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DETERMINATION OF CITRATE, CAMPHOR AND MENTHOL BY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
By
TSOI Yeung-pang
( 蔡 仰 鵬 )
A thesis submitted in partial fulfilment of
the requirement for the degree of
Master of Philosophy in
The Chinese University of Hong Kong
1994
Thesis Committee : Dr. H. F. CHOW
DR. Glen K. C. HUI, Chairman
Dr. O. W. LAU
Prof. T. S. West, External Examiner
ACKNOWLEDGEMENTS
工 wish to express my deepest gratitude to my supervisor, Dr. 0. W. Lau, for her support, guidance and encouragement throughout the project, and for her invaluable advice given in the preparation of this thesis.
I am also indebted to Dr. C. S. Mok for his helpful discussion and technical advice throughout this project.
My thanks are also due to Mr. K.C. Cheng, Ms W.N. Ho, Ms. Y.T. Leung, Mr. K.L. So, Mr. M.C. Wong, Ms. Y.O. Lam, Mr. C.M. Chan, and other staffs of the Chemistry Department for their support and prompt assistance.
Department of Chemistry The Chinese University of Hong Kong June 1994
TSOI Yeung-pang
f 1
ABSTRACT
High Performance Liquid Chromatographic ( HPLC )
methods for the determination of organic compounds having
no special functional characteristic for detection by
ultra-violet ( UV ) or fluorescence have been developed and
illustrated by two examples. The first example is the
determination of citrate in pharmaceutical preparations by
indirect photometric detection. The second example
involves indirect conductometric detection for the
determination of camphor and menthol.
The first method applied ion-interaction
chromatography, employing an indirect method of detection,
where salicylate is used as a co-anion to an ion-
interaction reagent ( IIR ) , tetrabutylammonium ion, and
produces a constant background of UV absorption.
The citrate in the sample was separated and determined
quantitatively by isocratic reversed phase HPLC on a column
(25 cm X 4.6 mm ) of Adsorbosphere C18 ( 5 /Lim ) with an
aqueous solution containing tetrabutylammonium iodide (
0.12 mM ) and sodium salicylate ( 0.15 mM ) as the mobile
phase ( 1.8 mL / min. )• Detection at 242 nm vs. air with
the polarity of the integrator reversed. The calibration
graph was linear from 20 to 200 /ig / ml ) . Recoveries
ranged from 95 to 106% for 8 samples.
• • 11
The second method employed an indirect conductometric
detection in the quantitative determination of camphor and
menthol in pharmaceutical products using a Adsorbosphere
cyano column ( 25 cm X 4.6丽,5/m ) with 4 mM sodium
citrate in 30 % aqueous acetonitrile as the mobile phase (
1 ml / min. ) with the polarity of the integrator reversed.
The calibration graph was linear from 0.05 to 1 mg / ml for
both compounds. Recoveries ranged from 95 to 105% for
camphor and 97 to 100% for menthol from 10 samples.
• • « 111
TABLE OF CONTENTS
Page
• I. Acknowledgements i II. Abstract ii III. Table of contents iv IV. List of Tables and Figures v
Chapter 1. Introduction 1 1.1 Modes of chromatography 1.2 Objective of the present study
References
Chapter 2. Instrumentation and theory 8 2.1 Instrumentation of HPLC 2.2 Theory of liquid chromatography
References
Chapter 3. Determination of citrate in pharmaceutical 21 preparations by HPLC using indirect photometric detection
3.1 Introduction 3.2 Review of the analytical methods 3.3 Theory of detection 3.4 Experimental 3.5 Results and discussion 3.6 Conclusion
References
Chapter 4. Determination of camphor and menthol 74 by HPLC using indirect conductometric detection
4.1 Introduction 4.2 Review of the analytical methods 4.3 Theory of detection
(
4.4 Experimental 4.5 Results and discussion 4.6 Conclusion
References iv
LIST OF TABLES AND FIGURES
1. List of Tables : Page
3.1 Intensity of the light source at different
wavelength 43
3.2 Peak area of 2ng citrate at different
background settings 45
3.3 Effect of co-anions in the mobile phase on the
sensitivity and retention of citrate 48
3.4 The absorbance of TBAI and co-anions 49
3.5 Signal and retention at different TBAI and NaSa
concentrations in the mobile phase 54
3.6 Signal of 2 /xg citrate at various wavelengths 56
3.7 Data for the calibration of citrate 58
3.8 Precision test of the proposed method 60
3.9 Description of pharmaceutical preparation 61
3.10 Assay of content of citrate in drug sample 63
3.11 Recovery test for the proposed method 67
4.1 Effect of conducting species in the mobile phase
on the sensitivity and retention on camphor and
menthol 88
4.2 Concentration effect of background conducting
species on peak intensity and retention time 90
4.3 Concentration effect of acetonitrile on
retention time 92
4.4 Data for the calibration of camphor 93 4.5 Data for the calibration of menthol 95
V
4.6 Precision test of the proposed method 97
4.7 Description of pharmaceutical preparation 99
4.8 Assay of the contents of camphor and menthol in
drug sample 100
4.9 Results of recovery test for the proposed
method 103
2. List of Figures :
2.1 General instrumentation for a high performance
liquid chromatograph 8
2.2 Six-port sample injection valve 12
3.1 The paired-ion model 31
3.2 The dynamic ion-exchange model 31
3.3 The ion-interaction model 33
3.4 Retention of an oppositely charged sample 33
3.5 Intensity of the detector light source vs
wavelength 44
3.6 Signal to noise ratio of 2 ng citrate at
different background settings 47
3.7 Absorption spectrum of 0.12 mM tetrabutyl-
ammonium iodide 50
3.8 Absorption spectrum of mobile phase containing ,
0.12 mM tetrabutylammonium iodide and 0.15 luM
sodium salicylate 50
3.9 Absorption spectrum of mobile phase containing
0.12 itiM tetrabutylammonium iodide and 0.15 mM
vi
r.
sodium benzoate 51
3.10 Absorption spectrum of mobile phase containing
0.12 mM tetrabutylammonium iodide and 0.15 mM
potassium hydrogen phthalate 51
3.11 Calibration graph for the determination
of citrate 59
3.12a Chromatograms of sample No. 2 ( left ) and
sample No. 3 ( right ) 64 3.12b Chromatograms of sample No. 4 ( left ) and
sample No. 5 ( right ) 65
3.12c Chromatograms of sample No. 7 ( left ). and sample No. 8 ( right ) 66
4.1 Calibration graph for the determination
of camphor 94
4.2 Calibration graph for the determination
of menthol 96
4.3a Chromatograms of sample No. 4 ( left ) and
sample No. 5 ( right ) 101
4.3b Chromatograms of sample No. 6 ( left ) and
sample No. 8 ( right ) 102
•) J,
vii
CHAPTER 1
INTRODUCTION
Liquid chromatography is the generic name used to
describe any chromatographic procedure in which the mobile
phase is a liquid. High performance liquid chromatography
( H P L C ) is probably the single most used analytical
technique today, surpassing even gas chromatography in use
for the separation and analysis of mixtures. This is due
mainly to the extensive versatility of the technique which
results from the fact that both stationary phase and mobile
phase interactions may be utilised to alter the selectivity
of the system. Modern liquid chromatography has the
advantages that the columns are reusable, sample
introduction can be automated and detection and
quantitation can be achieved by the use of continuous flow
detectors. These features lead to improved accuracy and
precision of analysis.
1.1 MODES OF CHROMATOGRAPHY
The exact mode of chromatography operating in a
given application is determined principally by the nature
of the column stationary phase and the mobile phase. It
must be stressed that , while there may be one dominant
mechanism, the modes are not mutually exclusive. HPLC
separation methods can be classified as :
1
1. Adsorption chromatography;
2. Partition chromatography;
3. Ion-exchange chromatography;
4. Exclusion chromatography.
1.1.1 Adsorption chromatography
Adsorption chromatography was introduced by Tswett^ and
Day The separation is based on the selective adsorption
of solutes on the active sites on the surface of the
adsorbent such as silica and alumina. The active sites on
silica are hydroxy1 groups. The eluent systems are usually
non-polar solvents containing a small amount of polar
additive called polar modifier. When the sample is applied
to the column packing, polar molecules with polar
functional groups will be attracted towards the active
sites and will subsequently be displaced by the polar
modifier molecules and will pass down the column to be
readsorbed on new sites. More polar molecules will be
retained more strongly and hence eluted more slowly by the
column. This kind of chromatography is commonly applied in
the separation of isomer like propellants, vitamins and
alkaloids .3-5
1.1.2 Partition chromatography
Partition chromatography can be divided into liquid-
liquid partition chromatography and organo-bonded partition
2
chromatography. This method was developed by Martin and
Synge^ and in both modes of partition chromatography,
separation is achieved by partition of an organic solute
between a liquid mobile phase and an organic liquid
adsorbed on , or chemically bonded onto, a solid support.
Liquid-liquid partition chromatography is seldom used today
because of the problems of solvent stripping and limited
hydrolytic stability. This technique is displaced largely
by organo-bonded partition chromatography, which is, in
fact, a modified mode of the liquid-liquid partition
chromatography, where the liquid stationary phase is
chemically bonded or organo-bonded to an insoluble matrix.
This method is further divided into normal phase where the
stationary phase is more polar than the mobile phase and }
vice versa in the case of reverse phase partition
chromatography.
The selectivity in reversed phase chromatography is
controlled mainly by eluent effects and it is therefore
unnecessary to have an extensive range of packing
materials. With different mobile phases used, different
form of retention mechanisms will be resulted. The
technique found its use in the separation of plasticizer,
saccharide and fatty acids.^ -
Ion interaction chromatography belongs to this class
of chromatography which is a modified mode of reversed bond
phase chromatography having the ability of ion-exchange
3
chromatography.
1.1.3 Ion-exchange chromatography
Ion exchange chromatography is performed with packing
materials which contain ionic functional groups. These are
either acid groups such as sulphonic or carboxylic acids
for the separation of cations and amine or quaternary
ammonium basic groups for the separation of anions.
The original definition of ion chromatography
referred to an ion exchange separation with conductometric
detection making use of a suppressor column to remove the
background conductance of the eluent. The latest trend
however, moves away from the use of suppressor columns and
the definition has been considerably broadened to include
any high performance ion exchange separation which uses a
low capacity resin and eluents of low concentration. Ion
chromatography has been one of the fastest growing areas of
HPLC in recent years. Again, silica based materials have
proved popular but recently several manufacturers have
produced polymeric ion exchangers with low exchange
capacity specifically for ion chromatography. The main
advantage of these materials is their pH stability which
allows weakly acidic or basic species to be chromatographed
in their ionic form. They also show different selectivity
from the silica ion exchangers.^
4
1.1.4 Exclusion chromatography
Exclusion chromatography, also called gel permeation
chromatography ( GPC ) , utilizes the selective diffusion of
solute molecules within the solvent filled pores of a
three-dimensional lattice. Small molecules will permeate
the pores while large bulky molecules will be excluded.
Thus separation is achieved principally on the basis of
molecular weight and size, with larger molecules being
eluted from the column more quickly.^ It is commonly used in
the analysis of biological materials and polymer.
1.2 OBJECTIVE OF THE PRESENT STUDY
The objective of this work is to develop HPLC methods
for the determination of organic compounds, which do not
require the analytes to have specific structural
properties.
The work is divided into two parts. The first part of
this work is to develop an HPLC method for the
determination of citrate, which is an organic anion, by
using ion-interaction chromatography, which is usually used
for the determination of inorganic ions. Also, the
detection method used in this project is by UV detection, J
which is uncommon for citrate because it is UV transparent.
The detection method is based on the decrease in UV
absorption of the eluent when the analyte is eluted out and
5
the peak is recorded as a negative peak. This method is
usually called indirect photometric detection. Details of
this work will be shown in Chapter 3 of this thesis.
The second part of this work is to develop an HPLC
method for the determination of camphor and menthol, which
are neutral compounds without UV chromophores, and hence
are non-conducting and UV inactive, by using indirect
conductometric detection. Details of this work will be
shown in Chapter 4.
6
REFERENCES
1. M. Tswett, Trav, Soc• Nat, Warsowie, 1903, 14, 6
2. D.T. Day, Science, 1903, 14, 1007
3. “ J.O. Doali and A. A. Juhasz, Anal. Chem., 1976, 48,
1859
4. M.E. Evenson and B.L. Warren, Clin. Chem., 1976, 22,
851
5. M.G.M. De Ruyter and A.P. Deheenheer, Clin. Chem.,
1976, 22, 1593
6. A.J.P. Martin and R.L.M. Synge, Biochem. J., 1941,
35, 1358-1368
7. N.E. Hoffmann and J.C. Liao, Anal• Chem., 1976, 48,
1104
8. M.T. Gilbert, High Performance Liquid Chromatography,
lOP Publishing Limited, Bristol, 1987, p.1818
9. A. Braithwaite and F.J. Smith, Chromatographic
Methods, Chapman and Hall, New York, 1985, p.414 I
7
CHAPTER 2
INSTRUMENTATION AND THEORY
2.1 OVERVIEW OF HPLC INSTRUMENTATION
The high performance liquid chromatograph comprises of
the following components' ( Figure 2.1 ) : (1) a solvent
reservoir for the mobile phase; (2) a solvent pump,
equipped with a damping unit if a pulasating action
results, to force the mobile phase through the
chromatographic system; (3) a sampling or injection devices
to introduce the sample into the column; (4) the saparation
column; and (5) a detector with recorder readout or other
data handling device.
GRADIENT DEVICE
I Thtrmojtated
r INJECTOR CHR0MATCX5RAM
B L r ° [ f i f^ESEfWOm PUMP / / RECORDER
# @ COLUMN DETECTOR
Figure 2.1 General instrumentation for a high performance liquid chromatograph
8
2.1.1 Mobile phase
The mobile phase employed for HPLC separations may
comprise water, aqueous buffer solutions, organic solvents
such as methanol and acetonitrile or a mixture of the
above. All solvents should be of high spectroscopic
purity, dust free, and should be degassed before use.
They should also, if UV detection is being employed, be
transparent to the wavelength for detection.
2.1.2 Solvent delivery system̂ ''*
‘Several features of the solvent delivery system must
be considered: precise delivery of solvent over a
relatively broad range; maximum pressure attainable; ,
compatibility with other components in the HPLC system;
compatibility with the choice of solvents; and noise level
in the detector resulting from any pulsations. The final
choice of pump will be interwoven with the type of
separation column, the detector employed, whether isocratic
or gradient elution is to be performed, the minimum
detectability limit desired, precision in quantitation, and
the cost of the packaged chromatograph.
Three main types of pumps are used in HPLC to propel
the liquid mobile phase through the system: (1) the
constant pressure type; (2) the syringe design, which
delivers a constant non-pulsating flow; and (3) the
9
reciprocating piston pump, which may be of the single, dual
or triple head design.
2.1.2.1 Constant pressure pumps
Constant pressure pumps deliver solvents via a small
head piston which is driven by a pneumatic amplifier. The
main advantages of these pumps are low cost, ability to
deliver high pressure and stability of flow during the
delivery stroke of the pump. Although pulseless, the
flowrate and, hence, the elution volume, can vary with the
changes in permeability of the column or the- viscosity of
the solvent leading to poor precision and accuracy of
analysis.
2.1.2.2 Syringe type pumps
Syringe type pumps work on the principle of positive
solvent displacement by a piston mechanically driven at a
constant rate in a piston chamber (250-500 ml capacity)
with the generation of a pulseless flow and with high-
pressure capabilities (200-475 atm). This overcomes the
major disadvantage of pneumatic amplifier type pumps, and
makes syringe pumps ideal to yeild reproducible retention
times. However, the major problem encountered is the design
of a suitable refill mechanism.
2.1.2.3 Reciprocatincf piston pumps
Reciprocating piston pumps are most commonly used in
HPLC since they permit delivery of a wide range of flow
10
rates and are relatively inexpensive. Reciprocating pumps
of the single piston design function by having a slow
solvent delivery cycle compared to rapid refilling of the
piston chamber. However, there is some pulsing of the flow
because of the finite time taken to fill the piston
reservoir, and also the fact that the initial part of the
delivery stroke is concerned with compression of the
solvent prior to pumping. Improved precision and smoothing
of flow is provided by twin-piston reciprocating pumps,
where the pistons are driven approximately 180° out of I
phase. i.
2.1.3 Sample introduction
The ideal sample introduction method should be able to
insert reproducibly and conveniently a wide range of sample
volumes into the pressurized column as a sharp plug with
little loss in efficiency. The injection system should
possess zero dead volume to prevent loss of resolution. }
Sample application techniques can be broadly classified as
either syringe or valve injections.
2.1.3.1 Syringe injection''
In the syringe injection method, a small ( 10 pi )
sample is introduced into the pressurized column with a
high-pressure syringe through a self-sealing elastomer
septum and directly on top of the column packing. The
method is limited to low pressure unless a double septa
11
design is employed. At higher pressures there is sample
leakage around the syringe plunger, and it is difficult to
maintain the septum leak proof and to insert the needle
into the pressurized system. Advantages include: low
initial cost, variable and small sample volumes and low
band spreading.
2.1.3.2 Valve injection'*
Valve injection of the sample is now the preferred and
accepted technique in modern HPLC, particularly for routine
and quantitative analysis. In this method a fixed-volume
loop is filled with the sample using a syringe, then the
eluent flow is directed through the loop to flush the
sample onto the column. For most analytical separations a
six-port valve is used with a 10 or 20 external loop, as
illustrated in Figure 2.2/
^ Meedle port ; ^ ^
Vents
Loop ^ ^ LOAD INJECT
I Figure 2.2 Six-port sample injection valve
12
2.1.4 Separation column^
Columns for analytical HPLC are typically 10-25 cm
long and with 4-6 mm internal diameter. The columns are
made of stainless steel to cope with the high back pressure
and are glass lined to prevent metal catalysis of solvent-
solute reactions at high column pressures. The tubing must
have a smooth, precision-bore internal diameter to ensure
that a well-packed column will not channel near the
wall/packing interface because of wall irregularities.
This would result in broader peaks and lower efficiency.
Straight columns are preferred. They are operated in the
vertical position with the flow being directed either up or
down through the packing. Connections to the columns are
made with low dead-volume fittings designed to eliminate
stagnant pockets of mobile phase.
Current practice utilizes column packing that lies in
the range from 3 to 7 /Ltm in diameter; occasionally up to 10
jLtm or higher, especially for exclusion chromatography. The
type of packing material used depends on the type of
separation mode utilised.
2.1.5 Detector
Detectors used in liquid chromatography should ideally
function with high precision, high sensitivity, high
stability and fast response to record rapid eluting peaks.
13
They should also have a wide linear dynamic range to ensure
good quantitative analysis and be easy to operate and
maintain. Unfortunately there is no universal detector
that meets all these criteria. Thus, it is necessary to
select a detector on the basis of the problem at hand so
that in doing a variety of separations, more than one
detector will be needed.
There are three basic types of detectors :
1. Detectors which monitor a specific property of the
solute not shared with the solvent e.g. UV absorbance and
fluorescence. Possession of such a property by the solute
affords its detection in the effluent.
2. Detectors which monitor a bulk property of the eluent e.g. refractive index; in this case the solute
modifies the base value of the property associated with the
solvent.
3. Desolvation/transport detectors which function by
separating the solvent from the eluent, thus allowing
subsequent detection by techniques such as flame
ionization?’^ or mass spectrometry.^
2.1.5.1 Selective property detectors
UV-visible photometers and spectrophotometers
An ultraviolet photometer operating at fixed
wavelengths of 254 or 280nin is one of the most widely used
detectors for HPLC. The advantages of this detector are
14
its relatively low cost, high sensitivity ( nanogram level
)achieved for many compounds of chemical and biological
interest provided that they absorb UV light, and its
insensitivity to changes in temperature, flowrate, and
mobile phase composition.
The variable wavelength detectors are an order of
magnitude less sensitive than the fixed wavelength
detectors but are considerably more versatile, since any
wavelength within the range of the detector may be
selected.
Detectors are now commercially available which allow
programmable wavelength switching during analysis, thus
optimizing sensitivity and selectivity. As many as 12
wavelengths can be selected. The systems use reversed-
optics geometry, i.e., the light from the source is
dispersed after passing through the sample by a holographic
grating on to an array of photodiode detectors. The
photodiode array is a row of detectors ( up to 400 )
mounted on a 1 cm silicon chip, each diode receiving a
different wavelength. The full spectra obtained from diode
array spectrophotometer are a useful aid to component
identification. The examination of the many spectra taken
during the elution of a peak gives information on the peak
homogeneity thus aiding accurate quantitation.
15
Conductivity detectors
The conductivity detector which measures the
electrical resistance of a conducting medium between two
electrodes in solution is usually used in ion
chromatography. The sensitivity is about 10 jug/ml using an
eluent of relatively high conductance. Reducing the eluent
conductance can increase the sensitivity by about two
orders of magnitude. The detection of ionic species in
HPLC eluents using conductivity detector is very difficult
because of high background eluent conductance. The
development of suppressed ion chromatographic systems^®
obviated this problem.
Electrochemical detectors
Electrochemical detectors can be used for the
detection of compounds which are electroactive. The
detection depends on the fact that electroactive species
undergo electrolysis at an electrode when a voltage is
applied and that such solutes may be detected by monitoring
the resulting current. The applicability of
electrochemical detectors is limited by the requirement
that the mobile phase must be electrically conductive.
This may often be achieved by adding buffer to the eluent.
There are still other selective property detectors
such as fluorescence detector, which is sensitive and
selective; infrared detector; atomic absorption detector
and radioactivity detector.
16
2.1.5.2 Bulk property detectors
Refractive index (RI) detectors
Refractive index detector is the commonest bulk
property LC detector and under properly controlled 1
conditions this is virtually a universal detector, although
it is less sensitive than the selective property detectors.
The detector response depends on the difference between the
refractive index of the pure mobile phase and that of the
mobile phase together with the solute eluting from the
column. Therefore, this type of detection is- sensitive to
fluctuations in pressure, temperature and composition.
However, it is an ideal universal detector because most
compounds modified the RI to some extent and both positive
and negative changes in the solvent RI can be detected.
2.1.5.3 Desolvation/transport detectors
. The detection using transport detectors is based on
the concept of physically separating the solvent, which is
necessarily volatile, from the non-volatile solute. It is
ideal for most gradient elution applications, the major
limitation being not applicable to systems with non-
volatile buffers. I
Examples of such detectors include the flame
ionization detector, electron capture detector and mass
spectrometric detector.
17
2.2 THEORY OF LIQUID CHROMATOGRAPHY
Chromatography is a dynamic separation system
consisting of two media, a stationary phase and a mobile
phase. When the solute molecules in the mobile phase come 會
into contact with the stationary phase, there is now
competition between the two phases for the solute
molecules, which depends on their physical properties and
affinity for the stationary phase. This process is termed
partition with each component distributed between the two
phases as they pass through the system. Since different
component molecules have different affinities, they will
proceed through the system at different speeds and hence
separation is achieved.
The molecular interactions leading to the distribution
of a component between the mobile phase and stationary
phase are attributed to a combination of polar forces
arising from induced and permanent electric fields and i.
London's dispersion forces which are influenced by the
relative molar masses of the solute and solvent.u
工ntermolecular forces predominate in chromatography with
polar and dispersion forces having a major contribution to
the overall interactions.
18
Solute retention is normally measured by the capacity
factor, k', which is defined by the expression :
= (Vr - Vo)/Vo = (tR - to) /to
where Vr, V。/ t^ , t。are respectively the retention volume
/ time of solute peak and unretained peak of the system at
constant flowrate of the mobile phase. The larger the value
of , the better the separation but longer the analysis
time and broader the peak/
The efficiency of a column is obtained from the number
of theoretical plates, N :
N = 16 ( tR/Wb )2 J (
where W^ is the base width of the solute peak.
The larger the value of N, the better the resolution
of the column and the value of N is between 2,000 to 20,000
for a good column.
19
*
REFERENCES
1. N. Hadden, F. Baumann, F.MacDonald, M. Munk, R. Stevenson, D. Gere, F. Zamaroni and R. Majors, Basic Liquid Chromatography, Varian Aerograph, 1971
2. L. Berry, and B.L. Karger, Anal. Chem., 1973, 45, 819
3. A. Braithwaite, and F.J. Smith, Chromatographic
Methods, Chapman and Hall, 1985, p.414
4 . M.T. Gilbert, High Performance Liquid Chromatography,
lOP Publishing Limited, Bristol, 1987, p.481
5. H.H. Willard, J.A. Dean, L.L. Merritt and F.A. Settle, Instrumental Methods of analysis, Litton Education Publishing Inc., p.1030
6. Rheodyne Inc. Bull. 106 Cotati, Calif.
7. M. Kreji, et al, J. Chromatogr., 1981, 218, 167
8. V.L. McGuffin and M. Novotny, Anal. Chem., 1981, 53, 946
9. T.E. Young and R.J. Maggs, Anal. Chim, Acta, 1967, 38, 105
10. H. Small, T.S. Steven and W.C. Bauman, Anal• Chem.,
1975, 47, 1801
11 A. B. Littlewood, Gas Chromatography Academic Press, 1970, p.71
20
CHAPTER 3
DETERMINATION OF CITRATE IN PHARMACEUTICALS
BY HPLC USING INDIRECT PHOTOMETRIC DETECTION
3.1 INTRODUCTION «
citric acid is a well known food ingredient, both to
consumers and food processors. Consumers know it as the
• predominant acid in oranges, lemons, and. limes, but
processors use it for its many functions in many foods.
Citric acid has complete acceptance by consumers as a
"natural" ingredient.丨
Citric acid is used as acidulant in beverages,
confectionary, effervescent salts, in pharmaceutical
syrups, elixirs, in effervescent powders and tablets, to
adjust the pH of foods. It is used in beverages, jellies,
jams, preserves and candy to provide tartness; in the
manufacturing of citric acid salts; as mordant to brighten
colours; in electroplating; in special inks; in analytical
chemistry for determining citrate-soluble P2O5; as reagent
for albumin, mucin, glucose, bile pigments
Citric acid is widely used as a metal deactivator or
chelating agent,3 and in combination with phenolic
antioxidants, it is used as synergistic antioxidant in
21
*
processing cheese. Also, detection of added acidulants in
juice drink would be indicative of adulteration provided
they were not declared on the label. The citric acid
content of these foods, therefore, determines their
acceptability and keeping quality. The accurate
quantification of citric acid in such foods is therefore
essential for quality control requirements and meeting
legal regulations.
\
22
3.2 BRIEF REVIEW OF THE ANALYTICAL METHODS FOR
THE DETERMINATION OF CITRIC ACID
3.2.1 Spectrophotometric methods
3.2.1.1 Enzymatic method Citric acid ( citrate ) is converted by citric
lyase to oxaloacetate and acetate. In the presence of the
enzymes malate and lactate dehydrogenase, oxaloacetate and
its decarboxylation product pyruvate are reduced to L-
malate and L-lactate, respectively, by reduced
nicotinamide-adenine dinucleotide ( NADH ) and
stoichiometric amount of NAD+ is produced. The NADH
consumed is measured at 340 nm with a spectrophotometer.^
This method is modified'' where a redox reaction
in which 2-( 4-iodophenyl )-3-( 4-nitrophenyl )-5-phenyl-
2H-tetrazoliuin chloride ( INT ) is reduced to NADH and then
to a red formazan, the concentration of which is
proportional to the original citrate concentration. The
method has good sensitivity and a short analysis time ( 10-
25 min ).
3.2.1.2 Derivatisation
Citric acid is derivatised to form a chromophore,
and the absorbance at the absorption maximum is measured.
23
An example is the modified method^ based on the
chromophore formed between pyridine, acetic anhydride and
citrate. The absorbance was measured at 428nm. Another
derivatisation agent is the acid alizarin black SN? and the
absorbance was measured at 603 nm vs. a blank prepared
similarly. The derivatisation technique gives a faster
analysis time than the enzymatic method while the enzymatic
method is more specific.
3.2.2 Titrimetric methods
Titrimetric method for the determination of
citric acid and citrate is based on complex formation
between copper ion and citrate ion in buffered solutions
(boric acid/borate or sodium hydrogencarbonate). The
equivalence point has been established with an indicator or
from potentiometric titration curves obtained with a
copper or a silicone rubber-based copper(II)-selective^
indicator electrode. The best reported precision was
about 0.9%. 01in9 employing a copper-selective electrode
titrimetric method where equal increments of copper(II)
solution were added stepwise and the sample solution was
kept at constant pH by simultaneously running a pH-
stat addition. Titration curves obtained at constant pH
can be linearized as described by Gran'o, so that the
precision of the results is increased considerably.
A dropping copper-amalgam electrode^^ was used
24
in conjunction with a SCE for the potentionmetric
titration of citrate with aq. 0.5M-CUSO4 in 0.IM-NaHCOj
medium (pH 8.3). The range of the method was 0. ImM to
0. IM-citrate and was used in the analysis of the citrate
content in orange and lemon juices.
3.2.3 Atomic Absorption Spectrometry (AAS)
A new indirect AAS method for determining citric
acid in soda water is investigated by Che et al. After
adding the CU3(P04)2 reagent to react with citric acid in the
sample solution, the unreacted reagent is isolated by .
centr if ligation, and Cu is then determined by AAS after
dissolving the reagent and the content of citric acid is
then calculated. The result is not affected by pH in the
range of 1 to 11. The only drawback for this method is
that it needs 20 hours to obtain a stable and maximum
signal of Cu.
3.2.4 High Performance Liquid Chromatography (HPLC) Methods
High performance liquid chromatography (HPLC)
affords a rapid and simple technique for analyzing
certain mixtures of organic acids. Many analytical
methods have been reported for the separation and
determination of organic a c i d s .歸 Three main methods are
commonly u s e d , 28 namely, ion-exchange chromatography,
solvophobic chromatography and reverse-phase chromatography
25
of the derivatized products.
3.2.4.1 Ion~exchanqe Chromatography
Ashoori6 applied this method to determine citric acid
in a wide variety of food products. The citric acid was
detected as a single peak in all samples analyzed with no
interference from other compounds. These results
indicated that the method is specific for citric acid.
Zhu27 used this method to determine saccharine and citric
acid in beverage.
3.2.4.2 Solvophobic Chromatography
The addition of acids or acidic buffers to the
mobile phase lowers the pH and suppresses the ionization of
the acidic functional groups of the solutes. Ionization
suppression-aided separations are therefore based on the
hydrophobicities of the solutes. The retention is the
result of hydrophobic interactions of the hydrocarbonaceous
moiety of the solute with the octadecyl chains of the
stationary phase. Relatively polar substances can be
separated on Cig-silica gel columns with neat aqueous
eluents of the appropriate pH. This technique is termed
hydrophobic chromatography or ion-supression
chromatography. ‘
A reversed-phase HPLC method described by
Coppola29 uses 2% aqueous potassium dihydrogen phosphate
solution adjusted to pH 2.4 with phosphoric acid for the
26
determination of organic acids (including citric acid) in
cranberry juice. Distler^® used sulphuric acid to adjust
the pH of the pure aqueous mobile phase for the separation
of short-chain carboxylic acids on Cig-silica gel. Marce^^
employed a direct method for the simultaneous determination
of organic acids (including citric acid) in fruit juices
and wines by isocratic reversed phase HPLC. The selection
of the experimental conditions (pH, ionic strength, flow
and temperature) has been carried out by optimizing the
resolution and time of analysis using a modified sequential
simplex method.
3 , 2 .4» 3 Reversed-phase chromatocfraphy of derivatized
products
Chemical derivatization has been required for the
selective and sensitive detection of analytes such as
biological or bioactive substances and metals in tissues,
body fluids, and pollutants. It also affords selectivity
in the separation of the analytes. Hence, growing numbers
of papers have been published recently reporting methods
and applications for derivatization in liquid
chromatography, especially high-performance liquid
chromatography. Review papers and monographs that have
appeared in the past several years give information on this
subject. 31 •
Recent progress in the chemistry of derivatization and
techniques of liquid chromatography has made possible trace
27
analysis of organic functional groups of low-molecular-
weight compounds and, with the aid of enzymes,
differentiation of certain functional group(s) from other
functional groups of the same type in large molecules such
as proteins.
Most chemical reagents reported so far for carboxylic
acids are not appropriate in terms of being undetectable
themselves by UV, fluorescence, and other detectors.
Nevertheless, some of them are useful in practice.
Carboxylic acids in beverages such as wines and other
commercial drinks or natural fruit juices were derivatized
with p-bromophenacyl bromide in 50% acetone-water (pH 7 to
8) containing 8.5 mM 18-crown-6 in boiling water for 75
min, separated on RP-18, and detected at 254 nm.^^
3.2.5 A brief comparison of the method
The AAS is an indirect method while the titrimetric
method and spectrophotometric methods for the determination
of carboxylic acids are lengthy. Hence, chromatographic
methods must be regarded as an attractive alternative.
However, as citric acid (or citrate) is UV transparent
making detection difficult. Nevertheless, this problem' can
be overcome by using ion-interaction chromatography. The
theory of detection of this technique will be described in
the next section.
28
3.3 THEORY OF DETECTION
3.3.1 Indirect photometric detection
Analyte which is ultraviolet transparent cannot be detected in the usual way and the method of indirect detection has been developed.32-35
In this kind of detection method, an anion ( in this
case, salicylate ion which is ultraviolet, UV, active ) is
used as co-anion to an ion-interaction reagent ( tetra-
butylammonium ion ) • At equilibrium, a constant background
of UV absorption is maintained when the co-anions
dynamically occupy all the sites in the primary layer of
the stationary phase.
When an analyte ion of the same charge as the co-anion
first enters the column, it will compete with the coanion
and displace it out of the primary layer to the mobile
phase and hence a positive peak is developed. After some
time when the analyte leaves the column, it will be
released from the primary layer to the mobile phase, and
there will be an excess of charge in the primary layer and
the co-anion in the mobile phase will enter the primary
layer in order to maintain the charge balance. Therefore,
the concentration of the co-anion in the mobile phase will
be decreased. • Thus, the UV detector responds to the
29 T
presence of the analytes by producing a negative peak in the baseline absorbance plot. The magnitude of this peak is directly related to the difference in the concentration and the molar absorptivity between the eluent and analyte species .34
3.3.2 Models for ion-interaction chromatography
Most applications of reversed-phase ion-pair
chromatography involve the addition of a long-chain alkyl
sulfonate ions to the mobile phase to give enhanced
separation of oppositely charged sample ions. The exact
mechanism to describe the ion-pair phenomenon is still
uncertain. There are three popular hypotheses. Two
models propose extreme situations and each covers a
substantial amount of chromatographic data. These two
proposals are the ion-pair model and the dynamic ion-
exchange model. A third view, which is broader in scope
than the previous two concepts, accommodates both extreme
views without combining the two models. This proposal is
the ion-interaction model.
3.3.2.1 The ion-pair model
The ion-pair postulate stipulates that the formation
of an ion pair̂ '̂ ( Figure 3.1 ) occurs in aqueous mobile
phase prior to its adsorption onto the bonded, hydrophobic
stationary p h a s e R e t e n t i o n is governed by the amount of
non-polarity of the "ion-pair", which determines the
30 *
o參 O眷
Figure 3.1 The paired-ion model
• : ion of +ve charge ^ : ion of -ve charge
^^^^^^^ : ion-pair reagent /^M^hT : sample molecule
/ ° ; j
Figure 3.2 The dynamic ion-exchange model
書 :ion of +ve charge O : ion of -ve charge
/vvWV^ : ion-pair reagent /SA/wn/^ : sample molecule
31
affinity to the stationary phase. A longer alkyl chain on
the pairing agent simply makes a less polar ion pair and
the retention of the pair increases as a result of its
greater affinity for the stationary phase.
3.3.2.2 Ion-exchange model
A second view stipulates an ion-exchange mechanism
In this hypothesis, it is the unpaired lipophilic alkyl
ions that adsorb onto the nonpolar surface and cause the
column to behave as an ion exchanger. This concept^^ is
depicted in Figure 3.2. The longer the chain length of the
ion-pairing reagent the more surface coverage of "ion-
exchanger" will occur and the longer will be the retention
of the ionic sample.
3.3.2.3 The ion-interaction model
Most recently, an ion-interaction mechanism has been
proposed by Bidlingmeyer, et a l , which is less restrictive
than the two previous models. This model is based upon
conductance measurements, which show that upon a series of
experiments involving neutral and charged samples injected
into systems containing positively and negatively charged
lipophilic compounds added to the mobile phase, ion-pairs
do not form in the mobile phase. Neither the ion-pairing
nor the ion-exchange model can explain the data in a
consistent way. Instead, the results suggest a retention
32
BULK ELUANT
SECONDARY\ , O LAYER \
\ P R I M A R Y / SURFACE LAYER
Figure 3.3 The ion-interaction model
• : ion of +ve charge
o : ion of -ve charge
/VSAAT : ion-interaction reagent
/
/ o \ o O . .
- c:> ;' \ :
Figure 3.4 Retention of an oppositely charged sample
molecule
/SAA/V^ : analyte ion
Dotted figure denotes that it has moved from that
position
33 r
*
mechanism that is broader in scope and is best described as
one of ion interaction. The ion-interaction mechanism does
not require ion-pair formation in either phase and is not
based on classical ion-exchange chromatography. The ion-
interaction mechanism assumes dynamic equilibrium of the
lipophilic ion resulting in an electrical double layer
forming on the surface. The retention of the sample
results from an electrostatic force due to the surface
charge density provided by the reagent ion and from an
additional "sorption" effect onto the nonpolar surface.
This has been considered by same workers as
"pseudoexchange". .1
In the ion一interaction model depicted in Figures 3.3
and 3.4, a layer of lipophilic ions from the ion-pair
reagent ( or ion-interaction reagent, IIR ) is absorbed
onto the nonpolar surface ( see Figure 3.3 ). Because these
lipophilic ions have the same charge, they are well spaced
from one another. Most of the surface area on the nonpolar
packing surface is unaffected with only a small fraction of
the surface area being coated with the ion-pair reagent.
A primary ion layer and an oppositely charged counter-ion
layer are formed on the top of the non-polar surface. This
is an electrical double-layer model. The adsorbed ions are
in dynamic equilibrium between the bonded phase and mobile
phase so that if the reagent concentration is increased in
the mobile phase, the amount of reagent ion adsorbed is
also increased, thus increasing the amount of charge on
34
the surface. Transfer of samples through this double layer
is affected by electrostatic and van der Waals forces. For
instance, an ionic organic sample of opposite charge to the
reagent ion is attracted to the charged surface.
Chromatographic retention results from this coulombic
attraction and from an additional "sorption" of the
lipophilic portion of the sample molecule ( Figure 3.4 )
onto the nonpolar surface. The net result is that a pair
of ions ( not necessarily an ion pair ) has been adsorbed
onto the stationary phase.
In ion-pair chromatography many parameters can be
varied in order to effect a separation. In addition to the
stationary and mobile phases and the type, size and
concentration of the counter ion, the pH is a very
important parameter as it determines the concentration of
the ionic form of the solutes.
I. .
Ion interaction chromatography has several
advantages over ion exchange chromatography in some
applications .45 A number of applications rely on the
ability to modulate the "capacity" of the
"pseudoexchanger" simply by altering one or more of the
following:
1. the concentration of the IIR in the mobile phase. 2. The lipophilicity of the IIR-in a homologous series
of IIRs: the longer the nonpolar chain the more the
35
IIR is adsorbed by the substrate.
3. The solvent polarity of the mobile phase - the
addition of water-miscible solvents such as
alcohols or acetonitrile will diminish the
interaction between the lipophile and the nonpolar
stationary phase.
This ability to raise and lower the capacity can be an
advantage in dealing with samples that contain ions with a
wide range of affinities. Furthermore, since large pore
substrates are used and the functionality of the
pseudoexchanger is readily accessible on the pore walls,
IIR methods are especially suitable for the chromatography
of large ions/^
i,
36
3.4 EXPERIMENTAL
3.4.1 Apparatus
3.4.1.1 The liquid chromatoqraph
The high performance liquid chromatographic system was
composed of a solvent delivery module ( Beckman llOB ) with
a controller ( Beckman 4 21A ) ; a system organizer ( Beckman
);an injection port ( Beckman Altex 210A Value ) ; an
adsorbosphere C18 column of length 250 mm, internal
diameter of 4.6 mm and particle size of 5 jtzm ( Alltech
Associates ), protected by a lOmm x 4. 6inm guard column
packed with the same material as the column; a detector
(Waters Lambda Max Model 481 LC Spectrophotometer ) and an
integrator ( Hitachi 833A Data Processor ).
3.4.1.1.1 Parameter setting of the detector
The following settings for the UV
detector were kept unchanged during the
experiment.
Parameter Settings
Response time 1
Coarse zero -0.3 AU
3•4•1•2 Spectrophotometer
All absorption spectra and absorption measurement
(for the counter check spectrophotometric method ) were
recorded by using a Spectrophotometer ( Hitachi Model
37
U-2000 ) with matched 1 cm quartz cells.
3.4.1.2.1 Operational conditions
Wavelength scanning for the compounds
were taken with the following settings :
Parameters Settings .
Data mode ABS
Start WL 340 nm
Stop WL 2 00 nm
Scan speed 200 nm/min
Response Medium
Baseline User
Lamp change 340 nm
VIS On
UV On
List interval 0.1 nm
Threshold 0.001
Sens 1
3 . 4 . 1 . 3 P H meter
A digital pH ( Jenway 3 02 0 ) meter was used for all
pH measurements. The meter was calibrated by using standard
buffer solutions of pH=4 and pH=7 solution before used.
3.4.1.4 Glassware
All volumetric flask and pipettes were of grade A and
grade B and were calibrated to relevant BS Standard before
use.
38 r
3.4.2 Reagents and materials All reagents used were of analytical reagent grade and
used without any further purification.
3.4.2.1 Water ( Ultra pure water )
All water used during the experiment was distilled
water purified by the Millipore Milli-Q50 ultra pure water
system ( Millipore, France ). The water was deionized and
filtered through the 0.1 /xm filter from the system. The
resistivity of water produced was 18 Mfl/cm.
I. 3.4.2.2 Aqueous stock solutions
3.4.2.2.1 Stock solution of 50 mM tetrabutylammonium
iodide ( TBAI )
50 mM of the aqueous stock solution of TBAI was
prepared by dissolving 9.2346 g of TBAI in 500 ml of ultra
pure water in a calibrated volumetric flask. The solution
was then filtered through a Millipore HA type filter of
0. 45 /Ltm pore size.
3.4.2.2.2 Stock solution of 200 mM sodium salicylate (NaSa) 200 mM of aqueous stock solution of NaSa was prepared
f -by dissolving 8.0063 g of NaSa in 250 ml of ultra pure
water in a calibrated volumetric flask. The solution was then filtered through a Millipore HA type filter of 0.45 fim
pore size.
39 f
3.4.2.2.3 Stock lOmg/ml sodium citrate solution lOmg/ml aqueous stock solution of sodium citrate was
prepared by dissolving 3.9268 g of tri-sodium citrate
dihydrate in 250 ml of ultra pure water in a calibrated
volumetric flask. The solution was then filtered through
a Millipore HA type filter of 0.45 jLtm pore size.
3.4.3 Aqueous mobile phase
Various mixtures of the mobile phase were prepared by
diluting appropriate volumes of the stock IIR and co-anion
solutions in suitable volume of ultra pure water. The
mobile phases so prepared were then filtered through a
Millipore HA type filter of 0.45 jum pore size to remove any
trace of solid particles. The filtered mobile phase was d
then degassed by shaking in an ultrasonic bath ( Branson
1200 ) for at least half an hour before use.
3.4.4 Column conditioning
The column was conditioned each time when a new mobile
phase was used. This normally took two hours to do so.
Whenever the new mobile phase was changed from one
system to another, the column had to be cleaned thoroughly
by 2 0 column volume of water ( this usually took two hour
at a flowrate of 1 ml / min ) followed by another 20 column
volume of 1 : 1 mixture of methanol and water so as to
40 t
ensure that the IIR and co-anion adsorbed on the column bed
were completely removed. 1
3.4.5 Sample treatment
Generally the liquid samples were diluted to
appropriate concentrations with the mobile phase. The
solid samples were first ground and then dissolved in the
mobile phase with shaking in an ultrasonic bath for fifteen
minutes. Any undissolved particles were filtered and the
solution was transferred to a calibrated volumetric flask
and made up to the mark with the mobile phase.
3.4.6 Sample introduction
Samples were introduced into the column through the
external loop injection valve by using a 1 ml luerlock
syringe ( Becton Dickson ) fitted with a 0.2 /m disposable
filter.
41
3.5 RESULTS AND DISCUSSION
The objective of this work was to find out the optimum
conditions for the determination of citrate using ion-
interaction chromatography. A calibration graph was then
obtained. A series of samples^Xxere then taken for
analysis to assess the practicability of the proposed
method. Finally the analytical results obtained were
compared with those obtained by an established method in
order to check the accuracy of the proposed method.
3.5.1 Checking of the detector light source
The intensity of the UV detector light source was
first checked to see whether the detector was functioning
properly. Pure methanol was used as the mobile phase at
a flowrate of 1 ml/min. The data and graph were shown in
Table 3.1 and Figure 3.5 respectively.
Figure 3.5 shows a spectral response curve similar to
common spectrophotometer light sources and we can conclude
that the detector is reliable.
{
\
42
Table 3.1 Intensity of the light source at different wavelength
Wavelength (nm) Intensity
26
12
^ ^
^
230 193
201 •
198
192
270 ^
^
290 2J_9
115
^ 171
^
330
^
3£0 ^ ^ ^
138
43
220.00 ]
170.00 一 / X
: \ . >N -
CD -
o 120.00 — c -L J -
70.00 -u 20.00 I I I I I I I I I I I I I I I I I I I I I I I I I I
160.00 260.00 360.00
Wavelength / nm
Figure 3.5 Intensity of the detector light source vs wavelength
44
3.5.2 Choice of background setting The mobile phase used was 0.60 mM TBAI together with
0.75 mM sodium salicylate at a flow rate of 1.8 ml/min. The
wavelength of detection was 248 nm.
An aliquot of standard citrate ( 2 jitg ) was injected at different background settings ( i.e. different absorption intensities of the mobile phase ) of the detector. The peak area so obtained together with the noise level and the signal to noise ratio ( S / N ) are shown in Table 3.2.
Table 3.2 Peak area of 2jLtg citrate at different background settings
BG SET Peak area Noise S/N
( X 103 counts) ( X 103 counts)
-0.10 130.181 48 2.712
-0.05 139.201 50 2.784
0.00 139.262 49 2.842
0.05 85.104 36 2.364
0.10 14.489 8 1.811
0.15 7.936 4 1.984
0.20 4.329 2 2.165
BG SET : background setting
S/N : signal to noise ratio
45
From Table 3.2, it was shown that the peak area
obtained varies greatly with the background setting chosen.
The setting that gives the highest S/N value is the one
that set at zero value. Therefore, the peak area
measurement should be done at zero background, or at least
near it, in order to have a higher sensitivity and hence
more accurate results.
Figure 3.6 shows graphically the relationship between
signal to noise ratio with different background settings.
46
3.00 q
2 . 8 0 二
? \
0 2.60 二 \
1 : \ .
一 2.40 - \
I 丨 \
� I \ z 2.00 - \ /
I : V y 1.80 £ ^
1 .60 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
-0.10 -0.00 0.10 0.20
Background setting ‘
Figure 3.6 Signal to noise ratio of 2fMg citrate at different background settings
47
3.5.3 Retention and peak area due to different co-anions
Three kinds of co-anions ( of 0.15 mM each ) were
mixed with tetrabutylammonium iodide of concentration of
0.12 mM to produce three mobile phases. They were then
passed in turn to the column for conditioning, and 2 /ig of
citrate was injected after equilibrium had been reached.
The peak area were recorded in each case and the results
are summarized in Table 3.3. Besides, the UV spectra from
340 to 200 nm of the IIR ( TBAI ) and different mobile
phase containing different co-anions were taken and shown
in Figures 3.7 to 3.10.
Table 3.3 Effect of co-anions in the mobile phase on the sensitivity and retention of citrate
Co-anion pH Peak area k'
(X 10^ counts)
Sa" 6.08 482.41 9.00
Bz- 6.15 210.66 15.03
HP" 4.68 155.76 10.58
Sa" , : salicylate
Bz" : benzoate
HP" : hydrogen phthalate
pH : pH of the mobile phase
: capacity factor of the eluted peak
Detection wavelength at 240 nm
Flow rate : 1.8 ml/min
48
The absorbance of 0.12 mM TBAI and mobile phase
containing different co-anions were measured at 240 nra and
the results are shown in Table 3.4.
Table 3.4 The absorbance of TBAI and co-anions
Absorbance
•12 mM TBAI 0.477
•12 mM TBAI + .15 mM NaSa 1.081
.12 mM TBAI + .15 mM NaBz 0.868
.12 mM TBAI + .15 mM KHP 1.308
.15 mM NaSa O . 604
.15 mM NaBz 0.391
.15 mM KHP 0.831
49
1.500 ,
ABS \ j \
0. 000 r - ^ ^ , :_, rim 200 240 280 320
Figure 3.7 Absorption spectrum of 0.12 mM tetrabutyl-ammonium iodide
1+500 j
ABS \
I 八 _ 0. 000 , _ — _ , \
nm 200 240 280 320
Figure 3.8 Absorption spectrum of mobile phase containing 0.12 mM tetrabutylammonium iodide and 0.15 mM sodium salicylate
50
1.500 ^
ABS \
0. 000 , • nm 200 240 280 320
Figure 3.9 Absorption spectrum of mobile phase containing 0.12 mM tetrabutylammonium iodide and 0.15 mM sodium benzoate
1;500 ^
ABS \
0. 000 , ^ _ _ ^ ^ •
nm 200 240 280 320
Figure 3•10 Absorption spectrum of mobile phase containing 0.12 mM tetrabutylammonium iodide and 0.15 mM potassium hydrogen phthalate
51 t
From Table 3.3, we can conclude that salicylate ion
gives the highest signal with the smallest retention.
The signal obtained in ion interaction chromatography
relies very much on the absorptivity and the amount of co-
anions present in the mobile phase. The reason why
salicylate gives the largest signal may probably be that it
is a monovalent ion. When the analyte ( citrate ion )
leaves the column, salicylate will replace the vacancy left
by the analyte in the primary layer in a one to one mole I
ratio. Since hydrogen phthalate will exist in both the
monovalent and divalent form, the replacement of the
analyte ions will be less than a one to one mole ratio.
Therefore, hydrogen phthalate gave a smaller signal than
the salycilate despite that hydrogen phthalate has a higher
molar absorptivity than that of salycilate.
For the co-anion benzoate, it gave a larger signal
than the hydrogen phthalate but a smaller signal than the
salicylate ion although it is also a monovalent ion. The
reason is possibly that benzoate possesses the lowest
absorptivity at 240 nm ( refer to Figures 3.7 to 3.10 or
Table 3.4 ) and therefore it gives the smaller signal than
that of salicylate.
52
t
The elution order for the citrate anions by the co-
anions was as follows: »
salicylate < hydrogen phthalate < benzoate
This is probably due to the reason that benzoate
possesses only one polar functional group ( -COOH ) whereas
hydrogen phthalate has two identical polar functional
groups ( -COOH ) , so that benzoate is less polar and is
attracted less strongly by the primary layer. As ion
interaction chromatography is a competition between the
analyte ions and the co-anions for sites in the primary
layer, the analyte ions are displaced comparatively less
easily by benzoate as compared with hydrogen phthalate.
Similar reasoning applies to the comparison between
benzoate and salicylate, which possesses two functioal
groups, namely, the -COOH group and the -OH group. When
compared with the hydrogen phthalate, salicylate is more
polar because the hydroxy 1 group is more polar than the
carboxylic acid group ( 一COOH ) and therefore held more
strongly by the primary layer and hence the analyte anions
is less strongly retained. ,
It is obvious that sodium salicylate should be chosen
as the CO一anion for the determination of citrate rather
than potassium phthalate or sodium benzoate because it can
produce the highest signal with a resonably short retention
time.
53
3.5.4 Choice of mobile phase concentration
The effect of the concentrations of TBAI and NaSa on
the signal and retention for the analyte was tested. For
simplicity, the same concentration ratio for the TBAI and
NaSa was used and 2 /ig of the analyte was injected for the
different mobile phases. The area counts and the retention
time for the analyte peak were recorded and the capacity
factors were then calculated. The results are shown in
Table 3.5.
Table 3.5 Signal and retention at different TBAI and NaSa concentrations in the mobile phase
Cone, of TBAI and NaSa Peak area Capacity
in the mobile phase (xlO^counts) factor
.12 mM TBAI + .15 mM NaSa 178.46 9.00
.24 mM TBAI + .30 mM NaSa 156.54 8.84
.36 mM TBAI + .45 mM NaSa 155.99 8.47
.48 mM TBAI + .60 mM NaSa 146.21 7.93
•60 mM TBAI + .75 mM NaSa 136.38 7.39 ”
.72 mM TBAI + .90 mM NaSa 112.64 6.88
Detection wavelength : 248 nm
Flow rate : 1.8 ml/min
54
From Table 3.5, it is apparent that the higher signal �
were obtained at lower concentration of TBAI and NaSa. With
increase in concentration of TBAI and NaSa in the mobile
phase, a decrease in retention of the analyte was observed.
It seems contradictary at first since an increase in
concentration of TBAI will cause an increase in the active
sites in the column an hence the analyte will be retained
longer in the column. However, the increase in the
concentration of the co-anion will increase the competition
between the analyte ions and the co-anions towards the
primary layer. In this case, it is apparent that the effect
of CO-anion outweighs the effect of the IIR and therefore
the analyte ions will be eluted out more easily with an
increase in the concentration of the co-anions.
It can be concluded that it is better to employ low
concentrations of both TBAI and NaSa for the analysis of
citrate since a high signal can be obtained within a
reasonable period of time. However, it is no good to test
lower concentrations. If the concentrations of TBAI and
NaSa in the mobile phase are too low, re-establishment of
equilibrium condition of the system may take a very long
time or becomes difficult, which will lead to failure in
the analysis. Hence, the optimun condition is a compromise
between sensitivity and a reasonable analysis time.
55
3.5.5 Choice of detection wavelength
The peak area was recorded and the absorbance was
measured at various wavelengths using a UV-visible
spectrophotometer. The results are shown in Table 3.6.
Table 3.6 Signal of 2 /ig citrate at various wavelengths
Wavelength (nm) Area (xlO^counts) Absorbance
239 468.87 1.249
240 421.83 1.081
242 352.18 0.763
246 244.96 0.333
248 178.46 0.291
254 20.36 0.080
256 4.09 0.066
258 24.13 0.061
260 51.19 0.061
280 387.35 0.312
296 669.79 0.564
310 321.31 0.298
Mobile phase : .12 mM TBAI + .15 mM NaSa
56 *
The best wavelength for analysis is normally at the
peak maximum, which is 296 nm for sodium salicylate, and
the observed signal was highest at this wavelength, as
shown in Table 3.6. However, 296 nm was used, the
correlation coefficient of the calibration graph was
0.9984, which is not satifactory. The wavelength 242 nm is
choosen, which gave a higher sensitivity and precision for
the analysis of citrate. The other reason for choosing 242
nm is that the maximum of the intensity vs wavelength for
the light source ( see Figure 3.5 ) occurs at this
wavelength and hence a better lamp stability, that will
reduce fluctuation of the detected signal.
3.5.6 Preparation of calibration graph
The calibration graph was obtained by plotting the
peak area against the corresponding concentration of
citrate, which was made by the appropiate dilution of the
standard stock solution. The data for the calibration
graph are list in Table 3.7 and the calibration graph is
shown in Figure 3.11.
57
Table 3.7 Data for the calibration of citrate
Concentration ( jug/ml ) Peak area (xlO^counts)
‘ 20.0 72.35
40.0 165.58
60.0 260.72
80.0 362.09
100.0 455.55
120.0 548.92
140.0 637.58
160.0 737.53
180.0 826.84
200.0 922.12
Linear working range : 20-200 jitg / ml
Slope : 4721 ml / jLtg
Intercept : -20.42
Correlation coefficient : 0.9999
Conditions of the chromatograph
Detection wavelength : 242 nm
Mobile phase : .12 mM TBAI + .15 mM NaSa
Flow - rate : 1.8 ml / min
Operating pressure : 3,000 psi
58 f
1000.00 q
; /
800.00 - /
? 丨 /
0 / (A / D 600.00 - /
J : / O 400.00 - /
; 丨 /
2 0 0 . 0 0 一 /
: / , . . .
Q.00 —tn~~I~~II~1n~~I~I~~I~I~II~~II~III~~I~I~I~I 0.00 100.00 200.00
Concentration ( ug / mL )
Figure 3.11 Calibration graph for the determination of citrate
59
3.5.7 Precision test for the proposed method
In order to test the reproducibility of the proposed
method using the optimised system setting and mobile phase,
aliquots of citrate with a known concentration are
injected ten times into the column. The relative standard
deviation ( R.S.D. ) of the peak area was calculated and
was found to be 1.2 % ( Table 3.8 ). This means that the
proposed method is stable and good for analytical purposes.
Table 3.8 Precision test of the proposed method
2 fig citrate injected Peak area (counts)
1 456,250
2 449,717
3 - 448,997
4 456,887
5 459,894
6 455,994
7 456,667
8 447,098
9 453,891
10 441,864
R.S.D. 1.2 %
60
3.5.8 Determination of citrate in pharmaceuticals
The pharmaceutical products for this project were
purchased from Watson's medicine department store and a
brief description of the samples are shown in Table 3.9.
Table 3.9 Description of pharmaceutical preparation
Sample Sample Brief description no.
1 Cooling ENO, Antacid; contains sodium chrysanthemum carbonate, citric acid,
flavoured sodium saccharin, etc.
2 Cooling ENO, Ditto lemon flavour
3 Aspro Clear Anti-pain; contains aspirin,sodium bi-carbonate and citric acid.
.4 Alka-Seltzer Ditto
5 Bufferin Anti-pain;contains aspirin, calicium carbonate, magnesium stearate, citric acid , etc,...
6 Piriton Expectorant linctus; contains Piriton, ammonium chloride, sodium citrate, citric acid, glycerin, etc.
7 Vicks Extra strength cough Formula 44 suppresent; contains sodium
citrate, dextromethorphan HBr, alcohol, etc.
8 Visine (A.O.) Tears drop; contains boric acid, sodium citrate, sodium chloride, etc.,
61
Each saraple was diluted to an appropriate
concentration with the moble phase, and 20 jul of the
diluted sample was injected into the column three times.
The amount of citrate was deduced from the calibration
graph. The results of the analysis are shown in Table 3.10
and the respective results obtained using the standard AOAC
method mentioned"* and the claimed label values, if any, are
also shown in Table 3.10 for comparison. Typical
chromatograms for some of the samples were shown in Figures
3.12a to Figure 3.12c. 擎
The contents of citrate found by the proposed method
agree well with the respective claimed label value, and
those obtained using the standard method except for sample
No. 3 and 7.
62
Table 3.10 Assay of content of citrate in drug sample
. citrate content Sample No. Proposed method A.O.A.C. Claimed
spectroscopic label ^ „ method value Amount Mean*
1 41.96 , 41.52 41.87 % 42.3 43.26 % 42.14 (0.76)
2 41.22 , 40.08 41.10 % 42.17 43.02 % 41.99 (2.34)
3 208 ,218 ,213 213 mg/tablet 196 207 (2.35)
4 1.14 , 1.10 1.17 g/tablet 1.21 1.20 1.27 (7.69)
5 84.2 , 83.0 83.3 mg 78.8 82.69 (0.96)
6 9.54 , 9.48 9.41 mg/ml 9.32 9.66 9.21 (1.19)
7 39.5 , 39.0 38.7 mg/ml 35.4 36.6 37.7 (2.33)
8 12.6 , 12.9 13.0 mg/ml 12.2 13.5 (3.85)
* Relative standard deviation, in percent, shown in parentheses.
I
63 t
t •(
<=c
I I '1 ,..r ! i'''; I .... “.'::'‘M' I'"' ••: •'.! ....•: •::•;• ’.j::..丨• ,. 广丨..丨丨厂,I. •.丨•
o:' ‘ • •‘, •.,. t .1 r 八 j\ J 寸....;•;;:;• o . . 0_. f ^^
\ : “ , “ ‘ “ . , ,
.-.I •I •…•:•_:‘ !,:| • i.r", ‘ Y. I. .• • •
丨 丨 : r ^ 丨:, :
I . ,:...• . . •.….I H I: . •:.
^ .». I I' \
t »
I- .• • • - I:..�
Figures 3.12c Chromatograms of sample No. 7 (left) and sample No. 8 (right)
64
‘ !:
,J
1.1" 1 I ..• “
_
• O C .. . . - . • "...1 .-.I ft ':f[ C. •••',. •••i ...., •..':. II ' n. "• • - •“ “
'•'•••I ‘::!;,! , " • I . ., '••;' ••” . .."• 11:. ‘ . .:•«• 卜 _ II -^ j
I'.. ‘ 1.
卜...丨 I..... I • a 1-丨::丨: ‘:!::
“ L I , •.-•.I 丨•.丨 -'."••I "..'I 丨,丨! “ ij. ,:...• -._.... 1:11 1.1
Figures 3.12c Chromatograms of sample No. 7 (left) and sample No. 8 (right)
65
1.1, •• •:, kX •::! “
t-
<=c t-1 \0 ‘ H ....J
- ‘::! — -.:| .,::' It , C::' -' 1.1.丨 . “ .. -̂.I •• “ 11:.. n. \ l! ' I ..
o . A 巧 • ‘ v l • ..I I ..:‘. ^̂ ^̂ ^̂ m
I..,.,, .... 广 1:1. ... r, ••I / •I •‘ .1
I « . ’• • 1 L y
r...
I...... I"' I.…‘ • •••• a:
•..I. a.:
. Ml I: I ••••H r I • •* I ._
•1 I ti I t „T “
.J.
Figures 3.12c Chromatograms of sample No. 7 (left) and sample No. 8 (right)
66
3.5.9 Recovery tests
In order to test the reliability of the method,
recovery tests were also carried out by spiking known
amounts of standard citrate to each sample before any
treatment and the percent recovery was then calculated.
The percent recovery of the proposed method ranged
from 95.0 to 106 percent ( see Table 3.11 ) and may be
considered very good.
Table 3.11 Recovery test for the proposed method
Sample Add//igmr^ Found/jiigmr* Recovery ( % )
1 80 77.8 97.3
2 80 76.0 95.0
3 80 76.5 95.6
4 80 77.3 96.6
5 80 84.0 105
6 80 79.0 98.8
7 80 84.8 106
8 80 77.7 97.1
a. average of duplicate result
67
3.6 CONCLUSION
An accurate, simple and efficient high performance
liquid chromatographic method for the determination of
citrate in pharmaceutical preparations has been developed.
The proposed method employs ion-interaction chromatography
with an indirect method of detection, where salicylate is
used as a co-anion to an ion-interaction reagent,
tetrabutylammononium ion, and produces a constant
background of UV absorption. The UV detector will respond
to the presence of citrate by producing a negative peak.
Hence, the method does not depend on any specific
functional group of the analyte. This method has been
applied successfully to many types of pharamceutical
preprations with good precision and accuracy.
68
REFERENCES
1. D.D. Duxsury, Food Processing, 1991, 5, 83
2. The Merck Index, 11th edition, 1989, 2328
3. Branen, Food Additives 1990, 144
4. Official Methods of analysis-AOAC, 1990, 746
5. S. Girotti, R. Budini, E. Gattavecchia and D.
Tonelli, Anal, Chim, Acta, 1981, 124, 215
6. H.E. Indyk and A. Kurmann, Analyst, 1987, 112, 1173
7. S.B. Salama and S. A. Awad, Microchem. J., 1988,
37(1), 13
8. M.F. El-Taras and E. Pungor, Anal, Chim, Acta, 1976,
82, 285
9. A. Olin and B. Wallen, Anal. Chim. Acta., 1983,
151, 65 �
10. G. Gran, Analyst, 1952, 77, 661
11. Analytical Abstract May 1988 5C16 & 5F41
12. L. Che, J. Wang, J. Liu and L. Zhang Fenxi Huaxue,
69 *
1990, 18(6), 575
13. R.J. Schwarzenbach, J• Chromatogr., 1982, 251, 339
14. A. Clement and B. Loubinoux, J. Liquid
Chromatography, 1983, 6(9), 1705
15. R.H. Evans, W.V. Van Soestbergen and K.A. Ristow,
J,A.O,A,C,, 1983, 66(6), 1517
16. S.H. Ashoor and M.J. Knox, J. Chromatogr,, 1984, 299,
288
17. D. Cox, P. Hanison G Jandik and W. Jones, Food
Technology, 1985, 7, 41
18. V. Hong and R.E. Wrolstad, J,A.O.A,C., 1986, 69(2),
208
19. E.D. Coppola and M.S. Stan, J,A.O.A.C., 1986,
69(4), 594
20. F. Caccamo; G. Carfagnini, A.D. Corcia'^Xxnd R.
Samperi, J. Chromatography, 1986, 362, 47
70
21. D.B. Gomis, M.J. Moran Gutierrez, M. D. Gutierrez
Alvarez and A. S. Medel, Chromatographia, 1987, 24,
347
22. E. Burke, S.R. Zimmerman, D.S. Brown and D.R. Jenke,
J. Chromatogr, Sc,, 1988, 26(10), 527
23. D.B. Gomis, M.J. Moran Gutierrez, M.D. Gutierrez
Alvarez and J.J. Mangas Alonso, Chromatographia,
1988, 25, 1054
24. M.C. Gennaro and P.L. Bertolo, J. Chromatography,
1989, 472(2), 433 25. W.R. Jones, P. Jandik and M.T. Swartz,
J.Chromatography, 1989, 473, 171
26. R.M. Marce, M. Calull, R.M. Manchobas, F. Bonull and
F.X. Rius, Chromatographia, 1990, 29, 54
27. Y. Zhu, L. Zhu and X. Zhuang, Fenxi Huaxue, 1990,
18(3), 263
28. E. Mentasti, M.C. Gennaro, C. Sarzanini, C. Barocchi
and M. Savigliano, J• Chromatography, 1985; 322, 177 (
29. E.D. Coppola, E.G. Conrad and R. Cotter, J,A,0.A,C,,
1978, 61, 1490
71
30. W. Distler, J. of Chromatography, 1978, 152, 250
31. Giddings, Editor, Advances in Chromatography Vol. 27
1987
32. B.P. Downey and D.R. Jenke, J. Chromatogr. Sci,,
1987, 2 5 , 519
33. H. Small and T.E. Miller, Anal. Chem., 1982, 54, 462
i • 34. N. Raghavan and D.R. Jenke, J. Chromatogr. Sci.,
1985, 22, 75
35. W^Xx. Barber and P.W. Carr, J. Chromatography, 1984,
301, 25
36. B.A. Bidlingmeyer, J. Chromatogr, Sci., 1980, 18(10),
525
37. D.P. Wittmer, N.O. Nuessle and W.G. Haney, Anal,
Chem ” 1975, 47, 1422
38. C. Horvath, W. Melander, I. Molnar and P. Molnar,
Anal. Chem., 1977, 49, 2295
39. C. Horvath, W. Melander, I. Molnar and P. Molnar,
J. Chromatography, 1976, 125, 281
72
40. J.L.M. Venne, J.L.H.M. Hendrikx and R.S. Deedler
J. Chromatography, 1978, 167, 1
41. J.C. Kraak, K.M. Jonker and J.F.K. Huber,
J. Chromatography, 1977, 142, 671
42. N.E. Hoffman, and J.C. Liao, Anal. Chem,, 1977, 49,
2231
43. P.T. Kissinger, Anal. Chem., 1977, 49, 883
44. B. A. Bidlingmeyer, S.N. Deming, W.P. Price, Jr .B.
Sachok and M. Petrusek, J. Chromatography, 1979, 186,
419
45. H. Small, Ion Chromatography, 1989, Plenum, • 1 I New York, p.54
t
73
CHAPTER 4
DETERMINATION OF CAMPHOR AND MENTHOL BY
HPLC USING INDIRECT CONDUCTOMETRIC DETECTION »
4.1 INTRODUCTION
Camphor is an excellent plasticizer for cellulose
esters and ethers. It is used in manufacturing of
plastics, especially for celluloid; in lacquers and
varnishes; in explosives; in pyrotechnics; as moth
repellent; in embalming fluids; as preservative in
pharmaceuticals and cosmetics.' Applied externally, camphor
acts as a rubefacient and mild analgesic and is employed in
ointment as a counter-irritant in fibrositis, neuralgia,
and similar conditions.2 Camphor Injection was used as a
restorative in collapse because of its stimulating effect
on the cerebral cortex and medullary vasomotor and
respiratory centres.^ A great range of preparations are
used to relieve upper respiratory tract congestion and
obstruction in simple infections. It is also used in
eardrops and earwax softeners as a weak antiseptic and a
mild anesthetic intended to suppress itching. At
concentrations of 0.1 - 3 %, camphor depresses cutaneous
receptors, thereby relieving itching and irritation. At
higher concentrations of 3 - 11 %, camphor acts as a
counterirritant because it stimulates cutaneous receptors.
74 t ‘ ;
Camphor is safe and effective for use as an external
analgesic at these concentrations but can be very dangerous
if ingested.4
Menthol possesses similar usage as camphor." It may
also be used safely in small quantities as a flavoring
agent and has found wide acceptance in candy, chewing gum,
cigarettes,cough drops, toothpaste, nasal sprays, and
liqueurs. Menthol may give rise to hypersensitivity
reactions including contact dermatitis. There have been
reports of instant collapse in infants following the local
application of menthol to their nostrils. Ingestion of
menthol is reported to cause severe abdominal pain,
vomiting, drowsiness and coma.^
Both camphor and menthol are UV inactive and cannot be
determined by HPLC using UV~detection. They are neutral
compounds and hence cannot be detected using direct
conductometric detection. The objective of this work is to
develop an HPLC method for the simultaneous determination
of camphor and menthol using indirect conductometric
detection.
’丨
、
75
4.2 BRIEF REVIEW OF THE ANALYTICAL METHODS FOR
THE DETERMINATION OF CAMPHOR AND MENTHOL
Few methods are reported in the literature for the
simultaneous determination of camphor and menthol. The
only popular method is the gas-liquid chromatographic
method using flame ionization detector ( FID ) and no
article reporting the determination by HPLC method probably
due to the fact that camphor and menthol are UV inactive
where UV detection is one of the most popular method of
detection.
4.2.1 Gas - liquid chromatographic method i
Most of the methods used for the simultaneous
determination of camphor and menthol depend on the gas-
liquid chromatographic separation by choosing different
stationary phases with FID and either helium or nitrogen as
carrier gas.̂ "'̂ All these methods require a careful choice
of column and therefore only a few columns can be used for
the simultaneous determination of these two compounds.
4.2.2 GC - FTIR spectrometry
Sample containing camphor and menthol has also been
reported to be separated by a fused-silica column coated
with SE-30 with helium as carrier gas, FID, and FTIR
spectrometric detection with a CdHgTe detector.This kind
of analytical method is not common due to the high cost of
instrumentation.
76
4.3 THEORY OF DETECTION
4.3.1 Theory of conductometric detection*"^^^
When electrolytes dissolve in solvents of high
dielectric constant such as water they dissociate into
their constituent ions and the solutions are electrically
conducting. The electrical properities of the solution
obey Ohm's law in that the resistance to current flow may
be defined as
V = iR ( Eq. 4.1 )
where V is the voltage and i is the current that flows
through an element of solution. The resistance R is a
function of temperature and the concentration of the
electrolyte. The resistance of an element is proportional
to its thickness, 1, and inversely to its cross-sectional
area, A. Therefore
R = al/A ( Eq. 4.2 )
where o is defined as specific resistance and is in ohm cm.
If several ions in a solution can conduct, they
can be thought of as conductors in parallel, with the net
resistance R of the composite being related to the
separate resistances Rj; R?, R3
77
1 / R = 1 / R I + I / R 2 + I / R 3 + … ( E Q . 4 . 3 )
Since the reciprocal of resistances are additive,
electrolytes can be conveniently treated by using the
conductance, G, which is the reciprocal of resistance. The
specific conductance, denoted k, is therefore
k = 1/a = 1/AR ( Eq. 4.4 )
The conductance of the ions produced by one gram
equivalent of electrolyte at any concentration can be
evaluated by considering a cell with electrodes placed one
cm apart but of sufficient area as to just enclose the
whole volume containing the one equivalent of electrolyte.
The conductance of such an assembly is termed the
equivalent conductance and is denoted r . Equivalent
conductance r and specific conductance k are related in
the following way
r = 1000 k / C ( Eq. 4.5 )
where C is the concentration of the electrolyte in
equivalents per liter, and has the unit of cm2 equiv' ohm''.
Combining equations ( 4.4 ) and ( 4 . 5 ) gives the following
equation which relates equivalent conductance to measured
conductance, G
G = r C / 1000 K ( Eq. 4.6 )
78
where K = 1 / A and is called the cell constant and G has
the unit of ohm"' or mho. If G is expressed in /imhos, equation ( 4 . 6 ) becomes
G = r C / 10-3 K ( Eq. 4.7 )
The equivalent conductance of an electrolyte may be
considered to consist of the ionic conductance of the
cation and that of the anion r"
R = T + + T"- ( Eq. 4.8 )
At infinite dilution, the ions migrate independently
of each other and the limiting equivalent conductance r� is
thus related to the limiting ionic conductance T+Q and T� b y
r 。 = T + O + T-� ( Eq. 4.9 )
事
In ion chromatography, the concentration of the
eletrolytes are usually in the range of millimole and
therefore can be considered as at infinite dilution.
Equation ( 4 . 7 ) then becomes
G = r � C / 10-3 K
= ( r + � + r � ) C / 10-3 K ( Eq. 4.10 )
Consider an electrolyte E which contains a single
79
species and which can dissociate partially to give anions
and cations. When E acts an element and under column
equilibrium conditions, the background conductance of the
eluent as indicated by the conductivity detector can be
deduced according to Fritz et as
GBackgrouncl = ( ^ + 厂。’̂ ) C^l^ / lO'' K ( E q . 4 . 1 1 )
where C^is the concentration of the eluent species and is
the fraction of the eluent species which is present in the * ionic form.
4.3.2 Indirect conductometric detection
The mobile phase employed for this work is a 30 %
aqueous acetonitrile containing a background conducting
species, in this case, trisodium citrate.
When the column is in equilibrium state, there
exists a dynamic equilibrium for the adsorbed citrate ions
and the sodium ions between the mobile phase and the active
sites of the polar cyano column. The interactive forces
are mainly polar interations with a little non-polar
interations being involved because of the ionic character
of the conducting species. Hence, a continuous background
signal is maintained.
Since camphor and menthol contain no charge, we can
assume that the interaction with the column active sites
80
for these compounds is mainly hydrophobic interaction.
When the sample containing camphor and menthol enter
the column, some of the active sites will be occupied by
these compounds and some citrate ions will be displaced and
leave the column. In order to maintain the charge balance,
it is believed that sodium ion will also be displaced, when
the analyte leaves the column, citrate ions and also sodium
ions from the mobile phase will re-enter the column and
hence a sudden drop of the background conductance will
result and a negative peak is therefore detected by the
detector.
«
81
4.4 EXPERIMENTAL
4.4.1 Apparatus
4,4.1.1 The liquid chromatograph
The high performance liquid chromatographic system was
composed of a solvent delivery module ( Beckman llOB ) with
an Free-flow pulse dampener ( Alltech Associates ) ; a
conductivity detector ( Wescan model 21511001 ) with a
temperature controller ( model 24 02 0001 ) and column
compartment ( model 26650051 ); an injection port (
Rheodyne ) with a 20 or 100 /il sample loop ; an
adsorbosphere CN column of length 250 mm, internal diameter
of 4.6 mm and particle size of 5 /xm ( Alltech Associates ),
protected by a lOmm x 4. 6min guard column packed with the
same material as the column; and an integrator ( Hitachi 1
833A Data Processor ).
4.4.1.1.1 Parameter setting of the detector
The following settings for the
conductivity detector were kept unchanged
during the experiment.
Parameter Settings
Detector Range 1
Column Temperature 30 °C
82 r
Peaks were detected as negative changes in
conductance, and the detector-integrator connections
were reversed in polarity to give positive display of
peak on the integrator.
4.4.1.2 The gas chromatograph for the counter check eras-
liquid chromatographic method
The standard method employed for counter checking the
sample components is the method currently in use by the
Hong Kong Government Laboratory in the daily analysis of
pharmaceutical products submitted to the Laboratory. ̂^
The instrument consisted of the following
components :
Gas Chromatograph : HP 5890 Series II
Column : Supelcowax TH 10 Fused Silica
Capillary column, 30 m, 0.53 mm
internal diameter, 1.0 jim film
thickness.
Detector : Flame Ionization Detector (FID)
Integrator : HP 3396A Integrator
83
4.4.1.2.1 Operational conditions
Carrier gas : Helium
Flow 一 rate : 7.5 ml / min
Injector Temp. : 23 0 °C
Detector Temp. : 250 �C
Column Temp. : Temp, programming
Initial Temp. : 70 °C
Hold : 0 min.
Ramp : 2 0 °C / min
Final Temp. : 170 �C
Hold : 38 min.
4,4.1.4 Glassware
All volumetric flask and pipettes were of grade A and
grade B and were calibrated to the relevant BS standard
before use.
4.4.2 Reagents and materials
All reagents used were of analytical reagent grade and used without further purification.
4.4.2.1 Water f Ultra pure water )
All water used during the experiment was distilled
water purified by the Millipore Milli-Q50 ultra pure water
84
system ( Millipore, France )• The water was deionized and
filtered through the 0.1 /m filter from the system. The
resistivity of water produced was 18 Mn / cm.
4,4.2.2 Standard solutions
10 mg/ml Camphor and menthol standard solutions were
prepared by dissolving exact amount of each compound into
the mobile phase and the solutions were further diluted to
the required concentrations by mobile phase.
4.4.3 Aqueous mobile phase
Various mixture of the mobile phase were prepared by-
dissolving appropriate amount salt into 30 % aqueous
acetonitrile solution. The mobile phases prepared were
then filtered through a Millipore HA type filter of 0.45 ^m
pore size to remove any trace of solid particles. The
filtered mobile phases were then degassed by shaking in an
ultrasonic bath ( Branson 1200 ) for at least half an hour
before being used.
4.4.4 Column conditioning
I
The column was conditioned each time when a new mobile
phase was used. This normally took two hours to do so.
Whenever the new mobile phase was changed from one
85 , r
system to another , the column had to clean thoroughly by
20 column volume of water ( this usually took two hour at
a flowrate of 1 ml / min ) followed by another 20 column
volume of 1 : 1 mixture of methanol and water so as to
ensure that the salts adsorbed on the column bed were I
completely removed.
4.4.5 Sample treatment
Generally the liquid samples were diluted to
appropriate concentrations with the mobile phase. For the
cream base samples, an accurate amount of sample was
weighed and dissolved in mobile phase by shaking in an
ultrasonic bath ( Branson 1200 ) for fifteen minutes. Any
undissolved particles were filtered and the solution was
transferred to a calibrated volumetric flask and made up to
the mark by mobile phase.
4.4.6 Sample introduction
Samples were introduced into the column through the
external loop injection valve by using a 1 ml syringe
fitted with a 0.2 jLtm disposable filter.
86
4.5 RESULT AND DISCUSSION
The objective of the following work was to find out
the optimum condition for the proposed liquid
chromatographic method. Calibration graphs were then
obtained from the standard camphor and menthol solutions.
A series of sample were then taken for analysis to evaluate
the practicability of the proposed method. Finally the
analytical results obtained were compared with those
obtained by a developed official method in order to check
the accuracy of the proposed method.
4.5.1 Choice of background conducting species
Different kind of conducting species of 1 mM were
mixed with 30 % aqueous acetonitrile to produce different
mobile phases. They were then passed in turn to the column
for conditioning, and 10 ixg each of camphor and menthol
were injected after equilibrium had been reached. The peak
area and retention time were then recorded in each case and
the results are summarized in Table 4.1.
I
87 *
Table 4.1 Effect of conducting species in the mobile phase on the sensitivity and retention on camphor and menthol
Retention time Conducting Peak area
• ( min ) species (1 mM) " “ “ Camphor Menthol Camphor Menthol
‘ Na Benzoate 1827 2658 6.86 7.22
K hydrogen 2896 4669 6.78 7.17 phthalate
Na salicylate 2097 2733 6.7-3 7.09
Na citrate 8226 12231 6.75 7.13
Na oxalate 4902 5171 6.76 7.13
Na sorbate 3339 3770 6.69 7.08
Na acetate 1363 2217 6.71 7.09
Na formate 3415 4878 6.73 7.09
Na tartrate 5498 7944 6.63 6.98
Trimethyl- 5964 7507 6.77 7.16 ammonium chloride t
Methylammonium 5108 8212 6.69 7.06 chloride
Na : sodium K : potassium
88
From Table 4.1, we can find that tri-sodium citrate
gives the highest signal with similar separation ability (
difference in retention between camphor and menthol is
equal to 0.4 minutes ) . Therefore, we can conclude that
trisodium citrate is amoung the best conducting species and
should be chosen as the conducting background species.
It is obvious that with different conducting species,
the retention ability differs very little as you can see
from the difference in retention between camphor and
menthol. It can be inferred that the retention mechanism
does not depend very much on the polar conducting species,
the major function of the species is to provide the
conducting background.
Tri-sodium citrate gives larger signal than that of
formate or acetate, it seems contradicting since formate
and acetate is smaller in size and possesses larger ionic
mobility and hence larger conductance. The reason for the
cause is that citrate possesses 3 sodium and when one
citrate ion enters the active site in the column bed, 3
sodium ions will follow in order to maintain the charge
balance and therefore result in a larger conductance
change.
i.
89
4.5.2 Effect of background conducting species concentration on peak intensity and retention time
Different concentrations of sodium citrate in 3 0 %
aqueous acetonitrile were prepared and 20 fig of camphor and
menthol were injected into the column after equilibrium has
been reached. The peak area and retention time were taken
and was summarized in Table 4.2.
Table 4.2 Concentration effect of background conducting species on peak intensity and retention time
Concentration Peak area Retention time
of sodium ( min )
citrate ( mM ) " ‘ ‘ “ Camphor Menthol Camphor Menthol
1 4831 6307 6.39 6.96
2 11672 15003 • 6.42 7.01
3 18299 24347 6.48 7.06
4 28968 35708 6.69 7.31
It can be seen from the table that higher the
concentration of the conducting species, larger the
detector response and this means that more conducting
species were displaced from the column by the analyte.
The effect of the concentrationas on retention time of
90
the analyte is not obvious. It only shows a little
increase in retention with increasing concentration of the
conducting species. One possible reason may be due to the
fact that as the concentration of the conducting species
increases, the mobile phase will become more polar and
therefore, the analyte are then pushed to the column bed
since it is less polar. As a result, the analyte are
retained more strongly as increasing conducting species
concentration. It should be stressed that this effect is
not prominent and the separation mechanism is based mainly
on the hydrophobic interation.
4.5.3 Effect of changing the water / acetonitrile ratio
The retention behaviour of camphor and menthol on the
column using mobile phases with different water /
acetonitrile ratios was studied and the results are shown
in Table 4.3. 1 mM of sodium citrate is employed.
From the table, we can observe that with increasing
acetonitrile content, the elution time will be decreased
and this suggested that hydrophobic interaction of the
analytes with the column bed is mainly due to hydrophobic
interaction between the analytes and the column bed. j
It is probably correct to choose a lower fraction of
acetonitrile in order to give a good separation but
actually we must consider the practicability because too
91
small a fraction of acetonitrile in mobile phase will have
problem in dissolving the sample in the mobile phase for
analysis. Therefore, 30 % acetonitrile was chosen as the
analytical condition.
Table 4.3 Concentration effect of acetonitrile on retention time
Concentration of Retention time ( min )
acetonitrile ( % ) Camphor Menthol
10 6.98 7.70
20 6.87 7.50
30 6.37 6.96
40 5.95 7.31
3.5.4 Preparation of calibration graphs
The calibration graphs for camphor and menthol were
obtained by plotting the peak area against the
corresponding concentration of camphor and menthol, which
was made by the appropiate dilution of the standard - stock
solution. The data for the calibration graphs are listed
in Table 4.4' and Table 4.5. The calibration graphs are
shown in Figure 4.1 and 4.2.
92
Table 4.4 Data for the calibration of camphor
Concentration ( /xg/ml ) Peak area ( x lO^counts )
50.85 8.889
101.70 17.904
203.40 34.663
406.80 65.631
610.20 97.988
813.60 129.530
1017.00 162.558
Linear working range : 50-1000 jug / ml
Slope : 157.9 ml / /xg
Intercept : 1.613
Correlation coefficient : 0.9999
Conditions of the chromatograph
Detection temperature : 3 0° C
Mobile phase : 4 mM NaCit. in 30 % mM
aqueous acetonitrile
Flow 一 rate : 1.0 ml / min
Operating pressure : 2,000 psi
93
2 0 0 . 0 0 〕
三 /
150.00 一 /
1 : / W 100.00 - /
! : / 50.00 - /
••00 —[―I~I~[―i~I~rn~I~I~|—I~|~1~I~1I~~[―Irn 0.00 500.00 1000.00
Concentration ( ug / mL )
Figure 4.1 Calibration graph for the determination of camphor
94
Table 4.5 Data for the calibration of menthol
Concentration ( jug/ml ) Peak area ( x lO^counts )
50.55 13.169
101.10 20.096
202.20 39.901
404.40 75.469
606.60 111.042
808.80 146.853
1011.00 182.643
Linear working range : 50-1000 /xg / ml
Slope : 177.2 ml / fig
Intercept : 3.53 0
Correlation coefficient : o.9999
Conditions of the chromatoaraph
Detection temperature : 3 0° C
Mobile phase : 4 mM NaCit. in 30 %
aqueous acetonitrile
Flow - rate : 1.0 ml / min
Operating pressure : 2,000 psi
95
200.00 -J ‘
/ 150.00 - J
I / 一 100.00 - /
! / 50.00 一 /
/ 0.00 I I I I I I I I I I I I I I I I I I I I I I I I I
0-00 500.00 1000.00
Concentration ( ug / mL )
Figure 4.2 Calibration graph for the determination of menthol
96
4.5.5 Precision test for the proposed method
In order to test the reproducibility of the proposed
method using the optimised system setting and mobile phase,
aliquots of camphor and menthol with a known concentration
are injected ten times into the column. The relative
standard deviation ( R.S.D. ) of the peak area for camphor
and menthol were calculated and were both found to be 0.9
% ( Table 4.6 ) . This means that the proposed method is
stable and good for analytical purposes.
•
Table 4.6 Precision test of the proposed method
20 ug Peak Area ( counts ) camphor/menthol
injected Camphor Menthol
1 28968 35708
2 29017 35999
3 28966 35567
4 28263 35678
5 . 28997 34967
6 29115 35632
7 28778 35890
8 28909 36087
9 28663 35478
10 28563 35778
R-S.D. 0.9 % 0.9 %
97
4.5.6 Determination of camphor and menthol in
pharmaceuticals
The pharmaceutical products for this project were
purchased from Watson's medicine department store and a
brief description of the samples are shown in Table 4.7.
Each sample was diluted to an appropriate
concentration with the mobile phase, and 20 /xl of the
diluted sample was injected into the column three times.
The amount of citrate was deduced from the calibration
graph. The results of the analysis are shown in Table 4.8
and the respective results obtained using the standard
• methodi3 and the claimed label values, if any, are also
shown in Table 4.8 for comparison. Typical chromatograms
for some of the samples were shown in Figures 4.3a to
Figure 4.3c.
The contents of citrate found by the proposed method
agree well with the respective claimed label value, and
those obtained using the standard method.
«
98
Table 4.7 Description of pharmaceutical preparation
Sample Sample Ingredient no.
1 White flower Wintergreen oil, menthol oil. Eucalyptus oil, camphor, etc.
2 Axe Brand Ditto universal oil
3 Kwan Loong Menthol, camphor, methyl oil salicylate, Eucalyptus oil,
white oil, etc.
4 Golden lion Ditto shield medicated oil
5 Tiger oil Ditto
6 Banjemin Camphor, spirit turpentine, Jaminton Eucalyptus oil, liquid healing oil paraffin.
7 Zheng Gu Shui Camphor, menthol, Croton Tigliim, Inula Cappa, etc.
8 Ammeltz Methyl salicylate, thymol, camphor, menthol, etc.
9 Radian-B Menthol, camphor, methyl salicylate, ammonium salicy-late, etc.
*
10 Vicks Menthol, camphor, . Eucalyptus oil, etc.
99
Table 4.8 Assay of content of camphor and menthol in drug sample
Sample Camphor and menthol content No.
Proposed Standard Label value method method
1 4.89 % ( C ) 4.81 % ( C )
14.6 % ( M ) 14.4 % ( M )
2 4.93 % ( C ) 4.91 % ( C ) 5 % ( C )
19.6 % ( M ) 20.5 % ( M ) 20 % ( M )
3 . 9.73 % ( C ) 10.1 % ( C ) 10 % ( C )
25.6 % ( M ) 25.9 % ( M ) 25 % ( M )
4 9.75 % ( C ) 9.89 % ( C ) 10 % ( C )
19.6 % ( M ) 19.2% ( M ) 20 % ( M )
5 17.0 % ( C ) 17.6% ( C ) 17.5% ( C )
7.7 % ( M ) 7.9% ( M ) 8.0% ( M )
6 2.96 % ( C ) 3.10 % ( C ) 3 % ( C )
nil % ( M ) nil ( M ) nil ( M )
7 2.04 % ( C ) 1.99 % ( C ) 2 % ( C )
2.89 % ( M ) 2.91 % ( M ) 3 % ( M ) 8 51.0 mg/ml 50.2 mg/ml 52 mg/ml
( C ) ( C ) ( C ) 51.5 mg/ml 51.3 mg/ml 52 mg/ml
( M ) ( M ) ( M ) 9 0.58 % ( C ) 0.58 % ( C ) 0.6 % ( C )
1.43 % ( M ) 1.39 % ( M ) 1.4 % ( M )
10 4.51 % ( C ) 4.53 % ( C ) 4.73 % ( C )
2.50 % ( M ) 2.49 % ( M ) 2.6 % ( M )
100
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Figures 3.12c Chromatograms of sample No. 7 (left) and sample No. 8 (right)
101
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Figures 3.12c Chromatograms of sample No. 7 (left) and sample No. 8 (right)
102
4.5.7 Recovery tests
In order to test the reliability of the method,
recovery tests were also carried out by spiking known
amounts of standard camphor and menthol to each sample
before any treatment and the percent recovery was then
calculated.
The percent recovery of the proposed method ranged
from 95 to 105 percent ( see Table 4.9 ) and may be
considered very good.
Table 4.9 Recovery test for the proposed method
Sample Amount added Amount found Percentage No. ( jLtg / ml ) ( jLtg / ml ) recovery 1 100 ( C ) 96.7 ( C ) 97 ( C )
200 ( M ) 197.8 ( M ) 99 ( M ) 2 100 ( C ) 95.9 ( C ) 96 ( C )
200 ( M ) 200.8 ( M ) 100 ( M ) 3 100 ( C ) 102.9 ( C ) 103 ( C )
200 ( M ) 198.0 ( M ) 99 ( M ) 4 100 ( C ) 96.9 ( C ) 97 ( C )
200 ( M ) 196.8 ( M ) 98 ( M ) 5 100 ( C ) 95.2 ( C ) 95 ( C )
200 ( M ) 193.7 ( M ) 97 ( M ) 6 100 ( C ) 99.6 ( C ) 100 ( C )
200 ( M ) 198.1 ( M ) 99 ( M ) 7 100 ( C ) 104.7 ( C ) 105 ( C )
200 ( M ) 199.6 ( M ) 100 ( M ) 8 100 ( C ) 98.0 ( C ) 98 ( C )
200 ( M ) 194.9 ( M ) 97 ( M ) 9 100 ( C ) 95.1 ( C ) 95 ( C )
200 ( M ) 191.9 ( M ) 96 ( M ) 10 100 ( C ) 103.9 ( C ) 104 ( C )
200 ( M ) 198.0 ( M ) I 99 ( M )
average of duplicate result
103
4.6 CONCLUSION
An accurate, simple and efficient high performance
liquid chromatographic method for the determination of
camphor and menthol in pharmaceutical preparations was
developed. The proposed method applied reversed bonded
phase chromatography, employing an indirect method of
detection, where sodium citrate is used as a conducting
species produces a constant background of conductivity.
The conductivity detector will respond to the presence of
camphor and menthol by producing negative peaks. Hence,
the method does not depend on any specific functional group
of the analyte. This method has been applied successfully
to many types of pharmaceutical preprations with good
precision and accuracy.
I.
104
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106
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