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Gradient elution, improved separations and analytical figures of merit in high performance liquid chromatography using electrochemical detection

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Title:
Gradient elution, improved separations and analytical figures of merit in high performance liquid chromatography using electrochemical detection
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Hadjmohammadi, Mohammad Reza, 1947-
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English
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xiii, 112 leaves : ill. ; 28 cm.

Subjects

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Chromatography ( jstor )
Electric current ( jstor )
Electrodes ( jstor )
Electrolytes ( jstor )
Elution ( jstor )
Glassy carbon ( jstor )
Solutes ( jstor )
Solvents ( jstor )
Surfactants ( jstor )
Voltammetry ( jstor )
Electrochemical analysis ( lcsh )
Liquid chromatography ( lcsh )
Voltammetry ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1983.
Bibliography:
Includes bibliographical references (leaves 107-111).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Mohammad Reza Hadjmohammadi.

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University of Florida
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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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GRADIENT ELUTION, IMPROVED SEPARATIONS
AND ANALYTICAL FIGURES OF MERIT IN
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
USING ELECTROCHEMICAL DETECTION







BY

MOHAMMAD REZA HADJMOHAMMADI


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

UNIVERSITY OF FLORIDA


1983































To my parents, brother and sisters














ACKNOWLEDGEMENTS


I would like to express my deepest gratitude to

Dr. John G. Dorsey for his acceptance, guidance, advice,

patience, concern and support throughout my work.

My sincere thanks are also due to Dr. J. D. Winefordner,

Dr. R. A. Yost, Dr. A. Brajter-Toth, Dr. C. M. Riley,

Dr. R. G. Bates, and Dr. J. L. Ward for their encouragement,

advice and support. I would also like to thank my colleagues

and friends in Dr. Dorsey's research group, all of whom have

contributed much to my progress and have made my course of

study pleasant and fruitful.

Acknowledgement is also due to the donors of the

Petroleum Research Fund, administrated by the American Chemical

Society, for support of this research and to Nelson H. C. Cooke,

Altex Scientific, for a gift of columns.

Sincere thanks are also due to Laura Griggs for her

patience, helpful hints, and expert typing.

Finally, mere thanks are not enough to my parents,

brother, and sisters, whose sacrifices for me can never be

repaid.


iii














TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS................. ....... ... ..... .... 111

LIST OF TABLES......................................... vi

LIST OF FIGURES. ....... ............................... vii

KEY TO ABBREVIATIONS... .... ................... ....... ix

ABSTRACT.... ........................................... xi

CHAPTER

ONE INTRODUCTION................................ 1

TWO THEORY AND BACKGROUND....................... 10
Basic Parameters in Chromatography..... 10
Capacity Factor (k')............... 10
Peak Resolution (Rs) .............. 11
Efficiency (E) .................... 11
Control of Separation in Liquid
Chromatography.................. 12
Gradient Elution (GE) ................. 13
Limit of Detection..................... 14
Principles of Electrochemical Detection 15
Ion-pair Chromatography............... 16
Micellar Chromatography................ 18

THREE EXPERIMENTAL................................ 21
Cyclic Voltammetry Systems ............. 21
Liquid Chromatography System........... 21
Electrochemical Detector............... 22
Pretreatment of Glassy Carbon Electrode 24
Reagents..................... ........... 26







Page


FOUR SIGNAL CHANGE AND BASELINE SHIFT USING
ELECTROCHEMICAL METHODS..................... 27
Change of Residual Current with DC, DP
and NP Voltammetry in Isocratic Elu-
tion HPLC ............................ 29
Effect of Background Electrolytes and
Gradient Elution on Baseline Shift
Using Amperometric Detection......... 37
Effect of the Electrode Material on
Baseline Shift Using Amperometric and
NRDP Voltammetry Methods ............. 43

FIVE A COMPARISON OF MICELLAR AND HYDROORGANIC
MOBILE PHASES USING AMPEROMETRIC DETECTOR... 46
Hydrodynamic Voltammogram in Micellar
and Hydroorganic Mobile Phases........ 47
Analytical Figures of Merit Comparison
between Micellar and Hydroorganic
Mobile Phases......................... 56
Gradient Elution and Selectivity in
Micellar Mobile Phase................ 69

SIX RAPID SEPARATION AND DETERMINATION OF THYRO-
MIMETIC IODOAMINO ACIDS BY GRADIENT ELUTION
REVERSE PHASE LIQUID CHROMATOGRAPHY WITH
ELECTROCHEMICAL DETECTION................... 85
Standard Solutions..................... 87
Preparation of T4 Tablet Solution and
Injectable T4 Sample................. 87
Gradient Elution LC/EC................. 87
Isocratic Separations.................. 91
Assay of T4 Preparations............... 98

SEVEN CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK. 103
Conclusions............................. 103
Suggestions for Future Work............ 104

REFERENCES... ...................... ................... 107
BIOGRAPHICAL SKETCH................................... 112














LIST OF TABLES


Page

1 Residual Current Change, AI, and Decay Current,
decay, in DC, DP and NP Voltammetry in Isocratic
Elution.. ....................................... 35

2 Normalized Baseline Shift (nA/mm2) during
Gradient Elution with No Sample Injection........ 44

3 Analytical Figures of Merit for Phenol............ 57

4 Analytical Figures of Merit for B-6 Vitamins..... 59

5 Analytical Figures of Merit for Polyaromatic
Hydrocarbons .................................... 61

6 Analytical Figures of Merit for T2, T3 and T4.... 100














LIST OF FIGURES


Page

1 Electrochemical Cell with Glassy Carbon Electrode 23

2 Residual Current Change and Decay Current with 5%
CH3CN............................................ 30

3 Residual Current Change and Decay Current with
50% CH3CN... .................................... 31

4 Residual Current Change and Decay Current with
95% CH3CN......................................... 32

5 Residual Current Change by NP Voltammetry........ 33

6 Specific Conductance vs. Percentage of Solvent B. 39

7 Baseline Shift during Gradient Elution with No
Sample Injection, Using an Amperometric Detector. 42

8 Chemical Structures of Compounds Used in Chapter
Five............................................. 48

9 Hydrodynamic Voltammogram for Phenol............. 50

10 Hydrodynamic Voltammogram for B-6 Vitamins........ 52

11 Hydrodynamic Voltammogram for Polyaromatic Hydro-
carbons.. ....................................... 54

12 Analytical Curves for Phenol..................... 63

13 Analytical Curves for B-6 Vitamins............... 65

14 Analytical Curves for Polyaromatic Hydrocarbons.. 67

15 Gradient Micellar Chromatogram for Separation of
Vitamin B-6...................................... 71

16 Gradient Micellar Chromatogram for Separation of
Phenolic Compounds...... ......................... 73

17 Gradient Micellar Chromatogram for Separation of
Phenolic Compounds and Gradient with No Injection 75

vii







Page

18 Effect of SDS Concentration on k'............... 77

19 Isocratic Micellar Chromatogram for Separation of
Vitamin B-6 ................... .................. 80

20 Isocratic Micellar Chromatogram for Separation of
Vitamin B-6..................... .................... 82

21 Isocratic Micellar Chromatogram for Separation of
Vitamin B-6...................................... 84

22 Baseline during Gradient Program with Blank
Injection.. ..................................... 90

23 Separation of Seven Thyromimetic Iodoamino Acids. 93

24 Thyromimetic lodoamino Acids Used in This Study.. 94

25 Cyclic Voltammogram.............. ............. 95

26 Hydrodynamic Voltammogram for T2, T3 and T4...... 97

27 Analytical Curves for T2, T and'T ....99
S............
28 Isocratic Separation of T2, T3 and T 1............ 01


viii













KEY TO ABBREVIATIONS


BPC Bonded-phase chromatography

CMC Critical micelle concentration

CV Cyclic voltammetry

DC Direct current

DP Differential pulse

EC Electrochemical detection or electrochemical
detector

GC Gas chromatography

GCE Glassy carbon electrode

GE Gradient elution

HDV Hydrodynamic voltammogram

HPLC High performance liquid chromatography

IP Ion-pair

LC Liquid chromatography

LC/EC Liquid chromatography with electrochemical
detection

LC/MS Liquid chromatography mass spectrometry

LDR Linear dynamic range

LLC Liquid-liquid chromatography

LOD Limit of detection

MSRTP Micelle-stabilized room-temperature phosphorescence

NMR Nuclear magnetic resonance

NP Normal pulse

NRDP Non-ramping differential pulse







PAH

PAR

RP

RP-HPLC


RP-LC

RP-LC/EC


SDS

WE


Polyaromatic hydrocarbon

Princeton applied research

Reversed-phase or reverse-phase

Reversed-phase high performance liquid chromatog-
raphy

Reversed-phase liquid chromatography

Reversed-phase liquid chromatography with electro-
chemical detection

Sodium dodecyl sulfate

Working electrode













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


GRADIENT ELUTION, IMPROVED SEPARATIONS
AND ANALYTICAL FIGURES OF MERIT IN
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
USING ELECTROCHEMICAL DETECTION

By

Mohammad Reza Hadjmohammadi

December 1983


Chairman: Dr. John G. Dorsey
Major Department: Chemistry



The goal of this work was an improvement and better

understanding of electroanalytical methods used as liquid

chromatographic detection techniques. Particularly, methods

which would allow the use of chromatographic gradient elu-

tion with electrochemical detectors were investigated. Both

amperometric and pulse techniques were investigated with

traditional hydroorganic mobile phases. The use of gradient

elution with micellar mobile phases was shown to allow com-

patibility with electrochemical detectors. A further com-

parison of analytical figures of merit was made between

hydroorganic and micellar mobile phases using electro:1emical

detection

A comparison of baseline shift during gradient elution

with amperometric and nonramping differential pulse (NRDP)







methods was performed using both glassy carbon and gold

electrodes. The composition of the mobile phase is virtu-

ally constant during one pulse with gradient elution, but

high residual current changes preclude routine use of this

technique. A more stable baseline was achieved with an

amperometric detector and a glassy carbon electrode, than

it was otherwise with NRDP method. A higher concentration

of phosphoric acid in the organic modifier as opposed to

equal concentrations in both modifier and water produced a

more stable baseline during gradient elution using a glassy

carbon electrode and an amperometric detector.

The hydrodynamic voltammograms and analytical figures

of merit for phenol, two B-6 vitamins, and polyaromatic

hydrocarbons were compared in micellar and hydroorganic

mobile phases. The limit of detection in both mobile phases

was comparable, whereas the upper limit of linear dynamic

range was greater in micellar mobile phases.

Gradient chromatograms for separation of phenolic com-

pounds and B-6 vitamins with an anionic surfactant and

phenolic compounds with a nonionic surfactant using ampero-

metric detection are shown. Selectivity of micellar mobile

phases toward B-6 vitamins changes with surfactant concen-

trations.

A rapid separation of thyroxine and related thyroid

hormones is shown using gradient elution and electrochemical

detection. A five minute isocratic separation of thyroxine

and three related hormones is also reported. Limits of


xii







detection are in the sub-nanogram range with an upper limit

of linear dynamic range of 500 to 1000 nanograms for these

compounds. Analysis of levothyroxine sodium tablets and

injectable intravenous samples is described.


xiii













CHAPTER ONE

INTRODUCTION



Chromatographic methods can be classified according to

a number of schemes. The major one is based on the state of

the mobile phase. If the mobile phase is a gas, the method

is called gas chromatography (GC). If the mobile phase is a

liquid, the method is named liquid chromatography (LC).

Liquid and gas chromatography are each divided according to

the nature of the stationary phase. When the solid station-

ary phase has adsorption properties, the process is called

adsorption chromatography, and when the stationary phase is

a liquid supported by an inert matrix, the process is called

partition chromatography.

Special classifications have also been introduced. For

example, chromatography can be classified according to

whether the stationary phase is present as a thin-layer, a

paper, or a column. Chromatography can alternatively be

classified with respect to the flow of the mobile phase;

this classification includes one-way, two-dimensional, and

radial chromatography. According to the mechanism of the

retention, i.e., the interaction between solutes and the

stationary phase, chromatographic methods are classified as

adsorption, partition, ion-exchange, and gel permeation.








Finally, the chromatographic technique may be classified

according to the kind of sample introduction onto the sta-

tionary phase and migration through the system. This gives

rise to development, elution, displacement and frontal anal-

ysis chromatography. The first two techniques are the most

common, while the last two methods are relatively special-

ized and, therefore, of limited value and use.

In 1941, Martin and Synge [1] were led to contrive a

scheme of liquid-liquid chromatography for separation of the

amino acids of a wool hydrolyzate with a countercurrent

extractor [2]. To improve the efficiency of the counter-

current method, Martin and Synge considered a means of

immobilizing one phase while the second phase flowed over

it in such a manner as to maximize the contact of the im-

miscible liquids at the interface. Martin believed this

would facilitate the rapid distribution, or partitioning, of

the solutes between the two phases. The efficiency of their

partitioning column was almost 104 times greater than that

of the countercurrent method [3].

The difference between modern liquid chromatography and

traditional column chromatography (whether adsorption, par-

tition or ion-exchange) involves improvements in equipment,

materials, technique, and the application of theory. Modern

liquid chromatography provides more convenience, better

accuracy, higher speed, and the ability to carry out diffi-

cult separations. At the beginning of the 1970's, the

modern form of liquid chromatography was named high pressure









liquid chromatography (HPLC). Later, the P for "pressure"

was replaced by P for "performance." The reason was the

appearance of microparticles that allowed researchers to

perform, at a lower pressure drop, the same efficient and

rapid analyses done with other supports.

In conventional liquid-liquid chromatography (LLC), the

stationary phase is a bulk liquid, mechanically held to the

support by adsorption. In recent years, organic phases are

chemically bonded to the support, leading to a separate LC

method called bonded-phase chromatography (BPC). Bonded-

phase chromatography is the most widely used in modern LC,

and many laboratories use BPC columns for their LC separa-

tion. In contrast to LLC, bonded-phase chromatography pack-

ings are quite stable because the stationary phases are

chemically bound to the support and cannot be easily removed

or lost during use. The availability of a wide variety of

functional groups in BPC packing allows for both normal-

and reversed-phase chromatography.

Polar BPC packing are used for normal-phase separa-

tions. Samples of moderate to strong polarity are usually

well separated by normal phase-chromatography. The mobile

phase in normal phase chromatography is a hydrocarbon sol-

vent such as hexane, heptane, or isooctane, plus small

amounts of a more polar solvent. The mobile phase strength

can be varied for a given application by varying the concen-

tration of the more polar solvent component. Reverse phase







BPC normally involves a relatively nonpolar stationary phase

(e.g., C8 or C18 hydrocarbon) used in conjunction with polar

(e.g., aqueous) mobile phases to separate a wide variety of

less polar solutes.

The general reaction for preparing bonded-phase pack-

ings on silica-based supports to produce siloxanes is:


ClSi(Me)2R
Si-OH + or -- Si-O-Si(Me)2R + HC1
R'OSi(Me)2R


where R is the desired organic moiety and, for reversed-

phase (RP) packing, is an n-alkyl chain (n = 2,8,18). This

reaction is based on silanol groups on the surface of the

siliceous support. Fully hydrolyzed silica contains about

8 pmol of silanol groups per m2. Because of steric hin-

drance, a maximum of about 4.5 pmol of silanol groups per m2

can be reacted at best [4], and an end-capping process

usually can be done to cover most of the residual silanols

by a reaction similar to the one above using chlorotrimethyl-

silane. The concentration of the organic moiety per m2 of

BPC packing depends upon the surface area of the packing

particles (e.g., pellicular or porous support). Siloxane

bonded-phase packing are available with pellicular or

totally porous supports. Bonded-phases of this type are

hydrolytically stable throughout the pH range 2-8.5. Due to

the different methods for the preparation of BPC packing

and shielding the residual silanol groups by end-capping,

the surface coverage and overall volume of the organic




5

stationary phase tends to show large differences from manu-

facturer to manufacturer, and even from lot to lot. Without

end-capping of the residual silanol groups, a mixed-retention

mechanism can result and lead to asymmetric peaks.

Increasing the alkyl chain length on BPC results in an

increase in selectivity and retention times [5,6] (when the

columns were compared) using the same mobile phase. The

study of chain length (C8-C22) of bonded organic phases

showed that the selectivity [7] depended on the chain length

of the bonded phase and on the molecular structure of the

solute. The same study showed that the utilization of long

chain phases made it possible to reduce the water content

of the water:methanol mobile phase, which increases the

efficiency and loading capacity. Hemetsberger et al. [8,9]

studied the behavior and the effect of structure of bonded

phases. Kikta and Grushka [10] studied the retention behav-

ior on alkyl bonded phases as a function of chain length,

surface coverage, solute type, mobile phase composition, and

temperature. Colin and Guiochon [11] compared the resolu-

tion of RPC packing (C6-C22). As a result, the shorter

length alkyl chain columns generally gave the worst resolu-

tion and efficiency. A comparative study on the separation,

efficiency under optimum mobile phase conditions with three

different mobile phases, and three groups of solutes on

three commercially available alkyl bonded phases (C2, C8 and

C18) was done by Haleem [12].

In high-performance liquid chromatography (HPLC), as in

all analytical methods, the trend is to do it faster and







cheaper, make it more selective and sensitive, and combine

it with other methods. Selection of column type in LC has

been more restricted because of the viscosities and solute

diffusivities in the liquid phase which are orders of mag-

nitude different from the values in GC. The most efficient

columns presently used in LC are those packed with totally

porous small particles (with particle size down to a few

micrometers). Although substantial reductions of the plate

height are achieved while decreasing the particle size,

there are some practical limits to this procedure. As

pointed out by Halasz [13], there are difficulties in the

uniform packing of very small particles, as well as the

problem with evolved heat of friction.

Microbore HPLC as now commercially available uses 1-

to 2-mm diameter columns. The next generation of columns,

studied only in research laboratories for the past few

years, may be inner-coated microtubular or packed microcap-

illary columns, just 50 pm in diameter. The advantage of

these would be hundreds of thousands of theoretical plates

in a very long length at low cost. Eluents from these col-

umns also could be fed into a mass spectrometer with low

interference from the solvent. These small diameter columns

often require new instruments or modifications of existing

instruments to meet new needs in injection, pumping, or

detection.

The rapid progress in HPLC places great demands on

detection techniques. Unfortunately, the highly developed







GC detectors are mostly useless at this time in LC because

of principal differences between LC and GC. An ideal detector

should be universal; however, the great diversity of systems

to be analyzed makes the construction of a sensitive univer-

sal detector impossible, and thus detectors monitoring vari-

ous physicochemical properties of substances are employed,

the optimal detection conditions being determined specific-

ally for each system. It is possible to measure either bulk

properties, which depend on the variation of the composition

of the system (e.g., refractive index, electrical conduc-

tance, etc.), or properties that selectively characterize

certain components in the mobile phase (such as the absor-

bance at a certain wavelength, fluorescence, electric current

at a certain electrode potential, etc.). The bulk property

detectors are universal detectors, and they require that

the properties of the solutes be substantially different

from those of the mobile phase to attain sufficient signal

changes during detection. The bulk property detectors are

usually less sensitive and subject to a higher noise than

the measurement of specific properties of the solutes, and

they are rarely compatible with gradient elution. On the

other hand, the measurement of specific properties requires

that the mobile phase yields the lowest possible signal

under the given conditions.

Currently available commercial LC detectors are based

on a number of detection principles, including absorbance,

fluorescence, refractive index, electrochemical reaction,





8

and mass spectrometry. A number of these detectors are

likely to be improved in the next few years. For instance,

new types of LC/MS interfaces will probably appear, and

currently available interfaces should be further refined.

Nuclear magnetic Resonance (NMR) detectors should material-

ize, and GC detectors are being seriously considered for

their applicability to LC detection. The development of

laser based detectors [14] for chromatography is in progress

and in many cases offer better sensitivity and selectivity

than conventional LC detectors. The complexity and expenses

of the laser based detectors have delayed the acceptance of

these detectors in the laboratory.

Attempts to use electrochemical detection of molecules)

in effluents from chromatographic columns were made long

before the advent of HPLC. The first papers dealing with

polarographic detectors were those of Drake [15] and

Kemula [16]. Present electrochemistry offers a large group

of methods that can be used for continuous detection of

substances [17]. The field of electrochemical HPLC has been

reviewed several times [17-19]. A survey of scientific

papers on LC detector usage during the 1980-81 period showed

that 4.3% of LC analyses were based on electrochemical

detectors [20].

The suitability of electrochemical detection to a given

problem ultimately depends on voltammetric characteristics

of the components) of interest in a suitable mobile phase

and a suitable working electrode surface. Electrochemical








detection is more limited with respect to the mobile phase

composition than other LC detection methods because of the

fact that a complex surface reaction which depends on the

medium is involved.

Direct electrochemical detection is not likely to be

useful in normal-phase chromatography since nonpolar organic

mobile phases are not well suited to many electrochemical

reactions. The HPLC stationary phases of choice clearly

include all ion-exchange and reverse-phase (RP) materials

since these are compatible with polar mobile phases contain-

ing some dissolved ions. The ionic strength, pH, electro-

chemical reactivity of the mobile phase and background elec-

trolyte, and presence of electroactive impurities (dissolved

oxygen, halides, trace metal ions) are all important con-

siderations.

The choice of electrode material is one of the important

considerations, because of the ruggedness, potential range,

residual current, and long-term stability requirements.

Electrode materials such as platinum, glassy carbon, gold,

and mercury films may work well in some cases but may be

disastrous in others. These electrodes are subject to com-

plicated surface renewal problems but are not mechanically

awkward devices such as the dropping mercury electrode.

With all limitations mentioned, liquid chromatography with

electrochemical detection (LC/EC) has three distinct

advantages for applicable systems, namely, selectivity,

sensitivity and economy.














CHAPTER TWO

THEORY AND BACKGROUND



Basic Parameters in Chromatography


Capacity Factor (k')

The capacity factor is equal to n /n where n and n
s m s m
are the number of moles of solute in the stationary and

mobile phase, respectively. Therefore, k' can be written

according to the following:


n [X] V KV t -t0
k' = -- = (1)
n [X] V V t
m mm m 0


where


[X] = concentration of solute X in the sta-
tionary phase

[XI = concentration of solute X in the mobile
m
phase

Vs = volume of the stationary phase

V = the total volume of the mobile phase within
the column

K = distribution constant

tR = retention time of solute X

t = time for mobile phase or other unretained
molecules to pass through column







Peak Resolution (Rs)

By convention, peak resolution, Rs, is defined as the

ratio of the distance between the two peak maxima (At) to

the mean value of the peak width at base (Wb).


S At 2At
R = (2)
s Wbl + Wb2 Wbl + Wb2
2


For two closely spaced peaks, one can assume that the two

peak widths are the same, and Wb2 can be used instead of the

mean.



Efficiency (E)

Efficiency of chromatography, E, is defined as peak

retention time divided by peak width at base [211.


tR
E = (3)
Wb


The theoretical plate number, N, contains the same informa-

tion as E.



N = = 2 16[ = 16E2 (4)

or


N = 5.54 (5)
W h


where a and Wh are the standard deviation and peak width at

half height of the peak, respectively. An equation derived





12

by Foley and Dorsey [22] can be used for the calculation of

the number of theoretical plates of skewed peaks in a chro-

matographic system (Nys).
sys

41.7(tR/W 0.1)2
N = (6)
sys B/A + 1.25


where tR, W0.1 and B/A are retention time, peak width at

10% of peak height, and asymmetry factor, respectively.



Control of Separation in Liquid Chromatography

The key to separating components of a mixture is to

control resolution.


k'
R = (a-1)/ 2- (7)



An increase of separation factor, a, which is the ratio of

two capacity factors, k'2/k'1, results in a displacement of

one band center relative to the other and a rapid increase

in Rs. Increasing the number of theoretical plates narrows

the bands while increasing the peak height. For early

eluting peaks, an increase in k' can provide a significant

increase in resolution, however, with increasing k', band

height decreases and separation time increases.

The available options for increasing, a, in order of

decreasing utility are: change of stationary phase, change

of temperature, and special chemical effects. Increasing

the number of theoretical plates can be done by increasing

column length and decreasing flow rate for a given column.





13

Capacity factor can be increased by increasing the volume

of stationary phase, decreasing the strength of mobile

phase, and decreasing temperature.



Gradient Elution (GE)


The most convenient way to separate a complex mixture

of solutes is to use gradient elution. Gradient elution in

LC is similar to temperature programming in GC, except that

the composition of the mobile phase is changing during

separation time. To do GE, one needs at least two different

solvents (binary gradient). One of the solvents has higher

eluent strength, and usually its percentage increases during

the gradient. Ternary gradients using three solvents are

sometimes used in LC. Multisolvent gradients are rarely

required in LC, and because of the complexity, one usually

avoids the use of multisolvent gradients.

The purpose of gradient elution is to resolve early

eluting bands and decrease the retention time of strongly

retained compounds in comparison to isocratic elution. To

achieve this, the gradient must start with a weak mobile

phase, and the strength of the mobile phase increases during

the chromatographic run. Because of decreasing retention

times for late eluting bands, these peaks are greatly

sharpened in gradient elution when compared to isocratic

elution, and sensitivity for these bands is therefore much

improved. Gradient elution increases the peak capacity for

a mixture that contains a large number of individual








components and improves the peak shapes for bands that would

tail in isocratic elution. The gradient shape for binary

solvents can be linear, concave, convex, or any other shape.

The appropriate gradient shape is dependent on LC methods,

sample, and solvent composition. The steepness of the gra-

dient is the mobile phase strength change with time.



Limit of Detection


The limit of detection represents the ability of an

analytical method for quantification of a chemical component

in terms of concentration or absolute amount. In most ana-

lytical methods, the limit of detection, CL, is defined

according to the following equation.


KSb
C K (8)
L m


where Sb and m are the standard deviation of the blank and

slope of the calibration curve, respectively. On a statis-

tical basis, K = 3 is the most appropriate number for cal-

culation of the detection limit. For more information about

detection limits, one is referred to articles by Kaiser [23]

and Winefordner [24].

To calculate the detection limit in LC, the peak to

peak noise is measured while mobile phase passes through the

column. In a normal distribution, peak to peak or random

noise in LC is considered to be 5 times the standard devia-

tion of the blank, as in equation (8). Detection limit in







LC is usually defined as 3 times the peak noise or 3/5 of

the peak to peak noise divided by the slope of the calibra-

tion curve. The latter was used for reporting detection

limits in this dissertation.



Principles of Electrochemical Detection


The most common detector in LC/EC is the amperometric

detector which measures the current at constant potential.

The amperometric detector is more sensitive and less complex

than the coulometric detector. In a coulometric detector,

the amount of electricity for complete electrochemical con-

version of the analyte is measured at constant potential.

The lower sensitivity of the coulometric detector is due to

geometrical requirements necessary for complete electrochem-

ical conversion in a flowing stream. The requirement of

larger surface area of working electrode causes higher back-

ground current and noise which reduces the signal to noise

ratio in comparison to amperometric detector.

The technique of electrochemical detection is based

on electroactivity of components eluted from the column.

Sometimes it is possible to detect nonelectroactive compo-

nents by pre- or post-column derivitization. Selecting the

applied working electrode potential is the primary require-

ment in amperometric detection. The applied potential

should be held at the minimum value at which the current

reaches the limiting-current plateau of the analyte

(Eplateau), however, in most of the cases, Eplateau is







different for different components of a mixture to be ana-

lyzed. In this case the analyst should choose the optimum

applied potential to analyze all components of interest in

the mixture. The applied or analytical potential for an

analyte can be determined by a hydrodynamic voltammogram

(HDV). In an HDV, current is measured versus applied poten-

tial for analyte injected into an LC amperometric detection

system. The Eplateau can be precisely identified by HDV

measurement under analytically useful LC conditions, but it

is a time-consuming process, due to the time required for

the baseline to stabilize after each change of electrode

potential. The time required for stabilization of the base-

line is dependent upon the mobile phase composition and flow

rate. In the case of a glassy carbon electrode, for a change

of 0.1 V in applied potential, it takes 15 to 30 min to get

a stable baseline. Cyclic voltammetry (CV) is a much faster

method for determining Eplateau, which usually has a higher

magnitude than the CV peak potential (E ) under typical

measurement conditions for slow electron transfer reaction.

Data from CV and Eplateau can be related via a simple

empirical equation [25].



Ion-pair Chromatography


The extraction of ionized solutes into organic phases

has been well known for a number of decades. To extract

ionized species from aqueous solution, an ion-pairing reagent

of opposite electrical charge is added to the aqueous phase







resulting in ion-pairing between the solute ion and pairing

ion. The resultant complex which has a net low electrical

charge or polarity can easily be extracted by an organic

phase. To separate ionic species by reversed-phase HPLC,

an ion-pairing reagent can be added to the mobile phase.

Ion-pairing reagents can also be used as a probe to detect

and quantify compounds which cannot be directly detected

[26,27]. The following scheme shows overall phase transfer

of ion pairs.


A+ +B I (A,B)
aqueous organic
phase phase


In the case of reversed-phase chromatography, the organic

and aqueous phases on the above scheme are considered to be

stationary and mobile phases, respectively. The most popular

ion-pair reagents for cationic solutes are long-chain alkyl

sulfonate ions which are usually added to the mobile phase

to enhance separation of oppositely-charged sample ions.

The exact ion-pairing mechanism for the separation of

ionic samples is still uncertain. Three popular hypotheses

are: (1) the ion-pair model, (2) the dynamic ion-exchange

model, and (3) the ion-interaction model. The ion-pairing

model stipulates that the formation of an ion-pair occurs in

the aqueous mobile phase which is in agreement with solvo-

phobic theory [28], while the dynamic ion-exchange model

States that unpaired lipophilic alkyl ions adsorb onto the

nonpolar stationary phase, causing the column to behave as







an ion exchanger [28]. The ion-interaction model is based

upon conductance measurements. It proposes that neither the

ion-pairing nor the ion-exchange model can explain the ex-

perimental data in a consistent way [28]. The ion-interac-

tion model assumes that a primary layer of lipophilic ion

covers the surface of the stationary phase which is in

dynamic equilibrium with the bulk eluent. In the vicinity

of this primary layer exists a secondary layer of opposite

charge creating an electrical double layer on the surface.

The retention of the ionic components is due to the electro-

static force between these ions and the primary layer, as

well as an additional sorptionn) effect onto the nonpolar

stationary phase.



Micellar Chromatography


It is well known that surfactants, detergents, or

surface active agents are amphiphilic molecules (i.e.,

molecules in which a hydrophobic tail is joined to a hydro-

philic head-group). Surfactants can be anionic, cationic,

nonionic, and zwitterionic. Above a certain concentration,

surfactant molecules associate in aqueous solution to form

large molecular aggregates of colloidal dimensions termed

micelles. The concentration threshold at which a surfac-

tant starts to form micelles is called the critical micelle

concentration (CMC), and the number of surfactant molecules

in a micelle is called the aggregation number. The aggrega-

tion number and the CMC differ from one surfactant to







another, and even for the same surfactant in different media.

At concentrations greater than the CMC, a dynamic equilibrium

exists between the surfactant molecules and micelles. The

general size and shape of the particular micelle depend on

the aggregation number.

The term normal micelles is used for surfactant aggrega-

tion in aqueous media. The hydrophilic head groups are

directed toward and in contact with aqueous solution to

form a polar surface, while the hydrophobic tails are

directed away from the water to form a central nonpolar core.

In nonpolar solvents, the surfactant aggregates are termed

reversed or inverted micelles. In these micelles, polar

head groups are concentrated in the interior of the aggre-

gates and hence form a central hydrophilic core, while the

hydrophobic tail moieties extend into and are in contact

with the bulk nonpolar solvent.

The solubilizing power of micellar systems is one of

its most important aspects. This refers to the ability of

micelles to solubilize a wide variety of solutes that are

insoluble or only very slightly soluble in the bulk solvent

alone. The solubilization of solutes in micellar systems is

a dynamic process and depends upon such factors as the tem-

perature, the nature of solutes, the surfactant concentra-

tion, and the type of micelles. The amount of solute

solubilized is usually directly proportional to the concen-

tration of micelles. The solubilization of a solute at a

micelle site is dependent upon the type of solute and the







nature of the micelle. In a normal micelle, a nonpolar

solute is thought to be located near the center of the

hydrophobic core,while an ionic solute is adsorbed on the

polar micellar surface.

According to the properties of micellar systems men-

tioned above, micellar solutions can be used as mobile

phases in HPLC. The normal micellar solution can be used

as the mobile phase in reversed-phase HPLC, while reversed

micellar solutions are compatible with normal-phase HPLC.

Equations for partitioning behavior of solutes with micellar

mobile phases in LC have been derived by Armstrong and

Nome [29]. From these equations,one can calculate the

partition coefficients of solutes between water and

micelles, between the stationary phase and water, and be-

tween micelles and the stationary phase. One of the draw-

backs of micellar mobile phases in RP-HPLC is its poor

efficiency in comparison to hydroorganic mobile phase,

however, Dorsey et al. [30] showed that by the addition of

3% of propanol and a temperature of about 400C, micellar

mobile phases can approach efficiencies of hydroorganic

mobile phases. Possible advantages of micellar mobile

phases over hydroorganic mobile phase are: (1) the unique

selectivity of micellar mobile phases toward different

types of solutes, (2) the economy when compared to hydro-

organic mobile phases, and (3) the simplicity of purifica-

tion of the crystalline surfactants compared to organic

solvents.













CHAPTER THREE

EXPERIMENTAL



Cyclic Voltammetry Systems


To find an approximate analytical potential for solutes

of interest, a CV-1A cyclic voltammetry instrument and an

electrochemical cell made by Bio Analytical Systems, Inc.

(West Lafayette, Indiana) were used. The working and ref-

erence electrodes were glassy carbon and Ag/AgCl, respec-

tively. Before running the CV experiments, the sample solu-

tions were purged for 20 min with helium. Cyclic voltammetry

was then carried out in an inert helium atmosphere. A

Plotamatic MFE-715 (MFE, Salem, New Hampshire) X-Y recorder

and digital voltmeter were used to record the cyclic voltam-

mograms.



Liquid Chromatography System


The solvent delivery unit used during the chromato-

graphic run was a Waters 6000 A (Waters Associates, Milford,

Massachusetts), an Altex model 322 gradient liquid chro-

matograph with two model 100 A pumps (Altex Scientific,

Berkeley, California), or a Spectra-Physics SP 8700 solvent

delivery system (Spectra-Physics, Santa Clara, California).







The injection valve was either an Altex 210 or Rheodyne 7125

(Rheodyne, Cotati, California) with 5, 10 and 20 pL loops.

Various columns--an Altex Ultrasphere octyl, 250 x 4.6 mm;

an Altex Ultrasphere ODS, 150x 4.6 mm; and Rainin Microsorb

octyl column, 150 x 4.6 mm--were employed.



Electrochemical Detector


The electronic controller was an LC-4 amperometric

controller from Bio Analytical Systems, Inc. or Princeton

Applied Research (PAR, Princeton, New Jersey) model 174

polarographic analyzer. The electrochemical cell used was

either from Bio Analytical Systems, Inc., with glassy

carbon as working electrode, or a porous membrane separator

with gold as the working electrode, a generous gift of

K. A. Rubinson. The latter working electrode was used only

in work of Chapter Four of this dissertation to compare

electrode material, while the former was used from

Chapter Four through Chapter Six. The reference electrode

for both cells was Ag/AgCl from Bio Analytical Systems,

Inc.

The electrochemical cell from Bio Analytical Systems,

Inc. [31], is a thin-layer cell, as shown in Figure 1. The

thin-layer cell, reference electrode compartment, clamp,

and waste and connecting tubes are preassembled as one unit.

Addition of a reference electrode to this unit completes

the detector cell. All of the detector cell components

have been machined to accept standard plastic tube end















Reference
Electrode








Auxiliary
Electrode ,


To Waste-


















Electrode Positioned
Downstream From Inlet
Port


Figure 1. Electrochemical Cell with Glassy Carbon Electrode


From LC
Column







fittings (-28 thread) as supplied by BAS, Altex, LDC,

Omnifit, and others; therefore, the detector cell is directly

compatible with commercial HPLC systems.

The porous membrane separator contained gold as the

working electrode [32]. The 0.5 mm gold wire of 5 cm length

was covered with porous polymer tubing which can be attached

to the outlet of the column in an LC system. The gold elec-

trode covered with porous membrane was cemented into a small

reservoir which contained an external electrolyte solution

(1 or 2 M KCl), auxiliary (Pt), and reference electrodes.

To prevent any damage to the porous membrane gold electrode,

it was soaked in 1 M KCl for several hours before running

mobile phase through it from an LC system.



Pretreatment of Glassy Carbon Electrode


It is well known that the sensitivity of glassy carbon

electrodes decreases with use. Decreasing activity at the

glassy carbon electrode surface can be due to adsorption of

analytes, mobile phase components, and byproducts of redox

reactions onto the electrode surface as well as formation

of electroactive species, such as carbonyl and hydroxy

groups, from components in the electrode material. The

adsorption of these components at the electrode surface may

form a polymeric film which would decrease electrode re-

sponse; however, whatever the source of deactivation might

be, it is necessary to clean and reactivate the electrode

surface by mechanical, chemical, electrochemical, or by a






combination of these methods. More details on pretreatment

and the study of the glassy carbon electrode surface are given

elsewhere [33-35].

A deactivated electrode surface produces a large resid-

ual current as well as a noisy baseline which affects the

accuracy of analytical application and increases the detec-

tion limits. For reactivation or pretreatment of a glassy

carbon electrode surface the following procedure has given

satisfactory results. This procedure would be used if

analytical potential is positive or anodic.


(1) The cell was dismounted and electrode surface was
washed with methanol.

(2) A few drops of a slurry of alumina [0.1 im, Gamal,
Grade B, Fisher Scientific Company (Fairlawn,
New Jersey)] in water were placed at the surface
of the electrode. The surface was then carefully
polished with Buehler LTD polishing paper (Evan-
ston, Illinois) for 2-3 min.

(3) The electrode surface was rinsed with methanol and
polished with the same polishing paper which was
soaked in methanol, and then the cell was assem-
bled.

(4) An anodic potential (0.1-0.2 V higher than that
of analytical potential) was applied for 3-4 min
while mobile phase passed through the electrochem-
ical cell.

(5) A cathodic potential (-0.8 V vs. Ag/AgCl) was
applied for 2-3 min while mobile phase passed
through the electrochemical cell.

(6) The steps of (4) and (5) were repeated, and then
the potential was set at the analytical potential.
The potential switch was turned off before chang-
ing from the anodic-applied potential to the
cathodic-applied potential or vice versa.







Reagents


Methanol and acetonitrile, HPLC grade (Fisher Scientific

Company, Fairlawn, New Jersey) were employed as organic

mobile phase components. Methanol, HPLC grade, was used as

the solvent for test solutes. Deionized water, another

mobile phase component, was purified by a Barnstead Nanopure

system (Sybron Corp.) The purified water then was irradi-

ated for 24 hr by ultraviolet light in a model 816 HPLC

reservoir (Photronix Corp., Medway, Massachusetts). The

other chemicals comprising the mobile phases were: (1) rea-

gent grade phosphoric acid (Scientific Products, McGaw Park,

Illinois), (2) sodium perchlorate and Brij-35 (Aldrich

Chemical Co., Milwaukee, Wisconsin), and (3) sodium dodecyl

sulfate (SDS; Fisher Scientific Co.). The reagents mentioned

above mostly were used as a component of the mobile phase

or dissolved in the mobile phase. Other specific chemicals

(e.g., sample solutes) will be mentioned in chapters related

to them.













CHAPTER FOUR

SIGNAL CHANGE AND BASELINE SHIFT
USING ELECTROCHEMICAL METHODS



In this chapter the following will be considered:

(1) change of residual current with ordinary DC voltammetry,

normal pulse (NP) voltammetry, and differential pulse (DP)

voltammetry in isocratic elution HPLC; (2) effect of the

background electrolyte and gradient elution on baseline

shift using amperometric detection; and (3) effect of the

electrode material on baseline shift using amperometric and

non-ramping differential pulse (NRDP) voltammetry methods.

Pulse techniques such as DP and NP can be used as LC

detection schemes if a constant pulse amplitude superimposes

over an initial potential. This means that there is no

potential ramp, and it is better to be called non-ramping

pulse techniques. The term "pulse techniques" has been

used instead of the term "non-ramping pulse techniques" in

the literature, however. Fleet and Little [36] were among

the first authors to be concerned with application of

pulse techniques as an HPLC monitoring method using a mer-

cury drop electrode (MDE). The idea was examined in prac-

tice by Swartzfager [37], using carbon paste as a working

electrode. The advantages of pulse systems in monitoring

have been discussed by Kissinger [19], who noted the







possibility of separating the monitored electrochemical sys-

tem from systems with more positive or negative half-wave

potentials and prolonging electrode service life by electro-

chemical cleaning. Electrochemical cleaning during the

pulse operation is the subject of a patent by Fleet [38]. A

versatile voltammetric detector with double polarization

pulse and semi-differential scanning for LC is reported by

Stastny et al. [39]. Pulse techniques also offer the advan-

tage of decreased dependence of the measured current on

flow-rate in comparison to amperometric techniques [37],

because the short potential pulse duration minimizes the

development of the diffusion layer of the electrode and

dependence of the thickness of this layer on flow-rate.

With all the advantages of pulse techniques (more selectiv-

ity, increased electrode stability, and less current depen-

dence on flow-rate) over amperometric techniques, these

techniques are limited by poor sensitivity and complex in-

strumentation. The residual current at a solid electrode is

reportedly high [17]. A study of the change in magnitude of

the residual current with different electrochemical methods

using isocratic elution and with amperometric method using

gradient elution was performed and will be reviewed. The

residual current change during gradient elution with ampero-

metric detection is a baseline shift due to the change in

the composition of the mobile phase.

The electrochemical equipment used was a model 174

polarographic analyzer, an LC-4 amperometric controller, and







an electrochemical cell either with a glassy carbon elec-

trode (GCE) or a porous membrane gold (Au) electrode.



Change of Residual Current with DC, DP and
NP Voltammetry in Isocratic Elution HPLC


In differential pulse (DP) voltammetry and normal pulse

(NP) voltammetry, the current is measured during a time in-

terval of the pulse when the ratio of faradaic current to

charging current is a maximum. The electrochemical cell

can be considered as an RC-circuit, and thus the charging

current decays exponentially. The purpose of these experi-

ments was a comparison of the residual current change versus

applied electrode potential with different electrochemical

methods using isocratic elution.

The residual current change and decay current on a

glassy carbon electrode with different percentage of acetoni-

trile as an organic modifier in water are shown in Figures 2

through 5. The data from these figures are reported in

Table 1. All mobile phases contained 0.04 M NaClO4 as a

background electrolyte. The potential was scanned from

0.55 V (E ) to 1.30 V (E ) versus Ag/AgCl reference elec-

trode.

The residual current change, AI = If Ii, is due to

the difference between the current at the final potential

(Ef) or the current at the end of the ramp, If, and the cur-

rent at the initial potential, I.. The residual current

change, AI, can be considered to be due to the residual

current difference at two different potentials. Decay













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current is the amount of current depleted at the end of the

ramp (Ef) within a short time. This current depletion might

be considered as the charging current and decreasing current

due to impurities. Since the charging current decays quick-

ly (10-4 to 10-3 sec), it is impossible to find out the

origin of decay current with a stripchart recorder.

The data in Table 1 show that on the DP mode the resid-

ual current change, AI, and decay current, idecay are in

the limit of measurement error (measurement device was a

ruler). This means that the currents sampled shortly before

the end of the pulse and shortly before the rise of the

pulse are almost the same, and no difference in charging

current is sampled. On the DC mode, there is some current

change during the scan (potential applied to the electrode

changes with time) and some decay current at the end of the

scan. Increasing the scan rate will increase both AI, and

idecay in the DC mode because the magnitude of charging

current increases as the scan rate increases. In the NP

mode, AI is much larger than that of the DC mode, while the

decay current is in the limit of measurement error. The

current varies linearly with the applied potential on the

working electrode (WE) in the NP mode. The larger magnitude

of AI in the NP mode as compared to the DC mode might be due

to the surface clean up of the working electrode which

occurs with pulsing. This cleanup increases the electrode

response.








Table 1.


Residual Current Change, AI, and Decay Current,
decay, in DC, DP and NP Voltammetry in Isocratic
Elution. Flow rate: 1 mL/min; column: Altex Ultra-
sphere ODS 150 x 4.6 mm; Ei = 0.55 V, Ef = 1.30 V
vs. Ag/AgCl; working electrode: glassy carbon; low
pass filter = 0.3 sec; pulse amplitude = 100 mV.


Solvent A =
Solvent B =


0.04 M NaC104 in water.
0.04 M NaC104 in acetonitrile.


Scan
Rate
mV/sec


Electrochemical
Mode

DC
DP
DC
DC
DP
DC
DP
NP


Figure


Mobile
Phase


2-a
2-b
3-a
3-b
3-c
4-a
4-b
5


5% B
5% B
50% B
50% B
50% B
95% B
95% B
95% B








Table 1 Extended


Drop Knocker
Time
(sec)

1
1
1
1
1
0.5
0.5
0.5


AI Measure-
ment Error
(iA)

0.75 0.02
0.02
0.65 0.01
0.82 0.01
0.01
1.41 0.02
0.02
7.36 0.08


decay measure-
ment Error
(pA)


0.16


0.12
0.34


0.10

+


0.02
0.02
0.01
0.01
0.01
0.02
0.02
0.08








Effect of Background Electrolytes and Gradient
Elution on Baseline Shift Using Amperometric Detection


In isocratic elution, the composition of the mobile

phase during the course of separation is constant; however,

isocratic elution sometimes is incapable of separating com-

plex samples which have a wide range of retention times or

k' values. One of the solutions to this problem is gra-

dient elution (GE). The necessity (of the presence) of a

background electrolyte and changing composition of the

mobile phase causes changes in viscosity, dielectric constant,

and conductivity of the mobile phase. The dependence of

the diffusion coefficient on viscosity, variation of the

conductivity, and changing condition of the working elec-

trode surface during gradient elution will cause a change in

the residual current.

Walden's rule [40] states "the product of the equiva-

lent conductivity (X) and viscosity of the solvent (n) for

a particular electrolyte at a given temperature should be

a constant."


S* n = constant (9)


In a binary gradient (solvents A and B), the percentage of

organic modifier (solvent B) increases in hydroorganic mo-

bile phase during the gradient course. Decreasing the po-

larity of the mobile phase during the gradient course

decreases the conductivity of the mobile phase if the con-

centration of background electrolyte in both solvents is





38

the same. If the concentration of background electrolyte

in solvent B is higher than that of solvent A, the total

concentration of background electrolyte in mobile phase

increases during the gradient course. An increase in the

concentration of background electrolyte during the gradient

course may compensate for decreasing the polarity and chang-

ing the viscosity of the mobile phase, which are important

factors in baseline stability. As a result of this discus-

sion, a more stable baseline should be achieved with higher

concentration of background electrolyte in solvent B as

compared to equal concentration of background electrolyte

in both solvents.

If one assumes a binary gradient in which the percen-

tage of solvent B increases during the gradient course, one

can measure the conductivity or specific conductivity vs.

the percent of solvent B in A+B. The specific conductivity

vs. the percentage of organic modifier (solvent B) is shown

in Figure 6 for H3PO4 and NaClO4 as background electrolytes.

The conductance measurement was done with a conductance

bridge of the Janz-McIntyre type [41]. The specific conduc-

tivity, K, was calculated from using equation (10).


0.126
K = specific conductivity = R (10)


The cell constant is 0.126 cm-1, and R can be calculated

from equation (11).


Rmeasured 10000
R 10000 R (11)
measured

















40 1E-4


* d ~.. __


,
..
4
.ni.i
F- -1


Figure 6.


Specific Conductance vs. Percentage of Solvent B


(a) Solvent A = 0.15% H3PO4 in H20

Solvent B = 0.20% H PO4 in CH OH


(b) Solvent A = 0.05 M NaCIO4 in H20

Solvent B = 0.05 M NaCIO4 in CH30H


c -4







In the case of H3PO4 as a background electrolyte, the spe-

cific conductance decreases with the increasing percentage

of the organic modifier, while in the case of NaClO4 it

passes through a minimum at about 65% of organic modifier

(solvent B).

Figure 7 shows the baseline shift during gradient elu-

tion (with no sample injection) using an amperometric detec-

tor and glassy carbon electrode, with H PO4 and NaClO4 as

the background electrolytes. The column is equilibrated

with solvent A, and gradient starts with holding solvent A

for 3 min, then linear ramp to 20% A and 80% B over 30 min.

The baseline shift, Ai, is the difference in current before

starting the gradient program and at the end of the gradient.

Figure 7-a is due to 0.15% H3PO4 in H20 and 0.20% H3PO4 in

CH3OH, with Ai = -9 n A, while Figure 7-b is due to 0.20%

H3PO4 in both H20 and CH3OH with Ai = -14 n A. The differ-

ence in Ai for Figures 7-a and 7-b is due to the concentra-

tion difference of H3PO4 in H20. The lower baseline shift

in Figure 7-a as compared to Figure 7-b is in agreement with

Walden's rule as discussed above. Figure 7-c is due to

0.05 M NaClO4 in both H20 and CH3 OH, with Ai = +6 n A.

Sodium perchlorate produces a smaller baseline shift because

it is completely ionized in both solvents. The peaks in

Figure 7 might be due to impurities in H20 or background

electrolytes which collect at the top of the column during

the early part of the solvent program and equilibration.

More examples of gradient elution with hydroorganic and
























04




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QC
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micellar mobile phases are given in Chapters Five and

Six.

The shift in baseline during gradient elution at a con-

stant applied potential might be due to different concentra-

tions of electroactive impurities, changing diffusion

coefficient of impurities with a change in the viscosity,

exhibition of different half-wave potential for specific

electroactive components, and effect of organic modifier on

the surface of the working electrode.



Effect of the Electrode Material on Baseline Shift
Using Amperometric and NRDP Voltammetry Methods


The baseline shift during gradient elution is evaluated

for glassy carbon and porous membrane gold electrodes. The

electrochemical methods used were amperometric and non-

ramping differential pulse (NRDP) voltammetry. In NRDP

method, the difference in current sampled at the end of the

pulse and shortly before the rise of the pulse is recorded.

Since the pulse time is very short, there is virtually no

change in the mobile phase composition with gradient elution

during one pulse. The normalized baseline shift (current

difference at the end and the start of gradient program

divided by the surface area of the working electrode) for

amperometric and NRDP voltammetry with their chromatographic

conditions are reported in Table 2.

Data in Table 2 show that the absolute amount of the

baseline shift in the amperometric method is much higher

for gold electrode as compared to glassy carbon electrode.








Table 2.


Normalized Baseline Shift (nA/mm2) during Gradient
Elution with No Sample Injection. Column: Altex
150x4.6 mm Ultrasphere ODS; E = 0.8 V vs. Ag/AgCl;
pulse amplitude = 100 mV; low pass filter = 3 sec.
Gradient program: initially 95% A, then linear
ramp to 5% A over 20 min, and finally holding for
5 min at 5% A.


Solvent A = 0.20% H PO4 in H20.
Solvent B = 0.20% H3PO4 in CH3OH.


Electrochemical
Method

Amperometric

NRDP

Amperometric


NRDP


Working
Electrode


Normalized Base-
line Shift
nA/mm2


Glassy Carbon

Glassy Carbon


Gold

Gold


-1.5


-116.0

-356.7

-152.8





45

This indicates that the residual current change with gradient

elution on the gold electrode surface is larger as compared

to glassy carbon electrode, which shows the gold is not an

appropriate working electrode under these conditions.

Although the absolute amount of the baseline shift in the

case of NRDP is also larger for the gold electrode as com-

pared to glassy carbon electrode, this gap is not as large

as in the case of amperometric method. This might be due to

cleanup of the electrode surface with pulsing.












CHAPTER FIVE

A COMPARISON OF MICELLAR AND HYDROORGANIC
MOBILE PHASES USING AMPEROMETRIC DETECTOR



In this chapter, the following will be considered:

(1) the comparison of the hydrodynamic voltammograms (HDV)

in micellar and hydroorganic mobile phases, (2) the compar-

ison of analytical figures of merit between micellar and

hydroorganic mobile phases, and (3) gradient elution and

selctivity with micellar mobile phases.

Ionic sufractants have been extensively used as ion-

pairing reagents in ion-pair chromatography in the past.

More recently, ionic surfactants and nonionic surfactants in

aqueous solution have been employed as the mobile phase in

RP-LC. The surfactant concentrations in the mobile phase

above the CMC have been shown to have properties similar to

conventional mobile phases for RP-LC [42]. Micellar solu-

tions have been used as media or matrices for room tempera-

ture phosphorescence [43], and the usefulness of micelle-

stabilized room-temperature phosphorescence (MSRTP) for

detection and quantification of aromatic molecules in HPLC

has been reported by Weinberger et al. [44]. Micellar solu-

tions have been used in electrochemistry to produce well-

defined redox waves for compounds that show only slight

shoulders or no waves in aqueous solutions [45]. Since the








redox voltammetry waves for organic compounds in micellar

solutions can be different from those of hydroorganic solu-

tions, three different classes of compounds were chosen in

this study. The structures of these three classes which

include various forms of B-6 vitamins (PLP,PL, PN, PNP and

PMP), phenolic compounds (phenol, hydroquinone, resorcinol,

catechol and o-cresol), and polyaromatic hydrocarbons

(anthracene and pyrene) are shown in Figure 8. The phos-

phate group in three of the B-6 vitamins can be partially

ionized. The extent of ionization is dependent upon the pH

of the mobile phase, however, in the low pH region, all

forms of B-6 vitamins will be protonated to produce cations.

A phosphoric acid buffer solution of pH 2.20 was used as the

background electrolyte in all mobile phases and in addition

generated an ionic site for ion-pairing of the nitrogenous

vitamins. With cationic solutes, sodium dodecyl sulfate

(SDS) in the mobile phase can act as an ion-pairing reagent

both below and above the CMC.



Hydrodynamic Voltammoqram in Micellar
and Hydroorganic Mobile Phases


Hydrodynamic voltammograms for phenol, two vitamins

(PLP and PL), and polyaromatic hydrocarbons (anthracene and

pyrene) with their experimental conditions are given in

Figures 9 through 11, respectively.

The signal achieved for the same amount of injected

phenol in the micellar mobile phase is greater than that of

the hydroorganic mobile phase when the applied potential is










HC=O


HO H CH: 0


H.C N H

Pyridcxal-5- phosphcae

PLP


pyridoxol

PL


CHo CH


CH2CH
CHtCH HO CH, O?-CH
Ii
YH 2 OH

H C N H

Pyridoxin.-5- phosp.c.a

PNP


U CHO -CP-OH
'I !OH

N N H H, C N H

Pyridoxina
Pyridoxcmine-5- phcsphc',a

PN PMP

Structures of B-6 Vitamins


on


01
OH


Hydroquinone

Structures


OH



o OR


OH


N^ ^


Resorcinol catcchol

of Phenolic Compounds


~8 ~
.7 2
6
N.5 NU ~


7 a


Anthracene Pyree
Pyrene
Structures of Polyaromatic Hydrocarbons



Figure 8. Chemical Structures of Compounds Used in Chapter
Five


0:I






Phenol


OH


o-Cresol
























Figure 9. Hydrodynamic Voltammogram for Phenol


Column: Altex Ultrasphere ODS, 150 x 4.6 mm
Flow Rate: 1 mL/min
Injection Volume: 20 pL (5 ppm)
Working Electrode: Glassy carbon electrode
Temperature: 300C


(o) Micellar mobile phase: 0.1 M SDS in
(3:97) (l-propanol:H20) + 5 x 10-2 M
NaClO4
(*) Hydroorganic mobile phase: (60:40) (H20:
CH OH) + 5 x 10-2 M NaCIO4
































































0,8 0,9


1.0 1, 1.2
E(v)


250 r


200 -


i(nA)


150 I


100 I


50 L
0.


7


I I I






















Figure 10. Hydrodynamic Voltammogram for B-6 Vitaminsa


Micellar mobile phase: 0.1 M SDS in (3:97)
(l-propanol:H20), pH = 2.20 (used
H3P4)

(A) PLP (250 ppm)
(0) PL (250 ppm)


Ion-pair mobile phase: 5 x 10-3 M SDS in
(12:88) (l-propanol:H20), pH = 2.20
(used H3PO4

(o) PLP (2000 ppm)
(*) PL (2000 ppm)

a All B-6 vitamins dissolved in 0.1 M HC1. Other con-
ditions as in Figure 9.

















1000 -







750 -





/
i (nA) 500 -

"I /





250 -







1,0 1.1 I.2 1.3 1.4
E (v)



















Figure 11. Hydrodynamic Voltammogram for Polyaromatic
Hydrocarbons


Column: Rainin microsorb octyl
Flow Rate: 2.5 mL/min


Micellar mobile phase: 0.1 M SDS in (3:97)
(l-propanol:H20), pH = 2.2 (used H PO4

(o) anthracene (50 ppm)
(*) pyrene (100 ppm)


Hydroorganic mobile phases:
(0) anthracene (50 ppm): (46.5:53.5)
(H20:CH3OH), pH = 2.20 (used H3PO4)

(A) pyrene (100 ppm): (43.2:56.8) (H20:
CH3OH), pH = 2.20 (used H3PO4)


a Anthracene and pyrene were dissolved in CH3OH. Other
conditions as in Figure 9.


































































0.8 0,9


iO I1I 1,2 1.3 1.4
E v)


500


400









300


i(nA)


200 -









00









0-
0,7







below 1.0 V, whereas above 1.0 V it is slightly greater in

the case of the hydroorganic mobile phase. According to

HDV, the best analytical potential for phenol is 1.1 V in

both mobile phases.

In the case of B-6 vitamins, the signal increases with

increasing potential in the same manner for both the micel-

lar mobile phase and the hydroorganic mobile phase which

contains small amounts of SDS as an ion-pairing reagent. The

signal for PLP and PL are greater in the micellar mobile

phase as compared with the ion-pairing mobile phase. At

first glance, the graph in Figure 10 shows that the signal

for PLP in the ion-pairing mobile phase is greater than that

in the micellar mobile phase at the same potential, however,

this is not the case because the absolute amount of injec-

tion of PL and PLP in the ion-pairing mobile phase is 8 times

greater than that in the micellar mobile phase. The poten-

tial 1.4 V might be chosen as an analytical potential for

vitamins.

Figure 11 shows the HDV for anthracene and pyrene in

both mobile phases. At potentials higher than 1.2 V, the

signal due to pyrene increases sharply, whereas the signal

due to anthracene decreases smoothly. One of the most im-

portant aspects of Figure 11 in comparison to Figures 9 and

10 is the unique selectivity response of polyaromatic hydro-

carbons with applied potential in both micellar and hydro-

organic mobile phases. This means that anthracene can be

measured in the presence of pyrene using a proper applied

potential. In order to compare analytical figures of merit







in micellar and hydroorganic mobile phases, the best possible

analytical potential to use for anthracene and pyrene would

be 1.1 V and 1.2 V, respectively.



Analytical Figures of Merit Comparison between
Micellar and Hydroorganic Mobile Phases


Analytical figures of merit such as limit of detection

(LOD), upper limit of linear dynamic range (LDR), sensitiv-

ity, correlation coefficient, and log-log slope, as well as

the experimental conditions for phenol, B-6 vitamins, and

polyaromatic hydrocarbons (PAH) in both micellar and hydro-

organic mobile phases, are reported in Tables 3 through 5,

respectively. Data in Tables 3 through 5 are derived from

Figures 12 through 14 (analytical or calibration curves),

respectively.

In order to compare analytical figures of merit for a

specific electroactive component in two different mobile

phase compositions using an amperometric detector, two con-

straints must be met. First, the flow rate should be nearly

the same for both mobile phases because the response of any

electrochemical detector is dependent on the rate of mass

transfer to the electrode surface [37]. Secondly, the

retention time of the electroactive component in both mobile

phases should be nearly the same because of the peak height

measurement. To get nearly the same retention time in both

mobile phases, the percentage of organic modifier in the

hydroorganic mobile phase was varied until the retention







Table 3. Analytical Figures of Merit for Phenol. Flow
rate: 1 mL/min; column: Altex Ultrasphere ODS,
150 x 4.6 mm; E = 1.1 V; working electrode:
glassy carbon electrode; injection volume: 20 lL.


Micellar mobile phase: 0.1 M SDS in 3% 1-propanol
+ 5 x 10-2 M NaCIO4.
Hydroorganic mobile phase: (H20:CH3OH)(60:40) +
5 x 10-2 M NaClO4.


Limit of Detection Upper limit of LDR
Mobile Phase (ppm) (ng) (ppm) (ng)

Micellar 0.0080 0.16 20a 400

Hydroorganic 0.0065 0.13 10 200


a 20 ppm was the most concentrated solution, so the upper
limit of LDR might be more than 20 ppm.







Table 3 Extended


Sensitivity
(nA/ppm) (nA/ng)

41.40 2.03

39.74 1.99


Correlation
Coefficient

0.9971

0.9973


Log-log
Slope

0.96

1.04


tR
(min)

5.0

3.8








Table 4.


Compound


Analytical Figures of Merit for B-6 Vitamins.
Column: Altex Ultrasphere ODS, 150 x 4.6 mm;
E = 1.4 V; working electrode: glassy carbon;
injection volume: 20 pL.


Micellar mobile phase: 0.1 M SDS in 3% 1-propanol
with pH = 2.20 (used H3P04).
Ion-pair mobile phase: 5 x 10-3 M SDS in 12%
1-propanol with pH = 2.20 (used H3PO4).


Mobile
Phase


Limit of Detection Upper Limit of LDR
(ppm) (ng) (ppm) (nq)


Micellar

Micellar


Ion-pair


PL Ion-pair


PLP

PL


PLP


0.316

0.236


0.0726


6.32

4.72


1.45


300

300


100


6000

6000


2000


0.0852 1.70


100 2000








Table 4 Extended


Sensitivity
(nA/ppm) (nA/ng)

3.80 0.170

5.09 0.255


4.96

4.22


0.248

0.211


Correlation
Coefficient

0.9999

0.9995


0.9999

0.9999


Log-log Flow Rate
Slope (mL/min)

0.99 1.0

1.00 1.0


0.95

0.95


1.1

2.0


tR
(min)

2.8

7.8


2.8

7.8





61


Table 5. Analytical Figures of Merit for Polyaromatic Hydro-
carbons. Flow rate: 2.5 mL/min; column: Rainin
microsorb octyl, 150 x 4.6 mm; injection volume:
20 jiL.


Micellar mobile phase: 0.1 M SDS in (3:97) (1-pro-
panol-H20), pH = 220 (used H3PO4)


Limit of
Detection


Compound


Anthracene


Pyrene


Anthracene


Pyrene


Mobile Phase


Micellar

Micellar


Hydroorganica

Hydroorganic


(ppm) (ng)

0.0933 1.87

1.77 35.4


0.0846 1.69


Upper Limit
of LDR
(ppm) (ng)


250

200


5000

4000


50 1000


a Hydroorganic mobile phase (used only for anthracene):
(46.5:53.5) (H20:CH30H); pH = 2.20 (used H3PO4); ionic
strength = 3.59 x 10-2 M.

b Hydroorganic mobile phase (used only for pyrene):
(43.20:56.80) (H20:CH3OH), pH = 2.20 (used H3PO4).





62

Table 5 Extended


Sensitivity


(nA/ppm)


0.707

0.667


3.545


(nA/ng)

0.0354

0.0338


0.177


Correlation
Coefficient

0.9996

0.9990


0.9906


1.2 9.3


Log-log
Slope

0.97

0.97


0.96


E
(V)

1.1

1.2


1.1


tR
(min)

13.7

9.3


13.7
















1000









750
/







500









250









4 8 12 15 20

C(ppm)

Figure 12. Analytical Curves for Phenol

(o) in micellar mobile phase
(*) in hydroorganic mobile phase

Experimental conditions are the same as Table 3.


























Figure 13. Analytical Curves for B-6 Vitamins

Micellar mobile phase:
(A) PLP
(0) PL

Ion-pair mobile phase:
(o) PLP
(*) PL

Experimental Conditions are the same as
Table 4.






















2000 r


1500 H


i(rnA)


1000 I


500 -


200

C(ppm)


300


400


vI ii






















Figure 14. Analytical Curves for Polyaromatic Hydrocar-
bons


Micellar mobile phase:
(o) anthracene
(*) pyrene


Hydroorganic mobile phase:
(0) anthracene


Hydroorganic mobile phase:
(A) pyrene


Experimental conditions are the same as
Table 5.



















400 r


300 1


i(nA)


200


100 1


C(ppm)


200


300







time of the electroactive component nearly matched that of

the micellar mobile phase.

In the case of phenol, the LOD is approximately the

same in both mobile phases, whereas the upper limit of the

LDR in the micellar mobile phase is at least twice that of

the hydroorganic mobile phase. For B-6 vitamins there is

not much difference in the LOD in both mobile phases, while

the upper limit of LDR in micellar mobile phase is 3 times

that of the hydroorganic mobile phase. The LDR for anthra-

cene is 5 times greater in the micellar mobile phase as

compared to the hydroorganic mobile phases, while the LOD

is approximately the same in both mobile phases. It is hard

to determine the precise analytical figures of merit for

pyrene in the hydroorganic mobile phase because, as

Figure 14 shows, the analytical curve is practically non-

linear in that concentration range. Pyrene, however, should

be similar to anthracene, and, as shown in Figure 14, the

upper limit of the LDR for the micellar mobile phase is much

greater when compared to the hydroorganic mobile phase.

The greater upper limit of LDR in the micellar mobile

phase as compared to the hydroorganic mobile phase may be

due to the higher ionic strength (resulting from the pre-

sence of ionic surfactants as well as background electro-

lyte), in the micellar mobile phase [19,46] or due to the

nature of surfactants. Increasing the ionic strength of the

mobile phase decreases the IR gradient across the electrode

face and between the electrode and the bulk solution. Sur-

factants or surface active agents can change the interfacial







properties. Hence, the interfacial properties of the micel-

lar mobile phase with working electrode will be quite dif-

ferent from those of hydroorganic mobile phase.



Gradient Elution and Selectivity in Micellar Mobile Phase


Gradient elution with the micellar mobile phase is

primarily used for the same purpose as it is with the hydro-

organic mobile phase. Gradient elution may also be used to

effect a change in selectivity because micellar mobile

phases have unique selectivities at different concentrations.

Armstrong and Henry [42] have used gradient flow rates with

a UV detector. The gradient micellar chromatogram, obtained

with an amperometric detector using an anionic surfactant

(SDS) and a nonionic surfactant (Brij-35) are shown in

Figures 15 through 17. Figure 15 shows the separation of

B-6 vitamins with SDS, and Figures 16 and 17 show separation

of phenolic compounds with SDS and Brij-35, respectively.

Micellar mobile phases have been shown [47] to offer

control over selectivity in liquid chromatography as the

concentration of surfactant changes in the mobile phase.

The selectivity of the micellar mobile phase toward B-6

vitamins is shown in Figure 18. The elution order of PL and

PMP changes above and below a certain concentration range

of SDS, whereas they coelute in this range. The void volume

of the column, determined by water injection, slightly

decreased as the concentration of SDS increased in the

mobile phase. For an Altex Ultrasphere ODS 25 x 4.6 mm, a












Figure 15. Gradient Micellar Chromatogram for Separa-
tion of Vitamin B-6


Solvent A: (3:97) (1-propanol:H20),
pH = 2.20
Solvent B: 0.2 M SDS in (3:97)(1-propanol:
H20), pH = 2.20 (used H3PO4)


Gradient program: started with 25% solvent
B and held at 25% B for 8 min, then
ramped to 100% B over 8 min and contin-
ued with 100% B


Flow rate: 1 mL/min; temperature: 300C;
working electrode: glassy carbon elec-
trode; E = 1.3 V


Column: Altex Ultrasphere ODS, 250 x 4.6 mm;
precolumn: 15 x 4.6 mm, packed manually
with 25-40 pm silica gel


Injection volume: 10 iL


Peaks are as follows: (1) PLP (200 ppm),
(2) PL (200 ppm), (3) PMP (400 ppm),
(4) PNP (400 ppm), (5) PN (400 ppm)
































200 nA


24 12

t(min)














Figure 16. Gradient Micellar Chromatogram for Separa-
tion of Phenolic Compounds


Gradient program: started with 25% solvent
B and held at 25% B for 4 min, then
ramped to 100% B over 8 min and con-
tinued with 100% B


Injection volume: 13 pL; E = 1.1 V


Peaks are as follows: (1) hydroquinone
(10 ppm), (2) resorcinol (8 ppm),
(3) catechol (20 ppm), (4) phenol
(18 ppm), (5) o-cresol (arbitrary con-
centration)


Other conditions as Figure 15.














































N)


I


8 12 6
t(min)












Figure 17. Gradient Micellar Chromatogram for Separa-
tion of Phenolic Compounds and Gradient
with No Injection


Solvent A: (3:97)(1-propanol:H20),
pH = 2.20 (used H3PO4)
Solvent B: 0.1 M Brij-35 in (3:97)(1-pro-
panol:H20), pH = 2.20 (used H3PO4)


Gradient program: started with 20% sol-
vent B and held at 20% B for 4 min,
then ramped linearly to 100% B over
6 min and continued with 100% B


Flow rate: 1 mL/min; temperature: 300C;
working electrode: glassy carbon
electrode


Column: Altex Ultrasphere ODS, 150 x
4.6 mm


Injection volume: (a) 20 iL; (b) none


Peaks are as follows: (1) hydroquinone
(10 ppm), (2) resorcinol (8 ppm),
(3) catechol (20 ppm), (4) phenol
(18 ppm), (5) o-cresol (arbitrary
concentration); these are for (a)






























4 2 loo nA









5



(a)





(b)

I |i
16 8 0
t(min)




























Figure 18. Effect of SDS Concentration on k'


Mobile phase: (3:97)(1-propanol:H20) with
different concentrations of SDS,
pH = 2.20 (used H PO4)


Flow rate: 1 mL/min; temperature: 300C;
working electrode: glassy carbon;
E = 1.3 V


Column: Altex Ultrasphere ODS, 250 x
4.6 mm


(Q) PLP
(o) PL
(x) PMP
(A) PN and PNP















1,20





1.00 x

0



0.80





0.60





0,40





0.20





0.00 O





-0,20 '''
-1.40 -1.20 -1.00 -0.80 -0.60
log [SDS] (mM)







change in concentration of SDS from 0.05 M to 0.15 M or more

in the mobile phase caused the void volume to change from

2.20 mL to 2.00 mL. The adsorption isotherm of surfactants

on ODS-hypersil is reported by Knox and Hartwick [48]. The

surfactant concentrations chosen were below the CMC and one

SDS concentration supposedly above the CMC was also used.

The adsorption isotherms shown by these researchers are in

methanol:water (20:80) solution which results in a different

CMC from that of pure water. It is doubtful they ever

reached the CMC for their system. There is no literature

adsorption isotherm available for SDS above the CMC.

Figures 19 through 21 show the isocratic micellar chromato-

grams for B-6 vitamins and their selectivity with three dif-

ferent concentrations of SDS in the mobile phase.

















Figure 19. Isocratic Micellar Chromatogram for Separa-
tion of Vitamin B-6


Mobile phase: 0.05 M SDS in (3:97)(1-pro-
panol:H20), pH = 2.20 (used H3PO4)


Flow rate: 1 mL/min; temperature: 300C;
working electrode: glassy carbon elec-
trode; E = 1.3 V


Column: Altex Ultrasphere ODS, 250 x 4.6
mm; precolumn: 15 x 4.6 mm, packed
manually with 25-40 pm silica gel


Injection volume: 10 pL


Peaks are as follows: (1) PLP (200 ppm),
(2) PL (200 ppm), (3) PMP (400 ppm),
(4) PNP (400 ppm), (5) PN (400 ppm)





















S


20
t(min)


4 ;5
1


200oo nA


40


Ji


w-


U


























Figure 20. Isocratic Micellar Chromatogram for Separa-
tion of Vitamin B-6


Mobile phase: 0.1 M SDS in (3:97) (1-pro-
panol:H20), pH = 2.20 (used H3PO4)


Other conditions are as in Figure 19.










4;5


2;3


2oo nA


20 10 0
t(min)




























Figure 21. Isocratic Micellar Chromatogram for Separa-
tion of Vitamin B-6


Mobile phase: 0.2 M SDS in (3:97)(1-pro-
panol:H20), pH = 2.20 (used H3PO4)


Other conditions are as in Figure 19.























L


2oonA


t (min)


4;5


1


i


J












CHAPTER SIX

RAPID SEPARATION AND DETERMINATION OF THYROMIMETIC
IODOAMINO ACIDS BY GRADIENT ELUTION REVERSE PHASE LIQUID
CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION



The mechanism of action of the thyroid hormones, T3 and

T4, is of considerable interest in part because of the

amazing diversity of thyroid hormone effects. These agents

influence the metabolism of almost every class of foodstuff.

They exert profound effects on many enzymes and on almost

all organ systems, and they play an integral role in the

complex biological processes involved in growth and differ-

entiation [49]. The time-consuming procedures in USP mono-

graphs have been used for analysis of levothyroxine sodium

tablets [50] and liothyronine sodium tablets [511. The as-

say of the major thyroid hormones T3 and T4 also is done by

wet analysis [52-54], radioimmunoassay [55-58], chemical

derivatization followed by gas chromatography with electron

capture detection [59,60], thin-layer chromatography [61-63],

paper chromatography [64,65], electrophoresis [66], and gas

chromatography/mass spectrometry [67]. Thin-layer and paper

chromatography as well as the electrophoretic procedures do

not have good limits of detection for this kind of analysis.

Although the gas chromatographic procedures are sensitive,

they require the isolation of the iodoamino acids in a pure

form, which must then be converted to a volatile derivative








for chromatographic analysis. The radioimmunoassay proce-

dures are impractical for a small number of samples and have

the added problem of disposal of the radioactive wastes.

Liquid chromatography would normally be the method of

choice for these analyses because of the poor volatility of

the compounds. In fact, HPLC methods for separation of pure

iodoamino acids have appeared in the literature [68-72].

Recently, T3 and T4 tablets have been analyzed by reverse

phase LC using UV detection [73], and a gradient elution

separation of sixteen thyromimetic iodoamino acids has been

reported [74]; however, the low molar absorptivity of these

compounds at 254 nm precludes their determination at trace

levels. Detection at 220 nm improves this situation some-

what [74], and a clever catalytic post-column detection

scheme has also been shown [75]. Most recently, application

of amperometric electrochemical detection to these compounds

has been shown to give excellent limits of detection [76],

as has dansyl derivatization and subsequent fluorescence

detection [77].

The purpose of this study was to demonstrate the use-

fulness of gradient elution techniques with electrochemical

detection for the separation of seven thyromimetic iodoamino

acids. A rapid isocratic separation of TO, T2, T3 and T4,

as well as analysis of T4 both in tablets and injectable

intravenous samples is presented.







Standard Solutions


The compounds Tyr, MIT, DIT, TO, T2, T3 and T4 were

purchased from Sigma Chemical (St. Louis, Missouri) and

were stored in a freezer. Standard solutions were prepared

by dissolving appropriate amounts of each compound in meth-

anol containing 1% ammonium hydroxide and were stored in a

refrigerator.



Preparation of T4 Tablet Solution
and Injectable T4 Sample


Twelve tablets (1.5676 g) containing levothyroxine

sodium were dissolved in 20 mL of 0.01 M sodium hydroxide

using an ultrasonic bath. The sample solution was heated at

600C for 3 min, shaken for 3 min, and then filtered through

F2406-9 (S/P) filter paper. Before chromatographic injec-

tion, this solution was again filtered with a Rainin

(Rainin Instruments, Woburn, Massachusetts) HPLC sample

filter syringe using a 0.45 pm nylon-66 membrane filter.

The injectable sample was present as a powder and was pre-

pared by dissolving in 5 mL 0.9% sodium chloride solution.

This resulted in a clear solution which was then filtered

with the sample filter syringe.



Gradient Elution LC/EC


Amperometric electrochemical detectors are generally

considered incompatible with gradient elution




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INGEST IEID EGPSYTB6V_029780 INGEST_TIME 2011-08-24T12:45:52Z PACKAGE AA00003432_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



GRADIENT ELUTION, IMPROVED SEPARATIONS
AND ANALYTICAL FIGURES OF MERIT IN
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
USING ELECTROCHEMICAL DETECTION
BY
MOHAMMAD REZA HADJMOHAMMADI
A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLOPIDA
1983

To my parents, brother and sisters

ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to
Dr. John G. Dorsey for his acceptance, guidance, advice,
patience, concern and support throughout my work.
My sincere thanks are also due to Dr. J. D. Winefordner,
Dr. R. A. Yost, Dr. A. Brajter-Toth, Dr. C. M. Riley,
Dr. R. G. Bates, and Dr. J. L. Ward for their encouragement,
advice and support. I would also like to thank my colleagues
and friends in Dr. Dorsey's research group, all of whom have
contributed much to my progress and have made my course of
study pleasant and fruitful.
Acknowledgement is also due to the donors of the
Petroleum Research Fund, administrated by the American Chemical
Society, for support of this research and to Nelson H. C. Cooke,
Altex Scientific, for a gift of columns.
Sincere thanks are also due to Laura Griggs for her
patience, helpful hints, and expert typing.
Finally, mere thanks are not enough to my parents,
brother, and sisters, whose sacrifices for me can never be
repaid.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
KEY TO ABBREVIATIONS ix
ABSTRACT xi
CHAPTER
ONE INTRODUCTION 1
TWO THEORY AND BACKGROUND 10
Basic Parameters in Chromatography 10
Capacity Factor (k1) 10
Peak Resolution (Rs) 11
Efficiency (E) 11
Control of Separation in Liquid
Chromatography 12
Gradient Elution (GE) 13
Limit of Detection 14
Principles of Electrochemical Detection 15
Ion-pair Chromatography 16
Micellar Chromatography 18
THREE EXPERIMENTAL 21
Cyclic Voltammetry Systems 21
Liquid Chromatography System 21
Electrochemical Detector 22
Pretreatment of Glassy Carbon Electrode 24
Reagents 26
IV

Page
FOUR SIGNAL CHANGE AND BASELINE SHIFT USING
ELECTROCHEMICAL METHODS 27
Change of Residual Current with DC, DP
and NP Voltammetry in Isocratic Elu¬
tion HPLC 29
Effect of Background Electrolytes and
Gradient Elution on Baseline Shift
Using Amperometric Detection 37
Effect of the Electrode Material on
Baseline Shift Using Amperometric and
NRDP Voltammetry Methods 4 3
FIVE A COMPARISON OF MICELLAR AND HYDROORGANIC
MOBILE PHASES USING AMPEROMETRIC DETECTOR... 46
Hydrodynamic Voltammogram in Micellar
and Hydroorganic Mobile Phases 47
Analytical Figures of Merit Comparison
between Micellar and Hydroorganic
Mobile Phases 56
Gradient Elution and Selectivity in
Micellar Mobile Phase 69
SIX RAPID SEPARATION AND DETERMINATION OF THYRO-
MIMETIC IODOAMINO ACIDS BY GRADIENT ELUTION
REVERSE PHASE LIQUID CHROMATOGRAPHY WITH
ELECTROCHEMICAL DETECTION 8 5
Standard Solutions 87
Preparation of T4 Tablet Solution and
Injectable T^ Sample 87
Gradient Elution LC/EC 87
Isocratic Separations 91
Assay of T^ Preparations 98
SEVEN CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK. 103
Conclusions 103
Suggestions for Future Work 104
REFERENCES 10 7
BIOGRAPHICAL SKETCH 112
v

LIST OF TABLES
Page
1 Residual Current Change, AI, and Decay Current,
'’'decay' ;’'n D<~' an(^ NP Voltammetry in Isocratic
Elution 35
2 Normalized Baseline Shift (nA/mm2) during
Gradient Elution with No Sample Injection 44
3 Analytical Figures of Merit for Phenol 57
4 Analytical Figures of Merit for B-6 Vitamins 59
5 Analytical Figures of Merit for Polyaromatic
Hydrocarbons 61
6Analytical Figures of Merit for T^, T^ and T^.... 100
vi

LIST OF FIGURES
Page
1 Electrochemical Cell with Glassy Carbon Electrode 23
2 Residual Current Change and Decay Current with 5%
CH3CN 30
3 Residual Current Change and Decay Current with
50% CH3CN 31
4 Residual Current Change and Decay Current with
95% CH3CN 32
5 Residual Current Change by NP Voltammetry 33
6 Specific Conductance vs. Percentage of Solvent B. 39
7 Baseline Shift during Gradient Elution with No
Sample Injection, Using an Amperometric Detector. 42
8 Chemical Structures of Compounds Used in Chapter
Five 48
9 Hydrodynamic Voltammogram for Phenol 50
10 Hydrodynamic Voltammogram for B-6 Vitamins 52
11 Hydrodynamic Voltammogram for Polyaromatic Hydro¬
carbons 54
12 Analytical Curves for Phenol 63
13 Analytical Curves for B-6 Vitamins 65
14 Analytical Curves for Polyaromatic Hydrocarbons.. 67
15 Gradient Micellar Chromatogram for Separation of
Vitamin B-6 71
16 Gradient Micellar Chromatogram for Separation of
Phenolic Compounds 7 3
17 Gradient Micellar Chromatogram for Separation of
Phenolic Compounds and Gradient with No Injection 75
Vll

Page
18 Effect of SDS Concentration on k' 77
19 Isocratic Micellar Chromatogram for Separation of
Vitamin B-6 ; 80
20 Isocratic Micellar Chromatogram for Separation of
Vitamin B-6 82
21 Isocratic Micellar Chromatogram for Separation of
Vitamin B-6 84
22 Baseline during Gradient Program with Blank
Injection 90
23 Separation of Seven Thyromimetic Iodoamino Acids. 93
24 Thyromimetic Iodoamino Acids Used in This Study.. 94
25 Cyclic Voltammogram 95
26 Hydrodynamic Voltammogram for 1^, T^ and T^ 97
27 Analytical Curves for T^, T^ and'T^ 99
28 Isocratic Separation of T2, T^ and T^ 101
viii

KEY TO ABBREVIATIONS
BPC
CMC
CV
DC
DP
EC
GC
GCE
GE
HDV
HPLC
IP
LC
LC/EC
LC/MS
LDR
LLC
LOD
MSRTP
NMR
NP
NRDP
Bonded-phase chromatography
Critical micelle concentration
Cyclic voltammetry
Direct current
Differential pulse
Electrochemical detection or electrochemical
detector
Gas chromatography
Glassy carbon electrode
Gradient elution
Hydrodynamic voltammogram
High performance liquid chromatography
Ion-pair
Liquid chromatography
Liquid chromatography with electrochemical
detection
Liquid chromatography mass spectrometry
Linear dynamic range
Liquid-liquid chromatography
Limit of detection
Micelle-stabilized room-temperature phosphorescence
Nuclear magnetic resonance
Normal pulse
Non-ramping differential pulse
ix

PAH
Polyaromatic hydrocarbon
PAR
Princeton applied research
RP
Reversed-phase or reverse-phase
RP-HPLC
Reversed-phase high performance liquid chromatog¬
raphy
RP-LC
Reversed-phase liquid chromatography
RP-LC/EC
Reversed-phase liquid chromatography with electro
chemical detection
SDS
Sodium dodecyl sulfate
WE
Working electrode
X

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
GRADIENT ELUTION, IMPROVED SEPARATIONS
AND ANALYTICAL FIGURES OF MERIT IN
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
USING ELECTROCHEMICAL DETECTION
By
Mohammad Reza Hadjmohammadi
December 1983
Chairman: Dr. John G. Dorsey
Major Department: Chemistry
The goal of this work was an improvement and better
understanding of electroanalytical methods used as liquid
chromatographic detection techniques. Particularly, methods
which would allow the use of chromatographic gradient elu¬
tion with electrochemical detectors were investigated. Both
amperometric and pulse techniques were investigated with
traditional hydroorganic mobile phases. The use of gradient
elution with micellar mobile phases was shown to allow com¬
patibility with electrochemical detectors. A further com¬
parison of analytical figures of merit was made between
hydroorganic and micellar mobile phases using electro::} emical
detection
A comparison of baseline shift during gradient elution
with amperometric and nonramping differential pulse (NRDP)
xi

methods was performed using both glassy carbon and gold
electrodes. The composition of the mobile phase is virtu¬
ally constant during one pulse with gradient elution, but
high residual current changes preclude routine use of this
technique. A more stable baseline was achieved with an
amperometric detector and a glassy carbon electrode, than
it was otherwise with NRDP method. A higher concentration
of phosphoric acid in the organic modifier as opposed to
equal concentrations in both modifier and water produced a
more stable baseline during gradient elution using a glassy
carbon electrode and an amperometric detector.
The hydrodynamic voltammograms and analytical figures
of merit for phenol, two B-6 vitamins, and polyaromatic
hydrocarbons were compared in micellar and hydroorganic
mobile phases. The limit of detection in both mobile phases
was comparable, whereas the upper limit of linear dynamic
range was greater in micellar mobile phases.
Gradient chromatograms for separation of phenolic com¬
pounds and B-6 vitamins with an anionic surfactant and
phenolic compounds with a nonionic surfactant using ampero¬
metric detection are shown. Selectivity of micellar mobile
phases toward B-6 vitamins changes with surfactant concen¬
trations .
A rapid separation of thyroxine and related thyroid
hormones is shown using gradient elution and electrochemical
detection. A five minute isocratic separation of thyroxine
and three related hormones is also reported. Limits of
Xll

detection are in the sub-nanogram range with an upper limit
of linear dynamic range of 500 to 1000 nanograms for
compounds. Analysis of levothyroxine sodium tablets
injectable intravenous samples is described.
xiii
these
and

CHAPTER ONE
INTRODUCTION
Chromatographic methods can be classified according to
a number of schemes. The major one is based on the state of
the mobile phase. If the mobile phase is a gas, the method
is called gas chromatography (GC). If the mobile phase is a
liquid, the method is named liquid chromatography (LC).
Liquid and gas chromatography are each divided according to
the nature of the stationary phase. When the solid station¬
ary phase has adsorption properties, the process is called
adsorption chromatography, and when the stationary phase is
a liquid supported by an inert matrix, the process is called
partition chromatography.
Special classifications have also been introduced. For
example, chromatography can be classified according to
whether the stationary phase is present as a thin-layer, a
paper, or a column. Chromatography can alternatively be
classified with respect to the flow of the mobile phase;
this classification includes one-way, two-dimensional, and
radial chromatography. According to the mechanism of the
retention, i.e., the interaction between solutes and the
stationary phase, chromatographic methods are classified as
adsorption, partition, ion-exchange, and gel permeation.
1

2
Finally, the chromatographic technique may be classified
according to the kind of sample introduction onto the sta¬
tionary phase and migration through the system. This gives
rise to development, elution, displacement and frontal anal¬
ysis chromatography. The first two techniques are the most
common, while the last two methods are relatively special¬
ized and, therefore, of limited value and use.
In 1941, Martin and Synge [1] were led to contrive a
scheme of liquid-liquid chromatography for separation of the
amino acids of a wool hydrolyzate with a countercurrent
extractor [2]. To improve the efficiency of the counter-
current method, Martin and Synge considered a means of
immobilizing one phase while the second phase flowed over
it in such a manner as to maximize the contact of the im¬
miscible liquids at the interface. Martin believed this
would facilitate the rapid distribution, or partitioning, of
the solutes between the two phases. The efficiency of their
partitioning column was almost 104 times greater than that
of the countercurrent method [3].
The difference between modern liquid chromatography and
traditional column chromatography (whether adsorption, par¬
tition or ion-exchange) involves improvements in equipment,
materials, technique, and the application of theory. Modern
liquid chromatography provides more convenience, better
accuracy, higher speed, and the ability to carry out diffi¬
cult separations. At the beginning of the 1970's, the
modern form of liquid chromatography was named high pressure

3
liquid chromatography (HPLC). Later, the P for "pressure"
was replaced by P for "performance." The reason was the
appearance of microparticles that allowed researchers to
perform, at a lower pressure drop, the same efficient and
rapid analyses done with other supports.
In conventional liquid-liquid chromatography (LLC), the
stationary phase is a bulk liquid, mechanically held to the
support by adsorption. In recent years, organic phases are
chemically bonded to the support, leading to a separate LC
method called bonded-phase chromatography (BPC). Bonded-
phase chromatography is the most widely used in modern LC,
and many laboratories use BPC columns for their LC separa¬
tion. In contrast to LLC, bonded-phase chromatography pack¬
ings are quite stable because the stationary phases are
chemically bound to the support and cannot be easily removed
or lost during use. The availability of a wide variety of
functional groups in BPC packings allows for both normal-
and reversed-phase chromatography.
Polar BPC packings are used for normal-phase separa¬
tions. Samples of moderate to strong polarity are usually
well separated by normal phase-chromatography. The mobile
phase in normal phase chromatography is a hydrocarbon sol¬
vent such as hexane, heptane, or isooctane, plus small
amounts of a more polar solvent. The mobile phase strength
can be varied for a given application by varying the concen¬
tration of the more polar solvent component. Reverse phase

4
BPC normally involves a relatively nonpolar stationary phase
(e.g., CD or C.„ hydrocarbon) used in conjunction with polar
o io
(e.g., aqueous) mobile phases to separate a wide variety of
less polar solutes.
The general reaction for preparing bonded-phase pack¬
ings on silica-based supports to produce siloxanes is:
CISi(Me)2R
Si-OH + or * Si-O-Si(Me)2R + HC1
R'OSi(Me)2R
where R is the desired organic moiety and, for reversed-
phase (RP) packings, is an n-alkyl chain (n = 2,8,18). This
reaction is based on silanol groups on the surface of the
siliceous support. Fully hydrolyzed silica contains about
8 ymol of silanol groups per m2. Because of steric hin¬
drance, a maximum of about 4.5 ymol of silanol groups per m2
can be reacted at best [4], and an end-capping process
usually can be done to cover most of the residual silanols
by a reaction similar to the one above using chlorotrimethy1-
silane. The concentration of the organic moiety per m2 of
BPC packings depends upon the surface area of the packing
particles (e.g., pellicular or porous support). Siloxane
bonded-phase packings are available with pellicular or
totally porous supports. Bonded-phases of this type are
hydrolytically stable throughout the pH range 2-8.5. Due to
the different methods for the preparation of BPC packings
and shielding the residual silanol groups by end-capping,
the surface coverage and overall volume of the organic

5
stationary phase tends to show large differences from manu¬
facturer to manufacturer, and even from lot to lot. Without
end-capping of the residual silanol groups, a mixed-retention
mechanism can result and lead to asymmetric peaks.
Increasing the alkyl chain length on BPC results in an
increase in selectivity and retention times [5,6] (when the
columns were compared) using the same mobile phase. The
study of chain length (C-C„„) of bonded organic phases
showed that the selectivity [7] depended on the chain length
of the bonded phase and on the molecular structure of the
solute. The same study showed that the utilization of long
chain phases made it possible to reduce the water content
of the water:methanol mobile phase, which increases the
efficiency and loading capacity. Hemetsberger et al. [8,9]
studied the behavior and the effect of structure of bonded
phases. Kikta and Grushka [10] studied the retention behav¬
ior on alkyl bonded phases as a function of chain length,
surface coverage, solute type, mobile phase composition, and
temperature. Colin and Guiochon [11] compared the resolu¬
tion of RPC packings As a result, the shorter
length alkyl chain columns generally gave the worst resolu¬
tion and efficiency. A comparative study on the separation,
efficiency under optimum mobile phase conditions with three
different mobile phases, and three groups of solutes on
three commercially available alkyl bonded phases (CCg and
C10) was done by Haleem [12].
In high-performance liquid chromatography (HPLC), as in
all analytical methods, the trend is to do it faster and

6
cheaper, make it more selective and sensitive, and combine
it with other methods. Selection of column type in LC has
been more restricted because of the viscosities and solute
diffusivities in the liquid phase which are orders of mag¬
nitude different from the values in GC. The most efficient
columns presently used in LC are those packed with totally
porous small particles (with particle size down to a few
micrometers). Although substantial reductions of the plate
height are achieved while decreasing the particle size,
there are some practical limits to this procedure. As
pointed out by Halasz [13], there are difficulties in the
uniform packing of very small particles, as well as the
problem with evolved heat of friction.
Microbore HPLC as now commercially available uses 1-
to 2-mm diameter columns. The next generation of columns,
studied only in research laboratories for the past few
years, may be inner-coated microtubular or packed microcap¬
illary columns, just 50 ym in diameter. The advantage of
these would be hundreds of thousands of theoretical plates
in a very long length at low cost. Eluents from these col¬
umns also could be fed into a mass spectrometer with low
interference from the solvent. These small diameter columns
often require new instruments or modifications of existing
instruments to meet new needs in injection, pumping, or
detection.
The rapid progress in HPLC places great demands on
detection techniques. Unfortunately, the highly developed

7
GC detectors are mostly useless at this time in LC because
of principal differences between LC and GC. An ideal detector
should be universal; however, the great diversity of systems
to be analyzed makes the construction of a sensitive univer¬
sal detector impossible, and thus detectors monitoring vari¬
ous physicochemical properties of substances are employed,
the optimal detection conditions being determined specific¬
ally for each system. It is possible to measure either bulk
properties, which depend on the variation of the composition
of the system (e.g., refractive index, electrical conduc¬
tance, etc.), or properties that selectively characterize
certain components in the mobile phase (such as the absor¬
bance at a certain wavelength, fluorescence, electric current
at a certain electrode potential, etc.). The bulk property
detectors are universal detectors, and they require that
the properties of the solutes be substantially different
from those of the mobile phase to attain sufficient signal
changes during detection. The bulk property detectors are
usually less sensitive and subject to a higher noise than
the measurement of specific properties of the solutes, and
they are rarely compatible with gradient elution. On the
other hand, the measurement of specific properties requires
that the mobile phase yields the lowest possible signal
under the given conditions.
Currently available commercial LC detectors are based
on a number of detection principles, including absorbance,
fluorescence, refractive index, electrochemical reaction,

8
and mass spectrometry. A number of these detectors are
likely to be improved in the next few years. For instance,
new types of LC/MS interfaces will probably appear, and
currently available interfaces should be further refined.
Nuclear magnetic Resonance (NMR) detectors should material¬
ize, and GC detectors are being seriously considered for
their applicability to LC detection. The development of
laser based detectors [14] for chromatography is in progress
and in many cases offer better sensitivity and selectivity
than conventional LC detectors. The complexity and expenses
of the laser based detectors have delayed the acceptance of
these detectors in the laboratory.
Attempts to use electrochemical detection of molecule(s)
in effluents from chromatographic columns were made long
before the advent of HPLC. The first papers dealing with
polarographic detectors were those of Drake [15] and
Kemula [16]. Present electrochemistry offers a large group
of methods that can be used for continuous detection of
substances [17]. The field of electrochemical HPLC has been
reviewed several times [17-19]. A survey of scientific
papers on LC detector usage during the 1980-81 period showed
that 4.3% of LC analyses were based on electrochemical
detectors [20].
The suitability of electrochemical detection to a given
problem ultimately depends on voltammetric characteristics
of the component(s) of interest in a suitable mobile phase
and a suitable working electrode surface. Electrochemical

9
detection is more limited with respect to the mobile phase
composition than other LC detection methods because of the
fact that a complex surface reaction which depends on the
medium is involved.
Direct electrochemical detection is not likely to be
useful in normal-phase chromatography since nonpolar organic
mobile phases are not well suited to many electrochemical
reactions. The HPLC stationary phases of choice clearly
include all ion-exchange and reverse-phase (RP) materials
since these are compatible with polar mobile phases contain¬
ing some dissolved ions. The ionic strength, pH, electro¬
chemical reactivity of the mobile phase and background elec¬
trolyte, and presence of electroactive impurities (dissolved
oxygen, halides, trace metal ions) are all important con¬
siderations .
The choice of electrode material is one of the important
considerations, because of the ruggedness, potential range,
residual current, and long-term stability requirements.
Electrode materials such as platinum, glassy carbon, gold,
and mercury films may work well in some cases but may be
disastrous in others. These electrodes are subject to com¬
plicated surface renewal problems but are not mechanically
awkward devices such as the dropping mercury electrode.
With all limitations mentioned, liquid chromatography with
electrochemical detection (LC/EC) has three distinct
advantages for applicable systems, namely, selectivity,
sensitivity and economy.

CHAPTER TWO
THEORY AND BACKGROUND
Basic Parameters in Chromatography
Capacity Factor (k1)
The capacity factor is equal to n /n , where n and n
are the number of moles of solute in the stationary and
mobile phase, respectively. Therefore, k' can be written
according to the following:
k'
n
s
n
m
[X] V
s s
[X] V
mm
KV
<
(1)
where
[X] = concentration of solute X in the sta¬
tionary phase
[X] = concentration of solute X in the mobile
m u
phase
Vs = volume of the stationary phase
V = the total volume of the mobile phase within
m the column
K = distribution constant
tD = retention time of solute X
I\
tg = time for mobile phase or other unretained
molecules to pass through column
10

11
Peak Resolution (Rs)
By convention, peak resolution, R , is defined as the
ratio of the distance between the two peak maxima (At) to
the mean value of the peak width at base (W^).
At
w + w
bl b2
2 At
Wbl + Wb2
(2)
For two closely spaced peaks, one can assume that the two
peak widths are the same, and can be used instead of the
mean.
Efficiency (E)
Efficiency of chromatography, E, is defined as peak
retention time divided by peak width at base [21].
E =
(3)
The theoretical plate number, N, contains the same informa¬
tion as E.
N =
ft 1
2
t
R
= 16
R
Ia
[Wbj
= 16E'
or
N = 5.54
-R
W,
l
(4)
(5)
where a and are the standard deviation and peak width at
half height of the peak, respectively. An equation derived

12
by Foley and Dorsey [22] can be used for the calculation of
the number of theoretical plates of skewed peaks in a chro¬
matographic system (NSyS)•
41-7(Vwo.it
Nsys - B/A + 1-25 161
where t , Wn and B/A are retention time, peak width at
K U • 1
10% of peak height, and asymmetry factor, respectively.
Control of Separation in Liquid Chromatography
The key to separating components of a mixture is to
control resolution.
R
s
h (ot—1) v/Ñ
(l + k'2)
(7)
An increase of separation factor, a, which is the ratio of
two capacity factors, k^/k'^, results in a displacement of
one band center relative to the other and a rapid increase
in Rg. Increasing the number of theoretical plates narrows
the bands while increasing the peak height. For early
eluting peaks, an increase in k' can provide a significant
increase in resolution, however, with increasing k', band
height decreases and separation time increases.
The available options for increasing, a, in order of
decreasing utility are: change of stationary phase, change
of temperature, and special chemical effects. Increasing
the number of theoretical plates can be done by increasing
column length and decreasing flow rate for a given column.

13
Capacity factor can be increased by increasing the volume
of stationary phase, decreasing the strength of mobile
phase, and decreasing temperature.
Gradient Elution (GE)
The most convenient way to separate a complex mixture
of solutes is to use gradient elution. Gradient elution in
LC is similar to temperature programming in GC, except that
the composition of the mobile phase is changing during
separation time. To do GE, one needs at least two different
solvents (binary gradient). One of the solvents has higher
eluent strength, and usually its percentage increases during
the gradient. Ternary gradients using three solvents are
sometimes used in LC. Multisolvent gradients are rarely
required in LC, and because of the complexity, one usually
avoids the use of multisolvent gradients.
The purpose of gradient elution is to resolve early
eluting bands and decrease the retention time of strongly
retained compounds in comparison to isocratic elution. To
achieve this, the gradient must start with a weak mobile
phase, and the strength of the mobile phase increases during
the chromatographic run. Because of decreasing retention
times for late eluting bands, these peaks are greatly
sharpened in gradient elution when compared to isocratic
elution, and sensitivity for these bands is therefore much
improved. Gradient elution increases the peak capacity for
a mixture that contains a large number of individual

14
components and improves the peak shapes for bands that would
tail in isocratic elution. The gradient shape for binary-
solvents can be linear, concave, convex, or any other shape.
The appropriate gradient shape is dependent on LC methods,
sample, and solvent composition. The steepness of the gra¬
dient is the mobile phase strength change with time.
Limit of Detection
The limit of detection represents the ability of an
analytical method for quantification of a chemical component
in terms of concentration or absolute amount. In most ana¬
lytical methods, the limit of detection, C , is defined
Li
according to the following equation.
C
L
KS.
b
m
(8)
where and m are the standard deviation of the blank and
slope of the calibration curve, respectively. On a statis¬
tical basis, K = 3 is the most appropriate number for cal¬
culation of the detection limit. For more information about
detection limits, one is referred to articles by Kaiser [23]
and Winefordner [24].
To calculate the detection limit in LC, the peak to
peak noise is measured while mobile phase passes through the
column. In a normal distribution, peak to peak or random
noise in LC is considered to be 5 times the standard devia¬
tion of the blank, as in equation (8). Detection limit in

15
LC is usually defined as 3 times the peak noise or 3/5 of
the peak to peak noise divided by the slope of the calibra¬
tion curve. The latter was used for reporting detection
limits in this dissertation.
Principles of Electrochemical Detection
The most common detector in LC/EC is the amperometric
detector which measures the current at constant potential.
The amperometric detector is more sensitive and less complex
than the coulometric detector. In a coulometric detector,
the amount of electricity for complete electrochemical con¬
version of the analyte is measured at constant potential.
The lower sensitivity of the coulometric detector is due to
geometrical requirements necessary for complete electrochem¬
ical conversion in a flowing stream. The requirement of
larger surface area of working electrode causes higher back¬
ground current and noise which reduces the signal to noise
ratio in comparison to amperometric detector.
The technique of electrochemical detection is based
on electroactivity of components eluted from the column.
Sometimes it is possible to detect nonelectroactive compo¬
nents by pre- or post-column derivitization. Selecting the
applied working electrode potential is the primary require¬
ment in amperometric detection. The applied potential
should be held at the minimum value at which the current
reaches the limiting-current plateau of the analyte
(E , . ) , however, in most of the cases, E , . is
plateau plateau

16
different for different components of a mixture to be ana¬
lyzed. In this case the analyst should choose the optimum
applied potential to analyze all components of interest in
the mixture. The applied or analytical potential for an
analyte can be determined by a hydrodynamic voltammogram
(HDV). In an HDV, current is measured versus applied poten¬
tial for analyte injected into an LC amperometric detection
system. The Ep^ateau can be precisely identified by HDV
measurement under analytically useful LC conditions, but it
is a time-consuming process, due to the time required for
the baseline to stabilize after each change of electrode
potential. The time required for stabilization of the base¬
line is dependent upon the mobile phase composition and flow
rate. In the case of a glassy carbon electrode, for a change
of 0.1 V in applied potential, it takes 15 to 30 min to get
a stable baseline. Cyclic voltammetry (CV) is a much faster
method for determining Ep^ateau' which usually has a higher
magnitude than the CV peak potential (E ) under typical
P
measurement conditions for slow electron transfer reaction.
Data from CV and Ep^ateau can be related via a simple
empirical equation [25].
Ion-pair Chromatography
The extraction of ionized solutes into organic phases
has been well known for a number of decades. To extract
ionized species from aqueous solution, an ion-pairing reagent
of opposite electrical charge is added to the aqueous phase

17
resulting in ion-pairing between the solute ion and pairing
ion. The resultant complex which has a net low electrical
charge or polarity can easily be extracted by an organic
phase. To separate ionic species by reversed-phase HPLC,
an ion-pairing reagent can be added to the mobile phase.
Ion-pairing reagents can also be used as a probe to detect
and quantify compounds which cannot be directly detected
[26,27]. The following scheme shows overall phase transfer
of ion pairs.
A+ + B
(A, B)
aqueous
phase
organic
phase
In the case of reversed-phase chromatography, the organic
and aqueous phases on the above scheme are considered to be
stationary and mobile phases, respectively. The most popular
ion-pair reagents for cationic solutes are long-chain alkyl
sulfonate ions which are usually added to the mobile phase
to enhance separation of oppositely-charged sample ions.
The exact ion-pairing mechanism for the separation of
ionic samples is still uncertain. Three popular hypotheses
are: (1) the ion-pair model, (2) the dynamic ion-exchange
model, and (3) the ion-interaction model. The ion-pairing
model stipulates that the formation of an ion-pair occurs in
the aqueous mobile phase which is in agreement with solvo¬
phobic theory [28], while the dynamic ion-exchange model
states that unpaired lipophilic alkyl ions adsorb onto the
nonpolar stationary phase, causing the column to behave as

18
an ion exchanger [28]. The ion-interaction model is based
upon conductance measurements. It proposes that neither the
ion-pairing nor the ion-exchange model can explain the ex¬
perimental data in a consistent way [28]. The ion-interac¬
tion model assumes that a primary layer of lipophilic ion
covers the surface of the stationary phase which is in
dynamic equilibrium with the bulk eluent. In the vicinity
of this primary layer exists a secondary layer of opposite
charge creating an electrical double layer on the surface.
The retention of the ionic components is due to the electro¬
static force between these ions and the primary layer, as
well as an additional (sorption) effect onto the nonpolar
stationary phase.
Micellar Chromatography
It is well known that surfactants, detergents, or
surface active agents are amphiphilic molecules (i.e.,
molecules in which a hydrophobic tail is joined to a hydro¬
philic head-group). Surfactants can be anionic, cationic,
nonionic, and zwitterionic. Above a certain concentration,
surfactant molecules associate in aqueous solution to form
large molecular aggregates of colloidal dimensions termed
micelles. The concentration threshold at which a surfac¬
tant starts to form micelles is called the critical micelle
concentration (CMC), and the number of surfactant molecules
in a micelle is called the aggregation number. The aggrega¬
tion number and the CMC differ from one surfactant to

19
another, and even for the same surfactant in different media.
At concentrations greater than the CMC, a dynamic equilibrium
exists between the surfactant molecules and micelles. The
general size and shape of the particular micelle depend on
the aggregation number.
The term normal micelles is used for surfactant aggrega¬
tion in aqueous media. The hydrophilic head groups are
directed toward and in contact with aqueous solution to
form a polar surface, while the hydrophobic tails are
directed away from the water to form a central nonpolar core.
In nonpolar solvents, the surfactant aggregates are termed
reversed or inverted micelles. In these micelles, polar
head groups are concentrated in the interior of the aggre¬
gates and hence form a central hydrophilic core, while the
hydrophobic tail moieties extend into and are in contact
with the bulk nonpolar solvent.
The solubilizing power of micellar systems is one of
its most important aspects. This refers to the ability of
micelles to solubilize a wide variety of solutes that are
insoluble or only very slightly soluble in the bulk solvent
alone. The solubilization of solutes in micellar systems is
a dynamic process and depends upon such factors as the tem¬
perature, the nature of solutes, the surfactant concentra¬
tion, and the type of micelles. The amount of solute
solubilized is usually directly proportional to the concen¬
tration of micelles. The solubilization of a solute at a
micelle site is dependent upon the type of solute and the

20
nature of the micelle. In a normal micelle, a nonpolar
solute is thought to be located near the center of the
hydrophobic core,while an ionic solute is adsorbed on the
polar micellar surface.
According to the properties of micellar systems men¬
tioned above, micellar solutions can be used as mobile
phases in HPLC. The normal micellar solution can be used
as the mobile phase in reversed-phase HPLC, while reversed
micellar solutions are compatible with normal-phase HPLC.
Equations for partitioning behavior of solutes with micellar
mobile phases in LC have been derived by Armstrong and
Nome [29]. From these equations, one can calculate the
partition coefficients of solutes between water and
micelles, between the stationary phase and water, and be¬
tween micelles and the stationary phase. One of the draw¬
backs of micellar mobile phases in RP-HPLC is its poor
efficiency in comparison to hydroorganic mobile phase,
however, Dorsey et al. [30] showed that by the addition of
3% of propanol and a temperature of about 40°C, micellar
mobile phases can approach efficiencies of hydroorganic
mobile phases. Possible advantages of micellar mobile
phases over hydroorganic mobile phase are: (1) the unique
selectivity of micellar mobile phases toward different
types of solutes, (2) the economy when compared to hydro-
organic mobile phases, and (3) the simplicity of purifica¬
tion of the crystalline surfactants compared to organic
solvents.

CHAPTER THREE
EXPERIMENTAL
Cyclic Voltammetry Systems
To find an approximate analytical potential for solutes
of interest, a CV-1A cyclic voltammetry instrument and an
electrochemical cell made by Bio Analytical Systems, Inc.
(West Lafayette, Indiana) were used. The working and ref¬
erence electrodes were glassy carbon and Ag/AgCl, respec¬
tively. Before running the CV experiments, the sample solu¬
tions were purged for 20 min with helium. Cyclic voltammetry
was then carried out in an inert helium atmosphere. A
Plotamatic MFE-715 (MFE, Salem, New Hampshire) X-Y recorder
and digital voltmeter were used to record the cyclic voltam-
mograms.
Liquid Chromatography System
The solvent delivery unit used during the chromato¬
graphic run was a Waters 6000 A (Waters Associates, Milford,
Massachusetts), an Altex model 322 gradient liquid chro¬
matograph with two model 100 A pumps (Altex Scientific,
Berkeley, California), or a Spectra-Physics SP 8700 solvent
delivery system (Spectra-Physics, Santa Clara, California).
21

22
The injection valve was either an Altex 210 or Rheodyne 7125
(Rheodyne, Cotati, California) with 5, 10 and 20 yL loops.
Various columns—an Altex Ultrasphere octyl, 250 x 4.6 mm;
an Altex Ultrasphere ODS, 150x4.6 mm; and Rainin Microsorb
octyl column, 150 x 4.6 mm—were employed.
Electrochemical Detector
The electronic controller was an LC-4 amperometric
controller from Bio Analytical Systems, Inc. or Princeton
Applied Research (PAR, Princeton, New Jersey) model 174
polarographic analyzer. The electrochemical cell used was
either from Bio Analytical Systems, Inc., with glassy
carbon as working electrode, or a porous membrane separator
with gold as the working electrode, a generous gift of
K. A. Rubinson. The latter working electrode was used only
in work of Chapter Four of this dissertation to compare
electrode material, while the former was used from
Chapter Four through Chapter Six. The reference electrode
for both cells was Ag/AgCl from Bio Analytical Systems,
Inc.
The electrochemical cell from Bio Analytical Systems,
Inc. [31], is a thin-layer cell, as shown in Figure 1. The
thin-layer cell, reference electrode compartment, clamp,
and waste and connecting tubes are preassembled as one unit.
Addition of a reference electrode to this unit completes
the detector cell. All of the detector cell components
have been machined to accept standard plastic tube end

23
Reference ^
Electrode
Auxiliary
Electrode
To Waste
Electrode Positioned
Downstream From Inlet
Port
From LC
Column
Figure 1.
Electrochemical Cell with Glassy
Carbon Electrode

24
fittings (%-28 thread) as supplied by BAS, Altex, LDC,
Omnifit, and others; therefore, the detector cell is directly
compatible with commercial HPLC systems.
The porous membrane separator contained gold as the
working electrode [32], The 0.5 mm gold wire of 5 cm length
was covered with porous polymer tubing which can be attached
to the outlet of the column in an LC system. The gold elec¬
trode covered with porous membrane was cemented into a small
reservoir which contained an external electrolyte solution
(1 or 2 M KCl), auxiliary (Pt), and reference electrodes.
To prevent any damage to the porous membrane gold electrode,
it was soaked in 1 M KCl for several hours before running
mobile phase through it from an LC system.
Pretreatment of Glassy Carbon Electrode
It is well known that the sensitivity of glassy carbon
electrodes decreases with use. Decreasing activity at the
glassy carbon electrode surface can be due to adsorption of
analytes, mobile phase components, and byproducts of redox
reactions onto the electrode surface as well as formation
of electroactive species, such as carbonyl and hydroxy
groups, from components in the electrode material. The
adsorption of these components at the electrode surface may
form a polymeric film which would decrease electrode re¬
sponse; however, whatever the source of deactivation might
be, it is necessary to clean and reactivate the electrode
surface by mechanical, chemical, electrochemical, or by a

25
combination of these methods. More details on pretreatment
and the study of the glassy carbon electrode surface are given
elsewhere [33-35].
A deactivated electrode surface produces a large resid¬
ual current as well as a noisy baseline which affects the
accuracy of analytical application and increases the detec¬
tion limits. For reactivation or pretreatment of a glassy
carbon electrode surface the following procedure has given
satisfactory results. This procedure would be used if
analytical potential is positive or anodic.
(1) The cell was dismounted and electrode surface was
washed with methanol.
(2) A few drops of a slurry of alumina [0.1 ym, Gamal,
Grade B, Fisher Scientific Company (Fairlawn,
New Jersey)] in water were placed at the surface
of the electrode. The surface was then carefully
polished with Buehler LTD polishing paper (Evan¬
ston, Illinois) for 2-3 min.
(3) The electrode surface was rinsed with methanol and
polished with the same polishing paper which was
soaked in methanol, and then the cell was assem¬
bled.
(4) An anodic potential (0.1-0.2 V higher than that
of analytical potential) was applied for 3-4 min
while mobile phase passed through the electrochem¬
ical cell.
(5) A cathodic potential (-0.8 V vs. Ag/AgCl) was
applied for 2-3 min while mobile phase passed
through the electrochemical cell.
(6) The steps of (4) and (5) were repeated, and then
the potential was set at the analytical potential.
The potential switch was turned off before chang¬
ing from the anodic-applied potential to the
cathodic-applied potential or vice versa.

26
Reagents
Methanol and acetonitrile, HPLC grade (Fisher Scientific
Company, Fairlawn, New Jersey) were employed as organic
mobile phase components. Methanol, HPLC grade, was used as
the solvent for test solutes. Deionized water, another
mobile phase component, was purified by a Barnstead Nanopure
system (Sybron Corp.) The purified water then was irradi¬
ated for 24 hr by ultraviolet light in a model 816 HPLC
reservoir (Photronix Corp., Medway, Massachusetts). The
other chemicals comprising the mobile phases were: (1) rea¬
gent grade phosphoric acid (Scientific Products, McGaw Park,
Illinois), (2) sodium perchlorate and Brij-35 (Aldrich
Chemical Co., Milwaukee, Wisconsin), and (3) sodium dodecyl
sulfate (SDS; Fisher Scientific Co.). The reagents mentioned
above mostly were used as a component of the mobile phase
or dissolved in the mobile phase. Other specific chemicals
(e.g., sample solutes) will be mentioned in chapters related
to them.

CHAPTER FOUR
SIGNAL CHANGE AND BASELINE SHIFT
USING ELECTROCHEMICAL METHODS
In this chapter the following will be considered:
(1) change of residual current with ordinary DC voltammetry,
normal pulse (NP) voltammetry, and differential pulse (DP)
voltammetry in isocratic elution HPLC; (2) effect of the
background electrolyte and gradient elution on baseline
shift using amperometric detection; and (3) effect of the
electrode material on baseline shift using amperometric and
non-ramping differential pulse (NRDP) voltammetry methods.
Pulse techniques such as DP and NP can be used as LC
detection schemes if a constant pulse amplitude superimposes
over an initial potential. This means that there is no
potential ramp, and it is better to be called non-ramping
pulse techniques. The term "pulse techniques" has been
used instead of the term "non-ramping pulse techniques" in
the literature, however. Fleet and Little [36] were among
the first authors to be concerned with application of
pulse techniques as an HPLC monitoring method using a mer¬
cury drop electrode (MDE). The idea was examined in prac¬
tice by Swartzfager [37], using carbon paste as a working
electrode. The advantages of pulse systems in monitoring
have been discussed by Kissinger [19], who noted the
27

28
possibility of separating the monitored electrochemical sys¬
tem from systems with more positive or negative half-wave
potentials and prolonging electrode service life by electro¬
chemical cleaning. Electrochemical cleaning during the
pulse operation is the subject of a patent by Fleet [38]. A
versatile voltammetric detector with double polarization
pulse and semi-differential scanning for LC is reported by
Stastny et al. [39]. Pulse techniques also offer the advan¬
tage of decreased dependence of the measured current on
flow-rate in comparison to amperometric techniques [37],
because the short potential pulse duration minimizes the
development of the diffusion layer of the electrode and
dependence of the thickness of this layer on flow-rate.
With all the advantages of pulse techniques (more selectiv¬
ity, increased electrode stability, and less current depen¬
dence on flow-rate) over amperometric techniques, these
techniques are limited by poor sensitivity and complex in¬
strumentation. The residual current at a solid electrode is
reportedly high [17]. A study of the change in magnitude of
the residual current with different electrochemical methods
using isocratic elution and with amperometric method using
gradient elution was performed and will be reviewed. The
residual current change during gradient elution with ampero¬
metric detection is a baseline shift due to the change in
the composition of the mobile phase.
The electrochemical equipment used was a model 174
polarographic analyzer, an LC-4 amperometric controller, and

29
an electrochemical cell either with a glassy carbon elec¬
trode (GCE) or a porous membrane gold (Au) electrode.
Change of Residual Current with DC. DP and
NP Voltammetry in Isocratic Elution HPLC
In differential pulse (DP) voltammetry and normal pulse
(NP) voltammetry, the current is measured during a time in¬
terval of the pulse when the ratio of faradaic current to
charging current is a maximum. The electrochemical cell
can be considered as an RC-circuit, and thus the charging
current decays exponentially. The purpose of these experi¬
ments was a comparison of the residual current change versus
applied electrode potential with different electrochemical
methods using isocratic elution.
The residual current change and decay current on a
glassy carbon electrode with different percentage of acetoni¬
trile as an organic modifier in water are shown in Figures 2
through 5. The data from these figures are reported in
Table 1. All mobile phases contained 0.04 M NaClO^ as a
background electrolyte. The potential was scanned from
0.55 V (E^) to 1.30 V (Ef) versus Ag/AgCl reference elec¬
trode .
The residual current change, AI = 1^ - 1^, is due to
the difference between the current at the final potential
(Ef) or the current at the end of the ramp, 1^, and the cur¬
rent at the initial potential, I^. The residual current
change, AI, can be considered to be due to the residual
current difference at two different potentials. Decay

i i 1 r~
0.55 0.75 0.95 1,15
E.
E vs. Ag / AgCI
Figure 2. Residual Current Change and Decay Current with 5% CH3CN
(a) DC voltammogram
(b) DP voltammogram
Other conditions are as in Table 1.

(c) DP
i 1 i r
0 55
0.75
0.95
1.15
E.
i
E vs. Ag / AgCI
E,
Figure 3.
Residual Current Change and Decay Current with 50% CH3CN
(a,b) DC voltammogram (c) DP voltanunogram
(jJ
Other conditions are as in Table 1.

OJ
NJ
I 1 1 1
0.55 0.75 0.95 1.15
Ei E vs. Ag/AgCI Ef
Figure 4. Residual Current Change and Decay Current with 95% CH3CN
(a) DC voltammogram
(b) DP voltammogram
Other Conditions are as in Table 1.

I 1 1 1—
0.55 0.75 0.95 1.15
E.
1 E vs. Ag /AgCI
Figure 5. Residual Current. Change by NP Voltammetry
Mobile phase: 95% CH^CN
Other conditions are as in Table 1.

34
current is the amount of current depleted at the end of the
ramp (E^) within a short time. This current depletion might
be considered as the charging current and decreasing current
due to impurities. Since the charging current decays quick¬
ly (10— 4 to 10-3 sec), it is impossible to find out the
origin of decay current with a stripchart recorder.
The data in Table 1 show that on the DP mode the resid¬
ual current change, AI, and decay current, i¿ecay' are an
the limit of measurement error (measurement device was a
ruler). This means that the currents sampled shortly before
the end of the pulse and shortly before the rise of the
pulse are almost the same, and no difference in charging
current is sampled. On the DC mode, there is some current
change during the scan (potential applied to the electrode
changes with time) and some decay current at the end of the
scan. Increasing the scan rate will increase both AI, and
idecay :''n the D(- moc^e because the magnitude of charging
current increases as the scan rate increases. In the NP
mode, AI is much larger than that of the DC mode, while the
decay current is in the limit of measurement error. The
current varies linearly with the applied potential on the
working electrode (WE) in the NP mode. The larger magnitude
of AI in the NP mode as compared to the DC mode might be due
to the surface clean up of the working electrode which
occurs with pulsing. This cleanup increases the electrode
response.

35
Table 1. Residual Current Change, AI, and Decay Current,
■’"decay' ;’'n D<~' DP an<^ NP Voltammetry in Isocratic
Elution. Flow rate: 1 mL/min; column: Altex Ultra¬
sphere ODS 150 x 4.6 mm; = 0.55 V, E^ = 1.30 V
vs. Ag/AgCl; working electrode: glassy carbon; low
pass filter = 0.3 sec; pulse amplitude = 100 mV.
Solvent A = 0.04 M NaClO^ in water.
Solvent B = 0.04 M NaClO^ in acetonitrile.
Figure
Mobile
Phase
Electrochemical
Mode
Scan
Rate
mV/sec
2-a
5%
B
DC
2
2-b
5%
B
DP
2
3-a
50%
B
DC
2
3-b
50%
B
DC
5
3-c
50%
B
DP
2
4-a
95%
B
DC
2
4-b
95%
B
DP
2
5
95%
B
NP
2

36
Table 1 - Extended
Drop Knocker
Time
(sec)
AI ± Measure¬
ment Error
(yA)
1decay
ment
± measure-
Error
(yA)
1
1
1
1
1
0.5
0.5
0.5
0.75 ± 0.02
± 0.02
0.65 ± 0.01
0.82 ± 0.01
± 0.01
1.41 + 0.02
± 0.02
7.36 + 0.08
0.16 + 0.02
± 0.02
0.12 ± 0.01
0.34 ± 0.01
± 0.01
0.10 ± 0.02
± 0.02
± 0.08

37
Effect of Background Electrolytes and Gradient
Elution on Baseline Shift Using Amperometric Detection
In isocratic elution, the composition of the mobile
phase during the course of separation is constant; however,
isocratic elution sometimes is incapable of separating com¬
plex samples which have a wide range of retention times or
k' values. One of the solutions to this problem is gra¬
dient elution (GE). The necessity (of the presence) of a
background electrolyte and changing composition of the
mobile phase causes changes in viscosity, dielectric constant,
and conductivity of the mobile phase. The dependence of
the diffusion coefficient on viscosity, variation of the
conductivity, and changing condition of the working elec¬
trode surface during gradient elution will cause a change in
the residual current.
Walden's rule [40] states "the product of the equiva¬
lent conductivity (X) and viscosity of the solvent (n) for
a particular electrolyte at a given temperature should be
a constant."
X * n = constant (9)
In a binary gradient (solvents A and B), the percentage of
organic modifier (solvent B) increases in hydroorganic mo¬
bile phase during the gradient course. Decreasing the po¬
larity of the mobile phase during the gradient course
decreases the conductivity of the mobile phase if the con¬
centration of background electrolyte in both solvents is

38
the same. If the concentration of background electrolyte
in solvent B is higher than that of solvent A, the total
concentration of background electrolyte in mobile phase
increases during the gradient course. An increase in the
concentration of background electrolyte during the gradient
course may compensate for decreasing the polarity and chang¬
ing the viscosity of the mobile phase, which are important
factors in baseline stability. As a result of this discus¬
sion, a more stable baseline should be achieved with higher
concentration of background electrolyte in solvent B as
compared to equal concentration of background electrolyte
in both solvents.
If one assumes a binary gradient in which the percen¬
tage of solvent B increases during the gradient course, one
can measure the conductivity or specific conductivity vs.
the percent of solvent B in A+B. The specific conductivity
vs. the percentage of organic modifier (solvent B) is shown
in Figure 6 for H^PO^ and NaClO^ as background electrolytes.
The conductance measurement was done with a conductance
bridge of the Janz-Mclntyre type [41]. The specific conduc¬
tivity, K, was calculated from using equation (10).
0.126
R
specific conductivity
(10)
K
The cell constant is 0.126 cm 1, and R can be calculated
from equation (11) .
^measured
X 10000
R
10000 - R
(ID
measured

K(fi) cm K(fi) 1 cm
39
4 0-
•—. cr-
-—1 ■—
30-
—' cr-
'2. S'
15
10
0{
(a)
B%
' ' tt
10
B%
Figure 6. Specific Conductance vs. Percentage of Solvent B
(a)
Solvent
A = 0.15% H.PO. in
3 4
h2o
Solvent
B = 0.20% H-.P0. in
3 4
CH30H
(b)
Solvent
A = 0.05 M NaC104
in H20
Solvent
B = 0.05 M NaClO.
4
in CH3OH

40
In the case of H^PO^ as a background electrolyte, the spe¬
cific conductance decreases with the increasing percentage
of the organic modifier, while in the case of NaClC>4 it
passes through a minimum at about 65% of organic modifier
(solvent B).
Figure 7 shows the baseline shift during gradient elu¬
tion (with no sample injection) using an amperometric detec¬
tor and glassy carbon electrode, with H^PO^ and NaClO^ as
the background electrolytes. The column is equilibrated
with solvent A, and gradient starts with holding solvent A
for 3 min, then linear ramp to 20% A and 80% B over 30 min.
The baseline shift, Ai, is the difference in current before
starting the gradient program and at the end of the gradient.
Figure 7-a is due to 0.15% H^PO^ in 1^0 and 0.20% H^PO^ in
CH^OH, with Ai = -9 n A, while Figure 7-b is due to 0.20%
H^PO^ in both and CH^OH with Ai = -14 n A. The differ¬
ence in Ai for Figures 7-a and 7-b is due to the concentra¬
tion difference of H^PO^ in I^O. The lower baseline shift
in Figure 7-a as compared to Figure 7-b is in agreement with
Walden's rule as discussed above. Figure 7-c is due to
0.05 M NaClO. in both H„0 and CH_OH, with Ai = +6 n A.
Sodium perchlorate produces a smaller baseline shift because
it is completely ionized in both solvents. The peaks in
Figure 7 might be due to impurities in H^O or background
electrolytes which collect at the top of the column during
the early part of the solvent program and equilibration.
More examples of gradient elution with hydroorganic and

Figure 7. Baseline Shift during Gradient Elution with No Sample
Injection, Using an Amperometric Detector
Gradient Program: 100% A for 3 min, then linear ramp
to 20% A, 80% b over 30 min
Flow rate: 2 mL/min; temperature: 30°C; working elec¬
trode: glassy carbon; E = 1.0 V vs. Ag/AgCl
Column: Altex Ultrasphere ODS 150 * 4.6 mm
(a)
Solvent
A = 0.15%
H3P°4
in
h2o
Solvent
B = 0.20%
H3P°4
in
CH30H
(b)
Solvent
A = 0.20%
H3P°4
in
h2o
Solvent
B = 0.20%
H3PO
4
in
CH3OH
(c)
Solvent
A = 0.05
M NaCIO
4
in H20
Solvent
B = 0.05
M NaCIO
4
in CH3OH

+ 6 n A
- 14 nA
- 9 nA
“1 1
30.0 20.0
time (min)
(C)
lO.OnA
10.0
0.0

43
micellar mobile phases are given in Chapters Five and
Six.
The shift in baseline during gradient elution at a con¬
stant applied potential might be due to different concentra¬
tions of electroactive impurities, changing diffusion
coefficient of impurities with a change in the viscosity,
exhibition of different half-wave potential for specific
electroactive components, and effect of organic modifier on
the surface of the working electrode.
Effect of the Electrode Material on Baseline Shift
Using Amperometric and NRDP Voltammetry Methods
The baseline shift during gradient elution is evaluated
for glassy carbon and porous membrane gold electrodes. The
electrochemical methods used were amperometric and non¬
ramping differential pulse (NRDP) voltammetry. In NRDP
method, the difference in current sampled at the end of the
pulse and shortly before the rise of the pulse is recorded.
Since the pulse time is very short, there is virtually no
change in the mobile phase composition with gradient elution
during one pulse. The normalized baseline shift (current
difference at the end and the start of gradient program
divided by the surface area of the working electrode) for
amperometric and NRDP voltammetry with their chromatographic
conditions are reported in Table 2.
Data in Table 2 show that the absolute amount of the
baseline shift in the amperometric method is much higher
for gold electrode as compared to glassy carbon electrode.

44
Table 2. Normalized Baseline Shift (nA/mm2) during Gradient
Elution with No Sample Injection. Column: Altex
150x4.6 mm Ultrasphere ODS; E = 0.8 V vs. Ag/AgCl;
pulse amplitude = 100 mV; low pass filter = 3 sec.
Gradient program: initially 95% A, then linear
ramp to 5% A over 20 min, and finally holding for
5 min at 5% A.
Solvent A = 0.20% H.PO. in H„0.
3 4 2
Solvent B = 0.20% H3PC>4 in CH3OH.
Normalized Base-
Electrochemical
Method
Working
Electrode
line Shift
nA/mm2
Amperometrie
Glassy Carbon
-1.5
NRDP
Glassy Carbon
-116.0
Amperometrie
Gold
-356.7
NRDP
Gold
-152.8

45
This indicates that the residual current change with gradient
elution on the gold electrode surface is larger as compared
to glassy carbon electrode, which shows the gold is not an
appropriate working electrode under these conditions.
Although the absolute amount of the baseline shift in the
case of NRDP is also larger for the gold electrode as com¬
pared to glassy carbon electrode, this gap is not as large
as in the case of amperometric method. This might be due to
cleanup of the electrode surface with pulsing.

CHAPTER FIVE
A COMPARISON OF MICELLAR AND HYDROORGANIC
MOBILE PHASES USING AMPEROMETRIC DETECTOR
In this chapter, the following will be considered:
(1) the comparison of the hydrodynamic voltammograms (HDV)
in micellar and hydroorganic mobile phases, (2) the compar¬
ison of analytical figures of merit between micellar and
hydroorganic mobile phases, and (3) gradient elution and
selctivity with micellar mobile phases.
Ionic sufractants have been extensively used as ion¬
pairing reagents in ion-pair chromatography in the past.
More recently, ionic surfactants and nonionic surfactants in
aqueous solution have been employed as the mobile phase in
RP-LC. The surfactant concentrations in the mobile phase
above the CMC have been shown to have properties similar to
conventional mobile phases for RP-LC [42]. Micellar solu¬
tions have been used as media or matrices for room tempera¬
ture phosphorescence [43], and the usefulness of micelle-
stabilized room-temperature phosphorescence (MSRTP) for
detection and quantification of aromatic molecules in HPLC
has been reported by Weinberger et al. [44]. Micellar solu¬
tions have been used in electrochemistry to produce well-
defined redox waves for compounds that show only slight
shoulders or no waves in aqueous solutions [45] . Since the
46

47
redox voltammetry waves for organic compounds in micellar
solutions can be different from those of hydroorganic solu¬
tions, three different classes of compounds were chosen in
this study. The structures of these three classes which
include various forms of B-6 vitamins (PLP,PL, PN, PNP and
PMP), phenolic compounds (phenol, hydroquinone, resorcinol,
catechol and o-cresol), and polyaromatic hydrocarbons
(anthracene and pyrene) are shown in Figure 8. The phos¬
phate group in three of the B-6 vitamins can be partially
ionized. The extent of ionization is dependent upon the pH
of the mobile phase, however, in the low pH region, all
forms of B-6 vitamins will be protonated to produce cations.
A phosphoric acid buffer solution of pH 2.20 was used as the
background electrolyte in all mobile phases and in addition
generated an ionic site for ion-pairing of the nitrogenous
vitamins. With cationic solutes, sodium dodecyl sulfate
(SDS) in the mobile phase can act as an ion-pairing reagent
both below and above the CMC.
Hydrodynamic Voltammogram in Micellar
and Hydroorganic Mobile Phases
Hydrodynamic voltammograms for phenol, two vitamins
(PLP and PL), and polyaromatic hydrocarbons (anthracene and
pyrene) with their experimental conditions are given in
Figures 9 through 11, respectively.
The signal achieved for the same amount of injected
phenol in the micellar mobile phase is greater than that of
the hydroorganic mobile phase when the applied potential is

48
HOO
H,C
Fyridcxcl-5- phosphate
PIP
PL
^ i
CH
ptjp
ch2ch
PPJ
CH, NH, 0
2 2 ii
CH CP 'OH
2 I
OH
Pyridoxcmine-5- phosphate
PMP
Structures of B-6 Vitamins
Phenol
Hydroquinone Resorcinol
catechol
o-Oesol
Structures of Phenolic Compounds
Anthracene pyrene
Structures of Polyaromatic Hydrocarbons
Figure 8. Chemical Structures of Compounds Used in Chapter
Five

49
Figure 9. Hydrodynamic Voltammogram for Phenol
Column: Altex Ultrasphere ODS, 150 x 4.6 mm
Flow Rate: 1 mL/min
Injection Volume: 20 yL (5 ppm)
Working Electrode: Glassy carbon electrode
Temperature: 30°C
(o) Micellar mobile phase: 0.1 M SDS in
(3:97) (1-propanol:H-O) + 5 x 10~“ M
NaCl04
(•) Hydroorganic mobile phase: (60:40)(H-O:
CH3OH) + 5 x 10-2 M NaC104 ¿

50
250
200
¡ ( nA ) 150
100
50 L-
0.7
—1 1 L
0,9 1.0 I.
0,3
E (v )
1.2

51
Figure 10. Hydrodynamic Voltammogram for B-6
Micellar mobile phase: 0.1 M SDS
(1-propanol:H~0) , pH = 2.20
W
(A) PLP (250 ppm)
(â–¡) PL (250 ppm)
Ion-pair mobile phase: 5 x 10“3
(12:88)(1-propanol:H2O), pH
(used H^PO^)
(o) PLP (2000 ppm)
( •) PL (2000 ppm)
Vitamins3
in (3:97)
(used
SDS in
2.20
a All B-6 vitamins dissolved in 0.1 M HC1. Other con¬
ditions as in Figure 9.

52
i (nA)

53
Figure 11. Hydrodynamic Voltammogram for Polyaromatic
Hydrocarbons3
Column: Rainin microsorb octyl
Flow Rate: 2.5 mL/min
Micellar mobile phase: 0.1 M SDS in (3:97)
(1-propanol:H20), pH = 2.2 (used H^PO^)
(o) anthracene (50 ppm)
(•) pyrene (100 ppm)
Hydroorganic mobile phases:
(â–¡) anthracene (50 ppm): (46.5:53.5)
(H20:CH30H), pH = 2.20 (used H3P04)
(A) pyrene (100 ppm): (43.2:56.8)(H„0:
CH3OH), pH = 2.20 (used H3P04>
a Anthracene and pyrene were dissolved in CH3 conditions as in Figure 9.

54

55
below 1.0 V, whereas above 1.0 V it is slightly greater in
the case of the hydroorganic mobile phase. According to
HDV, the best analytical potential for phenol is 1.1 V in
both mobile phases.
In the case of B-6 vitamins, the signal increases with
increasing potential in the same manner for both the micel¬
lar mobile phase and the hydroorganic mobile phase which
contains small amounts of SDS as an ion-pairing reagent. The
signal for PLP and PL are greater in the micellar mobile
phase as compared with the ion-pairing mobile phase. At
first glance, the graph in Figure 10 shows that the signal
for PLP in the ion-pairing mobile phase is greater than that
in the micellar mobile phase at the same potential, however,
this is not the case because the absolute amount of injec¬
tion of PL and PLP in the ion-pairing mobile phase is 8 times
greater than that in the micellar mobile phase. The poten¬
tial 1.4 V might be chosen as an analytical potential for
vitamins.
Figure 11 shows the HDV for anthracene and pyrene in
both mobile phases. At potentials higher than 1.2 V, the
signal due to pyrene increases sharply, whereas the signal
due to anthracene decreases smoothly. One of the most im¬
portant aspects of Figure 11 in comparison to Figures 9 and
10 is the unique selectivity response of polyaromatic hydro¬
carbons with applied potential in both micellar and hydro-
organic mobile phases. This means that anthracene can be
measured in the presence of pyrene using a proper applied
potential. In order to compare analytical figures of merit

56
in micellar and hydroorganic mobile phases, the best possible
analytical potential to use for anthracene and pyrene would
be 1.1 V and 1.2 V, respectively.
Analytical Figures of Merit Comparison between
Micellar and Hydroorganic Mobile Phases
Analytical figures of merit such as limit of detection
(LOD), upper limit of linear dynamic range (LDR), sensitiv¬
ity, correlation coefficient, and log-log slope, as well as
the experimental conditions for phenol, B-6 vitamins, and
polyaromatic hydrocarbons (PAH) in both micellar and hydro-
organic mobile phases, are reported in Tables 3 through 5,
respectively. Data in Tables 3 through 5 are derived from
Figures 12 through 14 (analytical or calibration curves),
respectively.
In order to compare analytical figures of merit for a
specific electroactive component in two different mobile
phase compositions using an amperometric detector, two con¬
straints must be met. First, the flow rate should be nearly
the same for both mobile phases because the response of any
electrochemical detector is dependent on the rate of mass
transfer to the electrode surface [37]. Secondly, the
retention time of the electroactive component in both mobile
phases should be nearly the same because of the peak height
measurement. To get nearly the same retention time in both
mobile phases, the percentage of organic modifier in the
hydroorganic mobile phase was varied until the retention

57
Table 3. Analytical Figures of Merit for Phenol. Flow
rate: 1 mL/min; column: Altex Ultrasphere ODS,
150 x 4.6 mm; E = 1.1 V; working electrode:
glassy carbon electrode; injection volume: 20 yL.
Micellar mobile phase: 0.1 M SDS in 3% 1-propanol
+ 5 x 10-2 M NaC104.
Hydroorganic mobile phase: (H~0:CH-.OH) (60:40) +
5 x IQ-2 M NaClC>4. J
Limit of
Detection
Upper limit
of LDR
Mobile Phase
(ppm)
(ng)
(ppm)
(ng)
Micellar
0.0080
0.16
20a
400
Hydroorganic
0.0065
0.13
10
200
a 20 ppm was the most concentrated solution, so the upper
limit of LDR might be more than 20 ppm.

58
Table 3 - Extended
Sensitivity
Correlation
Log-log
(nA/ppm)
(nA/nq)
Coefficient
Slope
(min)
41.40
2.03
0.9971
0.96
5.0
39.74
1.99
0.9973
1.04
3.8

59
Table 4. Analytical Figures of Merit for B-6 Vitamins.
Column: Altex Ultrasphere ODS, 150 x 4.6 mm;
E = 1.4 V; working electrode: glassy carbon;
injection volume: 20 yL.
Micellar mobile phase: 0.1 M SDS in 3% 1-propanol
with pH = 2.20 (used H^PO^).
Ion-pair mobile phase: 5 x 10-3 M SDS in 12%
1-propanol with pH = 2.20 (used H^PO^).
Compound
Mobile
Phase
Limit of
(ppm)
Detection
(ng)
Upper Limit
(ppm)
of LDR
(ng)
PLP
Micellar
0.316
6.32
300
6000
PL
Micellar
0.236
4.72
300
6000
PLP
Ion-pair
0.0726
1.45
100
2000
PL
Ion-pair
0.0852
1.70
100
2000

60
Table 4 - Extended
Sensitivity
(nA/ppm) (nA/nq)
Correlation
Coefficient
Log-log
Slope
Flow Rate
(mL/min)
tR
(min)
3.80
0.170
0.9999
0.99
1.0
2.8
5.09
0.255
0.9995
1.00
1.0
7.8
4.96
0.248
0.9999
0.95
1.1
2.8
4.22
0.211
0.9999
0.95
2.0
7.8

61
Table 5. Analytical Figures of Merit for Polyaromatic Hydro¬
carbons. Flow rate: 2.5 mL/min; column: Rainin
microsorb octyl, 150 x 4.6 mm; injection volume:
20 yL.
Micellar mobile phase: 0.1 M SDS in (3:97) (1-pro-
panol-H20), pH
= 22 0
(used H
3P°4>
Limit of
Detection
Upper
of
Limit
LDR
Compound
Mobile Phase
(ppm)
(ng)
(ppm)
(ng)
Anthracene
Micellar
0.0933
1.87
250
5000
Pyrene
Micellar
1.77
35.4
200
4000
Anthracene
Hydroorganic3
0.0846
1.69
50
1000
Pyrene
Hydroorganic'3
--
--
--
--
a Hydroorganic mobile phase (used only for anthracene):
(46.5:53.5)(H20:CH30H); pH = 2.20 (used H_PO.); ionic
strength = 3.59 x 10-2 M.
b Hydroorganic mobile phase (used only for pyrene):
(43.20:56.80) (H20:CH30H) , pH = 2.20 (used H3PC>4) .

62
Table 5 - Extended
Sensitivity
Correlation
Log-log
E
(nA/ppm)
(nA/nq)
Coefficient
Slope
(V)
(min
0.707
0.0354
0.9996
0.97
1.1
13.7
0.667
0.0338
0.9990
0.97
1.2
9.3
3.545
0.177
0.9906
0.96
1.1
13.7
1.2
9.3

63
1000
Figure 12. Analytical Curves for Phenol
(o) in micellar mobile phase
(*) in hydroorganic mobile phase
Experimental conditions are the same as Table 3.

64
Figure 13. Analytical Curves for B-6 Vitamins
Micellar mobile phase:
(A) PLP
(â–¡) PL
Ion-pair mobile phase:
(o) PLP
(•) PL
Experimental Conditions are the same as
Table 4.

65
C(ppm)

66
Figure 14. Analytical Curves for Polyaromatic Hydrocar¬
bons
Micellar mobile phase:
(o) anthracene
(•) pyrene
Hydroorganic mobile phase:
(D) anthracene
Hydroorganic mobile phase:
(A) pyrene
Experimental conditions are the same as
Table 5.

67
C(ppm)

68
time of the electroactive component nearly matched that of
the micellar mobile phase.
In the case of phenol, the LOD is approximately the
same in both mobile phases, whereas the upper limit of the
LDR in the micellar mobile phase is at least twice that of
the hydroorganic mobile phase. For B-6 vitamins there is
not much difference in the LOD in both mobile phases, while
the upper limit of LDR in micellar mobile phase is 3 times
that of the hydroorganic mobile phase. The LDR for anthra¬
cene is 5 times greater in the micellar mobile phase as
compared to the hydroorganic mobile phases, while the LOD
is approximately the same in both mobile phases. It is hard
to determine the precise analytical figures of merit for
pyrene in the hydroorganic mobile phase because, as
Figure 14 shows, the analytical curve is practically non¬
linear in that concentration range. Pyrene, however, should
be similar to anthracene, and, as shown in Figure 14, the
upper limit of the LDR for the micellar mobile phase is much
greater when compared to the hydroorganic mobile phase.
The greater upper limit of LDR in the micellar mobile
phase as compared to the hydroorganic mobile phase may be
due to the higher ionic strength (resulting from the pre¬
sence of ionic surfactants as well as background electro¬
lyte) , in the micellar mobile phase [19,46] or due to the
nature of surfactants. Increasing the ionic strength of the
mobile phase decreases the IR gradient across the electrode
face and between the electrode and the bulk solution. Sur¬
factants or surface active agents can change the interfacial

69
properties. Hence, the interfacial properties of the micel¬
lar mobile phase with working electrode will be quite dif¬
ferent from those of hydroorganic mobile phase.
Gradient Elution and Selectivity in Micellar Mobile Phase
Gradient elution with the micellar mobile phase is
primarily used for the same purpose as it is with the hydro-
organic mobile phase. Gradient elution may also be used to
effect a change in selectivity because micellar mobile
phases have unique selectivities at different concentrations.
Armstrong and Henry [42] have used gradient flow rates with
a UV detector. The gradient micellar chromatogram, obtained
with an amperometric detector using an anionic surfactant
(SDS) and a nonionic surfactant (Brij-35) are shown in
Figures 15 through 17. Figure 15 shows the separation of
B-6 vitamins with SDS, and Figures 16 and 17 show separation
of phenolic compounds with SDS and Brij-35, respectively.
Micellar mobile phases have been shown [47] to offer
control over selectivity in liquid chromatography as the
concentration of surfactant changes in the mobile phase.
The selectivity of the micellar mobile phase toward B-6
vitamins is shown in Figure 18. The elution order of PL and
PMP changes above and below a certain concentration range
of SDS, whereas they coelute in this range. The void volume
of the column, determined by water injection, slightly
decreased as the concentration of SDS increased in the
mobile phase. For an Altex Ultrasphere ODS 25 x 4.6 mm, a

70
Figure 15. Gradient Micellar Chromatogram for Separa¬
tion of Vitamin B-6
Solvent A: (3:97)(1-propanol:H?0),
pH = 2.20
Solvent B: 0.2 M SDS in (3:97) (1-propanol:
H20), pH = 2.20 (used H^PO^)
Gradient program: started with 25% solvent
B and held at 25% B for 8 min, then
ramped to 100% B over 8 min and contin¬
ued with 100% B
Flow rate: 1 mL/min; temperature: 30°C;
working electrode: glassy carbon elec¬
trode; E = 1.3 V
Column: Altex Ultrasphere ODS, 250 x 4.6 mm
precolumn: 15 x 4.6 mm, packed manually
with 25-40 ym silica gel
Injection volume: 10 yL
Peaks are as follows: (1) PLP (200 ppm),
(2) PL (200 ppm), (3) PMP (400 ppm),
(4) PNP (4 00 ppm) , (5) PN (4 00 ppm)

71
4; 5
t(min)

72
Figure 16. Gradient Micellar Chromatogram for Separa¬
tion of Phenolic Compounds
Gradient program: started with 25% solvent
B and held at 25% B for 4 min, then
ramped to 100% B over 8 min and con¬
tinued with 100% B
Injection volume: 13 yL; E = 1.1 V
Peaks are as follows: (1) hydroquinone
(10 ppm), (2) resorcinol (8 ppm),
(3) catechol (20 ppm), (4) phenol
(18 ppm), (5) o-cresol (arbitrary con¬
centration)
Other conditions as Figure 15.

73
loo nA
I £ r
18 12 6
t(mi n)
0

74
Figure 17. Gradient Micellar Chromatogram for Separa
tion of Phenolic Compounds and Gradient
with No Injection
Solvent A: (3:97)(1-propanol:H„0),
pH = 2.20 (used H3PC>4)
Solvent B: 0.1 M Brij-35 in (3:97)(1-pro
panol:H20), pH = 2.20 (used H^PO^)
Gradient program: started with 20% sol¬
vent B and held at 20% B for 4 min,
then ramped linearly to 100% B over
6 min and continued with 100% B
Flow rate: 1 mL/min; temperature: 30°C;
working electrode: glassy carbon
electrode
Column: Altex Ultrasphere ODS, 150 x
4.6 mm
Injection volume: (a) 20 yL; (b) none
Peaks are as follows: (1) hydroquinone
(10 ppm) , (2) resorcinol (8 ppm) ,
(3) catechol (20 ppm), (4) phenol
(18 ppm), (5) o-cresol (arbitrary
concentration); these are for (a)

75
(b)
"I
16
—r
8
t(min )
1
0

76
Figure 18. Effect of SDS Concentration on k'
Mobile phase: (3:97) (1-propanol:i^O) with
different concentrations of SDS,
pH = 2.20 (used H3PC>4)
Flow rate: 1 mL/min; temperature: 30°C;
working electrode: glassy carbon;
E = 1.3 V
Column: Altex Ultrasphere ODS, 250 x
4.6 mm
(â–¡)
PLP
(O)
PL
(*)
PMP
(A)
PN and PNP

log K
77

78
change in concentration of SDS from 0.05 M to 0.15 M or more
in the mobile phase caused the void volume to change from
2.20 mL to 2.00 mL. The adsorption isotherm of surfactants
on ODS-hypersil is reported by Knox and Hartwick [48]. The
surfactant concentrations chosen were below the CMC and one
SDS concentration supposedly above the CMC was also used.
The adsorption isotherms shown by these researchers are in
methanol¡water (20:80) solution which results in a different
CMC from that of pure water. It is doubtful they ever
reached the CMC for their system. There is no literature
adsorption isotherm available for SDS above the CMC.
Figures 19 through 21 show the isocratic micellar chromato¬
grams for B-6 vitamins and their selectivity with three dif¬
ferent concentrations of SDS in the mobile phase.

79
Figure 19. Isocratic Micellar Chromatogram for Separa
tion of Vitamin B-6
Mobile phase: 0.05 M SDS in (3:97) (1-pro-
panol:H20), pH = 2.20 (used H^PC^)
Flow rate: 1 mL/min; temperature: 30°C;
working electrode: glassy carbon elec
trode; E = 1.3V
Column: Altex Ultrasphere ODS, 250 x 4.6
mm; precolumn: 15 x 4.6 mm, packed
manually with 25-40 ym silica gel
Injection volume: 10 yL
Peaks are as follows: (1) PLP (200 ppm),
(2) PL (200 ppm), (3) PMP (400 ppm),
(4) PNP (400 ppm), (5) PN (400 ppm)

80
2oo nA
To
t(min)

81
Figure 20. Isocratic Micellar Chromatogram for Separa¬
tion of Vitamin B-6
Mobile phase: 0.1 M SDS in (3:97) (1-pro-
panol:H20), pH = 2.20 (used H^PO^)
Other conditions are as in Figure 19.

82
4;5

83
Figure 21. Isocratic Micellar Chromatogram for Separa¬
tion of Vitamin B-6
Mobile phase: 0.2 M SDS in (3:97) (1-pro-
panol:H20), pH = 2.20 (used H^PO^)
Other conditions are as in Figure 19.

84
t (min)
T
0

CHAPTER SIX
RAPID SEPARATION AND DETERMINATION OF THYROMIMETIC
IODOAMINO ACIDS BY GRADIENT ELUTION REVERSE PHASE LIQUID
CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION
The mechanism of action of the thyroid hormones, T^ and
T^, is of considerable interest in part because of the
amazing diversity of thyroid hormone effects. These agents
influence the metabolism of almost every class of foodstuff.
They exert profound effects on many enzymes and on almost
all organ systems, and they play an integral role in the
complex biological processes involved in growth and differ¬
entiation [49], The time-consuming procedures in USP mono¬
graphs have been used for analysis of levothyroxine sodium
tablets [50] and liothyronine sodium tablets [51]. The as¬
say of the major thyroid hormones T^ and T^ also is done by
wet analysis [52-54], radioimmunoassay [55-58], chemical
derivatization followed by gas chromatography with electron
capture detection [59,60], thin-layer chromatography [61-63],
paper chromatography [64,65], electrophoresis [66], and gas
chromatography/mass spectrometry [67] . Thin-layer and paper
chromatography as well as the electrophoretic procedures do
not have good limits of detection for this kind of analysis.
Although the gas chromatographic procedures are sensitive,
they require the isolation of the iodoamino acids in a pure
form, which must then be converted to a volatile derivative
85

86
for chromatographic analysis. The radioimmunoassay proce¬
dures are impractical for a small number of samples and have
the added problem of disposal of the radioactive wastes.
Liquid chromatography would normally be the method of
choice for these analyses because of the poor volatility of
the compounds. In fact, HPLC methods for separation of pure
iodoamino acids have appeared in the literature [68-72].
Recently, and tablets have been analyzed by reverse
phase LC using UV detection [73], and a gradient elution
separation of sixteen thyromimetic iodoamino acids has been
reported [74]; however, the low molar absorptivity of these
compounds at 254 nm precludes their determination at trace
levels. Detection at 220 nm improves this situation some¬
what [74], and a clever catalytic post-column detection
scheme has also been shown [75]. Most recently, application
of amperometric electrochemical detection to these compounds
has been shown to give excellent limits of detection [76],
as has dansyl derivatization and subsequent fluorescence
detection [77] .
The purpose of this study was to demonstrate the use¬
fulness of gradient elution techniques with electrochemical
detection for the separation of seven thyromimetic iodoamino
acids. A rapid isocratic separation of Tg, T2, T^ and T^,
as well as analysis of T^ both in tablets and injectable
intravenous samples is presented.

87
Standard Solutions
The compounds Tyr, MIT, DIT, TQ, and were
purchased from Sigma Chemical (St. Louis, Missouri) and
were stored in a freezer. Standard solutions were prepared
by dissolving appropriate amounts of each compound in meth¬
anol containing 1% ammonium hydroxide and were stored in a
refrigerator.
Preparation of Tablet Solution
and Injectable T^ Sample
Twelve tablets (1.5676 g) containing levothyroxine
sodium were dissolved in 20 mL of 0.01 M sodium hydroxide
using an ultrasonic bath. The sample solution was heated at
60°C for 3 min, shaken for 3 min, and then filtered through
F2406-9 (S/P) filter paper. Before chromatographic injec¬
tion, this solution was again filtered with a Rainin
(Rainin Instruments, Woburn, Massachusetts) HPLC sample
filter syringe using a 0.45 ym nylon-66 membrane filter.
The injectable sample was present as a powder and was pre¬
pared by dissolving in 5 mL 0.9% sodium chloride solution.
This resulted in a clear solution which was then filtered
with the sample filter syringe.
Gradient Elution LC/EC
Amperometric electrochemical detectors are generally
considered incompatible with gradient elution

88
techniques [78,17]. The necessity of the presence of a
background electrolyte and the dependence of the charging
or residual current on the exact composition of the mobile
phase has discouraged attempts to use this powerful liquid
chromatographic technique. Changes in the polarity and
dielectric constant of the mobile phase during a gradient
program yield steeply sloping baselines from the ever
changing charging current. Indeed, to our knowledge, the
only published report of gradient elution LC/EC used a
gradient of only 36-60% methanol [79] .
Addition of phosphoric acid to both constituents of
the mobile phase (f^O and CH^OH) provides background elec¬
trolyte and suppresses the ionization of the thyromimetic
iodoamino acids [80]. Both mobile phase constituents were
degassed with helium gas during chromatographic runs.
Initial attempts were made using equal concentrations of
background electrolyte in both the water and methanol reser¬
voirs. During a gradient from 0 to 100% methanol, a large
negative shift in background was noted, so the background
electrolyte concentration was increased in the methanol
reservoir. This then increased the concentration of back¬
ground electrolyte in the mobile phase as the gradient
progressed and somewhat lessened the effect of the decreas¬
ing polarity and dielectric constant on the residual cur¬
rent. Figure 22 shows the baseline change during a blank
injection and a gradient from 0 to 90% methanol with a
background electrolyte concentration of 0.15% H^PO^ in water

89
Figure 22. Baseline during Gradient Program with Blank
Injection
Solvent A: 0.15% H3P04 in H20
Solvent B: 0.20% H^PO^ in methanol
Gradient Program: initially 100% A, then
immediate linear ramp to 40% B over
8 min, to 60% B over 3 min, and to
90% B over 7 min.
Flow rate: 2.0 mL/min; working electrode:
glassy carbon electrode; E = 1.4 V vs.
Ag/AgCl
Column: Altex Ultrasphere ODS, 150 x 4.6
mm
Injection volume: 10 yL blank (1% ammo¬
nium hydroxide in methanol)

90
10 nA
TIME (min)
j
0

91
and 0.20% H..PO. in methanol. It should be stressed that
3 4
the potential of the working electrode during this gradient
program was +1.4 V and that lower working potentials should
show even less baseline shift. Also, no extraordinary
efforts were made to purify the water used, and some of the
peaks observed are undoubtedly from trace organic compounds
which had adsorbed at the top of the column. Figure 23
shows the rapid gradient elution separation of the 7 thyro-
mimetic iodoamino acids which are shown in Figure 24. The
peak at 3.5 min is from the ammoniacal methanol used to
dissolve the sample. This chromatogram demonstrates the
potentially powerful applications of gradient elution LC/EC.
Isocratic Separations
To maximize the signal-to-noise ratio of an electro¬
chemical detector, the applied potential should be held at
the minimum value at which the current reaches the limiting
current plateau of the analyte (Ep]_ateau^ ’ This potential
can be quickly estimated from cyclic voltammetry [25] and
can then be determined precisely from hydrodynamic voltam-
mogram (HDV) in which the current is measured vs. applied
potential point by point. A cyclic voltammogram for Tq [81]
is shown in Figure 25. Hydrodynamic voltammograms for T2,
T^ and T^ are shown in Figure 26. Each point is the average
signal from two, 5 yL injections of a 50 ppm solution
(0.25 yg/injection) at a flow rate of 1 mL/min. As seen in
Figure 26, a potential of 1.2 V is a reasonable potential

92
Figure 23. Separation of Seven Thyromimetic Iodoamino
Acids
Column: Altex Ultrasphere ODS, 150 x 4.6 mm
Injection: 10 yL of 20 ppm Tyr, 40 ppm MIT,
70 ppm DIT, 25 ppm T_, 60 ppm T2,
60 ppm , 200 ppm
Other conditions are as in Figure 22.

93
Tyr
TIME (min)

94
Thyromimetic Iodoamino Acids and Related Compounds
D,L-Tyrosine (Tyr)
M°—^ ^)—Q^:CMCOCH
MMj
3-Iodo-L-Tyrosine (MIT)
)=\
M" ^^~CII;CUCOOH
Nllj
3,5-Diiodo-L-Tyrosine (DIT)
HO— —CHjCll (VH« )COOH
D,L-Thyronine (Tq)
w,
HO—V ^-CH.CMCOOH
3,5-Diiodo-D,L-Thyronine (T2)
^ sh:
HO— 0 —' V- CH-CHCOCH
V- / ^ 7
3,3',5-Triiodo-L-Thyronine (T3)
HO —/ "V“ 0 —1/ ;— CX_CHCOOH
L-Thyroxine (T^)
HO“—0 \— CW.CHCOOH
/ N'
Figure 24. Thyromimetic Iodoamino Acids Used in This Study

95
Figure 25. Cyclic Voltammogram [81]
Sample: ; medium: 1 M H^SO^; concentration:
5 mg/25 mL; scan rate: 200 mV/sec; working
electrode: carbon paste electrode; reference
electrode: Ag/AgCl; instrument: CV-1A

96
Figure 26.
Hydrodynamic Voltammogram for Tand
Mobile phase: (70:30:0.2) (CH3OH:H20:H3P04)
Flow rate: 1.0 mL/min
Column: Altex Ultrasphere octyl, 250 x
4.6 mm
Injection: 5 yL of 50 ppm solution

50
40
30
20
10
0 i
97
1.2
1.3

98
for measurement of these compounds, and the other thyromi-
metic iodoamino acids were also found to produce large sig¬
nals at this potential.
Analytical calibration curves, current vs. concentra¬
tion, for T2, and are shown in Figure 27. Each point
is the average signal of two, 5 yL injections of standard
solutions with an applied potential of 1.2 V. The analyt¬
ical figures of merit for ' T3 an<3 T4 are given in Table 6.
As can be seen, the limits of detection are in the sub¬
nanogram range, and the LOD1s for Tyr, MIT, DIT and Tq
should be even lower, as the signal for these compounds is
greater than for an equal concentration of T3 or T4•
Figure 28 shows a rapid isocratic separation of Tq, T^r
and T^.
Assay of T^ Preparations
To demonstrate the usefulness of electrochemical detec¬
tion for these compounds, both T^ tablets and intravenous
solutions were analyzed. For the determination of T^, a
calibration curve was prepared using standard solutions.
Each standard was measured two times, and the average peak
height signal of these two measurements was used for the
calibration curve. The average signal of 5 measurements
was used for the unknowns. The average amount of L-thyroxine
per tablet was found to be 24.3 yg (25 yg/tablet claimed),
and the injectable solution was found to contain 704 yg
(500 yg claimed). The reason for this large excess is un¬
known .

I (nA)
Figure 27. Analytical Curves for T2, and
E = +1.2 V
Other conditions are as in Figure 26.

Table 6. Analytical Figures of Merit for and .
Flow rate: 1.0 mL/min; column: Altex Ultrasphere
octyl, 250 x 4.6 mm; injection: 5 pL of 50 ppm
solution; E = 1.2 V vs. Ag/AgCl.
Mobile phase: (70:30:0.2)(CH3OH:H20:H3P04).
Upper Limit of
Limit of Detection LDR Sensitivity T .. t
J Log-log R
Compound
(ppm)
(ng)
(ppm)
(ng)
(nA/ppm)
(nA/ng)
Slope
(min)
T4
0.026
0.13
200
1000
0.460
0.092
0.97
9.0
T3
0.012
0.062
100
500
0.984
0.197
0.98
7.0
T
± 0
0.014
0.074
100
500
0.805
0.161
1.00
6.0
100

101
T2
I I I I
6 4 2 0
time (mins.)
Figure 28. Isocratic Separation of T
Flow rate: 2.0 mL/min; E =
Other conditions are as in
T_. and T.
3 4
+ 1.2 V
Figure 26.

102
As amperometric detectors, particularly with glassy
carbon working electrodes, are known to undergo changes in
sensitivity with time, it is necessary to run two or more
standards daily to reestablish the slope of the working
curve. A study of reactivation methods for solid electrodes
used in LC/EC and flow injection analysis has recently been
made [33]. It is also necessary to prepare fresh standards
daily, as the compounds were found to slowly decompose,
with old standards showing a small peak eluting before the
peak.

CHAPTER SEVEN
CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK
Conclusions
A method for utilizing gradient elution techniques with
electrochemical detectors in both hydroorganic and micellar
mobile phases is described. This should greatly increase
the usefulness of this detection method and should serve to
shorten analysis time where electrochemical detection is the
method of choice. A comparison of analytical figures of merit
between hydroorganic and micellar mobile phases shows the
possibility of greater linear dynamic ranges for electroac¬
tive components in micellar mobile phases. The unique
selectivity of electrochemical detection can provide an
effective method to discriminate between coeluting components
in both micellar and hydroorganic mobile phases. The selec¬
tive detection is shown in hydrodynamic voltammograms for
anthracene and pyrene. Limits of detection in the nanogram
range in addition to the selectivity of micellar mobile
phase show the sensitivity of electrochemical detection and
versatility of micellar mobile phases for B-6 vitamins. A
very sensitive method (electrochemical detection) for thyro-
mimetic iodoamino acids is described. This sensitive method
can provide a limit of detection in the sub-nanogram to
103

104
picogram range, depending on the type of the thyromimetic
iodoamino acids. Rapid isocratic and gradient elution
separations of the thyromimetic iodoamino acids are also
reported.
Suggestions for Future Work
Future research in LC/EC looks promising due to the
versatility of the technique and the additional selectivity,
sensitivity, and signal enhancement found when micellar
mobile phases are used with LC/EC. Ideas for future
research are given in the following paragraphs.
Firstly, the separation of chemical compounds which
interact similarly with the stationary and mobile phases is
a major problem in HPLC; however, the selectivity of elec¬
trochemical methods at a properly applied potential will
provide the detection of one component in the presence of
another, if the signal of one component approaches zero at
that potential. This properly applied potential might not
be the analytical potential, which may cause a decrease in
detection sensitivity. One example might be the selective
detection of polyaromatic hydrocarbons (PAH) with ampero-
metric or pulse electrochemical methods, because these com¬
pounds are typically hard to separate.
Secondly, as mentioned in Chapter Five, surfactant solu¬
tions, especially cationic surfactants, can catalyze oxida¬
tive waves [45,82], The same literature also reported an
increase in the oxidation potential of water by up to 1 V

105
when platinum anodes were used in a surfactant solution
which was also 2 M sodium hydroxide; however, the literature
did not mention what occurs in the absence of 2 M sodium
hydroxide in the micellar solution. The advantages of
micellar solutions can provide the means for the detection
of many chemical compounds with high oxidative potentials
and can increase the sensitivity of compounds that produce
slight shoulders or no waves in aqueous 2 M sodium hydroxide
solutions. If one assumes this is true only in micellar
solutions with 2 M sodium hydroxide,however, one can add
sodium hydroxide after the components elute from the column
to get the benefit of this powerful detection technique.
Thirdly, the indirect determination of chemicals with
no detector response can be accomplished using sensitive
electrochemical methods. For example, sugars, steroids, and
alkyl ammonium compounds cannot be directly detected with
common liquid chromatographic detectors. The addition of
small amounts of an electroactive component to the mobile
phase will produce a negative peak for a nonelectroactive
component. The height of this negative peak will be propor¬
tional to the concentration of nonelectroactive component
band passing through a detector cell. Addition of an elec¬
troactive ion-pair reagent to the mobile phase will provide
a means of detection for ionic samples of the opposite
charge. For example, to separate and detect alkyl ammonium
compounds, an alkyl phenol sulfonate used as an ion-pair
reagent can provide the separation as well as detection by
RP-LC/EC.

106
Fourthly, a zwitterionic surfactant molecule contains
both cationic and anionic sites. Addition of the zwitter¬
ionic surfactants to the mobile phase will facilitate sepa¬
ration of cationic and anionic compounds in a mixture. A
mixture of cationic and anionic surfactants can be separated
by zwitterionic surfactants in the mobile phase.
Fifthly, as mentioned in Chapter Five, the upper limit
of LDR in micellar mobile phases was higher than in hydro-
organic mobile phases. The reasons mentioned were either
higher ionic strength in a micellar mobile phase or the
nature of surfactants. To discriminate between the two
reasons, one can compare the upper limit of LDR by setting
the ionic strength equal in both mobile phases, or by choos¬
ing nonionic surfactants with the same concentration of
background electrolyte in both mobile phases. Any observed
differences in the upper limit of LDR in two mobile phases
then will be due to the nature of surfactants.
Finally, surfactants can be chemically bonded to silica
gel to produce anionic stationary phase. A chemically
bonded anionic surfactant stationary phase can be used for
the separation of cationic compounds with a cationic micel¬
lar mobile phase, while a chemically bonded cationic surfac¬
tant can be used for separation of anionic compounds with an
anionic micellar mobile phase. The competition between the
ionic samples and surfactants in the mobile phase for sta¬
tionary phase causes the separation of ionic compounds in a
mixture.

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BIOGRAPHICAL SKETCH
Mohammad Reza Hadjmohammadi was born November 27, 1947,
in Iran. He attended primary and secondary schools there,
graduated from the Mashad University with a B. S. degree in
chemistry in 1972, served military service, and received
his masters degree in chemistry in 1975 from Tehran Univer¬
sity. He worked as an instructor at the Chemistry Department
of Abureihan University from 1975 to 1977. He came to the
University of Florida, Gainesville, Florida, U.S.A., in
September 1977, where he has taken some English courses and
is working toward the Ph.D. degree in analytical chemistry.
He is a student member of the American Chemical Society and
Analytical Chemistry Division.
112

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.
Dr.\J 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.
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.
Dr. Richard
Associate 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.
Dr. Anna Kira j ter-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.
Dr. Qhrl'stqpher M. Riley
AssistantProfessor of Pharmacy
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 Council, and was accepted
as partial fulfillment of the requirements for the degree
of Doctor of Philosophy.
Dean for Graduate Studies and
Research
December 1983

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