parameters which influence and even
determine the extent of the solubilization of the polar material.
These parameters include the structure of the surfactant, the physico¬
chemical property of the sol ubi 1izate, temperature, pressure, electric
fields and cosurfactants.
Considerable amounts of water can be solubilized by reverse mi¬
celles. This surfactant solubilized water is often called waterpool.
Initial water molecules are bound to the polar head groups, and their
motion is restricted. Additional water molecules occupy the core of
the micelle, and their properties resemble bulk water. A rapid ex¬
change takes place between these two kinds of water molecules.

The effective polarity, acidity and microscopic viscosity of the
water pools are expected to be substantially different from those in
bulk water.
Polar substrates are expected to be localized in water pools;
therefore, controlled amounts of surfactant-entrapped water in non¬
polar solvents provide a unique medium for interactions of polar sub¬
strate.

CHAPTER III
EXPERIMENTAL
Instrumentation
A Spectra-Physics 8700 (Santa Clara, California) liquid chromato¬
graph equipped with a model 7125 Rheodyne sample injection valve with
a 10 microliter loop, a model 153 Beckman UV detector (254 nm) with
an 8 microliter flow cell, and a model 1210 Linear strip chart re¬
corder were used. The columns used were: an Altex (Berkeley, CA)
Ultrasphere-Si (15 cm x 4.6 mm ID) 5 ym particle size and an Altex
Ultrasil-NH^ (25 cm x 4.6 mm ID) 10 ym particle size. Both columns
were thermostatted at 30°C ±0.2 using a water jacket and a Haake D1
water circulator.
Reagents
HPLC grade n-hexane (Fisher Scientific) was used as the solvent;
it was dried using Linde Molecular Sieves (Union Carbide) type 3A.
Reagent grade 2-propanol (Fisher Scientific) was used as organic
modifier and to wash the columns.
Reagent grade Aerosol 0T (dioctyl sodium sulfosuccinate, Fisher
Scientific) was used without further purification; it was dissolved in
hexane and then filtered with a Rainin solvent filtration apparatus
through a 0.45 ym membrane filter.
Water used in the mobile phase was purified using a Barnstead
Nanopure System (Symbron Corporation).
12

The solutes were used without further purification, and all the
solute solutions were prepared in n-hexane. The solutes were: 2,4-
dinitrotoluene, naphthol (Matheson Coleman & Bell) and phenol
(Mal 1inckrodt).
Reagent grade n-pentene (J.T. Baker) was used as an unretained
compound for the column's void volume calculation.
Procedure and Calculations
The appropriate weight of surfactant (Aerosol OT) was weighed
-4
and dissolved in hexane. The concentration range was from 1 x 10 M
to 1 x 10 1 M with alternate increments of 5x and 2x, respectively.
The columns were thermostated at 30°C. A silica gel precolumn was
used before the injector to prevent the silica in the column from
being dissolved by the surfactant. The columns were washed with 2-
propanol, and rinsed and stored in n-hexane.
A stock surfactant solution was prepared of 0.50M,and aliquots
_2
of 50 ml were taken and diluted with n-hexane to 5.0 x 10 M for the
study of the influence of mobile phase water content on the retention
_2
time of the solutes. In each of the six 5.0 x 10 M Aerosol 0T solu¬
tions, the volume percentage of water was varied (0%, 0.01%, 0.02%,
0.05%, 0.10%, 0.25%).
The capacity factor, k1, was used to compare the retention data
of the solutes and was calculated using the following equation:
V - V
k' = -V-5- (1)
o
where Vr = the retention volume of a given solute and is calculated
as the product of flow rate times retention time (ml), and VQ = the

column's void volume (ml). n-Pentene, assumed to be an unretained
solute, was used to calculate the void volume of the column.
The efficiencies of the columns were compared using the reduced
plate height, h, given by:
h = N^dp ^
where L = the column length, dp = the particle diameter, and N = the
18
plate number. The plate number, N, was calculated as follows:
N
41 •7
w0.1
B/A + 1.25
(3)
where t^ = the retention time of the compound (cm), Wq ^ = the peak
width at 10% of the peak height (cm), and B/A = the asymmetry factor.

CHAPTER IV
RESULTS AND DISCUSSION
Retention Changes with Surfactant Concentration
Figures 4 and 5 show the behavior of phenol and naphthol in
hexane/Aerosol OT mobile phases. These graphs show two linear compo¬
nents, one above and one below the CMC. Since the formation of mi¬
celles in nonpolar solvents occurs in sequential steps, it is diffi¬
cult to assign the CMC to one point. The intersection of the two
_3
linear components was found to be 3.5 x 10 MAOT in hexane. There-
-3 -3
fore, the concentration range from 1.0 x 10 M to 5.0 x 10 M A0T
in hexane was assigned as the CMC, in agreement with the formation
of reverse micelles as the multiple equilibrium model postulates. In
both columns, Ultrasphere-Si and Ultrasil-NH^, the first large change
in k‘ was observed in the same range of concentration, the CMC range.
In other words, the same value of CMC range for A0T in hexane was ob¬
tained for the two different columns and two different solutes. Table
I shows mean aggregation numbers and critical micelle concentrations
of Aerosol 0T in different hydrocarbon solvents.
At concentrations above the CMC range, the slope of the four
curves are slightly negative; with the Ultrasphere-Si column having a
greater negative slope. This can be attributed to the fact that the
silica surface has silanol groups that interact with polar compounds
15

A V
Figure 4. Effect of surfactant concentration on retention
using Ultrasphere-Si column. (0) naphthol, (I)
phenol.

A /
Figure 5. Effect of surfactant concentration on retention using
Ultrasil-NH0 column. (!) naphthol, (0) phenol.

lo
Table I
Mean Aggregation Numbers (n) and CMCs of
Aerosol OT in Different Hydrocarbon Solvents (from i4)
Solvent
Temperature
(°C)
Technique3
CMC
(M)
n
Cyclohexane
37
VPO
3.9
x 10"4
17
28 ± 4
LS
1.3
x 10'3
56
Benzene
37
VPO
3.5
x 10"4
13
RT
PA
2.2
x 10"3
-
28 ± 4
LS
2.7
CO
1
o
pH
X
23.6
20
TCNQ
2.0
x 10-3
-
cci4
25
VPO
1.6
«vt-
1
o
pH
X
17
37
VPO
4.0
x 10"4
17
20
TCNQ
6.0
X
I—*
o
1
-
Pentane
25
LS
4.9
<-
1
o
I—1
X
15
a VPO: vapor pressure osmometry
LS: light scattering
PA: positron annihilation
TCNQ: solubilization of 7,7,8,8-tetracyanoquinodimethane

i 'i
in a very strong way. Also, the polar heads of the surfactant can
interact with these active sites, and retention of polar compounds
will decrease as the number of active sites available is reduced by
the presence of free surfactant. In the Ultrasil-NH2 column, the
interaction of the polar surfactant head groups with the amino sta¬
tionary phase groups is less strong; therefore, the concentration of
free surfactant in the mobile phase for this column affects the re¬
tention of polar compounds to a lesser extent. Recently, Deming and
Tang proposed that addition of surfactant to the eluent reduces the
interfacial tension and thus decreases the retention time of the in-
19
jected samples.
Looking at Table II, one can observe a big change in k' value
-4
going from dry hexane to 5 x 10 M AOT in hexane mobile phases. Two
possible reasons for this change are (1) before the CMC range, the
surfactant is in a monomeric form, and the polar heads compete for
active sites in the stationary phase, and (2) the formation of small
aggregates at very low concentration of surfactant that can interact
with solutes. For both points, there is a decrease in the number of
active sites available at the stationary phase; therefore, a decrease
in the retention time of solutes is observed.
Above the CMC range, the k‘ of naphthol and phenol decrease very
rapidly as the formation of aggregates increases. The reason for this
behavior is that in a reverse micellar system a polar solute will par¬
tition to the polar core of the micelle spending less time interacting
with the stationary phase. This separation mechanism with reverse mi-
20
celles is similar to the one discussed by Armstrong and Nome for

U.\J
Table II
k' Value for Phenol and Naphthol in Dry Hexane and
5x10"^M AOT in Hexane for Ultrasphere-Si and Ultrasil-NH^ Columns
Column
Mobile Phase
k‘ phenol
k' naphthol
Ultrasphere-Si
dry hexane
72.53
47.50
5 x 10'4 M AOT in
hexane
27.89
20.30
Ultrasil-NH^
dry hexane
47.52
53.75
5 x 10'4 M AOT in
hexane
29.44
40.87

C. 1
micellar systems in reversed phase HPLC, or pseudo phase liquid
chromatography. In normal phase chromatography, the solvent strength
is varied in order to optimize k'. Increasing the strength of the
mobile phase by increasing the solvent polarity, a decrease in sample
k* value is observed. Preferred solvents used in normal phase to
increase the strength of the mobile phase are acetonitrile, ethanol,
ethylacetate, CHCl^, tetrahydrofuran and isopropyl ether.
Comparing micellar and organic mobile phases for normal phase
HPLC, one can say that (1) Organic mobile phases have somewhat lower
viscosity than micellar mobile phases, resulting in lower operating
pressures. For example, at a flow rate of 1 ml/min., 30°C, the pres¬
sure for 5/95 isopropanol/hexane mobile phase was 302 psig vs. 350
-4
psig for 5 x 10 M AOT in hexane mobile phase for Ultrasphere-Si
column. Table III shows the viscosity values for most common solvents
used in normal phase HPLC. (2) Surfactants are cheaper than the HPLC
grade organic solvents; an approximate cost per gallon of organic
solvent is thirty dollars vs. twenty dollars for 500 g of Aerosol 0T
(Table III). (3) Surfactants are non-toxic vs. some of the most
commonly used organic modifiers, for example tetrahydrofuran. (4)
Micellar mobile phases offer a different selectivity for separations
of compounds, and (5) For both mobile phases, a UV detector can be
used. The molar absorptivity coefficient for Aerosol 0T was calcu¬
lated to be 0.9986 L/mole cm at a wavelength equal to 254 nanometers
(Table III).

Lm i—
Table III
Viscosity, UV Cutoff and Price per Gallon for ??
Most Commonly Used Solvents in Normal Phase HPLC Frorri 5
UV
Solvent
Cutoff
(nm)
Viscosity
(cP, 25°C)
Price per
Gallon
Hexane
195
0.30
28.65
i-Propyl ether
220
0.38
27.85
Tetrahydrofuran
212
0.46
47.15
i-Propanol
205
1.9
24.80
Chloroform
245
0.53
29.85
Acetonitrile
190
0.34
55.85
Ethanol
210
1.08
26.75
Ethyl acetate
256
0.43
26.05

¿i
Efficiency
In looking for an optimum separation, one which gives adequate
sample resolution with a minimum of time and effort, one of the para¬
meters to be considered is column efficiency. The number of theo¬
retical plates, N, was calculated in order to compare the performance
of the system under the different experimental conditions studied.
Foley and Dorsey's equation was used to calculate the number of theo-
18
retical plates. (vide supra) This equation was preferred over the
most commonly used,
N = 5.54 (tr/w0>5)2 (4)
because the former corrects for the asymmetry of skewed peaks giving
more accurate values for N. The reduced plate height was calculated
by
h = H/dp (5)
where H is the plate height, and dp is the particle diameter.
Table IV shows the theoretical plate numbers, plate heights, re-
_2
duced plate height and asymmetry ratios for phenol in 5 x 10 M AOT
in hexane, 5/95 isopropanol/hexane and dry hexane mobile phases for
Ultrasphere-Si and Ultrasil-NH^ columns.
Better efficiencies were observed for 5/95 isopropanol/hexane
mobile phase for both columns. The reason for smaller values of N in
_2
5 x 10 M AOT is the higher viscosity of the micellar solution, hav¬
ing smaller diffusion coefficients, therefore, slower mass transfer.
This slow mass transfer results from the slower rate of movement of a
polar sample between the stationary phase and the interior of the

¿4
Table IV
Theoretical Plate Numbers (N), Plate Heights (H, mm), Reduced
Plate Heights (h) and Asymmetry Ratios (B/A) for Phenol in
5 x 10~2 M AOT in Hexane, 5/95 Isopropanol/Hexane and Dry
Hexane Mobile Phases for Ultrasphere-Si and Ultrasil-NH^ Columns
Ultrasphere
-Si
5
x 10"2 M AOT
5/95
in Hexane
Isopropanol/Hexane
Dry Hexane
N
3301
8174
3987
H (mm)
0.0454
0.0184
0.0376
h
9.08
3.68
7.40
B/A
1.50
1.14
2.02
Ultrasil
-NH
2
N
902
2409
-
H (mm)
0.277
0.104
-
h
27.7
10.4
-
B/A
2.18
1.22
-

micelle. Also, higher viscosity in the interior of the micelle in
comparison of the bulk solution contributes to slow the adsorption-
desorption interchange.
Greater values for asymmetry ratio in micellar system were ob¬
tained in accordance with the N values. Optimization of the micellar
system has to be done in order to be a feasible medium for separa¬
tion.
For Ultrasphere-Si columns, higher values of N were calculated
for the three systems studied. This can be explained in terms of a
smaller particle diameter, 5 pm vs. 10 pm for Ultrasil-NH^. It seems
that the fact of smaller particle diameter dominates over the longer
column for Ultrasil-NH^ and stronger active sites in Ultrasphere-Si.
Lower efficiency was observed for phenol in Ultrasphere-Si in
dry hexane mobile phase. This was expected since it is generally
accepted that with a dry mobile phase, at low water contents, much
lower efficiencies will be obtained because of the strong active
sites of the adsorbent surface, leading to slow adsorption-desorp¬
tion kinetics.^ The theoretical plate number for phenol in Ultrasil-
NH^ could not be calculated because of the long retention (more than
three hours).
In Szepezy, Combellas, Claude and Rosset's paper,^ very low and
rapidly changing values of efficiency at low water contents were
pointed out, indicating the necessity for selection and control of
the water content in the mobile phase. For this reason, the next part
of this experiment was performed.

Influence of Water Content on Retention
One of the disadvantages in using normal phase chromatography
is the changes in the chromatographic characteristics (capacity
ratio, selectivities, efficiencies) with increasing water content
of the mobile phase.
In order to observe the influence of the water content on reten-
_2
tion, plots of k' vs. volume percent of H^O in 5 x 10 M AOT/hexane
were drawn for both columns. The surfactant concentration in the
mobile phase was chosen above the CMC range to assure the existence
of micelles.
Figures 6 and 7 show the behavior of phenol, naphthol, and 2,4-
dinitrotoluene in Ultrasil-NH^ and Ultrasphere-Si columns. A smooth
change in k1 was observed at low water concentrations. For the amino
column, only a slight change was noticed; this can be attributed to
the fact that the silica surface is more active, (vide supra)
Retention values are quite sensitive to small changes in mobile
phase with low water concentration. In actual practice, it is diffi¬
cult to avoid small changes in mobile phase water content, for several
23
reasons;
-The water contents of the starting (supposedly dry) solvents
that comprise the mobile phase vary somewhat.
-The water is readily picked up or lost to the atmosphere, de¬
pending on its humidity and water content of the solvent.
-Further changes in mobile phase water content can occur as a
result of contact with the walls of intermediate containers, the
solvent reservoir, and so on.

%H20 in 5XIO'2 Maot'n hexane
Figure 7. Effect of water content variation on retention using an Ultrasil-NH?
column. (0) naphthol, (A) phenol and (X) 2,4-dinitrotoluene

5.0
1.0
i i
0 00001 0.02
0.05
01
0.20
025
Figure 6.
% H20 in 5 X I0"2 Maot in hexane
Effect of water content variation on retention using an Ultrasphere-Si column.
(0) 2,4-dinitrotoluene, (A) phenol and (X) naphthol

u -/
The importance of controlling the water content at low water
concentration in order to obtain reproducibility of k' values has
r 7 po
been pointed out by many authors. 5 Szepezy, Combellas, Claude
and Rosset demonstrated in their paper^ that for silica packing, a
small increase in water content corresponds to a large decrease in
k1 values. Their k' values change up to one order of magnitude for
dibutylphthalate varying from 100 to 600 ppm of water.
The solubility of water in hexane at 20°C, 1 atm, is 0.0111 g
24
water per 100 g of hexane, or 0.0073 percent V/V.
Using reversed micellar system, a maximum change of 38 percent
in k1 was observed at low water compositions (Table V). This small
change in k' values as the water content is increased can be ex¬
plained by the fact that reverse micelles tend to solubilize a large
amount of water in their interior. The first molecules of water are
attached to the counterion (sodium) which connects, via two hydrogen
bridges to two sulfonate molecules, forming a trimer. Additional
water molecules occupy the core of the micelle, and polar substrates
are expected to be localized in this water pool. Small changes in
the amount of water do not affect the retention, since almost all the
water in the system is restricted to the interior of the micelle.
Instead, it has been concluded that small amounts of water are a pre¬
requisite to the formation of closed, concentration independent sur-
25
factant aggregates. Large amounts of water cause the micelle to
swell, increase the mean aggregation number and to assume a different
shape; therefore, it will be interesting to find out the amount of
water that the micelle can hold without drastic changes in the k1
value.

3U
Table V
Percent of
Varies from
and from 0.0
in 5 x
k' Values Changes as the Water Content
0.0 to 0.25 Percent for Ultrasphere-Si
to 1.0 Percent for Ultrasil-Nl^ Columns
10“2 M A0T in Hexane Mobile Phase
% Change
l Change
% Change
Column
Phenol
Naphthol
2,4-Dinitrotoluene
Ultrasphere-Si
11.28
10.14
32.53
Ultrasil-NH^
24.03
19.81
38.27

CHAPTER V
CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK
Reverse micellar systems are a promising mobile phase for normal
phase HPLC, as normal micellar systems have been shown to be in re¬
versed phase HPLC. As the concentration of surfactant in the mobile
phase is increased, the retention times of polar compounds decrease.
Drastic reductions in k1 values for surfactant concentrations above
the critical micellar concentration range were observed. Only small
changes in capacity ratio values for polar solutes were obtained at
low water contents in the mobile phase. Due to the fact that the
amount of water in the mobile phase is critical in order to obtain
reproducible retention values in normal phase chromatography, an
addition of a small amount of water in micellar mobile phases may be
a feasible means to obtain reproducible k' values. Further studies
should be done in this area. It will be interesting to look for the
maximum amount of water than can be held by the micellar mobile phase
without an abrupt change in k' value.
Lower efficiencies were obtained for micellar mobile phases be¬
cause of the higher viscosity of these mobile phases, leading to
slower mass transfer. Raising the temperature or adding small amounts
3
of polar organic modifiers may be a good approach to improving effi¬
ciencies for reverse micellar mobile phases, and should be investi¬
gated.
31

The selectivity (a), or relative retention of solutes, has a
considerable effect on resolution. The value of a should be as large
as possible, other factors being equal. The use of a micellar mobile
phase and changing temperature are two options that can be studied in
the future in order to increase a, thereby improving resolution.
A similar study can be performed using a cyano column and more
polar solutes in order to clarify the retention mechanism of polar
compounds in reverse micellar systems.
Aerosol OT is an anionic surfactant. A study of different anionic
surfactants, cationic surfactants and nonionic surfactants will give
different types of aggregation systems from which the separation mecha¬
nism can be observed.

REFERENCES
1. R.E. Majors. Recent advances in HPLC packing and columns. J.
Chromatoqr. Sci. 18, 488-511 (1980).
2. D.W. Armstrong and S.J. Henry. Use of an aqueous micellar mo¬
bile phase for separation of phenols and polynuclear aromatic
hydrocarbons via HPLC. J. Liq. Chromatoqr. 3, 657-662 (1980).
3. J.G. Dorsey, M.T. DeEchegaray and J.S. Landy. Efficiency en¬
hancement in micellar liquid chromatography. Anal. Chem. 55,
924-928 (1983).
4. S.R. Abbot. Practical aspects of normal-phase chromatography.
J. Chromatoqr. Sci. 18, 540-550 (1980).
5. L.R. Snyder and J.J. Kirkland. "Introduction to Modern Liquid
Chromatography," 2nd Ed., John Wiley and Sons, New York, 1979,
pp. 272-277.
6. L. Szepezy, C. Combellas, M. Claude and R. Rosset. Influence
of water content of the mobile phase on chromatographic perform¬
ance in adsorption chromatography. J. Chromatoqr, 237, 65-78
(1982).
7. L.R. Snyder. "Principles of Adsorption Chromatography," Marcel
Dekker, Inc., New York, 1968, pp. 157-159.
8. J.J. Kirkland. "Modern Practice of Liquid Chromatography,"
John Wiley and Sons, New York, 1971, pp. 215-218.
9. R.W. Yost, L.S. Ettre and R.D. Conlon. "Practical Liquid Chromo-
tography," Perkin Elmer Corporation, New York, 1980, pp. 60-62.
10. D.W. Armstrong. Pseudophase liquid chromatography: applications
to TLC. J. Liq. Chromatogr. 3, 895-900 (1980).
11. D.W. Armstrong. Application of pseudophase liquid chromato¬
graphy (PLC). Amer. Lab. 13, 14-20 (1981).
12. K.A. Dill and P.J. Flory. Proc. Natl. Acad. Sci. USA 78, 676-
680 (1981).
13. M.T. DeEchegaray. Effect on temperature on the elution of non¬
ionic solutes in micelles containing mobile phases for reverse
phase HPLC, Master's Thesis, University of Florida, Gainesville,
1982, p. 8.
33

*/T
14. H.F. Eicke. Surfactants in nonpolar solvents aggregation and
micellization. Topics in Current Chemistry 87, 1-83 (1980).
15. B. Lindman and H. Wennerstrom. Micelles amphiphilic aggrega¬
tion in aqueous solution. Topics in Current Chemistry 87, 85-
145 (1980).
16. J.H. Fendler. Interaction and reactions in reversed micellar
system. Acc. Chem. Res. 9, 153-161 (1976).
17. K.L. Mittal. "Micellization, Solubilization and Microemul¬
sions," _1, Plenum, New York, 1977, pp. 429-454.
18. J.P. Foley and J.6. Dorsey. Equations for calculation of
chromatographic figures of merit for ideal and skewed peaks.
Anal. Chem. 55, 730-737 (1983).
19. M. Tang and S.N. Deming. Interfacial tension effects of non¬
ionic surfactants in reversed-phase liquid chromatography.
Anal. Chem. 55, 425-428 (1983).
20. D.W. Armstrong and F. Nome. Partitioning behavior of solutes
eluted with micellar mobile phase in liquid chromatography.
Anal. Chem. 53, 1662-1666 (1981).
21. L.R. Snyder and J.J. Kirland. "Introduction to Modern Liquid
Chromatography," 2nd Ed., John Wiley and Sons, New York, 1979,
pp. 248-250.
22. Rainin Catalog, 1983, pp. 124-125.
23. L.R. Snyder and J.J. Kirland, "Introduction to Modern Liquid
Chromatography," 2nd Ed., John Wiley and Sons, New York, 1979,
pp. 374-376.
24. C. Black, G.G. Joris and H.S. Taylor. The solubility of water
in hydrocarbons. J. Chem, Phys. 16, 53 (1948).
25.H.F. Eicke and H. Chisten. Is water critical to the formation
of micelles in apolar media? Helv. Chim. Acta. 61, 2258-2263
(1978).

BIOGRAPHICAL SKETCH
Maria A. Hernandez Torres was born in Mayaguez, Puerto Rico,
on May 29th, 1958. She received her elementary and high school edu¬
cation at the Colegio de La Milagrosa, Mayagüez, Puerto Rico. In
1980, she completed her Bachelor of Science degree in chemistry at
the University of Puerto Rico Mayaguez Campus. She is now pursuing
a Master of Science degree in analytical chemistry at the University
of Florida.
35

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 thesis for the degree
of Master of Science.
John'sG. Dorsey, Chairman
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 thesis for the degree
of Master of Science.
This thesis was submitted to the Graduate Faculty of the Depart¬
ment of Chemistry in the College of Liberal Arts and Sciences and to
the Graduate School, and was accepted for partial fulfillment of the
requirements of the degree of Master of Science.
December 1983
Dean for Graduate Studies and
Research

UNIVERSITY OF FLORIDA
3 1262 08556 7534



UNIVERSITY OF FLORIDA
3 1262 08556 7534



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