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CHIRAL CHRQIATOGSAPHY OF EIAITIOIBRIC CARDIOVASCULAR
AID OTHER DRUGS
by
GILLIAI A KIIGSTOI BSc
A thesis submitted for the degree of Doctor of Philosophy of the
University of Surrey
The Sobens Institute
University of Surrey
July 1990
S0HHABY
Since the enantioiaers of a number of racemic drugs have been found
to have different activities or modes of action, enantioselective analysis '
is becoming more important. A number of different approaches to
chromatographic chiral resolutions have been evaluated for their ability
to resolve the enantiomers of racemic beta blacking drugs.
Three chiral HPLC columns were investigated; a cyclodextrin phase, an
(S)~3,5-dinitrobensoylphenylglycine phase and a protein phase. The oc»
acid glycoprotein phase successfully resolved atenolol, alprenolol,
metoprolol, oxprenolol, propranolol and verapamil with 0.0IM phosphate
buffer eluents modified with either acetonitrile or isopropanol. The <R>-
3,5-dinitrobenzoylphenylglycine phase was used with eluents of
isopropanol in hexane and resolutions of propranolol, oxprenolol,
metoprolol, alprenolol and pronethalol were achieved after formation of
the 1- or 2-naphthamide derivatives, although no separations were
achieved for the underivatised samples. The cyclodextrin phase was also
found to be unsuccessful in resolving underivatised samples of
propranolol and verapamil. However preliminary results indicate that
resolutions are passible after the formation of their trifluoroacetyl
derivatives. The cyclodextrin phase was also successfully used to
resolve the enantiomers of chlorpheniramine and the geometric isomers of
clomiphene.
-■ 1 -
In addition to the chiral HPLC stationary phases, a (+)~1Q~
camphorsulphanic acid mobile phase additive was investigated, although
this was found to be completely unsuccessful.
Finally the use of a chiral diamide GLC column was investigated. This
was not suited to the analysis of beta blockers, even after
derivatisation, although derivatised amino acids were well resolved.
The use of computer modelling to predict the degree of separation of
enantiomers was also investigated for the <E)-3,5-dinltrobensoyl
phenylglycine phase, with the interaction energies between the phase and
both isomers of each compound calculated for the most stable
conformation. From a comparison with the experimental results, it was
shown that this approach to prediction was unsuccessful,
ACKIGWLEDGEHEITS
I would like to thank my supervisors; Dr D Stevenson and Professor J.V.
Bridges for their guidance and support throughout this study, and also
the Science and Engineering Research Council for their financial
assistance.
I am particularly grateful to Mr R.J. Briggs for all his advice and help,
and to all my friends and collegues at the Robens Institute for helping
to make this work more enjoyable.
Finally I would like to thank my parents, family and Mr A.J. Montan for
their continued love and encouragement, and without whom this work would
not have been completed.
_ 3 _
COITEITS
SUMMARY 1ACKfGFLEDGEMMTS 3COITEITS 4CHAPTER 1 IMTRQDUCTIOff TO CHIRALITY1.1 Chirality 71.2 Chirality in Pharmacology 11
1.2.1 Pharmacodynamics 131.2.2 Drug Disposition 161.2.3 Drug Elimination IS1.2.4 Drug Interactions 20
1.3 Analytical Methods in Pharmacology 211.3.1 Infra Red Spectroscopy 24'1.3.2 luclear Magnetic Resonance Spectroscopy 241.3.3 Mass Spectrometry 251.3.4 Electronic Spectroscopy 251.3.5 Radioimmunoassay 281.3.6 Chromatography 291.3.7 Polarography 311.3.8 Isotope Dilution Analysis 321.3.9 Isotope Derivative Analysis 32
1.4 Methods for Enantioselective Analysis 321.4.1 Crysallisation Techniques 341.4.2 Isotope Dilution Analysis 371.4.3 Kinetic Techniques 381.4.4 Differential licrocalorimetry 401.4.5 Enantioselective H R Spectroscopy 411.4.6 Chiral Chromatographic Techniques 431.4.7 Enantioselective Radioimmunoassay 45
1.5 Problems with Enantioselective Analysis 461.6 Sample Preparation 47
- 4 -
CHAPTER 2 CHROMATOGRAPHIC SEPARATIONS2.1 Historical Aspects 502.2 Theory 512.3 Chromatographic Techniques 602.4 Separation of Enantiomers 632.5 Enantioselective Stationary Phases for HPLC 66
2.5.1 Type 1 Phases 692.5.2 Type 2 Phases 722.5.3 Type 3 Phases 762.5.4 Type 4 Phases 79
2.6 Chiral Mobile Phases for HPLC 802.7 Chiral GLC 842.8 Chiral TLC 892.9 Chiral Detection 902, 10 Model Compounds 91
CHAPTER 3 MATERIALS AID METHODS3.1 Reagents
3.1.1 Model Compounds 963.1.2 Derivatising and Ion Pair Reagents 963.1.3 General Reagents 97
3.2 Solvents3.2.1 Mobile Phase Solvents 973.2.2 General Solvents 98
3.3 Equipment3.3.1 General Laboratory Equipment 983.3.2 Chromatography Equipment 99
3.4 Stationary Phases 1003.5 Test Conditions 1013.6 Derivatisation Procedures
3.6.1 Formation of Acyl Derivatives for GLC 1023.6.2 Formation of Naphthamide Derivatives 102
- 5 -
CHAPTER 4 RESULTS AID DISCUSSICM4.1 Achiral Method Development and Validation for Verapamil
4.1.1 Introduction 1054.1.2 Chromatography 1054.1.3 Extraction Procedure 1134.1.4 Validation 1204.1.5 Conclusion 123
4.2 Enantiomeric Resolution by Ion Pair Chromatography4.2.1 Introduction 1264.2.2 Retention Studies 1264.2.3 Discussion 133
4.3 Enantiomeric Resolution Using a Chiral GLC Phase4.3.'l Introduction 1404.3.2 Experimental 1404.3.3 Discussion 150
4.4 Enantiomeric Resolution Using a Cyclodextrin HPLC Phase4.4.1 Introduction 1534.4.2 Testing the Column 1544.4.3 Resolution of Propranolol 1574.4.4 Resolution of Chlorpheniramine 1644.4.5 Resolution of Clomiphene 1694.4.6 Discussion 175
4.5 Enantiomeric Resolution Using a "Pirkle" Type Phase4.5.1 Introduction 1774.5.2 Resolution of Propranolol 1784.5.3 Resolution of Propranolol Maphtharaide 1824.5.4 Computer Modelling 194
4.6 Enantiomeric Resolution Using an AGP Phase4.6.1 Introduction 2104.6.2 Retention Studies 210
CHAPTER 5 DISCUSSIOW 221
BIBLIOGRAPHY 230REFERENCES 233
- 6 -
CHAPTER 1
IITRODUCTIQI TO CHIRALITY
1.1 Chirality
Two or more compounds whose structures differ only in the three
dimensional arrangement of the constituent atoms are classified as
stereoisomers, Such isomerism can be either enantiomeric or
diastereomeric, depending on whether the isomers are related as abject
and nonsuperimposable mirror image or not; enantiomers are those isomers
which are so related, whereas diastereomers are not.
The study of chirality was initiated at the beginning of the nineteenth
century, when, following the discovery of the polarisation of light by
Malus in 1808, work by Blot, Arago and Fresnel showed that certain
materials, in particular quarts, caused a rotation of the plane of plane
polarised light. Detailed examination of quarts showed that the crystals
were not symmetrical and that two forms were present which were mirror
images of each other. On separation it was found that one caused the
plane of polarisation to rotate to the right, while the other caused
rotation to the left. Further research was limited and did not explain
the rotatory effects of other liquid substances such as camphor, oil of
turpentine and sugar solutions,
In 1848, the research of Pasteur [13 was centred on tartaric acid, a
compound of unknown structure with the formula C.+HeOe. Tartaric acid
was known to exist in two forms, one which was optically inactive, and
another which caused clockwise rotation of the plane of polarisation,
The crystals of the dextrorotatory form were found to be asymmetric, but
the recrystallisation of the optically inactive form, instead of yielding
crystals of a different, symmetrical structure as expected, showed an
approximately equal mixture of the same left and right handed crystals as
found for the other form [23. On separation, the right handed crystals
showed dextrorotation of polarised light, while the left handed crystals
showed laevorotatory effects. This proved that compounds with
asymmetric crystals could exist in two enantiomeric forms, each having an
equal and opposite effect on polarised light, and that compounds may be
optically inactive because of compensation between the opposite rotations
if both forms are present in equal quantities.
Although Pasteur’s work was centred on the idea of crystalline
dissymmetry, the concept of asymmetric molecular structures was not
introduced until many years later, after ICelcule had developed a theory
based on the existance of a tetravalent carbon atom in 1865. Prom this,
Van't Hoff [33 and Le Bel [43 independently postulated that if the four
valencies of the carbon atom were arranged in a tetrahedral manner, then
only when all four substituents were different could the structures exist
as nonsuperimposable mirror images (see fig. i.l>, and exhibit optical
isomerism.
An Example of a Pair of Enantiomers
Figure 1.1
The link between molecular asymmetry of compounds and their ability to
rotate the plane of plane polarised light is now well established and
such compounds are termed as chiral. Since it is the overall molecular
symmetry which determines the chirality of a molecule, an asymmetric
carbon atom is not a necessary prerequisite to optical activity, Hence,
although the majority of enantiomeric molecules contain one or more
asymmetric carbon atoms, some do not have a well defined chiral centre,
with the asymmetry being caused by, for example, a substituted cyclic
system. Molecules which are not optically active are termed achiral,
while those where chirality may be achieved by means of a single
chemical transformation are described as prochiral C5L
- 9 -
Although enantiomeric pairs of molecules are completely different
spatially, they have identical physical properties such as melting and
bailing points, solubilities, chromatographic characteristics and spectra.
In fact, generally the only difference between the enantiomers is in their
ability to rotate the plane of plane polarised light, each isomer having
equal and apposite rotatory powers. The energy difference between the
two enantiomers is so small as to make any distinction between them
virtually Impossible except in a chiral environment. In this case
attractive and repulsive forces such as covalent or ionic bonds, which
are spatially dependant, cause the physical and chemical properties of
the enantiomers to differ more markedly. If therefore, a pair of
enantiomers of one compound, A+and A--, are reacted with a single
enantiomer of a different compound, B-<», then the products, A.+B+- and A~B-».,
will be diastereomers, which have significant differences in both
physical and chemical properties.
There are several different styles of nomenclature applied to
differentiate between enantiomers C6], with the simplest using the prefix
<+> for enantiomers which rotate the plane of polarisation to the right,
in a clockwise direction, and (-) for the antipodes which rotate the
plane to the left, in an anticlockwise direction. Associated with this
are the designations d- and 1~ which stand for dextrorotatory, or
rotation to the right, and lae vara tat ory, or rotation to the left
respectively. These should not be confused with the notation D- and L-,
which was the original system introduced by Fischer, and later expanded
by Rosanoxf [71, for discrimination between enantiomers. This was based
on the arbitrary assignment of a particular spatial formula to the
- 10 -
dextrorotatory glyceraldehyde enantlamer which was then designated D~
glyceraldehyde, with the enantiomeric laevorotatory form having the
opposite spatial arrangement and being termed L-glyceraldehyde. From
this, all compounds which could be directly or indirectly transformed to
D-glyceraldehyde were prefixed D-, and all those chemically related to L-
glyceraldehyde were prefixed L-, This system is limited in application,
as it can only be used for compounds which can be related to
glyceraldehyde.
The third system in common use was introduced by Calm, Ingold and Prelog
in 1951, and relies on a set of sequence rules C81. Priorities are
assigned to the substituents on the chiral centre, based initially on the
atomic number of the adjacent atoms, and the molecule is viewed with the
substituent of lowest priority, or atomic number, pointing away. If the
priorities of the substituents decreases clockwise about the chiral
centre, then the configuration is termed R-, and conversely, if the
priorities decrease in an anticlockwise direction, the molecule is
prefixed S-. This is more widely used than the D~ and L- nomenclature
since it may be applied to any molecule, and not just those with
structures similar to glyceraldehyde, and is also more easily applied to
diaster earners and molecules with more than one chiral centre.
1,2 Chirality in Pharmacology
One field where stereochemical considerations are of prime importance is
that of pharmacology, the study of the actions and interactions of
chemicals, or drugs, within the body. Despite a high degree of external
- 11 -
anatomical symmetry, the body is in fact an intensely chiral environment,
and the majority of the chemical reactions essential to a living system
are stereospecific [93. Although the origins of optical isomerism are
still the subject of much debate, such stereospecificity appears to have
been acquired very early in the evolutionary process [10,113, and while
two contrasting theories dispute whether optical Isomerism originated
with the creation of the universe or whether it occurred as a consequence
of the biological evolution of life, the fact that the stability of DIA
requires that the component proteins are L-amino acids [12,133 indicates
that stereoselectivity was of critical importance in the development of
life.
As the action of a drug depends on a number of steric interactions, such
stereoselectivity is a crucial factor in the determination of the
pharmacological and toxicological effects, The study of pharmacology can
be subdivided into several sections including pharmacodynamics, concerned
with the physiological and biological action of drugs, drug disposition,
concerning the distribution, and transport mechanisms within the body, and
metabolism or elimination of the drug from the body. All these aspects
are linked, and factors which affect one will have an influence on the
others. It has long been accepted that stereochemistry is an important
factor in pharmacodynamics, due to the chirality of the receptor sites,
but it is onfy recently that stereochemical effects have been considered
important in the mechanisms involved in the transport and metabolic
elimination of drugs, and in order to determine the effect of the
molecular structure on the action of the drug, the steric interactions in
each of the processes must be individually evaluated.
- 12 -
1,2.1 Pharmacodynamics
The interaction of drugs with living systems at a molecular level is
not yet clearly understood, but two types of interaction, specific and nan
specific, have been postulated. In non specific interactions, the drug
exerts a general effect on a particular aspect of cell function, and since
it has been found that changes in the structure of the drug have little
effect on its activity, the process is unlikely to be stereospecific.
However, with specific interactions, the drug acts at a specific drug
receptor or active enzyme site within the cell, and minor structural
alterations in the drug have large effects on the activity at the site of
action, Research in this area has led to the development of a model of a
drug receptor site consisting of a three dimensional arrangement of
chemical groups on a macromolecular cell component, and hence it can be
seen that specific spatial arrangements of groups within the drug
molecule are required if the molecule is to "fit" properly in the receptor
site, Enantiomers of a given drug, having different spatial
configurations of groups would therefore appear as separate compounds at
the receptor site, leading to marked differences in both the type and
degree of activity, as shown in tables 1.1 and 1.2.
Although the interaction between the drug and receptor may be reversible
or irreversible, the response produced by the drug-receptor complex is a
function of a number of factors including the number of receptor sites
occupied by the drug molecule and also the efficacy or intrinsic activity
of the drug.
- 13 -
Enantiomers With Different Types of Activity
Compound <+>/R <->/S REF
Bupivicane anaesthetic anaesthetic, 14vasoconstrictor
DMBB (Barbiturate) convulsant depressant 15
Ketamine hypnotic CNS stimulant 16
lorphinan analgesic antitussive 17
Indacrinone diuretic, uric uricosuric 18acid retention
Timalol reduces intraocular reduces intraocular 19pressure pressure, J3 adrenergic
antagonist
Table 1.1
- 14 -
Enantioraere With Different Degrees of Activity
Compound Most Active Enantiomer REF
Ibuprofen (S> 20
Propranolol <S)/(~) 21
Hexobarbital (S)/<+) 22
Warfarin (S)/(~) 23
Verapamil (~) 24
Terbutaline (-) 25
Fenfluramine (-) 26
Table 1.2
- 15 -
In order for any drug receptor interaction to occur, the drug must
first be present at the site of action. Any factors influencing this, for
example the rate and extent of absorption, transport to and distribution
at the site of action and elimination of the drug will affect the rate,
magnitude and duration of the response to the drug.
In order for the drug to enter the bloodstream, the molecules must
traverse at least one cell membrane. This is normally achieved by
passive diffusion, although in some cases active transport, occurs.
Passive diffusion is based on the principle that the drug molecules,
present in aqueous solution on one side of the membrane, dissolve in and
diffuse across the lipid membrane and out into the aqueous bloodstream on
the other side. For the process to occur, the drugs must be soluble in
the both aqueous and lipid phases, and the rate of transport is dependent
on the concentration gradient across the membrane, and hence the lipid
affinity. Although factors such as pH have a large effect on the rate
and extent of diffusion, the process is governed by solubilities, a
function which is not stereospecific, and hence passive diffusion is
unlikely to be enantioselective.
Amino acids, sugars, and some salts and vitamins can traverse a membrane
against a concentration gradient by a process of active transport. A
number of mechanisms have been postulated to explain this process, the
oldest involving a mobile carrier which ferries the substrate from one
side across the membrane, rotating as it does so, to present the
substrate at the opposite face. Another model was based on a transfer
1,2.2 Drug D isp osition
- 16 -
channel or chainlike arrangement of sites in the membrane, with which the
substrate sequentially combines until it has traversed the membrane. The
more modern explanation is that transport is catalysed by the proteins
spanning the lipid bilayer undergoing conformational changes to allow the
passage of specific substrates. The channels act as small pumps, moving
the material from one side to the other. In each case, the spatial
arrangement of the substrate molecule will affect the passage, and hence
the mechanism is stereospecific, as demonstrated by the more rapid
absorption of L-amino acids than D-amino acids,
Once the drug has been absorbed into the bloodstream, it is transported
to the various tissues reversibly bound to the plasma proteins, in
particular human serum albumin and oci acid glycoprotein, which act as a
mass transport system for the drug. The drug is then taken up by tissue,
by either passive or active transport, at a rate proportional to the rate
of blood flow to the area. At equilibrium, the drug is partitioned
between the plasma protein, plasma water and the tissue, with the
distribution between the three determined by lipid solubilities and
affinities.
Since the response to a drug is determined by its interaction with tissue
receptor sites, the rate at which the drug can reach the receptor
determines the rate of response of the drug, and is influenced by the
flow of blood to the organ, The magnitude of the response is, however,
based on the free drug concentration at the receptor site. Since plasma
protein binding is stereospecific, this could lead to an increased
response for a particular enantiomer of a racemically administered drug
- 17 -
if the other is preferentially hound by the plasma proteins. Examples of
this include warfarin, where the <S)/<~) enantiomer binds more strongly
to human serum albumin than the (S)/(-f) enantiomer [273, as do the (S)
enantiomers of substituted l,4--3:>eiisadiazepines such as oxazepam [283
1.2.3 Drug Elimination
Drugs are eliminated from the 3)ody by excretion, predominantly from
the kidneys, but also from other organs such as the gut, skin, lungs,
sweat and salivary glands. Although in some cases the drug is eliminated
in an unchanged form, it is more common for a drug to undergo a
metabolic reaction prior to excretion. The process of metabolism
increases the polarity of the lipid soluble drugs, thus increasing the
rate of elimination. Lipid soluble drugs are widely distributed
throughout the body with only a small proportion found in plasma, and
hence plasma clearance is slaw. In addition to this, the high
lipophilicity promotes the reabsorption of the drug,
Metabolism may occur in all tissues, but in general the major metabolic
reactions occur in the liver and may be considered in two stages, Phase
1 metabolism, or biotransfarmatian, involves the introduction of new
functional groups within the molecule by oxidation, reduction or
hydrolysis reactions, thus increasing the polarity. However, although the
metabolite is less lipophilic than the parent compound, it is not
necessarily less active. Phase 11 metabolism involves the conjugation of
the original drug or metabolite with a small endogenous molecule, for
example an organic acid, to produce a highly water soluble compound which
is generally pharmacologically inactive,
- 18 -
Since metabolism is caused by various microsomal enzyme systems, the
metabolic reactions are generally stereospecific, and hence the rate of
plasma elimination of one enantiomer may be considerably different to its
antipode. This, in turn, will influence the concentration of each isomer
at the site of action of the drug causing possible differences in the
magnitude of the receptor response. Examples of drugs which exhibit such
enantiomeric differences in clearance rates include propranolol [29] and
verapamil [30], for which the (-) enantiomer is cleared faster than the
(+) isomer, and hexobarbital [31] and acenocoumarol [32], where the (8)
form is cleared more rapidly than the (R), The <R) isomers of 2-phenyl
propionic acids such as ibuprofen are inverted to the more active (S)
enantiomers and hence the <R) isomers have faster clearance rates [333.
The metabolic pathways may also differ between enantiomers, with one
isomer forming a totally different and possibly more active metabolite to
the other. There are also a number of cases where one enantiomer forms
a toxic metabolite, and examples of this include raephenytoin, where the
metabolite of the <R) isomer has the major therapeutic effect and that of
the <S) enantiomer is thought to be a bone marrow toxin [343, and
prilocaine, a local anaesthetic whose <R) enantiomer is hydrolysed to
form a toxin causing methemoglobinemia [353.
The process is further complicated by metabolic transformations, which
can introduce chirality to a previously achiral or prochiral drug,
rendering further metabolic pathways subject to stereochemical
considerations. These transformations can occur in phase 1 metabolism by
reactions at either the prochiral centre or at a group on the prochiral
- '19 -
centre, or as a result of conjugation of an achiral drug molecule with a
chiral endogenous molecule in phase 11 metabolism.
1.2.4 Drug Interactions
It has long been known that certain drugs interact with one another,
and if present in combination, may produce either beneficial or adverse
results. The mechanisms of such interactions may take several forms and
occur at each stage of the pharmacological process. Drugs which are
transported by the plasma proteins may displace one another from protein
binding sites, causing increased blood levels of the displaced species,
and competition may also occur at the tissue receptor sites, increasing
the concentration of one drug-receptor complex at the expense of the
other. The action of the drug metabolising enzyme systems may be
induced by certain compounds, thus accelerating their metabolism and that
of other* drugs, or inhibited if two drugs compete for the same metabolic
pathway, resulting in one delaying the metabolism of the other.
Interactions also occur in the actions of enantiomeric drugs, where one
isomer has an opposite effect to its antipode, There are a number of
examples of this including pincenadol, where the (+) isomer is an opiod
agonist and the (-) enantiomer has a competitive antagonist action [363,
and isopropylnoradrenaline, active on av adrenergic receptors, where the
<-) isomer is the agonist and the (+) isomer the antagonist C373.
20 -
1,3 Analytical Methods in Pharmacology
The role of analytical chemistry in pharmacology and routine drug
monitoring has long been accepted, and this has led to an extensive range
of techniques being available to measure drug concentrations in various
biological specimens. However, the necessity of finding rapid and
accurate methods of analysis for enantiomeric compounds is becoming
increasingly important, particularly in pharmacology, where evidence
exists to show that many drugs, produced as racemic mixtures, are
stereoselective in their action.
Until recently, the problem of optical isomerism in drugs has been
largely ignored, occasionally with tragic consequences such as the
Thalidomide disaster, where it was discovered that the teratogenic effect
was due to the S (~) form while the R<+) isomer was responsible for the
therapeutic effect [381, The resolution of enantiomeric molecules is not
an easy task and therefore progress has been slow. Although this is due
in part to the lack of analytical methodology for the determination of
the enantiomeric composition of chiral molecules in a biological matrix,
it is also due to the fact that, as a whole, stereochemical considerations
in the in-vivo pharmacological activity of drugs have received a low
priority.
The effects of stereospecificity, however, extends beyond the
pharmaceutical industry and is also apparent in agrochemicals, as many
herbicides, insecticides and fungicides are chiral and have been found to
show significant differences in biological activities between the
- 21 -
enantiomers C39-443, but as yet little has been done to prevent the
desecration of the environment associated with the use of vast quantities
of, for example, a biologically inactive pesticide enantiomer [45].
Until recently, the pharmacological parameters of enantiomeric drugs have
been determined by studying each enantiomer separately, but this approach
neglects the possibility that the two enantiomers may interact causing
the racemate to have a different effect, either quantitatively or
qualitatively, than either of the pure isomers. This has recently been
demonstrated by a study of the antiarrythinic agent, disopyramide [46],
the two enantiomers of which, when administered in a pure form, have
identical properties but the administration of the racemate causes d~
disopyramide to have a longer half life and a smaller volume of
distribution than 1-disopyramide.
In addition to the previous examples, there are many other cases of
enantioselectivity in the overall pharmacological action of drugs, whether
due to stereoselective metabolism, distribution or receptor binding, and
these include amphetamine [47,48], phensuxiraide [493, glutethimlde E503,
oxyphenonium [513 and the benzodiazepinones [523. These are just several
examples from a much more extensive list, which is more fully presented
in a book edited by Ariens [93. A particularly interesting situation
occurs in the case of the synthetic opiates such as methadone [533, for
which there are l̂ etween three and five stereoselective opiate receptor
sites, each triggering a different response.
At the present time, the majority of drugs are produced by non
stereospecific synthesis which results in the formation of a racemic
mixture. In order to produce an enantiomerically pure form of the active
isomer, either the synthesis of the compound must be altered to ensure
stereoselectivity or the racemate must be resolved into the composite
enantiomers and the inactive form discarded, both of which are usually
difficult and correspondingly costly options. Due to economic
considerations, the introduction of drugs preseibed in an enantiomerically
pure form is likely to be a slow process and will require the development
of accurate methods of stereoselective analysis, both to establish the
pharmacological response of the enantiomers of chiral drugs and to
monitor the optical purity of such compounds.
A fundamental assumption in pharmacology is that, for any given drug, the
magnitude of the response is directly related to the number of receptor
sites occupied by the drug molecules. However it was not until the early
1940's that it was recognised that the number of occupied receptor sites
could be more reliably correlated to the plasma concentration [54,551,
assumed to be proportional to that at the site of action, than to the
administered dose of the drug, as had previously been thought. Analysis
of plasma drug concentrations, therefore, became increasingly important
and led to the development of various methods for routine drug monitoring
in biological fluids,
There are a number of different methods available for chemical analysis,
and the choice of technique depends on factors such as the nature of the
- 23 -
analyte, the sample matrix, the expected concentration range, any possible
interferences and the information required from the analysis.
In general, drug analysis involves mainly quantitative methods, although
it is sometimes necessary to perform qualitative analysis to identify for
example, a metabolite. The three main techniques used for such structural
analysis are:
1.3.1 Infra Red Spectroscopy
If a molecule is irradiated with infra red radiation then this will
cause the interatomic bonds to oscillate, The frequency of the vibrations
and hence the energy required to produce such vibrations is
characteristic of each individual bond, Therefore, if a spectrum of the
absorption of energy at each frequency is obtained, then it is possible
to determine which bonds are present and thus elucidate the molecular
structure [561.
1.3.2 luclear Magnetic Resonance Spectroscopy
Certain nuclei, when placed in an external magnetic field, exist in
one of two possible quantum spin states. If irradiated with energy of
radio frequencies, an interchange between the spin states, known as
resonance, can occur resulting in the absorption of those frequencies
corresponding to the energy difference between the two spin states,
Although this energy difference is dependant on the strength of the
external field, it is also affected by the magnitudes of local magnetic
fields caused by the electrons of neighbouring nuclei in a process known
as shielding. Since differing adjacent nuclei shield to different degrees,
- 24 -
the frequencies at which resonance occurs for a sample is indicative of
its molecular structure [57 3. The technique of nuclear magnetic
resonance is limited to nuclei with spin, in particular where the spin
quantum number, I=JA or 1, and is most commonly used with 1H and 13C
although 1 ®F, 31P and :S4Si may also be used.
1.3.3 Mass Spectrometry
This is one of the most specific, and also most expensive
qualitative techniques in routine use. It is based on the principle that,
if a sample is bombarded with, for example, a stream of high energy
electrons, then fragmentation and ionization of the sample molecule will occur. These fragments are detected according to their mass to charge
ratio. Possible molecular structures can therefore be deducted from a
study of the fragmentation pattern and the relative abundance of each
fragment [583.
Due to a greater demand, there are many more techniques available for the
quantitative analysis of a sample than for structural determinations,
although not all are suitable for the routine analysis of drugs in
biological fluids, The methods most widely used include the following:
1.3.4 Electronic Spectroscopy
If a sample is irradiated with electromagnetic radiation, the energy
of the incident photons may be transferred to the sample molecules,
raising the electrons from the ground state to an excited state. This
process is known as absorption, and since the energy absorbed must be
- 25 -
equal to the energy difference between the excited and ground states,
only radiation of specific frequencies or wavelengths can be absorbed.
Ultraviolet and visible absorption spectroscopy [591 relies on measuring
the absorption or percentage transmission of radiation at one particular
wavelength, usually near the wavelength of maximum absorption of the
compound of interest, of an unknown sample. The concentration of the
sample can then be determined by comparison with a calibration curve,
prepared by measuring the response of standards of known concentrations
at the same wavelength.
As the technique depends on the characteristic absorption of individual functional groups or chromophores within the molecule, the method is not
particularly specific; two different compounds sharing common functional
groups, in particular a drug and the metabolite, will have similar
absorption spectra and hence the presence of one will interfere with the
measurement of the other. The second disadvantage of this technique is a
lack of sensitivity due to the problems involved in measuring small
changes in transmitted radiation against a background of intense incident radiation.
Fluorescent spectroscopy is not concerned with the energy absorbed in
exciting the molecule, but with that emitted as it decays back to the
ground state. In most molecules, the energy emitted is at an identical frequency to that absorbed and hence cannot be measured, However with
molecules that fluoresce, same of the excess vibrational energy of the
excited state is lost through interm a lecular collisions, and thus the
- 26 -
radiation emitted on decay to the ground state is at a lower frequency to
that absorbed [603.
This makes the technique much more specific, as it becomes less likely
that two compounds will absorb and emit radiation at the same
frequencies. Fluorescence is also more sensitive, since a low
concentration of a fluorescent sample corresponds to a low signal, which
can be readily amplified, whereas a low concentration of an absorbent
material corresponds to a very small reduction in the maximum signal, the
amplification of which increases the noise level. In addition to this,
the photomultiplier tube can be located perpendicular to the excitation
source within the detector, thus being more shielded from incident
radiation than in an absorbance spectrometer. As with absorption
spectroscopy, the intensity of the emitted radiation of an unknown sample
is related to the concentration by comparison to a calibration curve
produced by measuring the intensity of emission from standards of known
concentrations.
Phosphorescence is similar in principle to fluorescence, but is much less
widely used as an analytical technique. This is due to a number of
reasons, not least of which is that the number of compounds which exhibit
phosphorescence is small, and very low temperatures <8010 are required for significant emission. Since the process of phosphorescence involves
a spectroscopically forbidden transition, the decay to the ground state is slow and the compound will continue to emit radiation for some time after
the Initial excitation, limiting its analytical applications C613.
- 27 -
This technique is based on competition between labelled and
unlabelled forms of the analyte for a limited number of antibody binding
sites [62], If a known amount of radiolabelled analyte is added to an
unknown sample and allowed to equilibriate with a small amount of
antisera, then some of the antibody binding sites, Ab, will be occupied by
the labelled antigen, Ag*, and some by the unlabelled antigen, Ag, As
there are less antibody binding sites than antigen molecules, some of
both the labelled and unlabelled antigen will remain free, or unbound to
the antibody, as shown overleaf:
1.3,5 Radioimmunoassay
Ab + Ag + Ag*
Once the antibody bound fraction is separated from .the free fraction by
any suitable technique, the distribution of radioactivity between the two
fractions can be related to the amount of radiolabelled antigen originally
added in order to calculate the amount of unlabelled antigen in the
sample.
Radioimmunoassay methods can be applied to a large number of compounds
providing that antibodies can be raised, and is, in general, a very
sensitive and simple technique, requiring only minimal sample preparation in many cases, The disadvantages lie in the time and cost of raising the
antisera, and also in the risk of interferences arising from metabolites
- 28 —
which, being similar in structure to the antigen, also compete for the
antibody binding sites,
There are several other immunoassay techniques which do not require the
use of radiochemicals. These include enzyme immunoassays [633, where an
enzyme, such as peroxidase, is used as a marker in place of the
radiolabel, and the reactivity of the enzyme fractions are determined by
an appropriated enzyme reaction, and fluorescent immunoassays [643, where
a fluorescent dye is used to label the antigen. In both cases, the
determinations can often be carried out without performing the separative
step necessary for radioimmunoassay.
1.3.6 Chromatography
Although this is probably one of the most commonly used techniques
for the analysis of drugs in biological fluids [65-673, it is, however, only a means of separation and must therefore rely on some additional
form of detection system for quantitative analysis.
A chromatographic separation is based on the idea that, if a sample is
passed through a stationary phase under the influence of a mobile phase,
then partition will occur between the stationary and mobile phases
resulting in differing components of the sample being retained to different degrees. The rate at which individual components move through
the column is dependent on the relative affinity of each for the two
phases, and hence the component which is retained least strongly in the
stationary phase will be eluted first, and that most strongly retained,
- 29 -
eluted last. The separation is therefore dependant on the nature of the
stationary and mobile phases, and the interaction between the two,
Although the range of applications is very wide, the importance of the
technique lies with the enhanced sensitivities achieved with
chromatographic detection systems in comparison to that achieved by
using a similar detector alone. Since the compounds are separated one
from the other by the chromatography step the detectors used are,
theoretically, only responding to individual components, thus reducing the
possible interferences and therefore increasing the sensitivity.
There are a number of chromatographic techniques based on the nature of the phases involved, with the three main areas outlined below:
(i) High Performance Liquid Chromatography (HPLC), in which the
stationary phase is linked to a solid support and packed into tubular
columns through which a liquid mobile phase is pumped at a constant rate.
Both the stationary and mobile phases can be altered to achieve the
separation.
(ii) Gas Liquid Chromatography <GLC), in which separation occurs between
the stationary phase, usually a high boiling point liquid coated onto an
inert solid support, and an inert carrier gas at high temperatures. In
this case the stationary phase and temperature can be altered to effect
the separation, and the samples must be easily volatalised and not
decompose at high temperatures. To conform to these criteria, samples
sometimes require some form of prior derivatisation.
- 30 -
(iii) Thin Layer Chromatography (TLC), involves thin layers of stationary
phase coated onto inert backing supports, such as glass plates, placed in
a reservoir of mobile phase, which migrates across a flat surface. Both
stationary and mobile phases can be adjusted, and better separations can
sometimes be achieved by using 2-dimensional TLC, where the sample is
run in one direction with one solvent system and then run perpendicular
to this with a second solvent system. An extension of TLC is High
Performance (HP) TLC, which uses stationary phases ox a smaller particle
size and a narrower particle size distribution, hence increasing
sensitivity, selectivity and resolution.
1.3.7 Polarography
Polarography is an electrochemical technique used for the
quantitative analysis of those organic or inorganic compounds which can
be either oxidised or more commonly, reduced, in solution by an electric
current [683. If a fixed potential is applied across two electrodes, one
of which was classically a dropping mercury electrode, then the current
flowing between them is a function of the concentration of the analyte in
the sample. The applied potential is characteristic of the sample
molecule, being determined by the number and nature of reducible or
oxidisable bonds, but again drug analysis may suffer from interference 3>y
metabolites, which are likely to contain similar electroactive groups.
Thus the technique is most widely used after some form of separation
stage, such as chromatography.
- 31 -
1.3.8 Isotope Dilution Analysis
If a radio label led form of the analyte is thoroughly mixed with the
sample, and the total analyte separated and purified, using conventional
methods, then the specific activity of the purified material can be
related to that of the radiolabelled analyte added, to give an accurate
estimation of the concentration of the analyte in the sample. The method requires fairly large amounts of sample, and so is not commonly used in
drug monitoring,
1.3.9 Isotope Derivative Analysis
This is a similar technique involving the reaction of the sample
with a radiolabelled reagent, thus rendering the compound more sensitive
to radiodetectors, As the radiolabelled reagent is normally present in
excess, problems may arise in the separation of the compound of interest
from the unreacted reagent and any other side products that may have
been formed, prior to detection. Once again this technique is only rarely
used for routine drug analysis,
1.4 Methods for Enantioselective Analysis
In order to determine the pharmacological effects of enantiomeric drugs,
it is important to develop methods capable of measuring the plasma
concentration of each enantiomer of a racemic drug. Since the properties
of a pair of enantiomers are identical, none of the previously described
classical methods of analysis are directly applicable to chiral
determinations.
- 32 -
At the beginning of the century, the only method available for
enantiomeric determinations aside from Pasteur's crystallisation
techniques, was polarimetry, This involved a comparison between the angle of rotation of the plane of plane polarised light caused by the
unknown mixture and that caused by the pure enantiomer. This method was
not particularly accurate or sensitive, and prompted further work on the
development of other methods. There are now a number of techniques available for the resolution of enantiomers and the method of choice
depends on a number of criteria such as the scale of the resolution, the
availability of reagents and the information required from the analysis.
For large scale preparative resolutions, crystallisation is the most
suitable choice. Other techniques such as isotope dilution analysis,
kinetic resolutions and differential microcalorimetry provide information
on the optical and hence the enantiomeric purity of the sample, This is
equivalent to the percentage composition of the enantiomeric mixture, and
hence, if the absolute concentrations of each enantiomer are required, the
total concentration of the analyte in the sample must either be known or
determined in a prior analysis. These methods are also based on a
comparison between one particular property of the unknown mixture and that of the pure racemate, and thus are dependant on the availability of
a suitably pure sample of the racemate,
Chromatographic techniques are more suited to the routine analysis of
large numbers of samples, and, in addition to the determination of optical
purity, may be used for the determination of the absolute concentrations
of each analyte enantiomer in the sample. Muclear magnetic resonance
- 33 -
methods can also be used in this way, as can the relatively new technique
of enantioselective radioimmunoassay.
Although these techniques can all be used to determine the relative concentrations of each enantiomer in a mixture, the only way that the
absolute configurations of enantiomeric molecules can be ascertained is
by using X-ray crystallography techniques [69,703. This is limited,
however, to structural determinations of compounds in a crystalline state, which may be different from that in solution, a more relevent factor in
biochemical and pharmacological studies.
Stereoselective determinations may be carried out on enantiomers or
diasterearaers with or without prior separation. Those methods which
employ some form of separation include crystallisation, chromatography
and stereospecific reactions, while those that do not involve separation
require simultaneous analysis of the stereolsomeric components and
include polarimetry, nuclear magnetic resonance spectroscopy, and more
recently, differential mlcracalorimetry.
1.4,1 Crystallisation Techniques
These techniques may be subdivided into two main groups depending
on whether the resolution is achieved directly, or indirectly via the
formation of diastereomers [713. Crystallisation is normally only used
as a preparative separation step, and the attainment of optical purity
is monitored by techniques such as nuclear magnetic resonance
spectroscopy, thin layer chromatography or gas liquid chromatography.
- 34 -
There are several different approaches to the direct resolution of
enantiomers [72], the simplest being the method used by Pasteur with
tartaric acid, of allowing a solution to crystallise then manually
separating the enantiomeric forms. A variation of this involves seeding
a supersaturated racemic solution with crystals of the pure enantiomers
[733. Crystals form around the seed and are of the same enantiomeric
structure of the seed crystal, thus eliminating the need for manual
sorting E743,
More commonly used is the technique of preferential crystallisation, first
observed by Gernes in 1866 C753, and later by Werner in 1914 [763. This
is based on the theory that a supersaturated solution of one enantiomer
will not crystallise in the presence of a crystal of its antipode, Thus,
if a racemic solution is seeded with a crystal of one enantiomer,
crystals of only that enantiomer will be farmed and the solution will be
enriched with respect to the other. This technique is limited to
compounds where the racemate is more soluble than the enantiomers.
The solubilities of optically active substances are affected by factors
such as the solvent matrix, and since these techniques are based on
solubility differences between the racemate and the pure enantiomers, the resolutions may be improved by the use of optically active solvents or
co-solutes [77-793.
The resolution of a racemic mixture of enantiomers is facilitated by the
formation of diastereomers which have several different properties that
- 35 -
may be used to achieve a separation. Once separated, each diastereomer
may be cleaved to yield the pure enantiomer together with the reactant.
The diastereomers, A+B-*. and A B+, are farmed by reacting the racemic
mixture, A±, with an optically pure resolving agent, B.+.. There are
numerous different types of resolving agent [803 and the choice of which
one to use depends largely on the chemical nature of the racemic mixture,
The most widely used methods involve the formation of diastereomeric
salts from chiral acids and bases, although Lewis acid-base molecular
complexes, inclusion complexes and transition metal complexes have also been used.
The diastereomers differ, one from the other, in many physical ways
including crystal structures, densities [8.13, melting points, enthalpies of
fusion and solubilities, and although in some cases the densities differ
enough to theoretically allow separation by centrifugation [823, it is the
differences in solubilities which is most often exploited to effect a
separation by crystallisation. It is generally the case that the greater
the difference in solubilities between the diastereomers the easier, and
more successful the separation [83-853.
The formation of diastereomers is not limited to dissociable salt
complexes, it is also possible to produce covalent diastereomeric
derivatives, such as esters and amides £863, However, due to the greater
bond strength of covalent compounds, consideration must be given to
prevent racemisation of the resolving agent or the mixture of
enantiomers, if the latter is not racemic, and also to the method of
- 36 -
cleavage of the covalent diastereoraers if the enantiomers are required in
a pure form.
1,4.2 Isotope Dilution Analysis
The technique of isotope dilution analysis as used to determine the
optical purity of an enantiomeric mixture is very similar to the
procedure for achiral determinations. A measured amount of radiolabelled racemic analyte is added and thoroughly mixed with the analyte in the
sample under test, and the total compound isolated from the sample. The
optical purity of the sample analyte may be established by using a
mathematical equation relating the mass and specific activity of the
added racemate and the specific activity of the reisolated mixture to the
mass of analyte initially in the sample [873. This method was first used
to determine the optical purity of D- and L- amino acids in 1940 by
Graff, Rittenberg and Foster [883, but was not recognised as a general
technique until 1955 after work by Berson and Ben Efraim C893,
Although this technique has been successfully used to determine the
optical purity of a number of compounds, each of the various practical
methodologies relies on the assumption that the mass of analyte initially
present in the sample is a known quantity. Since this is generally not
the case, then some form of prior analysis, whether by achiral isotope
dilution or a different method, is required to establish this, thus complicating the enantiomeric analysis. The technique also suffers from
the same disadvantage as it's achiral counterpart, that is that large
samples are required, which renders the method unsuitable to routine drug
monitoring.
- 37 -
A similar technique to isotope dilution analysis is that of the
radiotracer method. This is used for the determination of the optical
purity of samples of very high enantiomeric purity [90], Again, this
method relies on measuring various specific activities which can then be
related to the optical purity of the sample by a mathematical equation.
The disadvantage of this method is that, in order to solve the equation
and find the optical purity, it is necessary to know the approximate enantiomeric purity of the sample,
1.4.3 Kinetic Techniques
Kinetic resolution methods are based on the difference in reaction
rates of enantiomers with chiral reagents, the rates reflecting the
differences in activation energies of the diastereameric transition states
[911. If a racemic mixture of an analyte A±, is reacted with a non
stoichiometric quantity of an enantiomerically pure reactant, B+-, then the
two reactions will take place at different rates and hence the products,
and A-B-.-, will be formed in a ratio corresponding to the ratio of
the rate constants for each reaction, and the residual analyte will be
enriched with the enantiomer with the lowest rate constant,
The main experimental procedure is that of the double resolution method
[923, a relative method performed as a two stage determination, in which
an unknown optical purity of a substance is correlated to that of another
substance of known composition. Initially a known amount of the analyte is reacted with an insufficient quantity of an enantiomerically pure
reagent, and the specific rotation of the remaining analyte determined,
In a second resolution, a known quantity of the racemic reagent, is
- 38 -
reacted, with an insufficient quantity of the optically active analyte, and
again, the specific activity of the residual mixture, which consists of a
non-racemic mixture of reagent enantiomers, is determined. In both cases,
the specific rotations can be related to the respective quantities of
reactants and yields of reactions, and the two mathematical equations may
be solved simultaneously to give the absolute rotation of the analyte,
from which the optical and hence enantiomeric purity can be ascertained.
The main requirement for the success of the method is that the rotations
of the residual mixtures of both the analyte and reagent enantiomers are
fairly large, and thus the differences between the rate constants for each
pair of reactions are correspondingly large in order to achieve an
optical yield which is outside the range of experimental uncertainty,
However, since the yield of each reaction is incorporated into the equations, the reactant is not necessarily required to be optically pure,
Other kinetic resolution techniques are based on employing enzyme systems
[931, and depend on the availability of an enzyme which will react
quantitatively with one enantiomer in the presence of a large excess of
the other. The experimental procedures vary according to the
determination, but often involves a volumetric determination of the
amount of oxygen consumed or carbon dioxide evolved in the enzymatic
reaction. Although the range of applications is limited by the problems
which undoubtedly arise from finding a suitable stereospecific enzyme
system, the technique is very sensitive and it is not unusual to reach limits of detection of less than 0,1% optical impurity.
- 39 -
1.4.4 Differential Microcalorimetry
This technique, devised by Fauquey and Jacques in 1966 [94,951, is
based on the phase relationship between the component enantiomers in an
unkown mixture. Such a mixture can generally be classified as either a
conglomerate, defined as an equimolar mixture of crystals of pure
enantiomers, or a racemic compound, in which equal quantities of
enantiomers are present in a well defined crystal lattice [961, and this
determines the experimental procedures required to find the enantiomeric
purity of the unknown mixture.
The analysis may be performed either directly or indirectly and involves
measuring certain thermodynamic parameters with a differential scanning
microcalorimeter. The enthalpy of fusion and the melting point of the pure racemic mixture are then compared to the temperature of termination
of fusion of the unknown sample to yield the composition of the binary
mixture. The direct method is generally employed for mixtures of
enantiomers of intermediate purity and, since the precision is not high,
is often used to monitor the progress of p resolution. The indirect
method detects the presence of impurities which lower the melting point
by forming a eutectic mixture with the nearly pure substance, and is only
used for mixtures which are either of high enantiomeric purity or are
very close to being racemic [97-993, However the method is unable to
distinguish between whether the melting point is lowered due to the
presence of an enantiomeric impurity in a nearly optically pure sample or
whether it is due to a chemical impurity. The precision, however, is
high, especially with high purity samples.
- 4 0 -
In addition to the necessity for chemically pure samples, these methods
also require that the compounds are thermally stable, and do not racemise
at elevated temperatures,
1,4.5 Enantioselective Nuclear Magnetic Resonance Spectroscopy
This technique provides a means of measuring the optical purity of
a substance without prior separation of the enantiomers or diastereomers.
Such methods are based on the necessity for two variable parameters, one
of which is structurally dependant but is unaffected by the concentration
factors, and the other which, independently of structural considerations,
varies with the concentrations of the species present. In nuclear
magnetic resonance spectroscopy, these parameters correspond to the
chemical shift, a structurally determined variable, and the integrated
intensities of the resonances, which is indicative only of the relative concentrations present in the sample. The application of nuclear
magnetic resonance spectroscopy to enantiomeric determinations is
therefore based on obtaining a measurably nonequivalent, or anisochronous,
set of signals due to corresponding groups in the two isomers. These
signals should either be singlets or doublets and should not overlap or
interfere with the signals due to any other part of the molecule.
There are several means by which such nonequivalence may be secured, the
most obvious being by the formation of diastereomers [873, Since the
analysis does not involve a separation, the ease of recovery of the
enantiomers from the diastereomers need not be considered, and hence
covalent derivatives are more often used. The nonequivalence may either
arise from the enantiomeric portion of the diastereomers, or may be
- 41 -
introduced by the derivatising reagent. Thus the analysis may be
improved by the choice of derivatisation reagent,
The alternative to the formation of diastereomers prior to analysis is to place the enantiomeric mixture in a chiral environment, resulting in the
occurence of diasteromeric interactions which may differ in energy
sufficiently to cause peak splitting. In a very few cases 11003, the
enantiomers themselves create the chiral environment, the homochiral
interactions between the enantiomers differing significantly from the
heterochiral interactions, thus enabling immediate analysis when bath
enantiomers are present in a mixture of unequal amounts [101,1023.
In most cases however, the diastereomeric interactions have to be
externally introduced into the system. In 1965, Eaban and Mislaw
postulated that this could be achieved by the use of a chiral solvent
[1033, and this was supported with experimental evidence by Pirkle C1043. It is interesting to note that it is not necessary for the chiral solvent
to be optically pure, although the magnitude of the difference between the
chemical shifts of the signals due to corresponding groups on each
enantiomer is proportional to the optical purity of the solvent [1053,
The principal disadvantage of this method is that, even with solvents of
high optical purity, the degree of separation between the signals from
the two enantiomers is small, and often cannot be used for quantitative
determinations.
An increasingly more popular approach is to use a chiral lanthanide shift
reagents (LSR). A lanthanide shift reagent consists of a six coordinate
lanthanide metal complex, which, by expanding its sphere of coordination,
accepts further ligands, thus forming a paramagnetic lanthanide complex
with heteroatoms exhibiting some degree of Lewis basicity [106,1073. The
mast common lanthanide shift reagents are based on europium [108,1093,
and a review of the use of such shift reagents for the determination of the enantiomeric purity of a wide range of organic compounds is provided
l>y Sullivan C1103. Although the magnitude of the enantiomeric differences
in chemical shifts is much greater [1113, the mechanisms which give rise
to the anisochronous signals of compounds in the presence of lanthanide
shift reagents is similar to that which occurs with compounds in
optically active solvents, and, as in the case of optically active
solvents, it is not necessary that the shift reagent is optically pure,
but that again, the magnitude of the peak splitting is reduced by lower
purities. One limitation to the use of lanthanide shift reagents is that
the relative shifting of the signals due to each enantiomer is difficult
to predict, and, in cases where it is important to determine absolute
concentrations of each enantiomer, standards, enriched in first one enantiomer then the other, would be required to establish the relative
peak order for each signal.
Determinations of optical, and hence enantiomeric purity of compounds are
not limited to proton nuclear magnetic resonance, and the methods are, in
general, equally applicable to 13C, iaF and 31 P.
1.4.6 Chiral Chromatographic Techniques
Since the technique of chromatography combines a separative stage
with one of several detection systems and is also already widely used for
- 43 -
low level drug analysis, it is ideally suited to the quantitative
determination of enantiomers. Once a separation has been effected, using a suitable chromatographic system, then achiral detection of each
enantiomer may be performed by classical means. As with most of the
other techniques, the chromatographic resolution of enantiomers may be
achieved either directly or indirectly, regardless of the type of chromatography employed. Direct analysis requires that the chirality of
the solute molecule is recognised by the chromatographic sjrstem, either
by a chiral stationary phase or by a chiral eluent in the case of liquid
chromatography, while indirect analysis is again based on the formation
of diastereomers prior to the chromatographic separation,
Until fairly recently, the majority of chromatographic resolutions have
been performed indirectly, by the formation of diastereomeric derivatives
which could then be separated using an achiral system, There are several
disadvantages inherent in this approach, not least of which is that the
quantitative discrimination between the enantiomers is dependant on the
optical purity of the derivatislng reagent. It is also important to
recognise that the diastereomers, once produced and separated, da not
necessarily give identical detector responses,
There are two approaches to the direct chromatographic resolution of
enantiomers, the most versatile of which is the use of chiral stationary
phases (CSP>. Chiral separations on such phases are based on the
formation of transient diastereomeric complexes between each enantiomer
and an optically pure chiral selector bound to the stationary support,
Energy differences between the two enantiomer-chiral stationary phase
- 44 -
complexes cause differences in the retentions of each isomer and thus
effect the separation. A second possibility is to include the chiral
selector in the eluent, although this is limited to thin layer and liquid
chromatographic systems. The mechanisms involved are similar, with the
transient diastereomeric complexes farmed between the enantiomers and the
mobile phase additive. The separation is achieved via the differential
partition of the transient complexes between the mobile and stationary phases. In either case, there is evidence to show that calibration curves
obtained with one enantiomer cannot necessarily be used to quantitate its
antipode, and thus for any analysis the calibration should be performed
with standards containing known concentrations of both enantiomers from
which two separate curves are obtained.
1.4.7 Bnantioselective Radioimmunoassay
A relatively new and promising technique for the routine analysis of enantiomeric drugs and metabolites involves radioimmunoassay with
enantiospecific antisera, This is based on utilising the natural
enantioselectivity of a living system to raise antisera to one isomer
which will be inactive towards the other, thus mimicking the body's
inherent stereospecificity. Although the technique is not yet caramon
practice, it has been successfully been used for several studies on drugs
such as propranolol 11123, warfarin C1133 and an antimalerial agent [1143, The method does however suffer from similar problems of cost and
difficulty in producing the antisera as does the achiral method, possibly
to an even greater extent.
- 45 -
1.5 Problems with Enantioselective Analysis
There are a number of problems inherent to enantioselective analysis,
irrespective of the particular technique used, which are not applicable in
achiral analysis, and may therefore tend to be easily overlooked. One
such consideration is that, for those methods in which the enantiomers
are reacted with an optically pure reagent to form diastereoraers, the
purity of the derivatising reagent is of crucial importance to the quantitative results £1151. In addition to this, it is important to study
the derivatisation reaction mechanism and conditions in detail since the
derivatisation could cause partial racemisation of either the sample enantiomers or the derivatising reagent, particularly, for example, if the
reaction proceeds through a trigonal planar intermediate. The reaction
may also show some stereoselectivity in the rates of reaction, causing
some kinetic resolution effects if the reaction is not allowed to proceed
to completion, resulting in the enrichment of the mixture with the isomer
with the fastest rate of reaction 11163. These factors could all lead to
significant errors in the results of the determination, and thus must be
fully evaluated prior to the analysis.
Another interesting potential source of error in chiral analysis is known
as the enantiodifferentiating process El.173, in which a nan racemic
mixture may, despite the absence of a chiral environment, become enriched
in one enantiomer as a consequence of procedures such as solvent
extraction, chemical reactions and achiral chromatography. This arises
from dissymmetric intermolecular interactions between the two
enantiomers, which are possibly concentration dependent.
- 46 -
It is therefore important that the validation of any method, particularly
if used for routine drug analysis, should be carried out both with known
enantiomeric mixtures and also with the pure enantiomers, and that the
standards used for such validation are of the highest optical purity, If
the separate enantiomers are not available, or the purity is not
sufficient, then the method should be validated by comparing the results
with those obtained for the same analysis using a second technique which relies on different principles of resolution.
In addition to the points already mentioned, it is also important to
consider the sample matrix when choosing the method of analysis. With
techniques such as chromatography, impurities in the sample are not
important providing they do not co-elute with the compound of interest,
whereas far example with differential microcalorimetry, the presence of a
chemical impurity would affect the results to such a degree as to render
the technique unsuitable for anything other than chemically pure samples,
1,6 Sample Preparation
For any analysis of drugs in biological fluids, a certain amount of
pretreatment is required to extract the drug from the sample matrix, and
this is generally achieved by liquid extraction with an organic solvent.
This technique involves several stages, the first of which is to adjust
the pH of the sample to ensure that the drug is in an unionized form. An
organic extraction solvent is agitated with the sample and partitioning
of the drug between the aqueous and organic phases will occur. The
majority of the unionized drug will exist in the organic phase, which can
- 47 -
be separated from the aqueous layer and dried if required. For routine
drug analysis, it is generally necessary to concentrate the samples prior
to analysis, and this may be achieved by the evaporation of the organic
solvent to dryness whereupon the sample residue may be redissolved in a
much smaller volume of solvent.
There are several factors which must be investigated prior to the
extraction of samples, the most important of which is the choice of an
extraction solvent system, The partition of the drug between the aqueous
and organic phases is controlled, by the relative polarities of the
organic solvent system and the drug; a lipophilic drug will be readily
extracted with a nonpolar solvent whereas a more polar drug will require
a correspondingly more polar solvent, In general, since the majority of
interfering impurities in the sample matrix are polar, the extraction
system should be as nonpolar as is possible to extract the compound of
interest,
Other passible extraction procedures are available, the most papular of
which involves the use of solid phase cartridges, These are small
cartridges containing a bonded phase on a solid silica support. The
sample is drawn through the cartridge and the drug is adsorbed onto the
phase, which is then sequentially washed with various solvents to elute
the impurities, and finally eluted with a suitable solvent in a purified
form, The separation is again a function of partition effects between
the sample, the solid phase and the various solvents used.
- 48 -
The choice of which extraction procedure to use depends largely on the
analysis and the number and type of samples, Traditionally liquid-liquid
extraction has been the method of immediate choice, but more recently
solid phase extractions are becoming more popular and also more
economically competitive.
- 49 -
CHAPTER 2
CHROMATOGRAPHIC SEPARATIONS
2.1 Historical Aspects
Chromatography has been defined by IUPAC [118] as "a method used
primarily for the separation of the components of a sample, in which the
components are distributed between two phases, one of which is stationary
while the other moves. The stationary phase may be a solid or a liquid
supported on a solid or a gel, and may be packed in a column, spread as a layer or distributed as a film. The mobile phase may be either liquid ar gaseous".
The first chromatographic separations were made by Runge, who used the
method for testing the composition of dyes and bleaches by spotting the
mixtures onto a special paper, thus producing colour separations [119,1201
in 1850, and also by Groppelsroder, who developed a system using strips
of paper with one end dipped in aqueous solutions to separate coloured
materials in solution [1211 in 1861, A more comprehensive description of the separation process followed much later, when Tswett used a glass
column filled with a calcium carbonate adsorbent to separate plant
pigments into distinctive green and yellow zones [122,1231 in 1906, He
used the term chromatography to describe his separations, and defined the
process by the statement "chromatography is a method in which the
components of a mixture are separated on an adsorbent column in a
flowing system".
- 50 -
However, it was not until the early 1930's that adsorption chromatography
began to attract more widespread interest, and was used for a number of
separations [124,1253. Following this, a second type of chromatography,
based on partition of the sample [between two liquids, was developed by
Martin and Synge [126,1273. Columns of silica gel were initially used to
support a water stationary phase, although in 1944, filter paper
impregnated with the liquid stationary phase was used instead of the
silica column [1283. A similar principle was later applied to separations
involving gaseous mobile phases [1293, During this time, the technique of
thin layer chromatography was introduced by Ismailov and Schraiber [1303,
but the method was of little value until a more reliable mechanical means
of producing reproducible thin layers was devised by Stahl [1313.
2.2 Theory
The theoretical aspects of chromatography were first studied by Wilson
[1323, although prior to this an attempt had been made to establish
several different principles of separation by Tiaelius and Claessan
£133,1343. Much work was done to relate the column performance to
factors such as the stationary phase particle size and the diffusion
effects of the column £135,1363, cummulating in the development of a rate
theory to describe the separation process by van Deemter in 1956 £1373,
Chromatography involves the separation of the components of a mixture by differences in the retention of each compound. Such differences are
caused by differing partition coefficients for each compound. The
partition coefficient is defined as the ratio between the concentration of
- 51 -
the component in the stationary phase and that in the mobile phase, The
separation depends therefore on the nature of the stationary phase, the
mobile phase and the solute, and the interactions of each with the others.
There are a number of equations which may be used to describe such
interactions and relate their effect to the retention of the sample,
Possibly the mast important theoretical considerations for analytical
purposes are the resolution and efficiency of a separation. The ideal
chromatographic process is one in which the components of a sample form
narrow bands which are completely resolved from one another, although in practice, this rarely happens.
The column efficiency is a measure of the rate at which the solute molecules spread out as they travel through the column, or across a
thin layer chromatography plate, The efficiency, or number of theoretical
plates, I, of a separation is determined by a comparison between the
retention of a component, tF<, and its peak width, tw, or, as is more
common, it's peak width at half the peak height, twi* (as shown in figure 2.1), by:
I = 16 (trc/tw ):2 = 5.54<WW>*
However, it is sometimes better to determine the number of effective
plates for the column, as this gives a snore accurate measure of the
efficiency. The effective plate number is based on the corrected
retention of the solute, tw', and therefore takes account of the retention
of the unretained peak, to, since
- 52 -
II
- 53 ~
Figu
re
2.1
Thus, the effective plate number, Nett, is always less than the
theoretical plate number, and is given by:
W e f f = 5 , 5 4 <tR VtwMs)2
The effective plate number is a useful parameter for the comparison of
efficiencies of similar columns with the same stationary phase, however
it is more common to use the height equivalent to a theoretical plate, or
plate height, H, for such comparisons, since this is independent of the
length of the column, L:
H = L/N
Efficient separations are therefore characterised by sharp, narrow peaks
giving a high plate number and a low plate height.
A general theory of factors contributing to band spreading was developed
by Giddings [138] which assumes that the progress of molecules through
the column is a succession of random stops and starts and that
dispersion, and hence band broadening, increases with the number of
transfer steps. The plate height, H, is used to express
the resultant effect of the various band broadening processes via the van Deem ter equation:
t R‘ = t R-to
- 54 -
H = A + (B/u) + CsU + CmU
where u is the average linear velocity.
The first term represents the eddy diffusion, and relates to the variable
unequal paths of the molecules around the stationary phase or support
particles. This is a function of the mean particle diameter, d;
A = Xd
where X is a coefficient reflecting the column geometry and uniformity of
packing, The second term describes band broadening due to longitudinal
diffusion, and is related to the degree of obstruction, or hindrance to
diffusion of the column packing, K;
B = 2KDm
where Dm represents the solute diffusion in the mobile phase. This term
is more important in gas chromatography since the diffusion coefficients
of salutes in gases are much greater than those in liquids. The last
terms describe the resistance to mass transfer effects. Those effects
occurring at the solute-stationary phase interface are a function of the
diffusion coefficient of the salute in the stationary phase, D®, and the.
thickness of the film of stationary phase on the support, d®, and that
caused by the resistance of the packing material particles to radial mass
transfer is related to the particle diameter, d p , and the diffusion
coefficient of the solute in the mobile phase, Dm:
- 55 -
Cs = d:=:s /Ds- Cm — dp'"'1 /Dm
The average linear velocity, and hence the flow rate, must therefore be
adjusted to compromise between each factor in order to obtain the optimum
efficiency, and the variation of the plate height with the average linear
velocity is shown in figure 2.2
Band broadening effects, particularly in liquid chromatography, are not
limited to the column, and may occur throughout the rest of the system.
Although the injection and detection system contribute, the major factor
causing extra-column band spreading is the connecting tubing, with the
broadening effects, av, related not only to the length of the tubing, but
also to the radius [139,1403:
avs a l r A
The resolution is a measure of the degree of separation of successive
salute bands, and is given by the ratio of the separation of the peak maxima to the broadness of the peaks, or the mean of the peak widths:
R t;= : = 2 (treB" treA > / (tw A + tw 1 3 >
Since resolution is also dependent on the relative retentions of each
compound, and therefore the relative partition coefficients, It, a
separation or selectivity factor, a, can be defined, where
a = Kb /Ka
- 56 -
The Effect of Variation in the Mobile Phase Velocity
on the Column Efficiency
Plate Height <H>
Figure 2,2
- 57 -
However, as the partition coefficient for a given solute in a particular
stationary phase is generally unknown, the capacity factor, k', which may
be determined from the chromatogram, is more often used, thus
a = k * 7 W
The capacity factor is the ratio of the number of moles of solute in the
stationary phase to that in the mobile phase, and for any component,
k ' = < t R - t o ) / t o
The efficiency of the column also has an effect on the resolution of the
separation, since less band spreading results in a smaller peak overlap,
This relationship is summarised in the Purnell equation:
Ss = [1**74] [<a-l>/oc3 DteV dtk*')]
This clearly shows that the resolution can be improved either by altering
the thermodynamics, which will in turn affect the selectivity and capacity factors, or by changing the column conditions to give a higher
efficiency, The resolution can also be expressed as a function of the
effective plate number, since
I* ft = [kVCl+kOFN
- 58 -
Thus the Purnell equation becomes:
R s = CNb * / 4 3 [ < o £ - l ) / a l
Other factors which affect the resolution of a separation include the
temperature, since all the parameters which control the efficiency of the
column are temperature dependent, and also the capacity of the stationary
phase in relation to the amount of sample introduced. This will have an
indirect effect on both efficiency and resolution, since exceeding the capacity of the phase by sample overloading will result in poor peak
shape and a possible modification of the retention times. The sample
capacity is dependent an factors such as the surface area and available
volume of the stationary phase as well as its chemical nature.
Although the theoretical considerations such as efficiency and resolution
are common to all types of chromatography, each different technique
requires different considerations. For example, in liquid chromatography
there are a number of mechanisms by which a separation may be effected
whereas this is not the case for gas chromatography. However, the latter
often requires same form of derivatisatian prior to the analysis in order
to increase the volatility of the sample, and there are a number of
different reactions which may be used to achieve this,
- 59 -
2,3 Chromatographic Techniques
A liquid chromatographic separation may be classified into several
categories depending on the mechanism involved: for partition
chromatography the stationary phase is a liquid, adsorption
chromatography occurs with a solid stationary phase, for ion exchange chromatography the stationary phase carries a net charge and the
separation involves the exchange of one ionic species for another and in
exclusion chromatography the separation is achieved according to the different molecular sizes of the analytes. Of these different types,
partition is the most common, and may be carried out in one of two
modes; either reverse phase or normal phase depending on the polarities
of the stationary and mobile phases. Reverse phase chromatography
utilises a non polar stationary phase in conjunction with a polar eluent,
whereas for normal phase separations, a polar stationary phase is used
with a non polar eluent.
A typical high performance liquid chromatography system is shown in
figure 2.3, The injection system ma.y either take the form of a manual
loop injector or an autosampler, In each case a fixed volume loop is
filled with the required injection volume of sample and, upon injection,
the eluent flow is redirected through the loop and the sample carried
onto the column. The type of detection used depends on the nature of the
analyte and the concentration range of interest, Those most commonly <■
used are ultraviolet, fluorescent and electrochemical detectors, although
others include mass spectrometric, refractive index, electron capture and
infra red detectors.
- 60 -
Basic Components of a Typical HPLC System
Figure 2.3
- 61 -
There are significantly less modes of gas chromatography than liquid
chromatography, and the separation is achieved either through gas liquid
partition or gas solid adsorption, although use of the latter is less
common. In addition to packed column gas chromatography, where the
liquid phase is coated onto a solid support and packed into glass or
stainless steel tubular columns, gas liquid chromatography may also be
performed on capillary columns, where the inner surface of a long length
of capillary tubing is coated with the liquid stationary phase, thereby
increasing the efficiency.
The two main requirements for gas chromatography are that the stationary
phase must be stable at elevated temperatures and that the samples are
gaseous at the working temperature of the column. If the column
temperature is too high the stationary phase will begin to bleed, or be
stripped off the support resulting in the loss of performance of the
column, Hence there are maximum operating temperatures for each column,
and if the sample is not sufficiently volatile at temperatures below this,
then derivatisation prior to analysis will be required,
There are a number of different reactions which may be used to increase
the volatility of the sample, the three most commonly used being
silylation, involving the replacement of an acidic hydrogen with an
alkylsilyl group, acylation, which is used to derivatise alcohols, phenols
and amines using, for example, trifluoroacetic anhydride, and alkylation,
in which an alkyl group is added to an active functional group.
- 62 -
As in liquid chromatography, there are a number of detectors which may
be used in gas chromatography. The two types most commonly used are
flame ionization detectors and electron capture detectors, although
various other detection systems are also used and include katherametric,
conductivity, flame photometric and nitrogen phosphorus detectors in
addition to mass spectrometry, and again, the choice of which to use
depends on the sample under analysis and the concentration range.
It can therefore be seen that, owing to the large number of variable
parameters, chromatographic methods have a very wide range of
applications, and it is due to such versatility that the technique of
chromatography lends itself so readily to the analysis of optical isomers.
2.4 Separation of Enantiomers
As has previously been mentioned, the chromatographic analysis of
enantiomeric compounds may be performed by one of two approaches;
indirectly, via diastereomeric derivatisation prior to the analysis, or
directly, using a chiral chromatographic system.
The idea of separating enantiomers by the formation of diastereomeric
derivatives is not new, and was used before the development of
chromatography for resolution techniques such as crystallisation. Since
diastereomers are physically and chemically different, they are easily separable on achiral columns. However, in contrast to this, problems may
arise in the derivatisation stage. The enantiomeric purity of the reagent
- 63 -
is critical to the quantitative formation of the derivatives and, in
addition to this, if partial raceraisation or kinetic fractionation occurs during the reaction, the product diastereomers will not necessarily be in
the same ratio as the original enantiomers. Despite this, however, there
have been a large number of successful applications of this approach to
the separation of isomers, using both high performance liquid
chromatography [141-1561 and gas liquid chromatography [157-1621, and
also less commonly, thin layer chromatography [163,1641.
More recently, the use of chiral chromatographic systems for enantioselective analysis has became more prominent, generally involving
the use of chiral stationary phases. The separation is then achieved by
the formation of transient diastereomeric complexes between the
enantiomers and a chiral selector which is bound to the silica support.
Due to the different spatial arrangements of the composite atoms, the two
diastereomeric complexes will have slightly different internal energies
and stabilities, which will cause one enantiomer to be eluted in advance
of the other, and hence the degree of separation is largely dependent on
the magnitude of the energy difference between the two complexes. A
similar effect may be achieved by adding the chiral selector to the
mobile phase. Transient diastereomeric complexes are farmed between the
optically pure mobile phase additive and the solute enantiomers, which
can then be resolved on an achiral stationary phase.
Although the majority of work using this approach has taken place in the
last twenty years, the first direct chromatographic resolution was
reported in 1938, involving the partial resolution of an enantiomeric
- 6 4 -
chromium complex on an adsorption column of powdered quartz [165]. This
was fallowed by resolutions of racemic (±)-p-phenylene-bis-iminocamphor
[166,167] and Trogers base [168] on an adsorption column containing solid
(t)-lactose. Better separations were observed by Senoh [169] in 1951,
with the use of paper chromatography to analyse amino acids. The paper
was found to be chiral because of both the helicity of the cellulose and
the glucose content of the paper. This work was extended by Dalgleish,
who, by studying the effect of the structural variations on the resolution
and retention of amino acids, was able to postulate a mechanism by which
such stereoselectivity may occur [1701. He stated that three simultaneous
interactions between the chiral selector and the enantiomers are
necessary for chiral recognition, since any two could be equally well
maintained by either enantiomer. Although this was not the first
statement of the three point rule [171], it marked an important
development, and was later to become one of the most crucial factors in
the design of specific chiral stationary phases. The first practical
application of this theory was in the separation of the enantiomers of
dihydroxyphenylalanine, where models were used to show that a stationary
phase based an 1-a.rginine would allow three interactions with the 1-
isomer but only two with the d- isomer [172], This approach to the
design of enantioselective stationary phases has now become universally
accepted, and much work has been done to ascertain the mechanisms and
sites of interactions between the solute enantiomers and the various phases. Notable in this area is the work of Pirkle, who has established
a number of chiral stationary phases, and rationalised the separation
mechanisms for each.
- 65
The increased interest in chiral separations is reflected in the
development of a large array of chiral stationary phases for high
performance liquid chromatography, gas liquid chromatography and also
thin layer chromatography. Since liquid chromatographic methods are more commonly used for routine analysis, this has led to a wider range of
commercially available high performance liquid chromatography columns
than either gas liquid chromatography columns or thin layer
chromatography plates, although many separations have been achieved on
specifically designed and synthesised phases by all three methods,
2.5 Enantioselective Stationary Phases for HPLC
C o m r o e n x a U -
The firstAchiral stationary phase for high performance liquid
chromatography was introduced by Pirkle in 1981 [173], although
separations using enantiomeric phases had been achieved prior to this,
lore recently, a wide range of different chiral stationary phases have been developed, thus reversing the original problem of adapting the
analyte to suit the phase, to one of selecting the best phase for a
particular analysis. A selection of commercially available chiral
stationary phases for high performance liquid chromatography is
summarised in table 2.1 and the scope of such phases is the subject of a
number of reviews £174-178], In spite of the increasing popularity of
the commercially available phases, a considerable number of separations
have been achieved on novel synthesised phases, designed to suit a specific analyte or range of analytes.
- 66 -
Commercially Available Liquid Chromatographic Chiral Stationary Phases
PHASE TYPE
Chirace1 OA Chirace1 OB Chiracel OC Chirace1 OD Chirace1 OE Chirace1 OK Chiralpak OT Chiralpak OP Chiralpak WH Chiralpak WM Chiralpak ¥E Crownpak CR Enant i opac Resolvosil Jfucleosil Conbriotac LC-<R)"Urea Phenylglycine Leucine lapht hy1alani ne
Cellulose triacetate Cellulose tribenzoate Cellulose tris(phenylcarbamate)Cellulose tris(3,5-dimethylphenylcarbamate)Cellulose tribenzyl etherCellulose tricinnamatePalytriphenylmethylmethacrylatePoly <2-pyridyldiphenylraethylmethacrylate)Proline copper complexAmino acid copper complex
Crown ether oc-Acid glycoprotein Bovine serum albumin
Cellulose triacetate
<3,5-Dinitrobensoyl) phenylglycine <3,5-Dinitrobenzoyl) leucine
Table 2 . 1
- 67 -
SUPPLIER
Daicel/Baker
LKBHichroraHichromEDTSupelcoRegis/Baker/lHichrom
Despite of the wide range of phases available, it is still necessary, in a
number of cases, to derivatise the sample prior to the analysis. Such
derivatisation is generally performed in order to reduce the polarity of
the solute molecules, particularly for strong acids and bases, although it
may also be used in order to introduce specific functional groups to facilitate the required number of interactions, Distinction should be
made, however, between chiral derivatisation, leading to the formation of
diastereomers, and achiral derivatisation, which serves only to alter the
retention characteristics of a solute an a chiral stationary phase. As
has previously been mentioned, the former approach may give rise to a
number of problems when used for quantitative analysis, whereas the
latter, since it is performed with achiral reagents, does not suffer from
the same disadvantages.
With such a wide range of phases available, either commercially or by
synthesis, some form of classification was required in order to
rationalise the selection of a column for a particular analysis. In
response to this, a system was devised by Wainer based on the mechanisms
involved in the chiral resolution. Initially the system centred on four
classes C1793, but this was later expanded to five [1803, However, in the
latter scheme, the division between two of the categories, namely types 2
and 3, do not appear to be well defined, and therefore, for the purposes
of this discussion, the original classifications have been adapted.
- 68 -
The stereoselectivity of phases of this type is dependent on
specific polar interactions between the chiral selector and the salute
enantiomers, A minimum of three interactions are required, and these may
be hydrogen bonding, re-re interactions, steric effects and dipole dipole
interactions [181,1821
The most common commercially available columns of this type are the
Pirkle phases, which can be subdivided into two types; re-electron
acceptors such as the dinitrobenzoyl derivatives of phenylglycine and
leucine (see figure 2.4), and re-electron donors such as naphthylalanine.
These phases can be used for a wide range of compounds, but the analytes
often have to be derivatised prior to the analysis, both to provide
suitable interaction sites and to reduce the polarity. In order to
achieve good separations on a re-electron accepting phase the salute must contain a re-electron donating group, and conversely, a re-electron
accepting group is necessary for analytes separated on a re-electron
donating phase. Such reciprocity has been used, particularly by Pirkle, to develop a number of additional phases [183-1901.
The re-electron accepting phases are available in several forms, depending
on whether the banding of the phase to the silica support is ionic or
covalent, and each has markedly different properties. It is generally
difficult to assess which of the four phases will give the best
separation, and various studies have been made to compare the covalent
and ionic versions of the same phase [1911 and the phenylglycine and
leucine phases [192-1961. The Pirkle phases, particularly
2.5,1 Type 1 Phases
- 69 -
Examples of a Type 1 HPLC Phase
3,5-Dinitrobensoyl Phenylglycine
3,5-Dinitrobenzoyl Leucine
Figure 2.4
- 7 0 -
(3,5~dinitrobenzayl)-phenylglycine, have a wide range of applications
[197-1993 including various chiral pharmaceuticals [200-2063 and
enantiomeric polycyclic aromatic hydrocarbons and their metabolites [207-
2113,
Other commercially available chiral stationary phases of this type
include the Sumipax range, which, although used for several applications
[212-2153, is overshadowed by the success of the Pirkle phases. This is
also true for the LC-<5>-Urea phase marketed by Supelco.
Due in part to the ease of design of these phases, a large number of
separations are carried out on novel, or specifically synthesised phases
of this type, such as those based on hydantoin and arylalkylamlnes [2163,
which were developed to be re-electron donors by the reciprocity
principle. In addition to this, a number of other chiral selectors have
been used as chiral stationary phases [217-2263, including those based on
amide derivatives [227-2333 and urea derivatives [234-2363. A phase has
also been recently developed incorporating both a re-acidic <3,5-
dinitrobensoyl) phenylglycine group and a re-basic 6,7-dimethylnaphthyl
group C2373 which may prove to be mare widely applicable than either of
the two individual phases.
These types of phases have also been successfully used in conjunction
with supercritical fluid chromatography [238-2423, whereupon the low
viscosities of the supercritical fluids together with the correspondingly
higher diffusion coefficients allow the technique to be run under mare
optimal conditions than HPLC, thus improving the resolution and reducing
- 71 -
the analysis time. Other attempts to improve the separations include
using the chiral phase in series with a racemic or achiral column [2433,
and performing chiral derivatisations prior to the analysis [244,2451,
since diastereomers are more readily resolvable on a chiral phase than
enantiomers.
2.5.2 Type 2 Phases
The mechanism proposed for chiral stationary phases of this type
involves the entry of enantiomers into chiral cavities within the structure of the phase. Differences in the fit of the two enantiomers in
the cavities together with the possibility of hydrogen bonding and steric
interactions affect the stability of the inclusion complexes formed, and
result in the stereoselective retention of the enantiomers.
Phases of this type can be subdivided into three main categories;
cellulose or cellulose derivatives, cyclodextrins and synthetic polymers,
and although each shares a common separation mechanism, the retention
characteristics and range of applications of each is different.
The earliest phase of this type was cellulose, which has been used for a
number of resolutions, particularly of polar compounds such as amino
acids [246-2483. However, it was soon discovered that the derivatives have a higher resolving power, and are therefore more generally used.
The first derivative to be used was triacetylcellulose, a phase which in
spite of having low separation factors, is applicable to many racemates
[249-2523. This was then followed by the introduction of a wide range of
derivatives including cellulose tribenzoate [253-2573,
- 72 -
Cellulose Derivative HPLC Phases
cellulose tris(phenylcarbamate) C258-2603, cellulose tris(3,5-
dimet hylphenylcarbamate) [261,2621 and a number of other similar phases
[2631. The structures of the cellulose derivatives are shown in figure
2.5. Separations on these phases may be effected with either polar or
non polar eluents, although the samples may sometimes need to be
derivatised to avoid strong acids or bases. The phases may also be
prone to irreversible adsorbtion, and so biological samples should not be
injected directly onto the column.
Cyclodextrins are cyclic oligoglucose molecules which have structures
resembling open ended cones (see figure 2.6). The interior of the toroid
is relatively nonpolar E2643, while both the larger and smaller ends are
rimmed with secondary and primary hydroxyl groups respectively.
Stereoselectivity is achieved through the formation of inclusion
complexes, whereby the lipophilic part of the solute molecule fits into
the cavity and the other substituents about the chiral centre undergo
asymmetric interactions such as hydrogen bonding and steric interactions
with the polar rim. There are a number of factors affecting the binding
strength of the inclusion complexes [2651 but in general, the enantiomeric
solute will form two complexes of different stabilities, and hence one
isomer will be preferentially retained. There are three types of
cyclodextrin used for chromatography, depending on the number of glucose
units present, and hence the size of the cavity can be chosen to suit
the solute requirements. The most popular is JJ-cyclodextrin, consisting
of seven glucose units and tliis has been used for the resolution of a
number of pharmaceuticals [266-2703 and other compounds C271-2753
including metallocene enantiomers [2763. The other two cyclodextrins are
- 7 4 -
Inclusion Complex Formation with a Cycladextrin HPLC Phase
Figure 2,6
or-cycladextrin, which, comprising of six glucose units, is the smallest
and is mainly used for the separation of amino acid enantiomers
[277,2783, and V-cyclodextrin, which, with eight glucose units, is only
suitable for very large molecules. Cycladextrin phases can be used with
mobile phases of any polarity, they are most commonly used in the
reverse phase mode, and in fact, the formation of inclusion complexes is
only possible in aqueous solutions. The retention is controlled by
organic modifiers such as methanol and acetonitrile, the pH and ionic
strength of buffers and temperature, Although inclusion complexing is
the primary mechanism, interactions with the silica support, or the
connecting spacer can also affect the resolution, particularly with less
- 75 -
polar eluents, In addition to enantiomeric separations, many geometrical
isomers are easily resolvable on cyclodextrin phases [279,280]
The most common synthetic polymer phases are based on polymethacrylates,
Such phases are novel in that their chirality arises purely from the
helicity of the polymer structure. Two phases of this type, (+)-poly
(triphenylmethylmethacrylate) and <+)-poly (2-pyridyldiphenylmethyl
methacrylate) are commercially available, and show good selectivity for
many samples [281-2853, particularly those with a rigid nonplanar structure, although again, strong acids and bases should be avoided.
More recently, another phase, based on a crown ether chiral selector has
become available, which falls into this category. This phase was first
used by Cram and co workers in 1975 [2863, but did not find widespread application at that time. The phase is best suited to the resolution of
amino acids [2873 and other compounds with a primary amino group close
to the asymmetric centre. These phases are used in conjunction with an
aqueous acidic eluent, and the retention is largely dependent on the
hydraphobicity of the analyte, hydrophilic solutes being less strongly
retained than hydrophobic ones.
2.5.3 Type 3 Phases
Separations of this type are based on the principle of ligand
exchange chromatography and depend on the formation of diastereomeric
ternary complexes involving a transition metal, a single enantiomer of a
chiral molecule and the racemic analyte. Such phases were introduced by
Davankov in the early 1970's [2883, who used a stationary phase of 1-
- 76 -
praline immobilised onto chlaramethylpalystyrene loaded with either
capper, sine, nickel or cadmium ions to separate various amino acid
enantiomers, and later transferred to silica based columns by Giibits
[2893.
Commercially available columns of this type include Chiralpak WH and
Chiralpak WE (see figure 2.7).
The mast commonly used transition metal for ligand exchange is capper,
and the selector ligands are generally amino acids. The analytes are
limited to those which have polar functional groups with the correct
spacing which can simultaneously act as ligands to form coordination
complexes with the transition metal. Steric repulsion and additional
interactions such as hydrogen bonding within the complex cause the
diastereomers to differ in stability, thus effecting the separation, The
retention and resolution of the analyte may be regulated by changes in
the pH and the amount of organic modifier in the eluent, and increasing
the temperature also improves the resolution.
Ligand exchange chromatography is generally only suited to the resolution
of amino acids [290-2983, although separations of aminoalcohols and 2-
hydroxy acid enantiomers have also been achieved [299,3003. There are
several disadvantages to this technique, not least of which is the complexity of the mobile phase, which must contain small amounts of
copper salts to prevent a nett loss of the ion from the stationary phase.
In addition to this, chiral selectors only give good resolutions for a
- 77 ~
Ligand Exchange HPLC Phases
Chiralpak WE
N.C'
. c
0'\
0\ / 'Cu
Figure 2.7
- 78 -
limited number of compounds, and closely related enantiomers may prove
difficult to separate,
2.5,4 Type 4 Phases
The resolution mechanism on protein type phases is not fully
understood, but is based on the stereoselective binding of enantiomeric
compounds to plasma proteins, and is influenced by hydrophobic
interactions, steric effects and interactions of the polar groups.
The first use of a protein based column for chromatographic separations
was by Stewart and Doherty [3011, who used bovine serum albumin bound to
agarose for the separation of tryptophan enantiomers. This was further
developed by Allenmark, who succeeded in immobilising the protein on a silica support, thus improving the column stability [302], Similarly,
Hermansson developed a phase based on a i-acid glycoprotein bound to a
silica support [3033
Both phases are commercially available and are particularly suitable for
the analysis of enantiomeric pharmaceuticals, most of which require no
prior derivatisation, In general, albumin is the principle plasma binding
protein for weakly acidic drugs, and is used for a number of resolutions
[304-3063 particularly amino acids and their derivatives [307-3093.
Conversely, cc-i-acid glycoprotein is the major binding protein for basic
drugs, and is therefore widely applicable to the separation of a number
of enantiomeric pharmaceuticals [310-3153 including amino alcohols [316-
3183. A comparison of the enantioselectivity of both phases has been
made, together with several other similar phases E3193
- 79 -
Protein phases are compatible with aqueous mobile phases, but the
separations are sensitive to changes in chromatographic conditions such
as the pH and ionic strength of aqueous buffers, the concentration of
organic modifier in the eluent and the temperature. Although the
selectivity of such phases is excellent, the efficiencies are often poor, and, as the loading of the protein an the silica support is also lav/, the sample capacity is limited and the columns are easily overloaded.
2.6 Chiral Mobile Phases for HPLC
As an alternative to using chiral stationary phases, there exists, in
liquid chromatography, the possibility of including the chiral selector in
the mobile phase, thus allowing enantiomeric separations to be performed
on achiral phases. Such mobile phase additives have the advantage that
the chiral selector can be chosen from a wide range of suitable reagents,
and easily changed if necessary to improve the resolution, a flexibility
which is not readily achieved using a chiral stationary phase. In
addition to this, a reversal of the elution order of the enantiomers can generally be obtained by the use of the optical antipode of the chosen
selector.
The separation mechanisms involved in this approach are not so well
defined as for resolutions on chiral stationary phases, but are thought
to depend on the relative affinities of the chiral selector for the
stationary phase and the solute enantiomers, In extreme cases, the
selector may either be strongly adsorbed on the silica support, forming
in effect a chiral stationary phase, or involved in the formation of
- 80 -
diastereomeric complexes with the solute in the mobile phase, whereby the
separation takes place as a normal liquid chromatographic separation of
diastereomers. However, in practice, most separations fall between these
two extremes, and a combination of these mechanisms occurs, giving a
range of mixed retention modes.
The disadvantages of this technique are mainly centred on the detection
system, since the choice of mobile phase additive may limit the range of
detection methods which can be used, or vice versa, In some cases this
may be overcome by the use of post column derivatisation to introduce for
example, a fluorescent moiety into the salute complex. In addition to
this, since the enantiomers are eluted from the column as diastereomeric
complexes which, due to their different physical properties, may give
different detector responses, individual calibration curves are required
for each enantiomer. Following an from this, if the separations are
performed on a preparative scale, problems may arise in isolating the
optically pure solute enantiomers from the chiral selector.
One of the most commonly used methods for resolutions using chiral
mobile phase additives is by ligand exchange chromatography. This was
first used by LePage E3201 and Gil-Av E3213 to overcome the problems of
producing ligand exchange chiral stationary phases experienced at that
time, and was later the subject of a more detailed study by Davankov
£3221. The formation of ternary transition metal complexes is similar to
those achieved on a ligand exchange chiral stationary phase, with both
the chiral selector ligand and the transition metal dissolved in the
eluent, Such additives are generally used to resolve amino acids [323-
- 81 -
3333, although various amines have also 3:>een resolved in this way [334-
3363.
Chiral ion pair reagents can also be used as mobile phase additives to
resolve oppositely charged racemates by normal phase chromatography.
This technique was first used by Pettersson and Schill for the resolution
of amino alcohols using (-O-TO-camphorsulphonic acid as the chiral
counter ion [3373, and although further work centred on the resolutions of
amino alcohols [336,3393 and other compounds [340,3413 with 10-
camphorsulphonic acid, other ion pair reagents were soon investigated,
and quinnine and quinidine in particular were found to be suitable for
resolving acids E342-3443. Improved results have also been achieved
using N-benzoylcarbonyl-glycy1-proline (ZGP) C3453, Although the use of
chiral counter ions has proved to be successful in a number of cases,
such resolutions are not always easy to achieve, and are influenced by a
number of factors. Of crucial importance is the water content of the
mobile phase, with the recommended proportion being 80-90 ppm [3463.
Concentrations above this impair the resolutions, but small amounts of
water are necessary to deactivate the silica surface, which would otherwise adsorb the polar components too strongly. The surface
properties of the polar silica are also important and it has been found
that the hydrophilic properties of dial phases are best suited to
resolutions of this type [3463. In addition to this, the capacity ratios
of the enantiomers decrea.se with increasing counter ion concentrations,
due to competition between the solute complexes and the counter ions for
adsorption sites on the phase, and increasing the concentration of polar
components in the eluent causes a decrease in the retention, and hence
- 82 -
the resolution, of the enantiomers. It is interesting to note, however,
that the optical purity of the ion pair reagent affects only the
separation factor of the enantiomeric complexes, and therefore
quantitative analysis can still be achieved in situations were the chiral
counter ion is not completely pure [3473.
Enantiomeric resolutions can also be performed by the addition of
inclusion complexing agents to the mobile phase, and in fact, one of the
first resolutions involving the use of a chiral mobile phase additive was achieved using an eluent containing a chiral crown ether to separate
racemic amino esters C3483. Recently, however, the majority of
resolutions of this type use cyclodextrin as the inclusion complexing
reagent with reverse phase chromatography conditions. These inclusion
complexes are similar to those formed using a cyclodextrin stationary
phase, but the retention mechanism is more complicated. In some cases,
resolution is achieved by differences in the stability of the two
complexes, Implying that the cyclodextrin is adsorbed onto the the
achiral phase, while in others, the resolution is achieved by differences
in the interactions between the achiral phase and the part of the solute
enantiomer protruding from the cyclodextrin cavity [3493. As with the
cyclodextrin stationary phases, ^-cyclodextrin is the most commonly used additive [350-3523, although several studies have compared the
stereoselectivities of a~f j3~ and "tf-cycladextrins far the resolution of
chiral barbiturates and norgestrel enantiomers [353,3543
Other mobile phase additive include those developed to undergo
enantioselective hydrogen bond interactions with the chiral solute, Such
- 83 -
selectors, particularly N-acetyl-L-valine-tert butylamide, may be used for
normal phase separations [355-357], and are generally thought to be
strongly adsorbed onto the silica surface. Resolutions of racemic drugs
[358] and other compounds [3591 have also been achieved on achiral
stationary phases using buffered eluents containing a t-acid glycoprotein.
2.7 Chiral GLC
The first successful gas chromatographic resolution with an optically
active stationary phase was performed by Gil-Av in 1966 [3603, using glass capillary columns coated with N-trifluoroacetyl-L-isoleucine lauryl
ester to separate racemic N-trifluoroacetyl-amina acid esters. Further
work was done to establish a theoretical base for the separations [361-
3643, and also to determine the structural features responsible for the
stereoselective effects [365,3663, This knowledge, coupled with the need
to increase resolution, efficiency and thermal stability led to the
development of a number of new phases which were originally classified
into three types [367,3683.
The dipeptide phases, introduced by Gil-Av and Feibush, are used mainly
for highly volatile amino acid derivatives [369-3733. and derived from
these are the diamide phases which contain two thermally stable amide
functions around one asymmetric centre, thus giving greater
stereospeclficity [374-3833, The third type of phase, the N,N-carbonyl-
bis-(amino acid ester) phases, were originally developed to increase the
probability of a three point interaction around the asymmetric centre of
the phase and thereby improving the separations, and are sometimes
incorrectly called ureide phases [384-3893. The general structures of
- 84 -
each type of phase are shown in figure 2-8. More recently, a fourth type
of enantiomeric stationary phase has been introduced which involves the
use of chiral metal coordination compounds for the separation of
enantiomers by stereoselective complexation [390,3913. Hence this
technique is particularly suited to the resolution of those compounds
which do not possess the appropriate functional groups capable of
hydrogen bonding as required by the other phase types [392-4003.
Although there are a large number of possible phases for enantlospecific
gas chromatographic analysis, the number of commercially available chiral
stationary phases is limited, and mostly fall into the diamide category. The two most common optically active stationary phases are Chirasil-Val,
based on L-valine-tert-butylamide, and XE-60-S-valine-S-phenylethylamide,
both available from Chrompaek. Similarities exist between the structures
of the two phases, as shown in figure 2.9, although XE-60-S-valine-S-
phenylethylamide has two chiral centres compared to the single
asymmetric centre in Chirasil-Val, and the separation mechanisms for both
are based on hydrogen bonding of the solute enantiomers about the chiral
centre of the phase, Although Chirasil-Val has a slightly higher maximum
operating temperature than XE-60-S-valine-S-phenylethylamide, each phase
has an extensive range of applications. Both have been used to separate
amino acids [401-4073, carboxylic acids [408-4103 and alcohols and dials [411-4153, Chirasil-Val has also been used to separate a range of other
compounds [416-4223 such as tocainide [423-4253 and mephenytoin C4263,
while XE-60-S-valine-S-phenylethylaraide has been used for various
resolutions [427-4283 including aminoalcohols [429-4323, chiral ketones
[433,4343 and panthenol [4353, A comparison between the two phases is
Examples of Chiral GLC Phases
Dipeptide Phase
0
F , C C - N '
H
X
Ri
QX
. N
H
H
C
■cj
r2
Diamide Phase
R i
0
0X- N t
H
C '
O
r 2
'NHRq
3H
NtN-Carbonyl-bis-(Amino Acid Ester) Phase
0XH
O R '
■N-
A
0 XoIIc N i
IH
Figure 2.8
Commercially Available GLC Phases
XE-- 6 0-S- Val ine-S~ Pheny lethy lam ide
H
HoO C
0
Figure 2.9
- 87
given with respect to the resolution of the enantiomers of putrescine and
cadaverine analogues as their N-perfluoroacyl derivatives [4363
Two other chiral gas chromatographic stationary phases are Supelco SP300,
a phase based on n-lauryl-L-valine-tert-butylamide available from Supelco
Inc. as bath a liquid phase and a column packing, and RSL-007 which is
based on L-valine-tert-butylamide and available from Alltech Associates
Inc.. Although neither phase has found common use, RSL-007 has been used
for the resolution of fungicide enantiomers in a comparison with
Chirasil-Val [4-373 and the interactions of SP300 have been studied by
Feibush [4383 prior to its use for enantiomeric separations [439,4403.
Recently, a new range of stationary phases have become available,
involving chiral selectors based on cyclodextrin derivatives. The
separation mechanism of these phases is still the subject of debate as to
whether inclusion complexes are formed in a similar fashion to those
achieved in liquid chromatography [4413 or whether the separations are
effected by dipole dipole interactions C4423. Although it was previously
thought that an aqueous medium was required for inclusion complex
formation, the polar solvent of the liquid phase may also provide
sufficient energy differences to facilitate the formation of the
complexes, and the mechanism may be determined by the structure of the
solute. Despite the low operating temperatures of these phases, they have
been successfully used far a number of applications C443-4473, and are
likely to become more popular.
It is also interesting to note that, in a similar fashion to liquid
chromatography, an attempt has been made to include the chiral selector in with the gaseous mobile phase C4481.
2,8 Chiral TLC
Mention must also be made of the technique of thin layer chromatography
since, although the popularity of this approach is limited, almost
certainly due to the difficulties in both quantitation and automation,
there are a number of advantages, One such point is that thin layer
chromatography can be used as a. rapid and simple means of checking the
enantiomeric purity of a compound, but more importantly, thin layer chromatography may be used to provide a relatively inexpensive
preliminary assessment of a separation for high performance liquid
chromatography on a similar phase.
The only commercially available thin layer chiral stationary phase,
Chiralplate, is based on the principle of ligand exchange chromatography,
with the chiral selector, (2S,4R,2,RS)-4~hydroxy-l-(2-hydroxydodecyl>
proline, coated onto a silica support and impregnated with copper ions.
The plates have been used extensively for the separation of amino acids
[449-4511, and resolutions have also been achieved for penicillamine
[452,453], 3,4-dihydroxyphenylalanine C4543 and chiral dipeptides [4553.
Such applications are the subject of a recent review by Gunther [4563,
Other chiral stationary phases for thin layer chromatography include a
|3-cyclodextrin phase, used for the resolution of a number of optical,
- 89 -
geometrical and structural isomers £4571, and a Pirkle type
3,5-dinitrobenzoyl phenylglycine phase [458], the enantioselectivity of
which is tested using 2,2,2-trifluaro-l- (9-anthryl)ethanol.
Despite the emphasis on the use of chiral stationary phases, there is no
reason to assume that enantioselective thin layer chromatography could
not also be performed using chiral mobile phase additives in a similar
fashion to high performance liquid chromatography,
Thus, there is an extensive range of techniques by which the
chromatographic analysis of enantiomers may, in theory, be achieved.
However, stereoselective separations, in practice, are not generally easily
accomplished, and are often time consuming and expensive. The
sensitivity of the resolution to minor fluctuations in chromatographic
conditions and the complexity of any necessary pre-column derivatisation,
whether chiral or not, makes the application of such techniques to the
routine analysis of large numbers of samples more difficult still.
2.9 Chiral Detection
However, a more recent development has led to the possibility of
performing quantitative analysis of enantiomers without chromatographic
separation, This approach is based on the use of an ultra-violet
detector in series with a micropolarimeter. While the ultra-violet, or
other achiral detector, measures the total concentration of analyte, the
polarimeter measures the sum of the laevorotatory and dextrorotatory
isomers. By a comparison of the two, the concentrations of each
- 90 -
enantiomer can be established. The sample preparation and analysis can
be carried out as if for an achiral determination, thus eliminating many
of the aforementioned problems. Since the ChiraMonitor micropolarimeter
has only recently become commercially available, there are few reported applications as yet [459-4633, although this approach is likely to become
more popular in time, with the only limiting factor being the sensitivity
of the detector.
2.10 Model Compounds
However the primary aim of this work was to develop methods for the
enantioselective analysis of a number of cardiovascular drugs such as
verapamil, propranolol and other beta adrenoceptor blocking agents, Such
drugs are used in the treatment of angina, hypertension and arrhythmias,
and are generally marketed and administered as racemic mixtures. The
structures of the compounds chosen for this study are shown in figures
2.10 and 2,1 1 .
All beta blockers have at least one asymmetric centre in their molecular
structures and evidence exists to show that their activity is highly
stereoselective. In the case of propranolol, it has been found that the
(S)-(-)-Q) isomer is largely responsible for the receptor antagonist
properties [213 although in other aspects, such as their local anaesthetic
activity, both isomers have a similar potency which is generally greater
than that of the racemic compound. In addition to this, the <E) form is
preferentially metabolised in man leading to 30-60% higher plasma levels
of the mare active (S) form after oral
- 91 -
Structures of the Model Compounds
AtenololOH
0~CH,—CH-CHr N~CH(CH3'2
C K-C O N K
PractololOH ^
O-Ch^-CH-CHf-N-
ONH-COCH,
Oxprenolol?H •?
0-C H r CH-CH2-N-CH(CH3)20-CHj-CH-CH,
AlprenololOH H
O -C H f-C H -C H fN
'CHfCH^CHj
Propranolol
Figure 2.10
- 92 -
CH(CH3)2
~CH(CH3 )2
Structures of the Model Compounds
MetoprololOH H
O -C H r CH-CHj-fvl-CH (CB, \V
o
rCH2-C H 2-0 -C H 3
PronethalolOH HCH -C H 2- N - CHtCHj^
O I O '
Chlorpheniramine
3 '2
Clomiphene
VerapamilCH3 CH(CH3)2
•C H r C H r Is l-C H r CH,- CHr C-CN
O-CH 3
0-C H ,
0 ~CHi0-C hi
Figure 2.11
- 93 -
administration [464,4653. Similar results have also been found for
pronethalol, another adrenoceptive J3-receptor antagonist, where the (->
isomer has been shown to be 40 times as active as the (+) isomer [4663.
Verapamil is a calcium channel antagonist which is again administered as
a racemic mixture, in which the (1) isomer is 3-10 times more active than
the (d) form [243. It has also been found that stereoselective first pass
metabolism preferentially reduces the plasma levels of the more active
(1) verapamil following oral administration, thus increasing the
bioavailability of (d) verapamil [303.
Verapamil enantiomers, together with those of pronethalol and metoprolol,
have been successfully resolved by HPLC on a cyclodextrin column without
prior derivatisation [2703, but better separations have been achieved for
these and other J3 blockers using an al-acid glycoprotein phase [3173
although in some cases this requires the formation of oxasalidone
derivatives [4673. Propranolol has been resolved on a cellulose tris(3,5-
diraethyIpheny1-carbamate) phase
without derivatisation E4683 and on a <3,5-dinitobensoyl)-phenylglycine
column as an amide or an oxasolidone derivative [469,4703, The
oxazolidone have also been resolved by capillary GLC using an XE-60-L-
valine- (E)-oc-phenylethylamide phase [4313.
Other enantioselective separations have been achieved through the use of
both mobile phase additives [3393 and chiral derivatising reagents, with
the derivatives resolved by either HPLC [471-4733 or TLC [4743.
- 94 -
In addition to the aforeiaentioed cardiovascular drugs, it was occasionally
found necessary to use other model compounds to assess the column
performance or when repeating other published work. In particular,
enantiomeric resolution of the antihistaminic drug, chlorpheniramine was
attempted together with the separation of the geometric isomers of clomiphene, an anti-oestrogen drug.
CHAPTER 3MATERIALS AID METHODS
3.1 Reagents
3.1.1 Model CompoundsRacemic atenolol, alprenolol, oxprenolol, practolol and propranolol
were obtained, mainly as salts, from ICI Pharmaceutical Division, Alderly Edge. The (+) and <-) isomers of propranolol (Inderal) and pronethalol (Aldelin) were obtained from the same source, Racemic metoprolol tartrate was obtained from Geigy Pharmaceuticals, Macclesfield, Cheshire, and racemic verapamil hydrochloride was a gift from Dr. G. Mould, St. Lukes Hospital, Guildford,
Chlorpheniramine maleate was received as a racemic mixture from Dr. Stewart, University of Georgia, Athens, Clomiphene was supplied as a mixture of the cis and trans isomers from Sigma Chemical Company Ltd, Poole, Dorset.
3.1.2 Derivatising and Ion Pairing ReagentsAcetic anhydride was obtained from BDH. Ltd., Poole, Dorset, as was
trifluoraacetic anhydride. Heptafluorobutyric anhydride was supplied by Sigma. Both 1-naphthoyl chloride and 2-naphthoyl chloride were obtained from Aldrich Chemical Company Ltd., Gillingham, Dorset. Pyridine was used in several derivatisation procedures and was supplied by BDH Ltd.
The ion pair reagent, (+)-10-camphorsulphonic acid, was obtained from Sigma.
- 96 -
3.1.3 General ReagentsOf the compounds used to test column performance, phenol, o-cresol,
1-naphthol and ra-nitroaniline were obtained from Chemistry Stores, University of Surrey, m-dinitrobenzene was obtained from BDH Ltd., and 1,1,1-trifluoro-l- (9-anthryl)ethanol was supplied by Aldrich.
Sodium dihydrogen orthophosphate, disodiura hydrogen orthophosphate and orthophosphoric acid were obtained from BDH Ltd. as was triethylamine, while glacial acetic acid was supplied by lay and Baker, Manchester. Hydrochloric acid was obtained from FSA, Loughborough and sodium hydroxide was purchased from Sigma.
Standard solutions of buffers at pH 4 and 7 were used to calibrate the pH meter and were supplied by BDH Ltd,
3.2 Solvents
3,2,1 Mobile Phase SolventsHPLC grade methanol and acetonitrile were obtained from FSA,
HiPerSolv grade dichloromethane, propan-l-ol, propan-2-ol, butan-l-ol and hexane were from BDH Ltd. Ethanol and pentan-l-ol were also from BDH Ltd. and of AnalaR grade. Water was generally glass distilled using a Jencons 4 Plus auto still (Jencons Ltd,, Leighton Buzzard, Beds,), although in some cases purer water was obtained by reverse osmosis (Elga, High Wycombe, Bucks.)
All HPLC mobile phases were thoroughly degassed prior to use, This was achieved by ultrasonication of the eluent in a sonic bath (Mettler Electronics, California) for approximately 10 minutes.
- 97 -
3.2.2 General SolventsHeptane and chloroform HiPerSolv and ether and cyclohexane AnalaR
were obtained from BDH Ltd. Laboratory grade ethyl acetate and butanone were obtained from the same source as was pentan-2-ol and n-butyl acetate. HPLC grade acetone was supplied by FSA and toluene was received from May and Baker. The hydrocarbons C©, Ci o, Cis, C14. and Ci s were all purchased from Sigma.
3.3 Equipment
3,3,1 General Laboratory EquipmentAccurate weighing of stardards were carried out on a Sartorius
2024IP five place balance (Sartorius Ltd,, Belmont Surrey), while general weighings were made on an Ohaus Brainweigh B1500D top pan balance (Ghaus Europe Ltd., Cambridge), Quantitative liquid transfers were achieved using Gilson Pipetteman P5000, P1000, P200 and P20 pipettes (Gilson Medical Electronics, Yilliers-le-Bel, France)
Mixing of samples was normally carried out using a whirlimixer (FSA), while shaking for solvent extraction was either done on a home made horizontal shaker or a multitube vortexer (Scientific Manufacturing Industries, California). Centrifugation was generally performed at 2000rpm at subambient temperatures using a Mistral 6L centrifuge (MSE(Fisons), Crawley, Sussex). A heating block (Grants Instruments Ltd,, Cambridge) was used to achieve the elevated temperatures required for some derivatisation procedures.
Evaporation of organic extraction solvents was carried out using a home made solvent evaporator where the glass test tubes are individually supported on a thermostatically controlled heating block and the contents subjected to a steady steam of dry oxygen free nitrogen.
Where buffers were used, the pH values were measured and adjusted using an Orion glass electrode (Orion Research Inc., Boston) connected to a PTI-15 digital pH meter (EDT Instruments Ltd., Dover, Kent). While measuring pH
- 98 -
values, a magnetic stirrer (Voss Instruments Ltd., Maldan, Essex) was used to gently agitate the solution.
Ultraviolet scans of the model compounds were carried out using a Perkin Elmer Lambda 5 scanning spectrophotometer (Perkin Elmer Ltd., Beaconsfield, Bucks.). Fluorescent scans were similarly achieved using a Perkin Elmer LS-5 scanning fluorimeter.
Disposable round bottomed borosilicate glass test tubes and screw capped tubes were supplied by Corning, lew York, together with teflon lined caps. Flat bottomed 5ml glass tubes were obtained from LIP (Shipley, Yorkshire)
3.3.2 Chromatography EquipmentThe initial work was carried out using Beckman 110 pumps (Beckman
RIIC Ltd., High Wycombe, Bucks.), although the latter part of the study involved the use of a Severn Analytical SA6410B dual piston pump (Severn Analytical, Shefford, Beds,).
Sample injection was achieved by the use of a Rheodyne 7120 sample injection valve (Rheodyne Inc., Berkeley, California) together with a Hamilton HPLC syringe (Hamilton, Bonaduz, Switzerland). Typical injection loop volumes were 20/jtl, 50/j.I and lOQpl.
Ultraviolet detection was carried out using either a Pye Unicam LC-UV (Pye Unicam Ltd,, Cambridge) an LDC Spectromonitor III (Laboratory Data Control, Stone, Staffs) or a Cecil CE212 detector (Cecil Instruments, Cambridge), all of which were variable wavelength detectors, Fluorescent detection was achieved using a Schoefel FS970 fluorimeter (Kratos Ltd., Manchester)
The detector signal was generally recorded using a CR650 or CR652 chart recorder <JJ Lloyd Instruments Ltd., Southampton), although in some cases a Spectra Physics SP4270 computing integrator was used (Spectra Physics, San Jose, California). In addition, an Axxiom Chromatography Data System (Quadrant Scientific Ltd,, Gloucester) was also used for a short time.
- 99 -
GLC was carried out using either a Perkin Elmer Sigma 3B or a Perkin Elmer 8320 gas chromatograph. In both cases sample injection was achieved using a 1/jlI type B syringe (Scientific Glass Engineering, Milton Keynes) and flame ionization detection was used. All gases were supplied by BOC, London, with the exception of air which was generated by a Jun Air compressor.
3.4 Stationary Phases
A variety of different stationary phases were used for this study, including a 25cm LiChrosorb DIOL column, obtained from HPLC Technology, Macclesfield, Cheshire and Spherisorb ODS-1 columns, supplied by Anaehem, Luton, Beds,
Of the chiral HPLC columns, a 25cm j3-cyclodextrin phase, Cyclobond i, was supplied by Technicol Ltd., Stockport, Cheshire, while the Pirkle type 3,5- dinitrobenzoyl phenylglycine phases were obtained in both the ionic and covalently bound forms from Hichrom, Reading, Berks, In addition, one 25cm Chirasil 1 ionic 3,5-dinitrobenzoyl phenylglycine cartridge was donated together with the cartridge holder and fittings, from Alltech Associates, Carnforth, Lancs. All HPLC columns were approximately 4.6mm internal diameter.
Two GLC capillary columns were used in this study; a 50m BP1 bonded phase column with an internal diameter of 0.33mm supplied by Scientific Glass Engineering, and a Chirasil-L-Val 25m column supplied by Chrompack,Hiddelburg, Netherlands. The chiral capillary column had a film thickness of 0,13pm and an internal diameter of 0,22mm.
- 100 -
3.5 Test Conditions
Each column, both GLC and HPLC, was tested upon arrival under the manufacturers stated conditions to verify the claimed efficiency. A set of standard conditions were then established to enable the column performance to be monitored throughout its use. Due to the wide variety of columns used, no single set of conditions could be established to suit all phases and so individual test conditions were found for each phase,
In each case the efficiency is calculated for the last eluted peak. Althaug'h no absolute values for the minimum efficiency of each column were set, the performance was frequently monitored for significant drops in efficiency, whereupon the column was replaced.
LiChrosorb DIOLTest Mixture: m-dinitrobenzene and toluene in heptane Eluent: 1% pentan-2-ol in hexane @2.0 ml/rainDetection: UV 254nm
Spherisorb S5 ODS 1Test Mixture: acetone, phenol, o-cresol and 1-naphthol in eluent Eluent: 60% methanol in water @ 1.0 ml/minDetection: UV 254nm
Cyclobond 1Test Mixture: m-nitraaniline in eluentEluent: 50% methanol in water @1.0 ml/minDetection: UV 254nm
3,5-Dinitrobenzoyl Phenylglycine (ionic and covalent)Test Mixture: 1,1,1-trifluoro-1-(9-anthryl) ethanol in eluent Eluent: 10% propan-2-ol in hexane @0,6 ml/minDetector: UV 254nm
/
- 101 -
Chirasi1-L-ValTest Mixture: derivatised amino acids as supplied by ChrompackOven Temp. : 140"C isothermalCarrier: He, 10 psiDetection: FID
3.6 Derivatisation Procedures
3.6.1 Formation of Acyl Derivatives for GLCSeveral different derivatisation reactions were carried out in an
attempt to increase the volatility of the sample. Heptafluorobutyryl, trifluoroacetyl and acetyl derivatives were each formed by using the appropriate acid anhydride derivatising reagent within a common reaction procedure, as outlined below:
A known amount of the sample was dissolved in 100-2Q0jul of a solvent such as n-hexane or dichloromethane. A similar volume of the derivatising reagent was added and the mixture heated to between 50-100 °C for 30 minutes, The excess reagents were then evaporated under nitrogen, and the residue dissolved in a suitable solvent.
On several occasions pyridine was added to the reaction mixture together with the derivatising reagent in order to act as a catalyst for the reaction, and in some cases the reaction had to be stopped by the addition of a volume of water following the heating.
3.6.2 Formation of laphthamide Derivativeslaphthamide derivatives of the beta blockers were formed in order
to reduce the polarity of the sample molecules and also to provide additional sites for possible interactions with the 3,5-dinitrobenzoyl phenylglycine HPLC stationary phase. Although an acid anhydride would normally have been the first choice of derivatising reagent, since any excess anhydride is relatively easy to remove, in this case, due to the availability of reagents, 1-naphthoyl
- 102 -
chloride was used to derivatise the samples according to the procedure described below:
A fixed volume of a 2M sodium hydroxide solution was added to 2G0pl of an aqueous or methanolic solution of the sample, and a suitable volume of the derivatising reagent was added. The reaction mixture was vortexed for 2 minutes then the derivatives were extracted by vortex mixing for a further 2 minutes with a appropriate solvent such as chloroform. The samples were centrifuged for 5 minutes at 2000rpm and a known volume of the organic extract was transferred to a fresh tube and taken to dryness under nitrogen. The residue was then dissolved in a suitable solvent.
In several cases, pyridine was added to the reaction mixture in an attempt to increase the rate and yield of the reaction, since it acts as a scavnger for the excess hydrochloric acid produced by the reaction. Additionally, a methanolic solution of 2-naphthoyl chloride was occasionally substituted for 1-naphthoyl chloride as the derivatising reagent.
- 103 -
\
CHAPTER 4
RESULTS AID DISCUSSIGI
- 104 -
4.1 Achiral Method Development and Validation for Verapamil
4.1.1 Introduction
Literature surveys have shown that, while there are numerous methods
available for the determination of ? . . . drugs, the published
methodology for verapamil is limited, with most methods describing
procedures involving a back extraction into sulphuric acid. Attempts
were therefore made to establish a simple HPLC method for the analysis
of verapamil in human plasma, involving the minimum amount of sample
preparation work,
Verapamil, 5- <<3,4-dimethoxyphenethyl)methylamino)-2- (3,4-
dimethoxypheny 1 )-2-isapropylvaleronitrile, is a drug which inhibits the
slow inward transmembrane calcium currents and has anti-anginal, anti-
arrhythmic and anti-hypertensive effects. The important metabolic
reactions are N-dealkylation, giving mainly nor verapamil, and 0-
demethylation, Depending on the dose, the mean plasma concentrations
range between 30 ng/ml and 500 ng/ml.
4.1.2 Chromatography
The initial column selection was based not only on a study of the
molecular structure, but also on economic and safety considerations.
Columns used far routine assays should, wherever passible, be both robust
and compatable with the organic solvents which present the minimum
hazard to the health and safety of the analyst, To this end, a reversed
phase actadecyl silica column was chosen, and thus a polar mobile phase
was required.
- 105 -
Since verapamil is a basic drug, an aqueous buffer, modified with an
organic solvent, was selected as the eluent. In this way the retention of
the solute could be controlled by variation in either the pH of the buffer
or the eluent composition, A system was therefore set up consisting of a
12.5cm Spherisorb S50DS1 column and an aqueous phosphate buffer-organic
modifier mobile phase at a flow rate of 1.0 ml/rain.
A UV detection system was used to monitor the solute elution for the
initial development work, and the UV absorption scan of verapamil is
given in figure 4.1.1. This shows three maxima, at 204, 228 and 277 nra
respectively, with that at 228nm chosen for detection since, although it
is not the most intense, it avoids the difficulties caused by impurities
in solvents at low wavelengths, Standard solutions of verapamil in
methanol with concentrations between 20-200 pg/ml were used for the
retention studies, of which 20 pi was injected with a Rheodyne sample
injection valve.
Retention studies were carried out to determine the optimum
chromatographic conditions and the initial investigations involved
varying the nature and concentrations of the organic modifier in a 0,11,
pH 3 phosphate buffer mobile phase, Methanol and acetonitrile
- 106 -
The UV Absorption Scan of Verapamil in Methanol
Figure 4.1.1
- 107 -
were chosen for this, and their percentage composition in the eluent
varied between 40 and 80. For each eluent the salute retention was
measured and the capacity factor (as defined in Chap 2) was calculated,
producing the results shown in figure 4.1,2.
Further studies were performed to determine the effect of the pH on the
retention, using a 70% acetonitrile in buffer eluent. Attempts were
initially made using a 0.1M phosphate buffer, but at these concentrations
it was found that the- addition of acetonitrile caused a precipitation of the buffer at pH values above 5. Consequently the concentration of the
buffer was reduced to 0.0111 to enable the effect to be examined over the
wider pH range of the column, that is, in the case of ODS columns,
between 3 and 7.5, The results are shown in figure 4.1,3 and highlight an
additional way of controlling the solute retention, namely by varying the
ionic strength of the buffer. Hence this trend was also investigated,
keeping the composition constant at 70% acetonitrile and the pH constant
at 3. The results are shown in figure 4.1.4.
Although the retention time of the solute can be selected by the choice
of chromatographic conditions, there are several important factors which
must be considered. For any routine assay, while it is desirable to keep
the retention times as short as passible, the solute must not only be
well resolved from the solvent peak but must also allow for the elution
of the polar interferences in plasma extracts, which normally follow
immediately after the solvent peak. Hence, in general, the aim is to find
a system which will give a capacity factor of between 3 and 5. Goad
peak shape is dependant upon maintaining the maximum difference between
The Variation of the Retention of Verapamil with Eluent Composition
Capacity Ratio (k’)
% Organic Modifier
Figure 4.1.2 Column: 12.5cm Spherisorb ODS1 Eluent: Organic modifier in 0.1 M
phosphate buffer, pH 3.0 Flow Rate: 1.0 ml/min Injection Volume: 20 ul Detector: UV 228nm
Capacity Ratio (k’)
The Variation of the Retention of Verapamil withthe pH of the Buffer
pH of Buffer
Figure 4.1.3 Column: 12.5cm Spherisorb ODS1 Eiuent: 70% acetonitrile in
phosphate buffer,Flow Rate: 1.0 ml/min Injection Volume: 20 ul Detector: UV 228nm
- no -
Capacity Ratio (k1)
The Variation of the Retention of Verapamil withthe Ionic Strength of Buffer
Buffer Concentration (M)
Figure 4.1.4 Column: 12.5cm Spherisorb ODS1 Eluent: 70% acetonitrile in
phosphate buffer, pH 3.0 Flow Rate: 1.0 ml/min Injection Volume: 20 ul Detector: UV 228nm
- i n -
the nature of the stationary phase and that of the mobile phase, and thus
it was desirable to keep the percentage of water in the mobile phase as
high as possible. It can be seen from the results in figure 4,1.2 that,
for a given capacity factor value, the percentage of methanol required is
higher than that of acetonitrile and hence the latter was considered
preferable. From this an eluent of 70% acetonitrile in buffer was chosen.
As can be seen from figure 4.1.3, increasing the pH of the buffer causes
an increase in the retention of the solute, This is the expected trend,
since verapamil is a basic drug and is therefore ionisable. In its
unionised form, verapamil is fairly non polar, and thus has a high
affinity for the non polar C-i© stationary phase leading to long
retentions. However, at a low pH, the molecule exists in its ionised
form, which, due to its increased polarity, is less corapatable in the non
polar environment of the stationary phase and more soluble in the polar
eluent, resulting in shorter retention times,
A similar trend is also true of the ionic strength of the buffer where
increasing the concentration decreases the retention of the solute. Since
the extent of ionization of the solute depends only on the pH of the
eluent and the pKa value of verapamil, the retention should be the same with each strength of buffer investigated. However, as the buffer is
adjusted to the set pH and not the eluent, the pH of the weaker buffer
may be more affected by the addition of acetonitrile than the stronger
buffer. Hence the differences in retention may be caused more by a
variation in pH than ionic strength, although other explanations based on
the nature of the buffer are also possible,
In summary, it was established that, in order to attain a suitable
retention time an a 12.5cm Spherisorb S50DS1 column, an eluent of 70%
acetonitrile in aqueous phosphate buffer was selected. The ionic strength of the buffer was set at 0.1M and the pH at 3.0. At a flow rate of 1.0
ml/min, these conditions resulted in a capacity factor of 3.0 and a
retention time of about 4 minutes.
4.1.3 Extraction Procedure
Having determined the chromatographic conditions, the next stage of
the method development work was to find a suitable proceedure to extract
verapamil from an aqueous biological matrix. Investigations were
therefore carried out to find a liquid liquid extraction method which
would give good recoveries,
The initial considerations involved the choice of extraction solvent and
so experiments were carried out to compare the recoveries of a range of
solvents using an aqueous verapamil solution as a model for plasma, A
set volume of each solvent was used to extract a 1 ml aliquot of an
alkaline verapamil solution <150pg/ml> by vortex mixing for 30s. A
standard volume of each solvent was then taken to dryness under nitrogen
and redissolved in 500 pi acetonitrile. Recoveries were assessed by the
comparison of chromatographic peak heights with a recovery standard
using the previously described system. The results are shown in table4.1.1.
- 113 -
A Comparison of the Recovery of Verapamil with Different
Extraction Solvents
SOLVEIT PEAK HEIGHT <mm) % RECOVERY
Stock Solution 129 -Butanone 47 36. 4Heptane 135 104.4Ether 122 94,4Cyclohexane 101 78. 1Diehloromethane 136 105.2Ethyl Acetate 67 51.8Trichloromethane 104 80.5Hexane 135 104.4
Table 4.1,1 Column: 12.5cm Spherisorb ODS1Eluent: 70% Acetonitrile in 0.IM Phosphate
Buffer pH 3,0 Flow Rate: 1.0 ml/min Injection Volume: 20pl Detector: UV 228nm
The Effect of pH on the Recovery of Verapamil with Ether
pH OF PLASM PEAK HEIGHT (ram) % RECOVERY
: Solution 237 _
3 192 81. 14 216 91.25 221 93.36 240 101.47 220 92.98 218 92, 19 224 94,610 218 92. 111 231 97.612(NaOH) 220 92,9
Table 4.1.2 Column: 12.5cm Spherisorb 0DS1Eluent: 70% Acetonitrile in 0.11 Phosphate
Buffer pH 3.0 Flow Rate: 1.0 ml/min Injection Volume: 20p,l Detector: UV 228nm
Although recovery is the most important factor when selecting a solvent,
a number of other physical properties must also be taken into account. Ideally, the solvent chosen should be less dense than water, and hence
plasma, and be fairly volatile, to facilitate easy drying down for sample
concentration, It is also desirable to choose the most non polar solvent passible, since this will extract the minimum amount of the plasma interferences, which tend to be polar. From this, ether was chosen as
the extracting solvent,
As with the retention studies, the pH of the plasma may have an effect on
the recovery of verapamil into the organic solvent. At low pH values,
verapamil exists in its ionised form which, being more polar, will be
more soluble in the aqueous plasma phase than the organic phase, and thus
will not be extracted. Hence it may be important to maintain the pH
above a certain value in order to achieve good recoveries. This effect
was investigated by taking 1 ml aliquots of spiked plasma and ajusting
the pH of each tube with buffers to give a pH range between 3 and 12.
The verapamil was then extracted with ether by vortex mixing for 30s,
centrifuging for 5 mins, transferring the supernatant to fresh tube and
evaporating the solvent under nitrogen. The residues were reconstituted
in 500 pi of eluent and the recoveries for each pH value calculated on
the basis of peak height comparison by reference to a recovery standard.
The results, shown in table 4.1.2, indicate that the pH of the plasma does
not affect the recovery of verapamil to any great extent, although it was
noticed that the extracts were cleaner at pH values above 9, possibly
implying that at least some of the impurities in the plasma were acidic
in nature. All further extractions were therefore performed with the
addition of a sodium hydroxide solution to the plasma prior to the
extraction.
Having developed the basis of the above method the next stage was to
produce some standard curves covering the range of expected plasma
levels, in this case between 10 and 100 ng/ml. At these levels it was
thought that the UV detector used so far, would not be sufficiently
sensitive, and so was exchanged for a fluorimeter, The excitation
wavelengths for the fluorescence of a compound are the same as the UV
absorption wavelengths, and because fluorimetry is a more selective
detection technique, it was now possible, in this case, to use the more
intense, lower maxima of the three shown in figure 4,1,1. Hence an
excitation wavelength of 203nm was used in conjunction with a 32Gnm
emmission cut off filter,
Pooled plasma was spiked with a methanolic verapamil solution to give a
range of plasma standard of 0-100 ng/ml. Each standard was made
alkaline by the addition of 1 ml of 10% sodium hydroxide solution, and
the verapamil extracted with with 10 ml of ether by vortex mixing for 30
s and centrifuging for 5 minutes at 1000 rpm. The ether layer was
decanted off and dried down under nitrogen and the residue redissolved
in 1 ml of eluent. The standards were injected on to the previously
described chromatographic system with an injection volume of 100 pi, and
a standard curve was constructed from the resultant peak heights (see
figure 4.1.5).
- 117 -
A Standard Curve for Verapamil After anEther Extraction
Peak Height (mm)
Verapamil Concentration (ng/ml)
Figure 4.1.5 Column: 12.5 cm Spherisorb ODS1 Eluent: 70% acetonitrile in
phosphate buffer, pH 3.0 Flow Rate: 1.0 ml/min Injection Volume: 20 ul Detector: Fluorimetric, ex 203 nm
Since the standard curve is not linear, and the blank plasma standard
also gave a peak at the verapamil retention, the extraction praceedure is
obviously not entirely satisfactory. An unextracted standard curve, run
over a similar range to that used for the extraction gave a more linear
response indicating that the problem lies more with the extraction than
with the chromatography, A volume of ether similar to that used for the
extraction was evaporated under nitrogen and the residue redissolved and
injected onto the chromatography system. The interfering peak was
present in this sample and so is likely to originate from the ether.
It was therefore necessary to reconsider other extraction solvents, and
the possibility of back extraction into 0.1 1 hydrochloric acid was also
explored in an attempt to eliminate the interferences. In addition to
ether, heptane and 1,5% pentanol in hexane were also investigated, and in
each case, back extraction into acid significantly reduced the recovery to
unacceptable levels, A sample of each extraction solvent was dried down
under nitrogen and tested for interferences, and this again confirmed
that the ether used contained a coeluting impurity, as did the hexane.
Although it may have been possible to purify the ether by the process of
redistillation, heptane contained no such interfering impurities and in
addition gave reasonable recoveries, and was therefore used far all
further extractions.
Hence a second standard curve was generated using heptane as the
extraction solvent following the previously described procedure, the
results of which are shown in figure 4.1.6. This gave a linear response
(r=0.9991) with detection limits of around 10 ng/ml, although this was
- 119 -
largely due to the unusually high background noise levels of the detector
at this time. Such noise levels have not been evident in previous assays
and may be due to the deterioration of the deuterium lamp, and therefore
lower detection limits may be expected in future runs.
In conclusion, a summary of the method of extraction of verapamil from plasma is as follows:
1 ml aliquots of plasma were made alkaline with 0.1M sodium hydroxide,10 ml of heptane was added to extract the verapamil, and the tubes
vortexed for 30s and centrifuged for 5 minutes at 1000 rpm, 9 ml of
each heptane layer was taken to dryness in clean tubes, then redissolved
in a suitable volume of eluent,
4.1.4 Validation
The validation of this assay centred mainly an finding an
appropriate internal standard and assessing the day to day precision of the method.
There are several factors which are important in selecting a suitable
internal standard. Ideally the compound chosen for the internal standard
should be fairly similar in structure to the sample compound, and elute
close to the sample peak under the chosen chromatographic conditions. It
is also important to ensure that, not only is the internal standard
extracted under the predetermined procedure, but that the recoveries of
both the internal standard and the sample are similar,
- 120 -
A Standard Curve for Verapamil After aHeptane Extraction
Peak Height (mm)
Verapamil Concentration (ng/ml)
Figure 4.1.6 Column: 12.5 cm Spherisorb ODS1 Eluent: 70% acetonitrile in
phosphate buffer, pH 3.0 Flow Rate: 1.0 ml/min Injection Volume: 20 ul Detector: Fluorimetric, ex 203 nm
- 121 -
Several compounds were investigated for their suitability as internal
standards and it was decided to use propranolol, which elutes just before
verapamil on the chromatographic system described above. Propranolol
also has the advantage that it is rarely administered in conjunction with
verapamil, and so is unlikely to be present in patient plasma samples.
Experiments were carried out to ascertain the retention of propranolol in
relation to verapamil, and to determine the comparative detector response. Since the two peaks of interest elute fairly close together, the mobile phase was adjusted to 35% 0.11 phosphate buffer, pH 3,0 : 65% acetonitrile
to give a better separation. Under these conditions the retention times
of verapamil and propranolol were found to be 414 and 3 mins respectively at a flow rate of 1.0 ml/min. The optimum concentraion for the internal
standard should result in a peak height of approximately half that of the
top standard, and in this case was found to be 10 ng/ml.
The percentage recovery of both verapamil and propranolol was assessed
by extracting a number of samples containing the drug and the internal
standard and comparing the peak heights of the resulting chromatograms
with those obtained from similar recovery standards. Taking into account
the quantifiable lasses in the extraction proceedure, both verapamil and
propranolol gave recoveries of about 75% at 100 ng/ml.
The next stage was to perform a six day four level validation, On each
of six different days, a standard curve was constructed by extracting 1ml
aliquots of plasma, spiked to give a range of verapamil concentrations
(100 - 5ng/ml), and a constant propranolol concentration (lOng/ml). The
standard curve was then used to determine the verapamil concentration of
four blind spike samples.
A comparison of the results obtained at each spike level on the six days
was made in order to assess the day to day precision of the method, and
the results are shown in table 4,1,3:
As can be seen, the coefficients of variation range between 4.6% at the
60 ng/ml level and 8,6% at the 8 ng/ml level with the correlation
coefficients of each standard curve being greater than 0.99. Although
these results are acceptable in themselves, it should also be noted that
the plasma aliquots for bath the blind spikes and the standards were
spiked on a day to day basis, and therefore the results also reflect the
precision and accuracy of the spiking procedure. These values may be
improved by preparing batches of spiked plasma at each concentrations
prior to the commencement of the validation study, thus producing the
required number of identical standards and blind spikes,
4.1.5 Conclusion
In conclusion, a summary of the method of analysis of verapamil in
plasma is as follows:
1 ml of plasma was dispensed into a clean screw capped tube and 100 pi
of a 100 ng/ml propranolol solution was added. The plasma was made alkaline by the addition of 0.11 sodium hydroxide. 10 mis of heptane was
added to extract the verapamil, and the mixture vortexed for 30s and
centrifuged for 5 minutes at 1000 rpm. 9 ml of the heptane layer was
transferred to a clean tube and taken to dryness in clean tubes under
- 123 -
Validation Results fo r Verapamil
STANDARD CURVE SPIICE LEVEL <ng/ml)
DAY K SLOPE 60 35 15 8
1 0.9976 0. 0217 61. 1 32.7 '17. 1 10,22 0,9974 0. 0196 58.5 30.7 16.8 9.43 0,9962 0, 0229 61.6 36.8 16, 0 8.44 0.9943 0, 0205 64.6 37. 1 15.9 8.75 0.9974 0.0193 57.8 35.8 14.5 -6 0.9967 0.0209 57.4 33.3 15. 5 9, 3
IEAI 60, 4 34.5 15.9 9,2S, D. 2.79 2.63 0.94 0.78C, V. 4.6% 7.6% 5.9% 8.6%
Table 4.1.1 Column: 12.5cm Spherisorb QDSiEluent: 65% Acetonitrile in 0. IM Phosphate
Buffer pH 3.0 Flow Rate: 1.0 ral/min Injection Volume: 20pl Detector: Fluorimetric, X«>s=203nm
oxygen free nitrogen. The residue was then redissolved in a suitable
volume of eluent.
The chromatographic system consisted of a 12.5 cm Spherisorb 50DS column
with an eluent of 65% acetonitrile 35% O.lKt phosphate buffer adjusted to
pH 3.0, at a flow rate of 1.0 ml/min. A fluorescence detector was used,
set to an excitation wavelength of 203 nm and a 320 nm cut off filter.
The resulting chromatograms were recorded using a chart recorder.
Although enantiomeric resolution was neither achieved or expected with
this system, it was hoped to be able to transfer the method to a reverse
phase chiral column, such as cyclodextrin, to achieve separations at a
later stage,
A r\oo-^vs
- 125 -
4.2 Enantiomeric Resolution by Ion P a ir Chromatography
4.2.1 Introduction
The use of chiral additives in the mobile phase provides, in theory,
the easiest and cheapest method of achieving enantiomeric resolutions by
HPLC, by removing the need for costly and sometimes delicate chiral
stationary phases. Of the various types of additives available for such
use, chiral ion pair reagents are particularly suited to ionic or
ionisable compounds, and in particular 10~campharsulphonic acid has been
used by Pettersson and Schill for the separation of aminoalcohol
enantiomers E337L
Attempts were therefore made to evaluate this approach with respect to
the resolution of alprenolol and other beta blacking drugs, Initial efforts were centreed on repeating the work of Pettersson and Schill and
involved the use of a normal phase dial column in conjunction with an
eluent of 0.5% pentanol in dichloromethane containing 5.4ml (+>—10—
camphorsulphanic acid. However, as this proved to be unsuccessful, with
the solute being retained on the column, more detailed retention studies
were undertaken,
4.2.2 Retention Studies
The chromatographic system consisted of a 25 cm LlChrosarb DIOL column
in conjunction with a dichloromethane eluent containing small
concentrations of an alcoholic polar modifier. A variable wavelength UV
detection system was used to monitor the solute elution, set to 271 nm,
corresponding to the absorption maximum of alprenolol as shown in figure
4.2.1. A 250 pg/ml alprenolol solution was used for this work, of which 20 pi was injected using a Rheodyne sample injection valve.
The initial investigation involved varying the nature and concentration
of the alcoholic polar modifier and measuring the retention or capacity
factor and the efficiency with each different mobile phase, The results,
as shown in table 4.2.1, follow the expected trend of increasing capacity
factors with decreasing polarity of the eluent, since alprenolol is a
basic drug and is therefore more readily partitioned into a more polar
mobile phase. It can also be seen that the efficiencies improve as the
eluent becomes progressively more non polar, and this is consistent with
the increase in retention times.
Since the alprenolol peaks were becoming broader, due to the increased retention times with less polar eluents, the effect of adding small
quantities of glacial acetic acid to the mobile phase was studied. An
eluent of 1% propanol in dichloromethane was used and the acetic acid
content was varied between 0 and 0.3%. The results, as shown in table
4.2.2, indicate that the addition of acetic acid causes a decrease in
retention accompanied by an increase in the efficiency.
The acetic acid lowers the pH of the eluent which forces the alprenolol
to exist in a charged form which should be more strongly retained on the
polar stationary phase. However the increased polarity of the acidic
UV Absorption Scan of Alprenolol in Methanol
Figure 4.2.1
The Effect of Variation in the Polar Modifier on the Retention
of Alprenolol on a lorm al Phase Column
Polar Modifier k* I
5% Methanol 0
3.3% Methanol 0. 33 554
2.5% Methanol 0. 83 342
2% Ethanol 1.8 554
2% Propanol 2. 0 798
Table 4.2.1 Column: 25cm LiChrosorb DIOL
Eluent: Diehloromethane: polar modifier
Flow Rate: 3.0 ml/min
Detection: UV 271 nm
Injection Volume: 20 pi
- 129 -
% Acetic Acid k‘ N
The Effect of the Addition of G lad a l Acetic Acid on the
Retention of Alprenolol on a Normal Phase Column
0 6.6 320
0.2 2.6 554
0,3 2.2 630
Table 4.2.2 Column: 25cm LiChrosorb DIOL
Eluent: 1% Propanol in dichloromethane
Flow Rate: 3,0 ml/min
Detection: UV 271 nm
Injection Volume: 20 pi
The Effect of Variation in the Polar Modifier on the Retention of
Alprenolol with Eluents Containing Glacial Acetic Acid
Polar Modifier k1 I
1% Propanol 2.2 6301% Butanol 2.2 2960.5% Butanol 4,5 262
1% 2-Pentanol 2 < 2 2530.5% i-Pentanol 2. 4 266
0.5% 2-Pentanol 2.6 278
Table 2.3 Column: 25cm LiChrosorb DIOL
Eluent: Diehloromethane: polar modifier
Flow Rate: 3,0 ml/min
Detection: UV 271 nm
Injection Volume: 20 pi
mobile phase more than compensates for this, and hence the capacity
factors are reduced.
As this effect widens the range of alcoholic polar modifiers which can be
studied, capacity factors and efficiencies were again measured for a
range of mobile phases, each containing 0.3% glacial acetic acid. The
results are given in table 4.2.3
In contrast to the results in table 4.2.1, there seems to be no noticable
trend between the polarity of the modifier and the retention. This could
possibly be due to column deterioration since the experiments were
performed over a relatively long period of time. However, subgroups
within the table, such as the pentanol series, indicate that, as the
polarity decreases, the capacity factors and the efficiencies increase.
Since the efficiencies are low, attempts were made to monitor the effect
of variations in flow rate on both retention and efficiency on a number
of different eluent systems.
The results given in figure 4.2.2 show that while increasing the flow rate
from 1.0 ml/min to 3.0 ml/min increases the capacity factor, the
efficiency is reduced with each of the solvent systems used. This can be explained by the van Deemter equation (Chap. 2), relating flow dependant
band broadening effects to the plate height or efficiency of the
separations. As the flow rate increases the mass transfer
characteristics become more important and hence the efficiency is
The Effect of Variations in Flow Rate on the Capacity Ratio and Efficiency of the Diol Phase
Efficiency (N)
Flow Rate (ml/min)
Capacity Factor (k‘)5
-------A ....._____ ____ ___
Flow Rate (ml/min)1 % Butanol 0,5% Butanol 1 % Pentan-2-ol
- - Q - • A -
0.5% Pentan-2-ol 0.5%Pentan-1-ol
Figure 4.2.2 Column: 25 cm LiChrosorb DIOLEluent: dichloromethane : polar modifier Detection: UV 271 nm Injection Volume: 20 ul
- 133 -
Mt
decreased. It was therefore decided that all further work should be
performed using a flow rate of 1.0 ml/min.
The next stage of the study involves an investigation of the effect of
the addition of the (+)~10~camphorsulphonic acid ion pair reagent as a
chir^al mobile phase additive, From the previous results, a 0.5% butanol
in dichloromethane system containing 0,3% glacial acetic acid was chosen,
and run at a flow rate of 1.0 ml/min. Samples of alprenolol were injected, and the peaks compared to those obtained with a similar eluent
containing 6ml <+)~10~camphorsulphonic acid (see figure 4.2.3). In
addition to this, the experiment was repeated using eluents which did not
contain any acetic acid (figure 4.2.5). The results for this experiment are given in table 4.2.4.
If ion pairing had occurred between the (+)-10-camphorsulphonic acid and
the alprenolol, the ion pair complex would be more lipophilic than the
unpaired alprenolol and would thus be expected to be less retained on
the polar DI0L stationary phase, Since the capacity factors are
significantly reduced by the addition of the (f)-lO-camphorsulphonic acid
ion pair reagent with both eluent systems, ie with and without acetic acid in the mobile phase, this would appear to indicate that an ion pair
had, in fact, been formed between the alprenolol and the campharsulphonlc
acid, However neither eluent system gave any hint of chiral resolution.
- 134 -
The Effect of the Addition of Ion Pair Reagent on the
Retention of Alprenolal
Eluent k' I
0.5% Butanol 3,16 2890.5% Butanol + CSA 2.17 1840.5% Butanol + Acid 2.00 3710.5% Butanol + Acid + CSA 1.20 382
Table 4.2,4 Column: 25cm LiChrosorb DIOL
Eluent: Dichloromethane: polar modifier
Flow Rate: 1.0 ml/min
Detection: UY 271 nm
Injection Volume: 20 pi
The Effect of the <+)-10-Camphorsulphonlc Acid Mobile Phase
Additive on the Resolution of Alprenolol with Acidic EluentsI
Figure 4.2.3 Column: 25cm LiChrosorb DIQL
Eluent A: 0.5% butanol, 0.3% acetic acid in
dichloromethane
B: 0.5% butanol, 0.3% acetic acid in
dichloromethane containing 6mM (+)-10-
camphorsulphonic acid
Flow Rate: 3.0 ml/min
Detection; UV 271 nm Injection Volume; 20 pi
3k- VteSutC- oLa~m52- -Vcd
- 136 -
The Effect of the (-l-)-lO-Camphorsulphonic Acid Mobile Phase
Additive on the Resolution of Alprenolol
Figure 4,2.4 Column: 25cm LiChrosorb DIOL
Eluent A: 0.5% butanol in dichloromethane
B: 0.5% butanol in dichloromethane containing
6mM <+)-10-camphorsulphonic acid
Flow Rate: 3.0 ml/min
Detection: UV 271 nm
Injection Volume: 20 jyil
4.2.3 Discussion
The results for this section of work are, in general, very disappointing.
Although the retention studies followed the trends expected for a normal
phase system, the addition of the camphor sulphonic acid did not result
in any indication of enantioselective resolution.
The rationale behind this approach to chiral separations is that
diastereomeric ion pairs are farmed between the single counter ion
enantiomer and each of the solute enantiomers, and that these ion pairs
have structural differences which allow them to be differentially
distributed between the two phases and are thus resolved. Although the
mechanism of chromatography is not completely clear, it is known that
there must be at least three points of interaction between the solute and
the counter ion to produce ion pairs with sufficient structural
differences to effect such a separation. In this case, the three
interactions are thought to be electastatic interaction between the amino
and acid groups, hydrogen bonding between the hydroxyl group of the
salute and the carbonyl group of the counter ion and steric interactions
between the two ring systems.
As has been previously discussed, there are a number of factors affecting
such resolutions, the most important of which is the water content of the
mobile phase. Low concentrations of water in the eluent adversly
modifies the degree of resolution, possibly due to competition between the
water and the salute for hydrogen bonding sites an the counter ion.
Attempts were therefore made to repeat the final experiment using eluents
which had been dried over a molecular sieve prior to use, but this
resulted in the solute being retained an the column. Work by Petersson
[3463 suggests that a water concentration of around 80 ppm creates an
ideal compromise between the detrimental effects of water on
stereoselectivity and its necessity for the controlled elution of the
solute. However, without more specialised techniques, it was impossible
to monitor the water concentration of eluents in this study to such a
degree, and thus the fine control required for optimum resolutions was
unattainable.
The effect of the acetic acid in the eluent is more complicated.
Essentially, the acetic acid provides a means of controlling the retention
3:>y lowering the pH of the eluent and ionising the solute enantiomers,
thus promoting the ion pair formation. However, in addition to this,
there will also be competition between the acetic acid and the
camphorsulphonic acid for the formation of ion pairs with, the alprenolol
isomers, although in practice, this effect will be relatively small since the camphorsulphonic acid is the stonger of the two.
The resolution of the enantiomers is also dependent on the optical purity
of the camphorsulphonic acid, V7ith ion pair resolution, any optical
impurities in the chiral counter ion decrease the separation between the
diastereomers rather than affecting the quantitive results. The <+)-10-
camphorsulphonic acid used in this study was of unknown age and optical
purity, and this, together with the previous points and the gradual
deterioration of the column efficiency could all help to account for the
lack of positive results.
4.3.1 Introduction
The aim of this section of work was to provide an evaluation of a
GLC approach to enantioselective resolution using a chiral stationary phase. The first commercially available chiral GLC column was a diamide
phase based on valine hert butylamlde, known as Chirasil Val, and since
this was also the most readily available capillary column at the time, it
was chosen for this study.
Although the phase has been widely used to separate enantiomeric amino
acid derivatives and other small molecules, it's application to the
resolution of other compounds is limited. This study therefore centres
on the evaluation of this phase with respect to the enantiomeric
resolution of amino alcohols such as propranolol, metoprolol and alprenolol.
4.3.2 Experimental
The column chosen was a 25m x 0.22mm i.d. fused silica capillary
column coated with Chirasil-L-Val with a film thickness of 0.12 pm and the initial work involved setting and optimising the GC parameters to
give the best column performance. The column efficiency is related to
the linear gas velocity by the van Deemter equation and thus the inlet
pressure must be adjusted to give the optimum linear velocity for the
carrier gas used. The linear velocity can be calculated by relating the
column length to the elution time of an unretained compound such as a
4.3 Enantiomeric Resolutioin Using A Chiral GLC S ta tionary Phase
solvent or methane. For a helium carrier, the ideal velocity is between
15 - 25 cm/s and thus for a 25m column, the elution time should be 2 - 3
mires. Hence the inlet pressure for this system was set at 12 psi, giving
a retention time of 2.3 mins for dichloromethane at a temperature of 140°
C,
A sample of the test mixture, as supplied by the manufacturer, was
injected to verify the performance (see figure 4.3.1). The test sample
was a mixture of four derivatised amino acids; alanine, leucine, aspartic
acid and methionine and the efficiency, calculated from the last eluted
peak, was found to be approximately 79,000 plates. This compared
favorably with the 82,000 plates claimed on the test report.
The column was retested at regular intervals and it was interesting to
note that the retention times of the peak increased considerably after a
short period of time (see figure 4.3.2), This increase however, was
achieved without any noticable reduction in the efficiency.
The next stage of this work was to attempt to separate the enantiomers
of several small molecules such as bromobutane, butan-2-ol and pentan-2-
ol, neither of which require derivatisation to improve their volatility.
Although this was only a limited experiment, the initial results seemed to indicate that some resolution had taken place albeit with low
separation factors (see figure 4,3.3). Such low values would be expected
with the short resolution times, and would be difficult to improve at
normal GLC working temperatures. However, further studies
- 141 -
BGN
The Initial Testing of the Chirasil Val Column
rOt inf- • •
Column: 25ra Chirasil-L-Val
Oven Temp: 140 CC Isothermal
Carrier: Helium 10 psi
Detection: FIDSample: Amino Acid Test Mixture
1. D-Alanine
2. L-Alanine
3. D-Leucine
4. L-Leucine
5. D-Aspartic Acid
6. L-Aspartic Acid
7. D-Methionine8. L-Methionine
(X| LU-J
Figure 4.3,1
- 142 -
Further Testing of the C h irasil Val Column
Column: 25m Chirasil-L-Val
Oven Temp: 140°C Isothermal
Carrier: Helium 10 psi
Detection: FID
Sample: Amino Acid Test Mixture
1. D~Alanine
2. L~Alanine
3. D-Leucine
4. L-Leucine
5. D-Aspartic Acid
6. L-Aspartic Acid
^ 7. D-Methioninein
4rft-i-
12 3 A 5 6
Figure 4.3.2
Resolution of 2-Br'omobutane and 2-Butanol
■nco•N
Column; 25m Chirasil-L-Val
Oven Temp: 140*C Isothermal
Carrier: Helium 12 psir <T» r -
Detection: FID ,-,JSample; 1. 2-Butanol
2. 2-Bromobutane
cc•s>
ootxt 00
Figure 4.3.3
- 144 -
revealed that the two peaks for each injection were not similar in size
as would be expected for a racemic mixture, and hence, in the absence of
enantiomerically pure samples, it was impossible to determine whether
chiral resolution had occurred or whether the peak splitting was caused
by other factors.
In order to analyse larger compounds like the arainoalcohols by GLC, some
form of derivatisation is required to increase the volatility of the
samples. Although the beta blockers may be derivatised in a number of
different ways, acylation may be achieved by a relatively quick and easy
procedure and so was the preferred method for this study.
Initially, heptafluorobutyryl derivatives of propranolol, metoprolol and
alprenolol were formed according to the procedure described in chapter $3
Injections of each sample were made, and the preliminary results showed
two peaks for both propranolol and metoprolol (see figure 4.3.4). In both
cases the peaks were smaller than expected, even taking the 1/60 split
ratio into account, but this may have been caused by sample losses in the
derivatisation procedure, No peaks, other than the solvent peak, were
observed for the alprenolol injection, leading to the assumption that the
derivative had either not been formed or was retained on the column.
The experiment was then repeated with fresh heptafluorobytyryl
derivatives and similar results achieved. However, similar injections,
made after the derivatives had been stared at 4*C
- 145 -
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- 146 -
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- 147 -
Figu
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4.3.
4b
overnight, showed no peaks at all. This appeared to indicate that the derivatives were not stable
Since it has been suggested that the concentration of derivatising
reagent could have a large effect on the stability of the derivatives
[4243, experiments were performed to vary the amount of
heptafluorobutyric anhydride used in the derivatisation of both
propranolol and metoprolol in order to assess the storage stability.
However, on injection none of the derivatised samples were eluted from
the column within 1 hour.
After several unsuccessful attempts to repeat the initial experiments,
further efforts were centred on finding a more volatile derivative, To
this end trifluoroacetyl derivatives were investigated, as were acetyl
derivatives. However, in neither case were any of the samples eluted
form the column at any temperature.
One possible explanation for this is that the derivatives were not formed
under the reaction conditions used. This was investigated by replacing
the chiral column with a non polar BP1 phase. Propranolol, metoprolol,
alprenolol, atenolol, practolol and oxprenolol were all derivatised with
trifluoroacetic anhydride by an identical procedure to that used
previously, and injected onto the BP1 column, which was run with a
temperatur'e program of 110 °C for 2 mins then 5Vmin to 220 °C and held
for 15 mins. With the exception of practolol, peaks were observed for
all the compounds, although calculations showed that in each case the
elution temperature was in excess of 200°C.
- 148 -
Having verified the formation of the derivative, a comparison between the
two phases was made. For each column, a range of hydrocarbons was
injected, together with a selection of other compounds, under standard
conditions of a 130 *C isothermal oven temperature with a helium carrier
at 10 psi. The capacity factors for each phase, as shown in table 4.3.1,
indicate that the Chirasil Val phase is significantly more polar than the
BP1 phase.
A Comparison of Capacity Ratios for Different Samples
Between the Chirasil Val Phase and the BP1 Phase
Compound Chirasil Val BP1
C© 0. 11 1. 02
Cl O 0. 17 3.23
Cl a 0.27 9.75
Butanone 0. 17 0.23
Butanol 0. 16 0.36
Pyridene 0.20 0.70
Benzene 0, 19 0.72
Diehloromethane 0. 18 0.71
Table 4,3.1 Oven Temp: 130eC
Helium Pressure: 10 psi
Detection: FID
In theory, resolutions of the beta blockers should be passible with
the Chirasil Val column since there are sufficient interactions between
the phase and the derivative samples, The mast probable interactions
include hydrogen bonding between the tert butylamide carbonyl of the
phase and the hydroxyl group of the salute, and also between the amide
carbonyl of the derivative and the second amide hydrogen on the phase.
The third interaction is likely to be a steric hindrance by the isopropyl
group on the selector which will block the approach of the solute on one
side.
However, the initial results from this study showing the resolution of
the enantiomers of propranolol and raetoprolol, although encouraging,
appear to be inconsistent with the remainder of the study, In both
cases, the peaks have very long retention times and, in consequence, are
very broad. The lack of peaks when attempts were made to repeat the
work could possibly be explained by reference to the regular testing of
the column with the test mixture, whereby it was noticed that, prior to
repeating the propranolol and metoprolol derivatives, the retention times
of the test mixture increased considerably, from 10,9 mins to 14.0 mins
for D-methionine. Although this was not accompanied by a significant
decrease in efficiency, a similar increase in the retention of the
propranolol and metoprolol enantiomers could possibly cause sufficient
band broadening as to reduce such small peaks to a non detectable level,
Mare significant conclusions can be drawn from a comparison between the
chiral phase and the BP1 phase and, in general, McReynolds constants are
4,3.3 Discussion
used to provide an assessment of the polarity of different phases.
Although there are more, the first five constants are the mast important
and these are given for benzene, representing the aromatic and
unsaturated compounds, 1-butanol, representing the alcohols, 2-pentanone
for the carbonyls, nitrapropane for the other polar compounds and
pyridene, representing the heterocyclics. McReynolds constants are
determined by measuring the Kovats indices for each of the five
compounds on the phase of interest under standard conditions, and
subtracting the corresponding Kovats indices for the same samples as
determined on a non polar, squalane phase. The McReynolds constants for
Chirasil Val and the BP1 phase are given in table 4.3.2. Other systems
have expanded this approach, one of the most useful being the CP Index.
Under this system, the sum of the first five McReynolds constants for the
phase of interest is compared to that of the most polar phase, QV275,
glvig a ratio of between 0, for non polar squalane, and 100 for the
highly polar phase, A comparison between the BP1 and the chiral phase
shows that the chiral phase is the most polar with both systems,
This result can be used to relate the expected retention of the
derivatives on the chiral phase to that achieved on the BP1 phase by
assessing the polarity of the samples. Although derivatisation of the
aminoalcohols reduces their polarity significantly, they still retain
enough of their polar characteristics to be more strongly retained on the
more polar chiral phase. From the work done with the BP1 phase, it was
found temperatures in excess of 200°C were required to elute the
derivatives within a reasonable length of time. However, the maximum
operating temperature of Chirasil Val is 200 °C and the column suffers
A Comparison of the MeReynolds Constants and CP
Indices for Chirasil L Val and the BP 1 Phase.
IcReynoldis Constants
Phase 1 2 3 4 5 I CP
Squalane 0 0 0 0 0 0 0
BP1 15 53 44 64 41 217 5. 14
Chirasil-L-Val 24 264- 102 130 107 627 14.86
GV275 629 827 763 1106 849 4219 100
Table 4.3,2:
from a high bleed at temperatures above 170 °C. This implies that at the
lower temperatures the retention of the derivatives will be very long,
and again, the peak broadening effects of increased retention may make
detection of the peaks of interest difficult.
It was therefore concluded that this approach was not easily applied to
the enantiomeric separation of beta blockers, and any further work must
first overcome the problem of finding a procedure to give more volatile,
less polar derivatives, or alternatively find ways to improve the thermal
stability of the phase.
4.4 Enantiomeric Resolution Using a Cycladextrin HPLC Phase
4.4.1 .Introduction
The use of chiral stationary phases for the resolution of
enantiomers by HPLC is becoming increasingly more common, and of the
wide range of stationary phases available, the cycladextrin phase is
possibly the most versatile, being compatable with the polar eluents used
in reverse phase chromatography and generally requiring no prior sample
derivatisation. As such, the column initially appeared to be the most
suatable choice for the resolution of the polar beta blacking drugs.
As has previously been described, cyclodextrins have a toroidal
configuration, the size of which is dependent upon the number of glucose
units in the structure. Since resolutions are achieved through inclusion
complexing, the size of the cyclodextrin cavity must be comparable to
that of the compounds to be separated. For this study, a Cyclobond I, or
J3 cyclodextrin column was selected as its seven glucose units provide the
most appropriate sized cavity for the aromatic systems of the amino
alcohols.
The aim of this section of work was not only to develop enantioselective
methods for the analysis of beta blockers, but also to assess the
suitability of the phase with respect to other compounds.
An assessment of the column performance was made on receipt of the
column by measuring the efficiency of the phase under the recommended
test conditions using a sample solution of m-nitroani 1 ine, see figure
4.4.1. The efficincy was found to be considerably below that claimed in
the manufacturers specifications, and although efforts were made to
improve this, by checking that there was no dead volume throughout the
HPLC system, no significant increases were achieved
As it was initially thought that the cause of this problem was a fault in
this particular column, a second was obtained. The results from this
second column, however, mirrored those of the first. Communication with
the suppliers revealed that the mass transfer characteristics of the
cyclodextrin phase were relatively poor and an improvement in efficiency
could possibly be achieved by a reduction in both the injection volume
and also the sample concentration
An investigation was therefore carried out to measure the efficiency for
injection volumes between 1 and 10 pi under the standard test conditions,
the results of which are shown in figure 4.4.2. This shows that the
efficiency may be increased to 11000 plates by injecting Ipl of a 500
ng/ml solution, although further improvements were not achieved by
additional reductions in the sample concentration.
While such measures succeeded in increasing the efficiency of the
separation to a more acceptable value, very low injection volumes are
4.4.2 Testing the Column
Testing the Cyclobond Column
H---------------------------------------------- 15 0
Figure 4.4.1 Column: Cyclobond I
Eluent: 50% mehtanol in water
Flow Rate: 1 ml/rain
Detection: UV 254nm
Sample: m-nitroaniline
Injection Volume: Ipl
The Effect of Variations in Injection Volume on the Efficiency of the Cyclobond Phase
Efficiency (N)
Injection Volume (ul)
Figure 4.4.2 Column: 25cm Cyclobond 1Eluent: 50% methanol in water Flow Rate: 1.0 ml/min Detection: UV 254nm Sample: m-nitroaniline
- 156 -
not readily suited to quantitative analysis unless specialised injection
valves are used, since syringe injections into a larger loop is not
sufficiently precise without the presence of an internal standard. Band
broadening effects will also occur when the small sample volume is loaded
into the loop of a Rheodyne type valve unless the injection is made
immediately; a requirement which cannot be achieved when injecting using
an autosampler. It also has been found that the relationship between the
sample concentration and the retention time is not constant and that low
amounts of injected sample cause retention time increases. More
importantly, small injection volumes have the effect of increasing the
limit of detection, particularly if more concentrated samples cannot be
used.
4.4.3 Resolution of Propranolol
As the main aim of this section of work was to evaluate the
Cyclobond with respect to the enantiomeric resolution of beta blocking
drugs, propranolol was selected as a suitable model compound along with
verapamil. A system was set up using the Cyclobond I column in
conjunction with a UV detection system. Following the manufacturers
advice, a 50% methanol in water eluent was chosen as a starting point,
and injections were made of both propranolol and verapamil. As this
choice of mobile phase gave no hint of separation for either propranolol
or verapamil, retention studies were carried out to determine whether
resolution could be achieved with different mobile phase compositions.
The percentage of methanol was varied between 40 and 90% and the
capacity factors calculated for both compounds. The results, as shown in
figure 4.4.3, show that the capacity factors decrease as the percentage of
The Effect of Eluent Composition on the Retention of Propranolol and Verapamil on Cyclobond I
30 40 50 60 70 80 90 100% Methanol
Figure 4.4.3 Column: 25cm Cyclobond I Eluent: methanol in water Flow Rate: 1.0 mi/min Detection: UV 254nm
of organic modifier in the eluent increases, but at no time was there any
indication that enantioselective resolutions could be achieved under these
conditions.
The mobile phase was then changed to a buffered system using 1% aqueous
trlethylamine adjusted to pH 4.1 with glacial acetic acid, and an eluent
of 25% methanol in buffer was used far the next stage of this
investigation.
Injections of propranolol were made, and the initial results appeared
promising, with the chromatogram showing a distinct shoulder on the peak
of interest, see figure 4.4.4. This was at first thought to be due to
some degree of enantioselective resolution occurring, but further attempts
to Improve the separation by reducing the injection volume only resulted
in the shoulder changing it's position from the leading to the tailing
edge of the propranolol peak. Additional information was obtained by
collecting fractions of eluent from the split peak and reanalysing each
fraction separately. Five fractions were collected over the peak of
interest and each was dried down under nitrogen, reconstituted in a
suitable volume of eluent and reinjected. For each fraction, the retention
and peak shape was found to be identical indicating that the shoulder and
main peak were caused by the same solute, and optical resolution had not
occurred.
The split peaks are therefore likely to have been caused by column
overloading since, if too much analyte is injected, either by injecting a
concentrated solution or using too large an injection volume, it is
Resolution of Propranolol on a Cyclobond I Column
Figure 4.4.4 Column: Cyclobond I
Eluent: 25% methanol in 1% triethylamine
acetate buffer, pH 4.1
Flow Rate: 1.0 ml/min
Detection: UV 288nra
Injection Volume: 10 pi
- 160 -
possible that not all of the sample molecules will be able to form
inclusion complexes within the cyclodextrin cavities. In this case, the
remainder of the solute would be retained on the column by a different
mechanism, such as adsorption onto the silica support, and therefore have
a slightly different retention time to the included sample, thus giving
rise to split peaks. Since the column efficiency is fairly low, the
smaller of the two peaks is manifested as a shoulder an the larger peak.
As the capacity of the column for inclusion complexing is likely to
constant, the position of the shoulder depends upon the total amount of
sample; for less concentrated solutions, inclusion complexing is the major
interaction and that retained by other mechanisms gives rise to the
shoulder, while for high concentration samples, the majority of the sample
is retained by the additional mechanism and inclusion complexing becomes
the secondary interaction causing the shoulder.
The study was then transferred to a new Cyclobond I phase and further
investigations continued, The new column was, however, tested prior to
use as previously described and found to give no improvement in
efficiency over those tested earlier. Using an eluent of 25% methanol in
buffer, injections were made of all the amino alcohols under
investigation, including verapamil, Although peaks were observed for all
the compounds, the retentions of which are shown in table 4.4.1, none of
them showed any sign of resolution.
Attempts were then made to resolve the enantiomers by derivatising each
compound prior to analysis. Samples of all the amino alcohols,
- 161 -
The Retentions of Under!vatised Amina Alcohols
Sample k'
Metoprolol 1.92
Atenolol 0.23
Propranolol 2.31
Verapamil 5.54
Alprenolol 1. 08
Oxprenolol 0.54
Practolol 0.31
Table 4.4.1: Column: 25cm Cyclobond I
Eluent: 25% methanol in 1% triethylamine
acetate buffer, pH 4.1
Injection Volume: 10 pi
Detection: UV 230 nm
including verapamil, were derivatised by the previously described
proceedure to form acetyl and trifluoroacetyl derivatives. Two mobile
phase systems were used; 75% methanol in water and 60% methanol in
water, and injections of the two derivatives of each compound were made
with both eluents, The retentions far- each derivative were measured for
all the compounds and it was found that, with the exception of verapamil,
for each eluent system all the acetyl derivatives shared a common
retention time, as was likewise the case far all the trifluoroacetyl
derivatives. In addition to this it was found that, for each eluent, the
trifluoroacetyl derivatives were more strongly retained than the acetyl
derivatives. The occurrence of a common retention time for each
derivative, although unexpected, may possibly be explained by considering
the nature and configuration of the inclusion complex formed between the
solute and the cyclodextrin. Since all the amino alcohols under
investigation have different aromatic ring systems attached to a common
side chain (see figure 2.11), if the aromatic ring system was included in
the cyclodextrin cavity, then the retention times far each salute would
differ, as was the case for the underivatised compounds. Although this
is the accepted configuration of the inclusion complex, it does not
account far the results obtained, which can be more readily explained by
considering that the aliphatic side chain is included into the
cyclodextrin cavity in preference to the aromatic system. This
postulation is more feasible in the case of the trifluoroacetyl
derivatives, since it is known that chlorine atoms have a strong affinity
for the cyclodextrin cavity and therefore the same is likely to be true
for fluorine atoms. However, this does not follow for the acetyl
derivatives, and it can only be supposed that the reduction in polarity
of the derivatives, together with a possible size criteria make this
configuration more stable than if the aromatic portion was included.
In addition to this, it was found that several of the trifluoroacetyl
derivatives showed signs of possible enantiomeric separation, with split
peaks achieved for atenolol, propranolol and alprenolol. The experiment
was repeated using freshly prepared trifluoroacetyl derivatives and
injection volumes varied to ascertain whether the peak splitting could be
caused by column overloading, as was previously the case. The fresh
derivatives were also found to give split peaks and the chromatograms
for each compound were similar to those obtained previously, regardless
of the injection volume used. Since the two peaks for each compound were
of comparable size, it therefore seems likely that they are caused by
chiral resolution, although further work to verify this by using single
enantiomers was prevented both by time considerations and by a drop in
the efficiency of the Cyclobond column.
It was interesting to note that no such resolution was achieved with the
acetyl derivatives, possibly since they were not so strongly included
within the cycladextrin cavity.
4.4.4 Resolution of Chlorpheniramine
Since the column had failed to resolve any of the previously
mentioned compounds without prior derivatisation, it was decided to
attempt to resolve a different sample compound using a published method
in order to obtain some assessment of the column performance in
comparison to similar columns used elsewhere. Chlorpheniramine, an
antihistaminic drug whose structure is shown in figure 2.10, has been
resolved on the Cyclobond I phase by Armstrong and co workers [2703
using an eluent of 15% acetonitrile in 1% triethylamine acetate buffer, pH
4.1,
- 164 -
A similar system was therefore set up and found to be successful in
partially resolving the sample enantiomers, although the peaks were not
fully resolved (see figure 4.4.5). Further retention studies were carried
out, initially investigating the effects of the mobile phase composition
on both the capacity ratios and separation factor of the peaks,
The percentage of organic modifier in the eluent was varied between 5 and
20% at a flow rate of 0,6 ml/min, and the results are shown in table 4.4.2
These results show that the Cyclobond column behaves in a similar way to
a standard reverse phase ODS column, with the increase in organic
modifier causing a decrease in the retention of the compound. As would
also be expected the separation of the partially resolved peaks decreases
as the percentage of acetonitrile increases. The degree of resolution is
dependent on the retention time, since the more rapidly the compound is
eluted through the column, the less time there is for the subtle
interactions, necessary for enantioselective resolutions, to be manifested.
The effect of the mobile phase pH on the retention and resolution of the
solute was also investigated over the range 4 to 7 and it was found that,
as the pH was increased, the retention of the compound increased, and the
separation factor decreased. In addition, the effect of flow rates on the
resolution was investigated, since it had been suggested that the
efficiency of the separation could be significantly increased by using
lower flow rates [4751, Two eluent systems were used: 15% acetonitrile in
buffer pH 4.1 and 10% acetonitrile in buffer, pH 4.1, and flow rates were
varied between 1.0 and 0,4- ml/min. The capacity and separation factors
- 165 -
The Resolution of Chlorpheniramine
H-----------------------------------------------------------115 0
Figure 4.4.5 Column: Cyclobond I
Eluent: 10% Acetonitrile in 1%
trlethylamine acetate buffer, pH
Flow Rate: 1.0 ml/min
Detection: UV 261 nm
Injection Volume: 10 pi
- 166 -
The Effect of Mobile Phase Composition on the Resolution of
Chlorpheniramine
% Acetonitrile k * t k'^ a
20 1.25 - 1. 0
15 2.20 2.34 '1. 064
10 4.83 5. 19 1. 074
5 10.54 11,40 1. 082
Table 4.4.2: Column: 25cm Cyclobond I
Eluent: Acetonitrile in 1% trlethylamine
acetate buffer, pH 4.1
Injection Volume: 10 pi
Detection: UV 261 nm
were calculated and the results shown in table 4.4.3.
Since the two peaks were only partially resolved, it was not possible to
assess the efficiency of the separations, and therefore only the
separation factors were calculated. These show only minor variations and
do not fall into any particular pattern, and do not give any indication
of changes in efficiency, However, visual comparisons and calculations
based on projected peak shapes appear to show that the flow rate makes
The Effect of Flow Sate on the Resolution of Chlorpheniramine
Eluent Flow Rate k* -i k'a a
15% CHs.CN 1.0 2.25 2.42 1.0 76
15% CHaCI 0,6 2.29 2.43 1. 061
10% CHs.CN 0. 6 4, 75 5,10 1. 074
10% CHs.CN 0,4 4. 75 5. 10 1. 074
Table 4.4.3: Column: 25cm Cyclobond I
Eluent: Acetonitrile in 1% triethylamine
acetate buffer, pH 4,1
Injection Volume: 10 pi
Detection: UV 261 nm
- 168 -
little difference to the efficiency of the separations or to the degree of
resolution.
In conclusion, this section of work shows that at least partial
enantioselective resolutions are possible for some compounds.lt is
interesting to compare the results obtained in this section with those
reported for the published method. Although the efficiency of the column
was considered to be particularly low, a separation factor of 1.074 was
achieved using an eluent of 15% acetonitrile in 1% triethylamine acetate
buffer, pH 4.1, whereas under similar conditions, the separation factor
reported by Armstrong and co workers was 1.070. These results however,
do not take peak shape into consideration, and therefore the degree of
resolution; that is, whether the peaks are baseline separated or only
partially resolved, cannot be ascertained without a study of the
chromatograms. Since no chromatograms were shown in the published
report, further comparisons were not possible
4.4.5 Resolution of Clomiphene
In addition to enantioselective analysis, the cyclodextrin phases are
ideally suited to the resolution of structural isomers, since the
inclusion complex formation is a spatially dependent interaction.
Although such isomers have differing physical properties, they are often
not sufficiently different to facilitate easy resolution by conventional
reverse phase columns.
Clomiphene, an anti-oestrogen drug whose structure is shown in figure
2.10, is a compound which exhibits such geometric isomerism, existing in
- 169 -
cis- and trans- forms, and was therefore selected for this investigation.
A system was set up using a methanol trlethylamine acetate buffer mobile
phase and retention studies were carried out. Initial investigations
involved varying the mobile phase composition to determine the effect on
both retention and resolution of the enantiomers, The composition was
varied between 40 and 70% methanol in 1% trlethylamine acetate buffer, pH
4,1, at a flow rate of 1.0 ml/min and the results are shown in table
4.4.4.
Good resolutions of cis- and trans-cloraiphene were achieved using this
system (see figure 4.4.6) and both peaks were well separated with a
methanol concentration of up to 60%, and again, as the percentage of
organic modifier in the eluent increased, the retention and resolution of
cloraiphene decreased.
The effect of varying the buffer pH was also investigated by selecting a
50% methanol in 1% triethylamine acetate buffer eluent system and varjring
the buffer pH between 4 and 7. The results, given in table 4.4^, show
that the resolution of the isomers decrease as the pH of the mobile phase
increases. This is in marked contrast to the expected trend which would
indicate that as the pH increases, the retention of a basic compound
increases, and in general, the resolution increases in accordance with
retention increases. In this case the increase in retention with pH is
observed but it is accompanied by a decrease in the separation factor.
This can be explained by considering the interactions between the
cyclodextrin and the solute when an inclusion complex is formed. The
retention behaviour of each isomer is determined not only by the
- 170 -
Clomiphene
% Methanol k' t k ' & oc
The Effect of Mobile Phase Composition on the Resolution of
40
50
60
70
2.21
1.21
0.32
0.23
5.54
3. 00
0.79
2.51
2.48
2.46
1 .0 0
Table 4.4..Z: Column: 25cm Cyclobond I
Eluent: methanol in 1% triethylamine
acetate buffer, pH 4,1
Injection Volume: 10 pi
Detection: UV 245 nm
Resolution of Cis and Trans Clomiphene on the Cyclobond Phase
Figure 4.4.6 Column: Cyclobond I
Eluent: 50% methanol in 1% trlethylamine
acetate buffer, pH 6.0
Flow Rate: 1.0 ml/min
Detection: UV 245nm
Injection Volume: 10 pi
- 172 -
inclusion of the salute but also by hydrogen bonding interactions with
the primary and secondary hydroxyl groups positioned at the opening of
the cyclodextrin cavity. Since both clomiphene and chlorpheniramine are
basic compounds containing a tertiary amine group in their structures, in
a law pH environment they exist in an ionised form, with a proton on the
nitrogen atom available for hydrogen bonding with a hydroxyl group if
the structure of the isomer permits. However, at a higher pH, the
compounds exist in a free base form, and although they are retained in
the lipophilic cyclodextrin cavity longer, the scape for hydrogen bonding
with the cyclodextrin hydroxyls Is limited, and therefore the differences
in retention between the two isomers lessens, ultimately becoming
insufficient to resolve the two peaks.
Since previous attempts to resolve the isomers of clomiphene on a reverse
phase ODS column had been unsuccessful, this section of work highlights
the suitability of the cyclodextrin phase for separations of this type.
- 173 -
The Effect of pH on the Resolution of Clomiphene
Eluent pH k* , k ’s oc
4 1.21 3.00 2.48
5 0.96 2.11 2.20
6 1.68 3.21 1.91
7 3.25 5,71 1.76
&Table 4.4.2-!: Column: 25cm Cyclobond I
Eluent: 50% methanol in 1% triethylamine
acetate buffer
Injection Volume: 10 pi
Detection: UV 245 nm
It was initially hoped that this phase would provide the basis of a
relatively simple method to resolve the enantiomers of beta blockers
under reverse phase conditions. Although the success of this section of
work was limited in this respect, there are still a number of interesting
points which can be drawn from it.
The inability to resave the enantiomers of propranolol and the other
amino alcohols, in spite of being disappointing, was not altogether
unexpected, since Armstrong and co workers [2701 had found it necessary
to use two 25cm Cyclobond I columns in series in order to increase the
efficiency sufficiently to resolve the two isomers of metopralol and
propranolol.
The resolution of same of the amino alcohols as trifluoroacetyl
derivatives should ideally have been studied further, had not the column
efficiency dropped to below an unacceptable level. As far as possible,
samples of single enantiomers should be derivatised to form similar
derivatives and chromatographed, to ensure that the split peak obtained
for the racemic mixture is caused by the resolution of the enantiomers of
each compound.
4.4.6 Discussion
- 175 -
Other types of derivatives could also have been studied to ascertain
whether separations could be achieved for the remainder of the beta
blockers, and in addition, as many different aminaalcahols as passible
should be reacted to form each type of derivative and injected in an
attempt to characterise the mechanism of stereoselective resolution and
investigate the orientation of the solute within the cyclodextrin.
Comparisons can also be made between the resolutions of chlorpheniramine
and clomiphene. Only partial resolutions of chlorphenirame can be
achieved with conditions of 5% acetonitrile in buffer, whereas clomiphene
isomers can still be separated with eluents of 70% acetonitrile in buffer
and pH values of up to 7. Further information an the variation of
retention and resolution of these compounds with the pH of the mobile
phase could be obtained by selecting a range of compounds with different
hydrogen bonding capabilities and investigating the effect of eluent pH
an each.
It can therefore be concluded that, although the Cyclobond I phase did
not produce the desired separation, such resolutions were more readily
achieved by the formation of a trifluoroacetyl derivative prior to
chromatographic analysis. It must be noted, however that if precolumn
derivatisation reactions are necessary, one of the main advantages of the
phase is lost.
- 176 -
4.5 Enantiomeric Separations Using a "Pirkle'" Type Phase
4.5.1 Introduction
Literature studies have showed that, of all the commercially
available chiral stationary phases, the "Pirkle" columns have the widest
range of applications, and hence it was selected to investigate the
possibility of achieving enantiomeric resolution of the beta blocking
drugs,
There are, however, a number of "Pirkle" type columns, differing both in
the chiral selector and in the type of banding between the selector and
the silica support, and therefore factors such as the type of sample and
mobile phase must be considered in making a selection. Since the beta
blockers are re electron donors, they will be resolved best on a phase
which is a re electron acceptor, and thus the 3,5-dinitrobenzoyl
phenylglycine phase was selected. These are available with either an
ionic or a covalent bond between the chiral selector and the silica
support and the choice between the two is dependent on the type of
mobile phase required for the separation; ionic phases can only be used
with eluents of low polarity while covalently bonded phases may be used
with solvents of any polarity, including water.
Such phases work on the basis of having a minimum of three points of
interaction between the salute and the phase, and if such interaction
sites are not present in the sample, a derivatisation reaction can be
performed to add a suitable group to the molecule. However there are a
- 177 -
number of disadvantages inherent with derivatisation reactions, not least
of which is the increase in sample preparation time and also in
unavoidable sample losses.
4.5.2 Resolution of Propranolol
The primary aim of this study centred on developing a method for
enantiomeric separations without the need for derivatisation, and once
again propranolol and verapamil were chosen to be the model compounds.
Initial attempts to resolve the enantiomers using the suggested eluent of
10% isopropanol in hexane were found to be unsuccessful, with both
compounds being retained on the column for over an hour, and so further
retention studies were carried out. A covalent phase was chosen for this
work in order that the mobile phase could be varied between 100%
methanol and 100% hexane, and a system was set up using methanol :
isopropanol : hexane eluents and UV detection. Injections of both
compounds were made and the capacity ratios and any sign of chiral
separations were noted for each eluent system (see table 4.5.1).
With methanol and methanol : isopropanol mobile phases, both propranolol
and verapamil were eluted with the solvent peak, but in no case was there
any evidence of enantiomeric resolution for either compound with any of
the eluent systems investigated, although as the polarity of the solvent
decreased so the retentions increased until with 10% isopropanol in
hexane, the compounds were retained on the column. This was due to both
the polarity of the solute molecules and
- 178 -
The Variation of the Retention of Propranolol with
Mobile Phase Composition
Eluent k ‘ (Prapranolol) k'(Verapamil)
1:2:2 0. 100 1. 08
4:3:3 0.83 1.50
CO
}—i 1. 83 2.33
8:1:1 3. 00 4. 00
18:1:1 5.25 7.38
Table 4.5.1 Column: 25 cm <R)~3,5-dinitrobenzoyl phenylglycine
Eluent: hexane:isopropanol:methanol
Flow Rate: 1,0 ml/rain
Detection: UV, verapamil-232nm, propranolol~288nm
Injection Volume: 20 pi
- 179 -
possibly to a lack of sufficient suitable functional groups to interact
with the phase. Since the aminoalcohols are highly polar molecules, as
is the phase itself, they are not readily eluted from the column, with
eluents of law polarity. Yet increasing the polarity of the mobile phase
causes the solutes to be eluted too rapidly for enantioseparation to
occur.
Molecular models of both the phase and the solutes were used to identify
the possible interaction sites and establish whether they are sufficient
to permit chiral separation in the absence of polarity considerations.
For the purpose of this exercise, the 3,5 dinitrobenzayl phenylglycine was
considered to preferentially reside in the conformation in which the
amide hydrogen is in the plane of, and trans to the amide carbonyl
oxygen.
The possible interactions of propranolol are shown in figure 4.5.1 and
indicate that chiral selectivity should be possible since the solute
molecule has the required three interactions, spaced about both chiral
centres. Once the solute is immobilised on the phase, differences in
steric hindrances between the isomers will weaken the interactions far
one enantiomer, allowing chiral resolution. However, similar studies show
that verapamil is unlikely to be resolved on the 3,5-dinitrobenzoyl
phenylglycine phase since there is only one point of interaction between
the solute and the phase, namely the re-re interaction between the
activated aromatic group of verapamil and the deactivated dinitrobenzayl
group of the phase,
- 180 -
The
Inte
ract
ions
of
the
Phas
e wit
h P
ropr
anol
ol
«4
cv>n
X
Xo
\\
- 181 -
Since the previous section of work had proved to be unsuccessful,
some form of derivatisation of the samples was deemed to be necessary.
As the point of performing a derivatisation reaction was to introduce a
functional group which would provide a suitable site for interaction with
the phase when necessary, the choice of derivative was critical.
Successful resolutions have been achieved for a number of amines C4763
and aryl aminoalkanes [477] on this phase by the formation of their a-
naphthamide derivatives and hence a similar approach was taken with the
beta blockers. The formation of naphthamide derivatives from the
secondary amine solute (see figure 4.5,2) not only reduces the polarity,
but the re basic naphthyl ring system is able to form strong re-re
interactions with the re acidic 3,5-dinitrobenzoyl group,
This approach was eventually found to be very successful and resolutions
have been achieved for five of the seven compounds of interest (see
figure 4.5,3 and figure 4.5.9a-g). However, the initial attempts involved
the use of an ionic phase which gave no separations at all, and it proved
necessary to transfer the method to a covalently bound column of the
same phase before suitable chiral separations were attained. Such
covalent columns have been found to be considerably more robust and also
give significantly higher efficiencies when tested with 2,2,2-trifluoro-l-
(9-anthryl) ethanol under standard conditions, in this case 21700 plates
for the covalent column compared to 9000 plates for the ionic phase (see
figure 4.5.4). Thus all further work was carried out using covalent
phenylglycine columns.
4.5.3 Resolution of Propranolol Naphtkamide
The
Form
atio
n of
1-N
apht
ham
lde
Der
ivat
ives
92
33
WaCd
35£o
E%2
+co
XOX01X
CVJXCJ
IX X 0-0
ICM
X
wcdB
ai
1Bsooc d
H<C/3BP2
c/3w9ss1O.<2
se
CN
- 183 -
Figu
re
4.5.
2
Resolution of Propranolol as the laphthamide Derivative
on a 3,5-Dinitrobenzoylphenylglyclne Phase
O''o■HK>
V
vn
Figure 4.5.3 Column: covalent <R>-3,5-dinitrabensoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20pl
- 184 -
Test Chromatograms for the <R>-3,5-Dinitrobenzoyl
Phenylglycine Phase
30
Covalent Phenylglycine Column
30 . 0Figure 4.5.4 Column: <R)-3,5-dinitrobenzoylphenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20pl
Sample: 2,2,2-trifluoro-1-(9-anthryl) ethanol
- 185 -
The influence of the mobile phase composition on both retention and
separation was investigated using samples of propranolol derivative. The
mobile phase was varied between 8% and 20% isopropanol in hexane, and
the retention times and separation factors for each eluent showed the
expected trend that increasing the polarity of the mobile phase has the
effect of decreasing the retention, and also the resolution of the two
enantiomers (see table 4.5,2),
An attempt to assess the effect of the stucture of the analyte on the
retention and separation of the enantiomers was made by derivatising
samples of each of the other beta blockers and subjecting them to
analysis with a mobile phase of 10% isopropanol in hexane. Wherever
possible the individual enantiomers of each compound were also
derivatised and chromatographed, to ascertain that the two peaks were
definitely due to the two isomers, and in each case the (+)- isomer
eluted before the <->- isomer (see figure 4,5,9d~g>. Although the
derivatives of practolol and atenolol were retained on the column, the
retention times and separation factors of the others are shown in table
4.5.3.
From a study of the molecular structures, shown as their l~naphthamide
derivatives in figure 4,5.5., several trends can be established. Both
propranolol and pronethalol molecules contain two naphthyl groups, either
of which could potentially form the transient re-re bonds with the 3,5-
dinitrobenzoyl group. However, in the case of propranolol, the
naphthamide system is the least re-basic of the two, owing to the electron
withdrawing power of the carbonyl group, and thus the
- 186 -
The Effects of Variations in Mobile Phase Composition on the
Resolution of Propranolol laphthamide Derivatives on a Covalent
<R) 3,5-Dinitrobenzoyl phenylglycine Phase
% Isopropanol ki* fcs' a
8 25,32 27.40 '1. 082
10 20.51 22.20 1. 082
12 17.71 18.91 1. 068
14 14.20 15. 11 1. 064
16 12.20 12.89 1. 057
20 9.95 10.36 1. 041
Table 4.5,2 Column: covalent (R)-3,5-dinitrobenzoyl
phenylglycine
Eluent: isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nra
Injection Volume: 20/il
- 187 -
Retention of Aminoalcohols as their 1~ Naphthamide Derivatives
on a Covalent (R) 3,5-Dinitrobenzoylphenylglycine Phase
Analyte ki' ka* oc
Propranolol 22. 06 23.88 1. 082
Pronethalol 12.75 13.67 1. 072
Oxprenolol 16. 00 17. 00 1. 063
Alprenolol ooo 7.40 1. 058
Metoprolol 16.92 17.83 1. 054
Table 4.5.3 Column: covalent <R)-3,5~dinltrobensoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20jil
- 188 -
Structures of l-Iaphthamide Derivatives
ATENOLOL
OHi0-C H2-CH-CH2 - N-CH (CH, )2
r Vch2-cg.\ih2
PRACTOLOL
0-CH2*CH-CH2-N-CH(CH3LV
OXPRENOLOL
OHI0-CH2-CH-CH2-N-CH(CH352 q Y 0-CH2-CH V-o
ch2 A / \foYo1
ALPRENOLOL
OHg-CH 2-C H -C H 2-M-CH(CH3)2
f o i o l
PROPRANOLOL metoprolol
0-CH2-CH-CH2-N-CH{CH3).
PRONETHALOL
Figure 4.5.5
- 189 -
interaction occurs at the opposite end of the molecule, This is also
likely to be true for pronethalol, since again the carbonyl group is more
electron withdrawing than a hydroxyl. The differences in retention and
resolution between the two probably arises from differences in the
aminoalcohol chain length. With the aromatic end secured by re-re
interactions, the longer chain length of propranolol may enable the other
functional groups to approach in closer proximity and thus form stronger
hydrogen bonds than would be possible for pronethalol, In addition to
this the re-re interactions are likely to be stronger for propranolol.
Since none of the other beta blockers have a second naphthyl system in
their molecular structures, the re-re interaction between the solute and
phase occurs at the naphthamide group, The difference in retention and
resolution between oxprenolol and alprenolol is more interesting since to
all intents and purposes the major interactions of re-re and hydrogen
bonding should be similar for each. The difference between the two
structures lies in the substituted phenyl groups; that of oxprenolol being
more electron deficient than that of alprenolol. For this to affect the
retention and resolution the phenyl group must also be involved in the
phase-solute interactions, although it is not yet clear in what way. It
was also interesting to note that the two compounds, atenolol and
practolol, which were not eluted under these conditions both had amide
groups on the phenyl ring, but it is difficult to assess whether the long
retention is due to the formation of different interactions or whether, in
fact, the derivatisation has not been successful.
- 190 -
A further investigation of the effect of structural differences in the
solute was carried out by altering the derivatising reagent. Two similar
samples of propranolol were derivatised, one with 1-naphthoyl chloride
and the other with 2-naphthoyl chloride, as shown in figure 4.5.6. When
chromatographed under standard conditions the results were as shown in
table 4.5.4. The increase in both retention and resolution of the 2-
naphthamlde derivative is possibly more due to the different orientation
of the 2-naphthamide ring system causing less steric hindrance between
the solute-phase complex than to differences in any of the primary
interactions.
Retention of Propranolol Naphthamide Derivatives on a Covalent
<R) 3,5-Dinitrobenzoylphenylglycine Phase
Derivative ki 1 ks' a
1-Naphtharaide 17.4 18.2 1,046
2-Naphthamide 25.8 27.4 1.062
Table 4.5,4 Column: (R)-3,5-dinitrobenzoylphenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20pl
- 191 -
The
Form
atio
n of
2-N
apht
ham
ide
Der
ivat
ives
WsCtf2XV
0£XCL.<z1CN
+
_ Cn]
00 Xo
o
C\lro
XoXo
IX
. CMXo
X x 0-0
Standard Curves for Propranolol as its Naphthamide Derivative on a 3,5-Dinitrobenzoylphenylglycine Phase
Peak Area3,000,000
2,500,000 --
A
2 ,000,000
1,500,000 --
1,000,000
500,000 --
P
//
/
/
/
/
/ff s (+) Propranolol y=-23133 + 4181x, r=0.9993
y j T - a -A ' / (-) Propranolol y=-68544 + 5486x, r=0.9970
j s r
A-------- +■100 200 300 400
Standard Concentration (ug)500
Figure 4.5.7 Column: (R)-3,5-dinitrobenzoylphenylglycine Eluent: 10% Isopropanol in hexane Flow Rate: 1.0 ml/min Detection: UV 288 nm
Although several stages of the derivatisation proceedure were
investigated , the unavailability of a pure unextracted naphthamide
standard made an assessment of the yield or recovery of the method a
difficult task. However, several standard curves were assayed over the
range 1-0.01 mg/ml propranolol, and in each case the correlation
caeficients were not less than 0.99 (see figure 4.5.7), leading to the
assumption that the derivatisation proceedure was, at least, uniformly
quantitative over this range. Problems with the method occurred at
concentrations below this range due to an interference which swamped the
detector response to the enantiomeric derivative. This may have been
caused by the use of Teflon lined screw caps during derivatisation, since
Teflon is known to affect the reaction (478), or alternatively the
interference may be due to the excess derivatising reagent. This could
possibly be overcame by either solid phase extraction or column switching
techniques when the method is put into routine use for monitoring
enantiomeric plasma levels.
4,5.4 Computer Modelling
In an attempt to assess whether computer modelling facilities could
be used as a tool to predict the ability of a phase to resolve a given
compound, computer modelling studies were carried out by Dr D. Lewis,
Dept, of Biochemistry, University of Surrey, with a view to comparing the
computer predictions to the experimental data.
Representative structures were set up for both enantiomers of the
naphthamide derivatives of each amino alcohol, and each structure was
- 194 -
Inte
ract
ion
of (+
) P
ropr
anol
ol w
ith
(R)-
3,5-
Din
itrob
enzo
yl
Phe
nylg
lyci
ne
CDC
*o>
- § > c 2® EE r i> JO O Q .N CO C C X _n o2 o
.ts c c co
TD Q . in 2 CO C l
OC +
COCO
COco co“ LO
_ 0 0u-oCO o 30 0
oO )
i Z
E■o0 T o
*33T J
13T 3
o ’>E - o
0 o3Q . 0E •go ‘x
o o
< CD
- 195 -
Inte
ract
ion
of (-)
Pro
pran
olol
with
(R
)-3,
5-D
initr
oben
zoyl
P
heny
lgly
cine
- 196 -
A. C
ompu
ter
mod
elle
d in
tera
ctio
nsB.
Gui
de
to in
divi
dual
mol
ecul
es;
(R)-
3,5-
dini
trob
enzo
yl p
heny
lgly
cine
—
(-) pr
opra
nolo
l na
phth
amid
e
Figu
re
5.4.
8b
modelled against that of the (R)-3,5-dinitrobenzoyl phenylglycine chiral
selector, as shown in figure 4.5.8. Interactions, such as hydrogen
bonding, ti-ju interactions and steric hindrances, between the two
molecules were assessed, and the total interaction energy, a H, was
calculated for the most stable of all possible conformations far each
solute-phase pair. This process was repeated for all of the beta blacker
enantiomers and the results are shown in table 4.5.5.
The values far a H provide a measure of the stability of the solute-phase
complex and the more negative the number, the more stable the complex.
If such computer predictions are to be successful, it must therefore
follow that the more stable the solute-phase complex, the longer it is
retained on the column far any given conditions. Thus the degree of
resolution between two enantiomers can be ascertained by calculating the
difference, A(AH), between their interaction energies since this is
indicative of the difference in retention of each isomer, and the relative
elution order of the isomers predicted.
In order to test the validity of this prognosis, it is necessary to
compare the interaction energy values with the experimental results.
Such a comparison, is given in table 4.5.6 and highlights a number of
discrepancies between the predicted and experimental results,
It is difficult to rank the compounds into an elution order using the AH
values since a number of the enantiomers overlap, but it can be seen that
atenolol should have a retention similar to propranolol and that
practolol should be eluted between oxprnolol and metoprolol, whereas in
- 197 -
Interaction Energies Between (R)3,5 Dinitrobenzayl Phenylglyine
and the Enantiomeric Amino Alcohol Naphthamides
Analyte a H<-> AH<+> A(AH)
(kcal/raole) (kcal/raale) (kcal/mole)
Propranolol -24.483 -12.089 12.394
Metaprolol -8.263 -11.257 2,994
Atenolol -19.557 -17.281 2.276
Pranethalol -16.031 -14.784 1.247
Practolol -13.770 -13.029 0.741
Alprenolol -18.446 -18.549 0. 103
Oxprenalol -15.099 -15.022 0. 077
Table 4.5.5
- 198 -
w N n CO i fc o t o 10 i no o o o o
OJ, coCOcoCM
r̂coco S3
•ac(0W M—(0 o*> cE> 5c 2ILi O_ V)
E &T> 0 ■§(8 £ Stm *-* £o oO -
c £« **^ ° 3 C w •=
r . 0 E0 cc <? «S S
a
c001
€ Em —S -Q. 0“ a x
LUEoO<
X<L<3
iX<
COo
sCOoj
O)COoCM
Si 2! S
r*.3
CMCMO0
OOrL
CMCDCO
52 0o CD N.T— cn CMo CM CM
cn fN. cn3 0CM 0CM CMoCOi" X rL COt—
X<1
S3 sCD
cncnoCO
00S3CMCO
r̂-in0cn
o1̂.fN.co
O
Occok_Q.O
■O
COsz0co
ooc0aXO
ooc0k _
Q.<
_gowao3
ooc0
ooa0bmmCL
- 199 -
Table
4.
5.6
practice, neither was eluted from the column within an hour. Metoprolol,
which has the least negative values, implying that the solute-phase
complexes are the least stable, is actually the second most retained
compound, and in contrast, alprenolol is the least retained compound yet
has one of the most stable interaction energies.
Comparisons of resolutions are no more successful, with the energy values
showing that alprenolol and oxprenolol are unlikely to be separated,
whereas experimentally they are reasonably well resolved. Likewise,
metoprolol is the least well resolved, yet has the second largest
difference between the enantiomers. It is impassible to judge whether the'
predictions of the elution order of individual enantiomers is correct
since this was determined experimentally in only two cases, both of v/hich
agreed with the energy values.
Hence, as a means of predicting whether or not a separation can be
achieved for individual compounds on a given phase, computer modelling
appears to be largely unsuccessful and neither can it be used to predict
the separation of an unknown compound by reference to a known
separation. The reasons for this lack of success are based primarily on
the simplicity of the models used.
While the interactions between the stationary phase and each salute
enantiomer can be accurately calculated from such molecular models there
are a number of other important factors in liquid chromatographic
separations. As has been illustrated throughout the whole of this study,
the nature of the mobile phase is of paramount importance to a
separation, and resolution can be achieved or lost through the choice of
mobile phase. A chromatographic resolution is a result of a competition
between the solute-stationary phase interactions and solute-mobile phase
interactions and therefore it is not sufficient to make predictions based
on only one of these, Although one enantiomer may farm a significantly
more stable solute-stationary phase complex than the other, the increase
in retention that this should produce may be counteracted by the same
enantiomer forming a more stable solute-mobile phase complex than the
other.
Other factors which should be taken into account when attempting to
predict the success of a separation include the nature of the linkage
between the chiral selector and the silica support, the nature of the
residual silanol groups on the silica support and the phase loading.
Although such a simple modelling system is not well suited to liquid
chromatography systems, it may be more successfully applied to gas
chromatographic separations. In this case the nature of the mobile phase
is not varied and separations are achieved solely though solute-
stationary phase interactions and temperature effects. As this is more
akin to the model system used by the computer, the predicted values and
experimental results should be more closely correlated.
'This section of work shows that computer based molecular modelling
systems have the potential to predict the separation of enantiomeric
compounds on a given phase, thus aiding the selection of the best phase
to resolve the compound of interest. However it is important to be able
to model all the experimental condition in far more detail than at
present. The complexity of such a system could make it easier and even
mare economical to find the best phase for each particular separation by
an experimental trial and error approach.
- 202 -
Resolution of Alprenolol as the Naphthamide Derivative
on a 3,5~Dinitrobenzoylphenylglycine Phase
Figure 4.5.9a Column: covalent <R)-3,5-dinitrobenzoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20p.l
- 203 -
Resolution of Gxprenolol as the Naphthamide Derivative
on a 3,5-Dinitrobensoylphenylglycine Phase
K<
in in c-i o
I’lM.
\A/ V J \
Figure 4.5.9b Column: covalent (R)-3,5~dlnitrobensoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20pl
Resolution of Metoprolol as the laphthamide Derivative
on a 3,5-Dinitrobenzoylphenylglycine Phase
CM M3in oM3 N CM C-
A AI A-.. \
V
Figure 4.5.9c Column: covalent <R)-3,5-dinitrabensoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nm
Injection Volume: 20pl
- 205 -
<+> Propranolol as the Naphthamide Derivative
on a 3,5-Dinitrobenzoylphenylglycine Phase
Figure 4,5.9d Column: covalent (R)-3,5-dinitrobenzoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2,0 ml/min
Detection: UV 280 nm
Injection Volume: 20pl
- 206 -
<-) Propranolol as the Naphthamide Derivative
on a 3,5-Dinitrobenzaylphenylglycine Phase
Figure 4.5.9e Column: covalent (R)-3,5-dinitrobenzoyl
phenylglycine
Eluent: 10% Isopropanol in hexane
Flow Rate: 2.0 ml/rain
Detection: UV 280 nm
Injection Volume: 20pl
- 207 -
<-) Pronethalol as the laphthamide Derivative
on a 3,5-Dinitrobensoylphenylglycine Phase
Figure 4.5,9f Column: covalent <R)-3,5~dinitrobenzayl
phenylglycine
Eluent: 10% isopropanol in hexane
Flow Rate: 2.0 ml/min
Detection: UV 280 nra
Injection Volume: 20pl
- 208 -
<+> Pronethalol as the faphthamide Derivative
on a 3,5-Dinitrobenzoylphenylglycine Phase
Figure 4-.5.9g Column: covalent (R)-3,5-dinitrobenzoyl
phenylglycine
Eluent: 10% isopropanol in hexane
Flaw Rate: 2.0 ml/rain
Detection: UV 280 nm
Injection Volume: 20pi
- 209 -
4.6 Enantiomeric Resolutions Using an oti Acid Glycoprotein Phase
4.6.1 Introduction
In a final attempt to achieve enantiomeric resolutions of the beta
blockers without performing a precolumn derivatisation reaction, an ah
acid glycoprotein (AGP) phase was obtained, Since protein phases of this
type attempt to mimic the in-vivo stereoselective binding processes, such
phases should theoretically be capable of resolving numerous enantiomeric
drugs. Unfortunately, initial phases of this type had low capacities and
short lifetimes unless treated with the greatest of care. However, these
columns have now been improved by a patented process by which the AGP
is bonded to the silica support [311], thus Increasing the phase
stability.
The columns are used almost exclusively with mobile phases of aqueous
phosphate buffers with the addition of small amounts of organic modifiers
such as methanol, ethanol, propanol, isopropanol and acetonitrile, and is
stable within a pH range of 3.0 to 7.5, The suggested buffer
concentrations are between 0,011 and 0.035M and the
4.6.2 Retention Studies
Initially a system was set up with the AGP column and an eluent of
10% acetonitrile in 0.011 phosphate buffer, pH 7.0, using a UV detector
set at 225 ni. Injections were made of 0.01 mg/ml solutions of a number
of beta blockers and verapamil. This approach proved to be very
successful, giving baseline resolutions of both verapamil and oxprenolol.
- 2 1 0 -
R e t e n t i o n s t u d i e s w e r e t h e n c a r r i e d o u t t o i n v e s t i g a t e t h e e f f e c t o f
c h a n g e s i n m o b i l e p h a s e c o m p o s i t i o n o n r e t e n t i o n a n d r e s o l u t i o n , T h e
e l u e n t s w e r e v a r i e d b e t w e e n 1 0 0 % 0 . 0 1 1 p h o s p h a t e b u f f e r p H 7 , 0 a n d 5 % o f
e i t h e r a c e t o n i t r i l e o r i s o p r o p a n o l i n a s i m i l a r b u f f e r , a n d w i t h i n t h i s
v a r i a t i o n , s e p a r a t i o n s w e r e a c h i e v e d f o r a l l t h e c o m p o u n d s u n d e r
i n v e s t i g a t i o n w i t h t h e e x c e p t i o n o f p r a c t o l o l ( s e e f i g u r e 4 . 6 . 1 a - f > .
T h e c h a n g e s i n r e t e n t i o n a n d r e s o l u t i o n c a u s e d b y v a r i a t i o n s i n t h e
m o b i l e p h a s e c a n b e s e e n b y c o n s i d e r i n g t w o c o m p o u n d s f o r s i m p l i c i t y ,
f o r e x a m p l e a t e n o l o l a n d m e t o p r o l o l , T h e c a p a c i t y a n d s e p a r a t i o n f a c t o r s
f o r t h e s e a r e s h o w n i n t a b l e 4 . 6 . 1 a n d i n d i c a t e t h a t d e c r e a s i n g t h e
a m o u n t o f o r g a n i c m o d i f i e r c a u s e s ' r e t e n t i o n i n c r e a s e s a n d a l s o i m p r o v e s
t h e r e s o l u t i o n . T h e r e a r e n o c l e a r d i f f e r e n c e s b e t w e e n u s i n g a c e t o n i t r i l e
o r i s o p r o p a n o l a s t h e o r g a n i c m o d i f i e r , a n d t a b l e 4 . 6 . 2 s h o w s a
c o m p a r i s o n o f e a c h m o d i f i e r i n a n e l u e n t a t a c o n c e n t r a t i o n o f 5 % i n
b u f f e r . T h i s i n d i c a t e s t h a t w h i l e t h e u s e o f i s o p r o p a n o l i n p r e f e r e n c e t o
a c e t o n i t r i l e i n c r e a s e s t h e r e t e n t i o n f o r p r a c t o l o l a n d a t e n o l o l , i t h a s
t h e o p p o s i t e e f f e c t f o r m e t o p r o l o l a n d o x p r e n o l o l . S u c h e f f e c t s a r e
l i k e l y t o b e d u e t o d i f f e r e n c e s i n t h e h y d r o g e n b a n d i n g a b i l i t y o f t h e
t w o s o l v e n t s .
F u r t h e r s t u d i e s o n t h e e f f e c t o f b u f f e r p H a n d c o n c e n t r a t i o n o n
r e s o l u t i o n w e r e p r e v e n t e d b y a d e t e r i o r a t i o n i n t h e c o l u m n c o n d i t i o n ,
r e s u l t i n g i n p e a k s p l i t t i n g a n d n o r w a s i t p a s s i b l e t o d e t e r m i n e t h e
e l u t i o n o r d e r o f t h e e n a n t i o m e r s o f t h e a n a l y t e s . A l t h o u g h t h i s m a y h a v e
b e e n d u e t o i n c o r r e c t t r e a t m e n t o f t h e p h a s e , i t s t i l l g i v e s r i s e t o
u n c e r t a i n t i e s c o n c e r n i n g t h e s t a b i l i t y o f t h e p h a s e . I n s p i t e o f t h i s
R e t e n t i o n S t u d i e s f o r A t e n o l o l a n d M e t o p r o l o l o n a n
o h A c i d G l y c o p r o t e i n P h a s e
A t e n o l o l M e t o p r o l o l
E l u e n t k* i k ' a a k ' i k ' a : oc
1 0 % CHaCN 1 . 8 0 - - 2 . 3 - -
5 % CHaCN 1 . 9 - - 2 . 9 3 . 1 1 . 07
1 0 0 % B u f f e r 3 . 4 4 3 . 8 9 1 . 1 3 9 . 5 1 2 . 2 1 . 2 8
5 % I P A 2 . 3 3 - - 2 . 7 - -
T a b l e 4 . 6 . 1 C o l u m n : A G P
E l u e n t : O r g a n i c M o d i f i e r i n 0 . 0 1 M p h o s p h a t e b u f f e r
p H 7 . 0
F l o w R a t e : 0 . 9 m l / m i n
I n j e c t i o n . V o l u m e : 2 0 p i
D e t e c t i o n : U V 2 2 5 n m
A c e t o n i t r i l e I s o p r o p a n o l
The Effect of Variations in the Organic Modifier on the
Retention and Resolution of the Amino Alcohols
A n a l y t e 1 c ' i k ’ s o c k ' i k ' 2 a
A t e n o l o l 1 . 9 - - 2 . 3 3 -
M e t o p r o l o l 2 . 9 3 , 1 1 , 0 7 2 . 7 -
P r a c t o l o l 2 . 0 - - 2 , 1 -
O x p r e n o l o l 4 6 . 0 6 7 . 0 1 . 4 - 7 1 7 . 8 2 0 . 6 1 . 1 6
T a b l e 4 , 6 . 1 C o l u m n : A G P
E l u e n t : 5 % O r g a n i c M o d i f i e r i n 0 . 0 1 M p h o s p h a t e b u f f e r
p H 7 . 0
F l o w R a t e : 0 . 9 m l / m i n
I n j e c t i o n V o l u m e : 2 0 p i
D e t e c t i o n : U V 2 2 5 n m
h o w e v e r , t h e r e s u l t s a c h i e v e d w i t h t h i s p h a s e a r e t h e m a s t p r o m i s i n g o f
t h e e n t i r e s t u d y , a n d i n d i c a t e t h a t e n a n t i o s e l e c t i v e r e s o l u t i o n s a r e
p o s s i b l e w i t h o u t t h e n e e d f o r p r e c o l u m n d e r i v a t i s a t i o n p r o v i d e d t h e
c o r r e c t c h o i c e o f c h r o m a t o g r a p h i c c o n d i t i o n s i s m a d e .
T h e m a j o r p r o b l e m w i t h t h e s e p a r a t i o n s a c h i e v e d i n t h i s i n v e s t i g a t i o n
d o e s n o t i n f a c t l i e w i t h t h e r e s o l u t i o n o f t h e e n a n t i o m e r s b u t r a t h e r
w i t h t h e e f f i c i e n c y o f t h e s e p a r a t i o n , A l t h o u g h n o t t e s t e d w i t h a
s t a n d a r d t e s t m i x t u r e , t h e e f f i c i e n c y o f t h e c o l u m n i s f a i r l y l a w , a n d t h e
s a m p l e p e a k s a r e g e n e r a l l y m u c h b r o a d e r t h a n i s c o n s i d e r e d i d e a l . T h i s
c o u l d h a v e a m a j o r i m p a c t o n t h e d e t e c t i o n l i m i t s o f a n y a s s a y r u n o n
t h i s p h a s e s i n c e , a s t h e c o n c e n t r a t i o n i s r e d u c e d , t h e b r o a d p e a k s w i l l
r a p i d l y b e c o m e i n d i s t i n g u i s h a b l e f r o m t h e b a s e l i n e . U n l e s s t h i s c a n b e
o v e r c o m e b y t h e u s e o f m o b i l e p h a s e a d d i t i v e s o r p H c o n t r o l , t h e
a p p l i c a t i o n o f t h e p h a s e t o p h a r m a c o l o g i c a l a n a l y s i s , w h e r e t h e s a m p l e
c o n c e n t r a t i o n i s g e n e r a l l y v e r y l o w , i s l i m i t e d .
I n a d d i t i o n t o t h i s , t h e i n j e c t i o n o f b i o l o g i c a l e x t r a c t s o n t o t h e c o l u m n
c o u l d f u r t h e r r e d u c e t h e c o l u m n l i f e t i n e u n l e s s e x t e n s i v e c l e a n u p
p r o c e d u r e s w e r e u s e d . U n f o r t u n a t e l y , t h e c o l u m n d e g r a d a t i o n p r e v e n t e d t h e
i n v e s t i g a t i o n o f a n a l y t e s e x t r a c t e d f r o m p l a s m a ,
Resolution of Atenolol on an oh Acid Glycoprotein Phase
i i
F i g u r e 4 . 6 . 1 a C o l u m n : A G P
E l u e n t : Q . 0 1 M p h o s p h a t e b u f f e r , p H 7 . 0
F l o v / R a t e : 0 . 9 m l / m i n
D e t e c t i o n ; U V 2 2 5 n m
I n j e c t i o n V o l u m e : 2 0 p i
- 215 -
Resolution of Alprenolol on an odi Acid Glycoprotein Phase
JU Li
3 0
F i g u r e 4 . 6 . 1 b C o l u m n : A G P
E l u e n t : 5 % i s o p r o p a n o l i n 0 . 0 1 M
p h o s p h a t e b u f f e r , p H 7 . 0
F l o w R a t e : 0 . 9 m l / m i n
D e t e c t i o n : U V 2 2 5 n m
I n j e c t i o n V o l u m e : 2 0 p i
- 216 -
Resolution of Metoprolol on an oci Acid Glycoprotein Phase
I S
F i g u r e 4 . 6 . 1 c C o l u m n : A G P
E l u e n t : 0 . 0 1 M p h o s p h a t e b u f f e r , p H 7 . 0
F l o w R a t e : 0 . 9 m l / m i n
D e t e c t i o n : U V 2 2 5 n r a
I n j e c t i o n V o l u m e : 2 0 p i
- 217 -
Resolution of Oxprenolol on an a - \ Acid Glycoprotein Phase
a o o
F i g u r e 4 . 6 . I d C o l u m n : A G P
E l u e n t : 1 0 % a c e t o n i t r i l e i n 0 . 0 1 M
p h o s p h a t e b u f f e r , p H 7 . 0
F l o w R a t e : 0 . 9 m l / m i n
D e t e c t i o n : U V 2 2 5 n m
I n j e c t i o n V o l u m e : 2 0 p i
- 218 -
Resolution of Propranolol on an cct Acid Glycoprotein Phase
F i g u r e 4 . 6 . l e C o l u m n : A G P
E l u e n t : 5 % i s o p r o p a n o l i n 0 , 0 1 M
p h o s p h a t e b u f f e r , p H 7 , 0
F l o w R a t e : 0 . 9 m l / m i n
D e t e c t i o n : U V 2 2 5 n m
I n j e c t i o n V o l u m e : 2 0 p i
- 219 -
Resolution of Verapamil on an oc-i Acid Glycoprotein Phase
IS
F i g u r e 4 . 6 , I f C o l u m n : A G P
E l u e n t : 1 0 % a c e t o n i t r i l e i n 0 , 0 1 1
p h o s p h a t e b u f f e r , p H 7 . 0
F l o w R a t e : 0 . 9 m l / m i n
D e t e c t i o n : U V 2 2 5 n r a
I n j e c t i o n V o l u m e : 2 0 p i
CHAPTER 5
D I S C U S S I O N
I n s p i t e o f a n o b v i o u s n e e d f o r d e v e l o p i n g m e t h o d o l o g y f o r
e n a n t i o s e l e c t i v e r e s o l u t i o n s f o r a w i d e r a n g e o f c h i r a l c o m p o u n d s , t h e r e
i s s t i l l m u c h w o r k t o b e d o n e b e f o r e s u c h a n a l y s i s i s c a r r i e d o u t i n
r o u t i n e d r u g m o n i t o r i n g a n d b i o a v a i l a b i l i t y s t u d i e s .
A l t h o u g h s e p a r a t i o n s w e r e a c h i e v e d w i t h a n u m b e r o f t h e d i f f e r e n t
a p p r o a c h e s t a k e n i n t h i s s t u d y , i t i s u n f o r t u n a t e l y s t i l l t h e c a s e t h a t
t h e c h o i c e o f p h a s e m u s t b e c a r e f u l l y t a i l o r e d t o t h e c o m p o u n d o f
i n t e r e s t a n d t h e t y p e o f a n a l y s i s r e q u i r e d , s i n c e a s y e t t h e r e i s n o t h i n g
e v e n r e m o t e l y r e s e m b l i n g a u n i v e r s a l l y a p p l i c a b l e p h a s e
O f a l l t h e m e t h o d s i n v e s t i g a t e d i n t h i s s t u d y , t h e A G P c o l u m n p r o v i d e s
t h e b e s t r e s o l u t i o n s a n d , i n t h e c a s e o f t h e b e t a b l o c k e r s , d o e s n o t
r e q u i r e a n y f o r m o f p r e c o l u m n d e r i v a t i s a t i o n . H o w e v e r t h e e f f i c i e n c y i s
l a w , l e a d i n g t o b r o a d p e a k s w h i c h w i l l u l t i m a t e l y l i m i t t h e d e t e c t i o n
s e n s i t i v i t y , a n d t h e c o s t o f t h e c o l u m n i s h i g h i n c o m p a r i s o n t o m o s t o f
t h e o t h e r c o m m e r c i a l l y a v a i l a b l e p h a s e s . T h e c o l u m n l i f e t i m e h a s s t i l l
n o t b e e n r e l i a b l y d e t e r m i n e d , e s p e c i a l l y w h e n d e a l i n g w i t h r e l a t i v e l y
d i r t y s a m p l e s , s u c h a s p l a s m a e x t r a c t s , o n a r o u t i n e b a s i s , a n d t h i s m a y
b e a n o t h e r d r a w b a c k t o t h e w i d e s p r e a d u s e o f t h i s p h a s e .
't
T h e 3 , 5 - d i n i t r o b e n z o y l p h e n y l g l y c i n e p h a s e a l s o g a v e g o o d s e p a r a t i o n s f o r
t h e b e t a b l o c k e r s , a l t h o u g h n o t w i t h o u t t h e p r i o r f o r m a t i o n o f a s u i t a b l e
d e r i v a t i v e . S u c h d e r i v a t i s a t i o n s s e r v e t o i n c r e a s e t h e t i m e , c o s t a n d
p r e c i s i o n o f t h e a s s a y a n d c o u l d a f f e c t t h e r e l a t i v e p r o p o r t i o n s o f e a c h
e n a n t i o m e r b y a l l o w i n g i n t e r c a n v e r s i o n t o o c c u r . T h e d e t e c t i o n l i m i t s
a c h i e v e d f o r t h e r e s o l u t i o n o f t h e b e t a b l o c k e r s b y t h e f o r m a t i o n o f
n a p h t h a m i d e d e r i v a t i v e s w e r e a l s o a d v e r s l y a f f e c t e d b y a n i m p u r i t y ,
p r o b a b l y i n t r o d u c e d b y t h e d e r i v a t i s a t i o n p r o c e s s .
T h e c y c l o d e x t r i n p h a s e s t i l l r e m a i n s o n e o f t h e b e s t a p p r o a c h e s t o c h i r a l
r e s o l u t i o n i f o n l y t h e e f f i c i e n c y a n d m a s s t r a n s f e r c h a r a c t e r i s t i c s c o u l d
b e i m p r o v e d t o a m o r e a c c e p t a b l e l e v e l . T h e r e l a t i v e l y l o w c o s t o f t h e
p h a s e , t o g e t h e r w i t h i t s c o r a p a t a b i l i t y w i t h r e v e r s e p h a s e s o l v e n t s w o u l d
m a k e t h e c o l u m n a t t r a c t i v e f o r r o u t i n e a n a l y s i s , p a r t i c u l a r l y s i n c e
p r e c o l u m n d e r i v a t i s a t i o n , a l t h o u g h s u c c e s s f u l l y u s e d i n t h i s s t u d y , s h o u l d
n o t g e n e r a l l y b e n e c e s s a r y ,
T h e C h i r a s i l - V a l G L C p h a s e i s l i m i t e d i n i t s a p p l i c a t i o n b y i t s m a x i m u m
o p e r a t i n g t e m p e r a t u r e . E v e n a c c e p t i n g t h a t s o m e f o r m o f d e r i v a t i s a t i o n
i s n e c e s s a r y f o r g a s c h r o m a t o g r a p h y , i t i s s t i l l d i f f i c u l t t o i n c r e a s e t h e
v o l a t i l i t y o f l a r g e m o l e c u l e s l i k e t h e b e t a b l o c k e r s t o s u c h a n e x t e n t a s
t o e l u t e t h e m f r o m a p o l a r p h a s e a t t e m p e r a t u r e s l e s s t h a n 2 0 0 ’ C , I t
m u s t h o w e v e r b e a p p r e c i a t e d t h a t , w h i l e n o t s u i t a b l e f o r s u c h l a r g e
c o m p o u n d s , t h e c o l u m n g a v e e x c e l l e n t r e s o l u t i o n s o f t h e s m a l l e r a m i n o
a c i d s i n t h e t e s t m i x t u r e , w h i c h w e r e d e r i v a t i s e d p r i m a r i l y t o r e d u c e
t h e i r p o l a r i t y .
O f a l l t h e m e t h o d s i n v e s t i g a t e d , t h e u s e o f a ( + ) ~ 1 0 ~ c a r a p h o r s u l p h o n i c
a c i d m o b i l e p h a s e a d d i t i v e p r o v e d t o b e t h e l e a s t s u c c e s s f u l , a n d n o
r e s o l u t i o n s w e r e a c h i e v e d u s i n g t h i s a p p r o a c h . W h i l e i n t h e o r y t h e
m e t h o d i s r e l a t i v e l y e a s y a n d i n e x p e n s i v e , s i m p l y u s i n g a n a c h i r a l ,
n o r m a l p h a s e c o l u m n , s e p a r a t i o n s a r e s i n g u l a r l y d i f f i c u l t t o a c h i e v e s i n c e
t h e f a c t o r s w h i c h c o n t r o l t h e r e t e n t i o n a r e d i f f i c u l t t o r e g u l a t e
e f f e c t i v e l y .
A l t h o u g h t h i s w o r k d e a l s w i t h a s e l e c t i o n f r o m t h e r a n g e o f a v a i l a b l e
m e t h o d o l o g y , t h e s c o p e o f t h i s s t u d y i s s u c h t h a t m a n y h a v e b e e n o m i t t e d .
T h e n u m b e r o f c o m m e r c i a l l y a v a i l a b l e H P L C c o l u m n s i s r a p i d l y i n c r e a s i n g ,
a n d w h i l e t h e g e n e r a l c l a s s i f i c a t i o n i n t o V a i n e r ’ s p r e d e f i n e d f o u r g r o u p s
i s s t i l l a p p l i c a b l e , t h e v a r i e t y w i t h i n e a c h t y p e i s e x t e n s i v e .
A d d i t i o n a l G L C c o l u m n s a r e n o w o n t h e m a r k e t , i n c l u d i n g t h e r a n g e b a s e d
o n d e r i v a t i s e d c y c l a d e x t r i n , a l t h o u g h a s y e t , s i g n i f i c a n t i n c r e a s e s i n t h e
m a x i m u m o p e r a t i n g t e m p e r a t u r e s h a v e n o t b e e n a c h i e v e d .
T h e u s e o f m o b i l e p h a s e a d d i t i v e s m a y y e t p r o v e t o b e a r e a l i s t i c
a p p r o a c h a n d w o u l d m e r i t f u r t h e r i n v e s t i g a t i o n . T h e r a n g e o f p a s s i b l e
a d d i t i v e s i s e x t e n s i v e a n d i n c l u d e s m a n y o f t h e c h i r a l s e l e c t o r s u s e d a s
p h a s e s i n H P L C , s u c h a s c y c l o d e x t r i n , 3 , 5 - d i n i t r o b e n s o y l p h e n y l g l y c i n e a n d
A G P . I n a d d i t i o n t o a c h i e v i n g a r e s o l u t i o n , t h i s a p p r o a c h c a n a l s o b e
u s e d t o o b t a i n f u r t h e r i n f o r m a t i o n o n t h e s e p a r a t i o n m e c h a n i s m b y
v a r y i n g f a c t o r s s u c h a s t h e a d d i t i v e c o n c e n t r a t i o n , w h i c h a r e f i x e d
p a r a m e t e r s w h e n u s i n g a s i m i l a r H P L C p h a s e .
T h e t y p e o f a n a l y s i s a l s o h a s a b e a r i n g o n t h e c h o i c e o f m e t h o d < I f t h e
a n a l y s i s i s b e i n g p e r f o r m e d t o c h e c k t h e e n a n t i o m e r i c p u r i t y o f a
c o m p o u n d , t h e n i t b e c o m e s i m p o r t a n t t o b e a b l e t o c o n t r o l t h e e l u t i o n
- 223 -
o r d e r o f t h e e n a n t i o m e r s . I f t h e s a m p l e c o n s i s t s o f a s m a l l a m o u n t o f
o n e e n a n t i o m e r i n t h e p r e s e n c e o f a n e x c e s s o f t h e o t h e r , t h e n t h e f o r m e r
w i l l b e m e a s u r a b l e o n l y i f i t e l u t e s f i r s t . I f t h i s i s n o t t h e c a s e , t h e n
t h e s m a l l e r p e a k w i l l b e s w a m p e d b y t h e t a i l i n g o f t h e l a r g e r p e a k .
C h i r a l s e l e c t o r s w h i c h c o n s i s t o f s i n g l e e n a n t i o m e r s , s u c h a s t h e " P i r k l e "
p h a s e s a n d C h i r a s i l - V a l , c a n b e o b t a i n e d a s e i t h e r e n a n t i o m e r w h i c h
a l l o w s t h e e l u t i o n o r d e r t o b e r e v e r s e d , w h e r e a s w i t h c y c l o d e x t r i n o r
p r o t e i n p h a s e s t h i s r e v e r s a l i s n o t p a s s i b l e .
T h i s v a s t v a r i a t i o n h i g h l i g h t s t h e u s e f u l n e s s o f a s y s t e m w h i c h c o u l d b e
u s e d t o p r e d i c t t h e s u c c e s s o f e a c h a p p r o a c h f o r t h e r e s o l u t i o n o f a
g i v e n c o m p o u n d , w i t h o u t t h e n e e d t o t r y e a c h m e t h o d o r p h a s e
e x p e r i m e n t a l l y . O n c e t h e c o m p o u n d o f i n t e r e s t h a d b e e n s p e c i f i e d , s u c h a
s y s t e m w o u l d i d e a l l y i d e n t i f y o n e o r t w o c h i r a l s e l e c t o r s w h i c h w o u l d
p r o v i d e t h e b e s t r e s o l u t i o n s . T h i s w o u l d r e d u c e t h e t i m e s p e n t i n m e t h o d
d e v e l o p m e n t s t u d i e s a n d a l s o a l l e v i a t e t h e c o s t o f o b t a i n i n g a n u m b e r o f
e x p e n s i v e p h a s e s w h i c h m a y n o t b e s u i t e d t o t h e r e s o l u t i o n .
I t w a s f o r t h i s r e a s o n t h a t c o m p u t e r m o d e l l i n g s t u d i e s w e r e c a r r i e d o u t
a n d c o m p a r e d w i t h t h e e x p e r i m e n t a l l y d e t e r m i n e d r e s u l t s . I t w a s h o p e d
t h a t , i f s u c c e s s f u l , a d a t a b a s e c o n t a i n i n g m o d e l s o f a n u m b e r o f c h i r a l
s e l e c t o r s c o u l d b e e s t a b l i s h e d a n d , a s a n d w h e n n e c e s s a r y , i n d i v i d u a l
r a c e m i c c o m p o u n d s c o u l d b e m o d e l l e d a g a i n s t e a c h s e l e c t o r t o a s c e r t a i n
w h i c h p h a s e w o u l d p r o v i d e s u f f i c i e n t s i t e s o f i n t e r a c t i o n t o e n a b l e
c h i r a l r e s o l u t i o n t o o c c u r . H o w e v e r , t h e i n v e s t i g a t i o n s h o w e d t h a t t h i s
s i m p l i s t i c a p p r o a c h d i d n o t c o r r e l a t e w i t h t h e e x p e r i m e n t a l f i n d i n g s a n d ,
e v e n u s i n g t h i s s y s t e m , t h e l e n g t h o f t i m e r e q u i r e d t o s e t u p e a c h
- 224 -
m o l e c u l a r m o d e l i s e x t e n s i v e . T h i s m a k e s t h e i d e a o f e s t a b l i s h i n g a m o r e
r e a l i s t i c s y s t e m , i n c l u d i n g f a c t o r s s u c h a s t h e m o b i l e p h a s e a n d s i l i c a
s u p p o r t f o r e a c h p h a s e m o r e c o s t l y a n d t i m e c o n s u m i n g t h a n p e r f o r m i n g
e x p e r i m e n t a l m e t h o d d e v e l o p m e n t s t u d i e s , p a r t i c u l a r l y s i n c e t h e p r o t e i n
p h a s e s s u c h a s A G P w o u l d b e c o n s i d e r a b l y m o r e d i f f i c u l t t o m o d e l t h a n
t h e s i m p l e 3 , 5 - d i n i t o b e n z o y l p h e n y l g l y c i n e m o l e c u l e .
T h e f u t u r e t r e n d s i n c h i r a l s e p a r a t i o n s a r e d i f f i c u l t t o p r e d i c t ; t h e
r a n g e o f c o m m e r c i a l l y a v a i l a b l e c o l u m n s , b o t h f o r H P L C a n d G L C , i s
c o n t i n u a l l y i n c r e a s i n g , w i t h a n u m b e r o f p h a s e s b e i n g t a i l o r e d t o v e r y
s p e c i f i c s a m p l e t y p e s . W h i l e t h i s i s a c c e p t a b l e i n c a s e s w h e r e
l a b o r a t o r i e s r e g u l a r l y p e r f o r m a n a l y s i s o n a l i m i t e d n u m b e r o f s a m p l e
t y p e s , w h e r e w i d e r r a n g e s o f s a m p l e s a r e h a n d l e d , i t d o e s n o t h e l p t h e
p r o b l e m o f w h i c h p h a s e t o u s e . T h i s r a t i o n a l e a l s o h a s a n u m b e r o f
f i n a n c i a l i m p l i c a t i o n s , s i n c e v e r y s p e c i f i c c o l u m n s c a n o n l y e x p e c t t o
a c h i e v e l i m i t e d s a l e s f i g u r e s a n d t h e r e f o r e t h e c o s t t o t h e c u s t o m e r
r e m a i n s h i g h . I f a m o r e u n i v e r s a l p h a s e c o u l d b e f o u n d , e q u i v a l e n t f o r
e x a m p l e , t o t h e a c h i r a l O D S c o l u m n s , t h e d e v e l o p m e n t c o s t s c o u l d t h e n b e
s h a r e d o v e r a m u c h w i d e r m a r k e t , t h u s l o w e r i n g t h e p r i c e o f i n d i v i d u a l
c o l u m n s .
T h e u s e o f c o l u m n s w i t c h i n g t e c h n i q u e s f o r H P L C m a y a l s o b e c o m e m o r e
w i d e s p r e a d , i n v o l v i n g b o t h c h i r a l a n d a c h i r a l c o l u m n s . T h e s a m p l e i s
i n i t i a l l y i n j e c t e d o n t o a n a c h i r a l c o l u m n w h i c h s e p a r a t e s t h e c o m p o u n d o f
i n t e r e s t f r o m a n y o t h e r i m p u r i t i e s a n d a u t o m a t e d s w i t c h i n g m e c h a n i s m s
d i v e r t t h e e l u e n t f l o w t o a c h i r a l p h a s e a t t h e p o i n t o x e l u t i o n o f t h e
s o l u t e . I n t h i s w a y t h e m o r e e x p e n s i v e c h i r a l c o l u m n i s o n l y u s e d t o
s e p a r a t e t h e t w o e n a n t i o m e r s a n d i s p r o t e c t e d t o a l a r g e e x t e n t f r o m
i m p u r i t i e s f r o m t h e s a m p l e m a t r i x . T h u s t h e l i f e o f t h e c h i r a l p h a s e i s
e x t e n d e d a n d i n m a n y c a s e s r e s o l u t i o n m a y b e i m p r o v e d . P r o b l e m s w i t h
t h i s t e c h n i q u e m a y a r i s e w i t h t h e s o l v e n t c o m p a t a b i l i t y o f t h e t w o
c o l u m n s , a n d a l s o t h e r e s o l u t i o n i s s t i l l d e p e n d e n t o n t h e c o r r e c t c h o i c e
o f c h i r a l p h a s e .
A n o t h e r a l t e r n a t i v e t o t h i s i s t o u s e a n a c h i r a l c o l u m n f o r t h e a n a l y s i s
t o g e t h e r w i t h a c h i r a l d e t e c t o r t o p r o v i d e e n a n t i o m e r i c q u a n t i t a t i o n .
S u c h d e t e c t o r s a r e a l r e a d y c o m m e r c i a l l y a v a i l a b l e a n d a r e g e n e r a l l y u s e d
i n s e r i e s w i t h a U V d e t e c t o r , T h e t o t a l c o n c e n t r a t i o n i s d e t e r m i n e d b y
t h e U V s p e c t r o p h o t o m e t e r , w h i l e t h e c h i r a l d e t e c t o r a l l o w s t h e
e n a n t i o m e r i c r a t i o t o b e d e t e r m i n e d . A l t h o u g h s u c h d e t e c t o r s , i n t h e o r y ,
p r o v i d e t h e i d e a l s o l u t i o n t o c h i r a l a n a l y s i s , d o u b t s h a v e a r i s e n
c o n c e r n i n g t h e s e n s i t i v i t y . H o w e v e r , c o n t i n u e d d e v e l o p m e n t i n b o t h t h e
f l o w c e l l d e s i g n a n d t h e e l e c t r o n i c c i r c u i t r y s h o u l d i m p r o v e t h i s
s i t u a t i o n ,
T h e u s e o f c a p i l l a r y z o n e e l e c t r o p h o r e s i s h a s a l s o b e g u n t o a t t r a c t
c o n s i d e r a b l e i n t e r e s t a s a s e p a r a t i v e t e c h n i q u e , s i n c e i t p r o d u c e s r a p i d
s e p a r a t i o n s w i t h v e r y h i g h e f f i c i e n c i e s a n d i s t h e r e f o r e p o t e n t i a l l y
u s e f u l f o r c h i r a l r e s o l u t i o n s . T h e t e c h n i q u e i n v o l v e s c a p i l l a r i e s f i l l e d
w i t h a b u f f e r s o l u t i o n , a l o n g w h i c h a p o t e n t i a l i s a p p l i e d . C h a r g e d
s p e c i e s m o v e a l o n g t h e p o t e n t i a l g r a d i e n t a t d i f f e r i n g r a t e s , d e p e n d i n g
o n t h e i r c h a r g e a n d s i z e . E n a n t i o m e r i c r e s o l u t i o n s o f a n u m b e r o f a m i n o
a c i d s h a s b e e n a c h i e v e d b y i n c l u d i n g a c o p p e r c o m p l e x i n t h e b u f f e r
[ 4 7 9 3 , w i t h w h i c h t h e a m i n o a c i d s f o r m d i a s t e r e o m e r i c t e r n a r y c o m p l e x e s ,
- 226 -
c o m p l e x e s . I t i s a l s o p o s s i b l e t o a d d o t h e r c o m p o u n d s t o t h e b u f f e r s ,
i n c l u d i n g m i c e l l e s , a n d h e n c e i t m a y b e p o s s i b l e t o u s e t h e c h i r a l
s e l e c t o r s m o r e c o m m o n l y u s e d a s m o b i l e p h a s e a d d i t i v e s t o s e p a r a t e s a
w i d e r r a n g e o f e n a n t i o m e r s .
W i t h a l l t h e r a n g e o f t e c h n i q u e s a v a i l a b l e , t h e r e s o l u t i o n o f d r u g
e n a n t i o m e r s i s s t i l l a d i f f i c u l t a n d e x p e n s i v e t a s k . A l a r g e n u m b e r o f
t h e c h i r a l s e l e c t o r s , w h e t h e r f o r G L C , H P L C o r m o b i l e p h a s e a d d i t i v e s a r e
o n l y t o t a l l y e f f e c t i v e f o r t h e r e s o l u t i o n o f a m i n o a c i d s a n d a r e n o t w e l l
s u i t e d t o o t h e r c o m p o u n d s . O f t h e t e c h n i q u e s w h i c h c a n b e u s e d f o r d r u g
a n a l y s i s , t h e r e a p p e a r s t o b e i n h e r e n t d i s a d v a n t a g e s f o r e a c h i n d i v i d u a l
m e t h o d .
T h e r e i s a n u r g e n t n e e d f o r s o m e f o r m o f r e l i a b l e g u i d e t o w h i c h m e t h o d s
a r e s u i t e d t o w h i c h c o m p o u n d s , w h i c h h a s b e e n p a r t l y m e t b y a b o o k l e t b y
D r I . W . W a i n e r e n t i t l e d " A P r a c t i c a l G u i d e t o t h e S e l e c t i o n a n d U s e o f
H P L C C h i r a l S t a t i o n a r y P h a s e s " C 4 8 0 L T h i s d e t a i l s t h e s t r u c t u r a l
r e q u i r m e n t s o f s o l u t e s f o r e a c h p h a s e a n d p r o v i d e s a d d i t i o n a l i n f o r m a t i o n
o n s u i t a b l e e l u e n t s , t o g e t h e r w i t h r e f e r e n c e s t o l i t e r a t u r e s e p a r a t i o n s .
H o w e v e r t h e s c o p e o f t h e b o o k i s l i m i t e d t o H P L C s t a t i o n a r y p h a s e s a n d
d o e s n o t a t t e m p t t o i n c l u d e m o b i l e p h a s e a d d i t i v e s o r G L C s t a t i o n a r y
p h a s e s .
and separation is effected by differences in the stability of the pair of
- 227 -
I t i s a l s o t r u e t o s a y t h a t , i n a d d i t i o n t o t h e d i f f i c u l t y o f t e n
e x p e i ' i e n c e d i n r e p r o d u c i n g p u b l i s h e d r e s u l t s , m u c h o f t h e l i t e r a t u r e
r e p o r t s t h e s u c c e s s o f a m e t h o d b a s e d o n i t s a b i l i t y t o r e s o l v e t h e
e n a n t i o m e r s o f a p u r e c o m p o u n d . W h i l e t h i s i s a s t a r t , i t c a n n o t b e
a s s u m e d t h a t t h e m e t h o d w i l l b e e q u a l l y s u c c e s s f u l i n r e s o l v i n g t h e
c o m p o u n d i n a b i o l g i c a l f l u i d , w h e r e t h e r e a r e m a n y o t h e r i n t e r f e r e n c e s
w h i c h a f f e c t t h e b e h a v i o u r o f t h e m e t h o d ,
I n c o n c l u s i o n , i t m u s t b e r e m e m b e r e d t h a t e n a n t i o s e l e c t i v e a n a l y s i s i s
s t i l l a r e l a t i v e l y n e w t e c h n i q u e , a n d w h i l e i t i s b e g i n n i n g t o r e c e i v e a
r e l a t i v e l y h i g h p r i o r i t y , t h e r e i s s t i l l a l o n g w a y t o g o b e f o r e r e a c h i n g
t h e s t a g e w h e r e c h i r a l a n a l y s i s c a n b e , a n d i n d e e d i s p e r f o r m e d o n a
r o u t i n e b a s i s .
S 3
mam
B I B L I O G R A P H Y
N a t t a , G . a n d F a r i n a , M . , " S t e r e o c h e m i s t r y "
L o n g m a n , L o n d o n , ( 1 9 7 2 )
T e s t a , B . , " P r i n c i p l e s o f O r g a n i c S t e r e o c h e m i s t r y "
J I a r c e l D e k k e r , N e w Y o r k , ( 1 9 7 9 )
I C i s l o w , K . , " I n t r o d u c t i o n t o S t e r e o c h e m i s t r y "
B e n j a m i n , N e w Y o r k , ( 1 9 6 6 )
G u n s t o n e , F . B . , " G u i d e b o o k t o S t e r e o c h e m i s t r y "
L o n g m a n , N e w Y o r k , ( 1 9 7 5 )
J a c q u e s , J . , C o l l e t , A . a n d ¥ i l e n , S . H . ,
" E n a n t i o m e r s , R a c e m a t e s a n d R e s o l u t i o n s "
W i l e y I n t e r s c i e n c e , N e w Y o r k , ( 1 9 8 1 )
A r i e n s , E . J . , S o u d i j n , W . a n d T i m m e r m a n s , P . B . J t . W J I . , ( E d s . ) ,
" S t e r e o c h e m i s t r y a n d B i o l o g i c a l A c t i v i t y o f D r u g s "
B l a c k w e l l S c i e n t i f i c , O x f o r d , ( 1 9 8 3 )
G i r d w o o d , R . H . , ( E d . ) , " C l i n i c a l P h a r m a c o l o g y "
Bailliere Tindall, London, (1979)
- 230 -
C h a m b e r l a i n , J . , " A n a l y s i s o f D r u g s i n B i o l o g i c a l F l u i d s "
C R C P r e s s , F l o r i d a , < 1 9 8 5 )
G o r r o d , J . W . a n d B e c k e t t , A . H . , ( E d s . ) , " D r u g M e t a b o l i s m i n M a n "
T a y l o r a n d F r a n c i s , L o n d o n , ( 1 9 7 8 )
H a m i l t o n , R . J . a n d S e w e l l , P . A . , " I n t r o d u c t i o n t o H i g h P e r f o r m a n c e
L i q u i d C h r o m a t o g r a p h y "
C h a p m a n a n d H a l l , N e w Y o r k , ( 1 9 8 2 )
B r a i t h w a i t e , A , a n d S m i t h , F . J . , " C h r o m a t o g r a p h i c M e t h o d s "
C h a p m a n a n d H a l l , N e w Y o r k , ( 1 9 8 5 )
S i m p s o n , C . F . , ( E d . ) , " T e c h n i q u e s i n L i q u i d C h r o m a t o g r a p h y "
W i l e y H e y d e n , C h i c h e s t e r , ( 1 9 8 2 )
B r o w n i n g , D . R . , ( E d . ) " C h r o m a t o g r a p h y "
M c G r a w H i l l , L o n d o n , ( 1 9 6 9 )
A l l e n m a r k , S . G . , " C h r o m a t o g r a p h i c E n a n t i a s e p a r a t i o n ,
M e t h o d s a n d A p p l i c a t i o n s "
E l l i s H o r w o o d L i m i t e d , C h i c h e s t e r , ( 1 9 8 8 )
S o u t e r , R . W . , " C h r o m a t o g r a p h i c S e p a r a t i o n s o f S t e r e o i s o m e r s "
CRC Press, Florida, <1985)
- 231 -
B l a u , K . a n d K i n g , G . S . , ( E d s . ) , " H a n d b o o k o f D e r i v a t i v e s f o r
C h r o m a t o g r a p h y "
H e y d e n , L o n d o n , ( 1 9 7 7 )
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- 234 -
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- 235 -
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