Comparison of separation performances of amylose- and cellulose-based stationary phases in the high-performance liquid chromatographic enantioseparation of stereoisomers of β-lactams

Article abstract of DOI:10.1002/chir.20714

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of 19 β-lactams. The direct separations were performed on chiral stationary phases containing either amylose-tris-3,5- dimethylphenyl carbamate, (Kromasil

Full text of DOI:10.1002/chir.20714

CHIRALITY 22:120–128 (2010)
Comparison of Separation Performances of Amylose- and
Cellulose-Based Stationary Phases in the High-Performance
Liquid Chromatographic Enantioseparation of
Stereoisomers of b-Lactams
1
1
1
2
2
1
´
´
´
´
´
¨ ¨
†
*
ZOLTAN PATAJ, ISTVAN ILISZ, ROBERT BERKECZ, ENIKO FORRO, FERENC FULOP, AND ANTAL PETER
1Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged, Hungary
2Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Hungary
ABSTRACT
High-performance liquid chromatographic methods were developed for
the separation of the enantiomers of 19 b-lactams. The direct separations were per-
formed on chiral stationary phases containing either amylose-tris-3,5-dimethylphenyl
carbamate, (Kromasil1 AmyCoatTM column) or cellulose-tris-3,5-dimethylphenyl carba-
mate, (Kromasil1 CelluCoatTM column) as chiral selector. The different methods were
compared in systematic chromatographic examinations. The separations were carried
out with good selectivity and resolution. The AmyCoatTM and CelluCoatTM columns
appear to be highly complementary. The best separations of bi- and tricyclic b-lactam
stereoisomers were obtained with the AmyCoatTM column, whereas the 4-aryl-substi-
tuted b-lactams were better separated on the CelluCoatTM column. The elution
sequence was determined in all cases; no general rule could be established. Chirality
C
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22:120–128, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: column liquid chromatography; b-lactams; CelluCoatTM column;
AmyCoatTM column
INTRODUCTION
isomers were separated on different types of selectors as
follows: (R)-N-(3,5-dinitrobenzoyl)phenylglycine,13–16 tris-
carbamates of cellulose or amylose polysaccharide,17–22 b-
cyclodextrin,23,24 and macrocyclic glycopeptide.25,26 Jiang
et al.27 applied b-cyclodextrin-based capillary electrophore-
sis for the separation of the stereoisomers of b-lactam.
In this article, direct HPLC methods are described for
the enantioseparation of racemic b-lactam stereoisomers
(for the structures, see Fig. 1). The direct HPLC methods
rely on the use of amylose-tris-3,5-dimethylphenyl carba-
mate or cellulose-tris-3,5-dimethylphenyl carbamate-based
CSPs. For comparison purposes, most of the separations
were carried out at constant mobile phase compositions
and the separation performances of the amylose- and cellu-
lose-based CSPs were compared. The experimental data
allow a discussion of the influence of the mobile phase
composition and specific structural features of the analytes
on the retention and chiral recognition. The sequence of
elution of the enantiomers was determined by the cochro-
b-Lactams or penams have been widely used in a variety
of applications in a number of scientific areas, but espe-
cially in medicinal and synthetic chemistry. As the most
commonly used group of antibiotics currently available, b-
lactams function as lethal inhibitors of the growth of the
cell walls of pathogenic bacteria.1,2 Monocyclic b-lactams
can be used as antibiotic agents (e.g., monobactams), and
they are used as intermediates in the synthesis of b-amino
acids,3 short peptide segments,4 taxoid antitumor agents,5
alkaloids,6 and different heterocycles of biological and
medicinal interest.7 N-Peptidyl-substituted 2-azetidinones
obtained from 4-phenyl-2-azetidinone (4-aryl-substituted b-
lactams) were earlier evaluated as inhibitors of serine pro-
tease elastase and cysteine protease papain.8 A facile trans-
formation of 3,4-disubstituted 2-azetidinones to chiral 5,6-
dihydro-2-pyridones, which can serve as valuable chiral
intermediates for different piperidine and indolizidine alka-
loids and azasugars, was reported by Lee et al.9 Various
direct and indirect enzymatic10,11 and enantioselective syn-
theses12 of b-lactams have been described.
Since the biological activity of a b-lactam depends
strongly on its stereochemistry, there is a clear need for
analytical methods by which their enantiomeric purities
can be determined. High-performance liquid chromatogra-
phy (HPLC) on chiral stationary phases (CSPs) is an effec-
tive analytical tool for the resolution of chiral compounds
Contract grant sponsor: Hungarian National Science Foundation;
Contract grant number: K 67563.
*Correspondence to: Antal Pe´ter, Department of Inorganic and Analytical
Chemistry, University of Szeged, H-6720 Szeged, Do´m te´r 7, Hungary.
E-mail: apeter@chem.u-szeged.hu
Received for publication 5 December 2008; Accepted 5 February 2009
DOI: 10.1002/chir.20714
Published online 19 May 2009 in Wiley InterScience
on both analytical and preparative scales. b-Lactam stereo- (www.interscience.wiley.com).
C
VV
2009 Wiley-Liss, Inc.

HPLC ENANTIOSEPARATION OF b-LACTAMS
121
azabicyclo[6.2.0]decan-10-one (6), cis-9-azabicyclo[6.2.0]-
dec-4-en-10-one (7), cis-3,4-benzo-6-azabicyclo[3.2.0]hep-
tan-7-one (8), cis-4,5-benzo-7-azabicyclo[4.2.0]octan-8-one
(9), cis-5,6-benzo-8-azabicyclo[5.2.0]nonan-9-one (10), exo-
3-azatricyclo[4.2.1.02.5]nonan-4-one (11), and exo-3-azatri-
cyclo[4.2.1.02.5]non-7-en-4-one (12) were prepared in our
laboratory by the cycloaddition of chlorosulfonyl isocya-
nate to the corresponding cycloalkanes or cycloalka-
dienes.12,28 The racemic 4-aryl-substituted b-lactams, 4-
phenyl- (13), 4-(p-tolyl)- (14), 4-(p-fluorophenyl)- (15), 4-
(p-chlorophenyl)- (16), 4-(p-bromophenyl)- (17), 4-(o-
chlorophenyl)- (18), and 4-(m-chlorophenyl)-2-azetidinone
(19) were prepared from styrene or 2-, 3-, or 4-substituted
styrene by chlorosulfonyl isocyanate addition. Enantiopure
b-lactams were synthesized by a very efficient enzymatic
method through catalysis of the enantioselective ring
opening of b-lactams with H2O in diisopropyl ether by Lip-
olase (lipase B from Candida antarctica produced by sub-
merged fermentation of a genetically modified Aspergillus
oryzae microorganism and adsorbed on a macroporous
resin).29–33
n-Hexane, n-heptane, methanol (MeOH), ethanol
(EtOH), 1-propanol (PrOH), 2-propanol (IPA), 1-butanol
(BuOH), tert.-butanol (t-BuOH) of HPLC grade were pur-
chased from Sigma-Aldrich (St. Louis, MO), as were other
reagents of analytical reagent grade.
The eluents were degassed in an ultrasonic bath, and
helium gas was purged through them during the analyses.
Apparatus and Chromatography
The HPLC measurements were carried out on a Waters
HPLC system consisting of an M-600 low-pressure gradi-
ent pump, an M-996 photodiode-array detector, and a Mil-
lenium32 Chromatography Manager data system (Waters
Chromatography, Milford, MA). The chromatographic sys-
tem was equipped with a Rheodyne Model 7125 injector
(Cotati, CA) with a 20-ll loop.
The columns used for direct separations were amylose-
tris-3,5-dimethylphenyl carbamate (Kromasil1 Amy-
CoatTM) or cellulose-tris-3,5-dimethylphenyl carbamate-
Fig. 1. Structures of analytes. cis-6-azabicyclo[3.2.0]heptan-7-one (1),
cis-7-azabicyclo[4.2.0]octan-8-one (2), cis-7-azabicyclo[4.2.0]oct-3-en-8-one
(3), cis-7-azabicyclo[4.2.0]oct-4-en-8-one (4), cis-8-azabicyclo[5.2.0]nonan- based (Kromasil1 CelluCoatTM) CSPs, measuring 150 3
9-one (5), cis-9-azabicyclo[6.2.0]decan-10-one (6), cis-9-azabicyclo[6.2.0]-
dec-4-en-10-one (7), cis-3,4-benzo-6-azabicyclo[3.2.0]heptan-7-one (8), cis-
4.6 mm I.D. with a particle size of 5 lm, both from Eka
Chemicals AB (Bohus, Sweden). The dead-times (t0) of
the columns were determined by injecting 1.0 mM potas-
sium bromide dissolved in IPA. The columns were ther-
mostated in a Spark Mistral column thermostat (Spark
Holland, Emmen, The Netherlands). The precision of tem-
perature adjustment was 60.18C.
4,5-benzo-7-azabicyclo[4.2.0]octan-8-one
(9),
cis-5,6-benzo-8-azabicy-
clo[5.2.0]nonan-9-one (10), exo-3-azatricyclo[4.2.1.02.5]nonan-4-one (11),
exo-3-azatricyclo[4.2.1.02.5]non-7-en-4-one (12), 4-phenyl-2-azetidinone
(13), 4-(p-tolyl)-2-azetidinone (14), 4-(o-chlorophenyl)-2-azetidinone (15),
4-(m-chlorophenyl)-2-azetidinone (16), 4-(p-chlorophenyl)-2-azetidinone
(17), 4-(p-fluorophenyl)-2-azetidinone (18) and 4-(p-bromophenyl)-2-azeti-
dinone (19).
Stock solutions of the analytes (1 mg ml21) were pre-
pared by dissolution in the starting mobile phase.
matography of racemic analytes with enantiomers with
known absolute configurations.
RESULTS AND DISCUSSION
Two CSPs were utilized for the direct enantioseparation
of the 19 b-lactam stereoisomers in this study: the amy-
lose-tris-3,5-dimethylphenyl carbamate-based AmyCoatTM
EXPERIMENTAL
Chemicals and Reagents
The 12 racemic b-lactams, cis-6-azabicyclo[3.2.0]heptan- and cellulose-tris-3,5-dimethylphenyl carbamate-based Cel-
7-one (1), cis-7-azabicyclo[4.2.0]octan-8-one (2), cis-7-azabi- luCoatTM. Both columns were used in the normal-phase
cyclo[4.2.0]oct-3-en-8-one (3), cis-7-azabicyclo[4.2.0]oct-4- mode. Relevant separation data on compounds 1-19,
en-8-one (4), cis-8-azabicyclo[5.2.0]nonan-9-one (5), cis-9- including retention factors (k0), separation factors (a), and
Chirality DOI 10.1002/chir

122
PATAJ ET AL.
TM
Tables 1 and 2 on the CelluCoat
and AmyCoatTM
TABLE 1. Chromatographic data, retention factors (k0),
separation factor (a), resolution (RS), and elution
sequence for the direct separation of the stereoisomers
of b-lactams on CelluCoatTM CSP
CSPs, and in Figure 2 for compounds 2, 9, 11, and 13 on
the AmyCoatTM CSP with the n-heptane/IPA mobile phase
system, k0 strongly decreased with increasing alcohol con-
tent, whereas the changes in a and RS differed. For the b-
lactam analogs, slight changes in a were registered with
increasing IPA content, whereas RS changed in parallel with
k0, i.e., RS in most cases decreased with decreasing k0 (spe-
cial behavior was observed for analyte 2, where RS exhib-
ited a maximum curve as a function of the IPA content).
The concentration of the alcohol exerted a slight effect on
the enantioselectivity, i.e., the ratio of the nonchiral and chi-
ral interactions between the CSP and the analytes depend
only slightly on the concentration of the alcohol (Fig. 2).
The nature of the alcohol greatly influenced the reten-
tion and resolution. Figure 3 reveals that, on the Amy-
CoatTM and CelluCoatTM CSPs for analytes 2, 9, and 13
at constant alcohol concentration (0.39 mol l21 in the n-
hexane/alcohol mobile phase system), the change in k0
did not appear to correlate with the carbon number of the
alcohols. From the six examples (Fig. 3), it can be con-
cluded that MeOH gives smaller k0 on the AmyCoatTM
Mobile
phase
Elution
Analyte
k10
a
RS
sequencea
1
a
b
c
a
b
c
a
b
a
b
c
a
c
a
b
a
b
c
a
a
a
a
a
a
b
a
a
b
a
a
b
c
a
b
a
b
1.28
1.71
4.96
1.54
2.10
4.82
2.11
3.26
1.71
2.63
5.92
1.63
4.79
1.54
2.58
1.72
2.79
6.40
2.50
2.23
2.50
1.24
1.47
3.30
5.72
2.55
3.03
6.13
3.33
3.64
7.40
11.94
3.67
6.74
3.96
7.75
1.00
1.00
1.00
1.05
1.05
1.06
1.09
1.09
1.00
1.00
1.02
1.00
1.03
1.05
1.00
1.04
1.00
1.00
1.31
1.11
1.52
1.15
1.13
1.00
1.05
1.20
1.10
1.11
1.12
1.00
1.04
1.07
1.06
1.09
1.06
1.09
0.00
0.00
–
–
–
0.00
b
2
1R,6S
1R,6S
1R,6S
1S,6R
1S,6R
–
–
1S,6R
–
1R,7S
1R,5S
–
n.d.
–
–
b
b
3
4
1.15
0.95
0.00
0.00
b
5
6
7
0.00
b
b
0.00
0.30
0.00
0.00
4.00
1.45
7.40
1.55
1.50
0.00
1.30
2.80
1.40
1.85
1.85
0.00
0.65
1.45
1.15
1.60
0.90
1.45
8
9
10
11
12
13
1S,5S
1R,6R
1R,7R
TABLE 2. Chromatographic data, retention factors (k0),
separation factor (a), resolution (RS) and elution
sequence for the direct separation of the stereoisomers
of b-lactams on AmyCoatTM CSP
1S,2R,5S,6R
1R,2R,5S,6S
–
R
R
R
R
S
–
R
R
R
R
R
R
Mobile
phase
Elution
Analyte
k10
a
RS
sequencea
14
15
1
a
b
c
a
d
a
a
a
a
a
a
a
a
a
c
a
a
c
a
c
a
a
b
a
a
a
1.65
2.81
6.74
1.74
9.82
1.97
1.74
1.81
1.49
1.66
2.46
2.39
3.79
2.00
7.58
1.95
2.40
7.55
2.21
7.75
2.01
2.33
4.97
2.55
2.43
2.60
1.24
1.34
1.21
1.00
1.06
1.09
1.13
1.14
1.40
1.25
1.98
1.30
1.56
1.02
1.09
1.07
1.02
1.08
1.05
1.04
1.21
1.00
1.04
1.13
1.09
1.15
2.40
3.50
2.60
0.00
0.85
0.90
1.50
1.45
3.75
2.70
11.3
3.80
1R,5S
1R,5S
1R,5S
–
1R,6S
1S,6R
1S,6R
1S,7R
1S,8R
1S,6R
1S,5S
1S,6S
1S,7S
–
16
17
2
3
4
5
6
7
8
9
10
11
18
19
Column, CelluCoatTM; mobile phase, a. n-heptane/IPA 5 90/10 (v/v), b.
n-heptane/IPA 5 95/5 (v/v), c. n-heptane/IPA 5 97/3 (v/v); flow rate,
0.5 ml min21; detection, 205 nm; temperature, ambient; t0, 3.88 min.
aConfiguration of the first-eluted enantiomer.
8.65
b
1.65
1S,2R,5S,6R
1S,2S,5R,6R
bSign of separation.
12
13
0.80
b
–
R
R
R
S
–
R
R
R
R
1.25
0.45
0.75
2.75
0.00
0.65
1.80
1.25
2.15
resolutions (RS) for each analyte with several mobile
phases are given in Tables 1 and 2. On the CelluCoatTM
column with a mobile phase of n-heptane/IPA (90/10 (v/
v), the stereoisomers of all the analytes were retained, but
only slight or no enantioseparation was observed for the
analytes of 1, 2, 4, 5-7, 13, and 17 (Table 1). On Amy-
CoatTM with the same mobile phase, the stereoisomers of
all the analytes were retained again and most of the stereo-
isomers were baseline separated. (The only exceptions
were 2, 11, 13, 14, and 16, Table 2.) However, by
changing the mobile phase composition most of the analy-
tes were baseline separated on both CSPs.
14
15
16
17
18
19
Column, AmyCoatTM; mobile phase, a. n-heptane/IPA 5 90/10 (v/v), b.
n-heptane/IPA 5 95/5 (v/v), c. n-heptane/IPA 5 97/3 (v/v), d. n-hep-
tane/IPA 5 98/2 (v/v); flow rate, 0.5 ml min21; detection, 205 nm; tem-
perature, ambient; t0, 3.90 min.
aConfiguration of the first-eluting enantiomer.
The alcohol content of the mobile phase strongly influ-
enced the chromatographic behavior. As can be seen in
Chirality DOI 10.1002/chir
bSign of separation.

HPLC ENANTIOSEPARATION OF b-LACTAMS
123
Fig. 2. Effects of IPA content on retention factor of first-eluting enantiomer (k0I ), separation factor (a) and resolution (RS) for analogs 2, 9, 11, and
13 on AmyCoatTM CSP. Chromatographic conditions: mobile phase, n-hexane/IPA 5 98/2–80/20 (v/v); flow rate, 0.5 ml min21; detection, 205 nm, (n)
k0; (l) a; (~) RS.
CSP and higher k0 on the CelluCoatTM CSP. The enantio-
For most of the analytes investigated, a structure-reten-
selectivity did not change dramatically when the different tion relationship was observed on both stationary phases.
alcohols were applied in the same molar concentration. With increasing number of carbon atoms attached to the
Wang and Winslow34 found that the changes caused in b-lactam ring (analytes 1-7), k0 usually increased (Tables
the CSP structure by the different alcohols may affect the 1 and 2). This increase in the normal-phase mode can be
chiral selectivity of the CSP, depending on the size and attributed to the increased steric effect (bulkiness) of the
structure of the analyte. The influence of the nature of analytes. For analytes 11 and 12, the k0 values on the Cel-
alcohol on resolution exhibited a large effect but no gen- luCoatTM CSP were somewhat lower, whereas and on the
eral rule could be established. When separation was AmyCoatTM CSP, they were somewhat higher than that of
observed sometimes, the application of alcohols with analyte 5, which also contained a seven-membered con-
bulky and branched side-chains, such as PrOH, IPA, densed ring. It is generally recognized that, at the supra-
BuOH, and t-BuOH resulted higher RS but the selectivity molecular level, the lamellar arrangement of the polysac-
seemed to be the same or sometimes even lower. Prob- charide chains provides a multitude of chiral cavities, and
ably the selector swells more in these solvents resulting therefore multiple interaction sites.35 In the case of the
better mass-transfer kinetics and higher RS values.
tris-3,5-dimethylphenyl carbamate-based CSPs, the polar
Chirality DOI 10.1002/chir

124
PATAJ ET AL.
Fig. 3. Effects of the nature of the alcoholic modifier on the retention factor of the first-eluting enantiomer (k0I ), the separation factor (a) and the reso-
lution (RS) for analytes 2, 9, and 13 on AmyCoatTM and CelluCoatTM columns. Chromatographic conditions: mobile phase, n-hexane/alcoholic modifier
(see text); alcoholic modifiers, MeOH, EtOH, PrOH, IPA, BuOH and t-BuOH; flow rate, 0.5 ml min21; detection, 205 nm.
carbamate residues are located inside, whereas the hydro- groups and dipole–dipole interactions involving the
phobic aromatic groups are outside the polymer chain. ꢀꢀCO¼¼ moiety. Besides these polar interactions, p–p
The enantiomers can therefore interact with the carbamate interactions between the phenyl groups of the CSP and an
groups via H-bonding with the ꢀꢀNHꢀꢀ and ꢀꢀCO¼¼ aromatic group of the solute may play some role in chiral
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF b-LACTAMS
125
Chirality DOI 10.1002/chir

126
PATAJ ET AL.
Fig. 5. Separation of minor enantiomers of 4, 5, 11, and 12 when it is present in an excess of the major isomer. Chromatographic conditions: col-
umn, AmyCoatTM for 4 and 5, CelluCoatTM for 11 and 12; mobile phase, n-heptane/IPA 5 90/10 (v/v); detection, 205 nm; flow rate, 0.50 ml min21
temperature 258C.
;
recognition. The three-ring systems of 11 and 12 prob- ylose-based CSP are the more favored; probably 11 and
ably fit better sterically into the amylose cavity than into 12 have stronger achiral interactions with AmyCoatTM
the cellulose cavity, and thus the interactions with the am- CSP. However, the chiral recognition and chiral interac-
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF b-LACTAMS
127
tion seems to be more favored on CelluCoatTM CSP
CONCLUSIONS
(Tables 1 and 2). The comparison of the retention behav-
ior of analytes with the same ring number, but not contain-
ing or containing a double bond (i.e., 2 vs. 3 and 4; 6 vs.
7; and 11 vs. 12), or the comparison of analytes not con-
densed with or condensed with an aromatic ring (1 vs. 8;
2 vs. 9; and 5 vs. 10) drew attention to the importance of
p–p interactions in the retention. Introduction of a double
bond or an aromatic ring into the molecules increased the
k0, and this increase in most cases (especially for the con-
densed aromatic ring) being accompanied by increases in
selectivity and resolution (Tables 1 and 2).
HPLC methods were developed for the separation of the
enantiomers of 19 b-lactams. The direct separations were
performed on CSPs containing either amylose-tris-3,5-
dimethylphenyl carbamate (AmyCoatTM column) or cellu-
lose-tris-3,5-dimethylphenyl carbamate (CelluCoatTM col-
umn) as chiral selector. By variation of the chromato-
graphic parameters, the separation of the stereoisomers
was optimized; as a result, baseline resolution was
achieved for the b-lactams in at least one chromatographic
system. The AmyCoatTM and CelluCoatTM columns appear
to be highly complementary. The elution sequence was
determined in all cases.
The largest k0 values of 8-10 and especially 13-19 in
the mobile phase n-heptane/IPA 5 90/10 (v/v) are possi-
bly due to the enhanced p–p interactions between the
phenyl ring of the analytes and the 3,5-dimethylphenyl
ring of the selector (Tables 1 and 2). A further tendency
could be observed for analytes 15, 18, and 19. At con-
stant mobile phase composition, k0 increased in the
sequence fluorine-chlorine-bromine. The size and polariz-
ability of the halogen substituted molecules increase in
the sequence fluorine-chlorine-bromine. Probably the po-
lar interaction between the CSP and the molecule
increases when fluorine was substituted by chlorine or
bromine resulting in larger k0. The larger size of the ana-
lyte may contribute to the retention by the increased
steric effect (bulkiness) as was observed for analytes 1-7
with increasing number of carbon atoms attached to the
b-lactam ring. The position of the chloro substituent in
analytes 16, 17, and 18 exerted marked effects on the
chromatographic behavior. At constant mobile phase
composition, the k0 values for these analytes were similar
on the CelluCoatTM and AmyCoatTM CSPs, but as con-
cerns chiral recognition the para-chloro substituted ana-
log was separated on both CSPs with similar enantiose-
lectivity, whereas for the orto-chloro substituted analog
CelluCoatTM CSP and for meta-chloro substituted analog
AmyCoatTM CSP proved to be much more efficient
(Tables 1 and 2).
In all cases when separation occurred, the sequence of
elution was determined, but no general rule could be
established for the stereoisomers of the b-lactams on ei-
ther the amylose- or the cellulose-based phase; interest-
ingly, a difference in elution sequence between Amy-
CoatTM and CelluCoatTM CSPs was observed for analytes
5, 6, 9, 10, and 12. This highlights the importance of
identification of the stereoisomers in each chromato-
graphic run.
Selected chromatograms for the enantioseparation of
analytes 1-19, evaluated on the CelluCoatTM or Amy-
CoatTM CSPs are depicted in Figure 4. Depicted chromato-
grams with RS values RS > 2.0 allow the determination of
enantiomeric impurity even impurity eluted second. If the
separation presented insufficient for enantiomeric purity
assessment resolution can be increased by decreasing of
the eluent strength of the mobile phase or by changing
the type of alcohol and the column (CelluCoatTM or Amy-
CoatTM). Figure 5 depicts separation of minor enantiomers
eluting second of 4, 5, 11, and 12 when it is present in
an excess of the major isomer (LOD < 0.5%).
ACKNOWLEDGMENTS
I.I. wishes to express his thanks for a Bolyai Ja´nos Post-
doctoral Research Scholarship supporting his work. A.P. is
grateful to Eka Chemicals AB (Bohus, Sweden) for the
generous gift of the Kromasil1 columns.
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