A new N-acyl derivative of (S)-cysteine for quantitative determination of enantiomers of amino compounds by HPLC with a precolumn modification with o-phthalaldehyde

Article abstract of DOI:10.1023/B:RUBI.0000043781.72807.f5

N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enantiomers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was compared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctionalized amines and amino alcohols. After the NAC-modification, the resulting diastereomeric isoindoles were difficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols. The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition, allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the amino component and the thiol reagent favored the diastereomer separation. This method was used for determination of the absolute concentration of individual enantiomers of amines in the course of stereoselective enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of penicillin acylase.

Full text of DOI:10.1023/B:RUBI.0000043781.72807.f5

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 5, 2004, pp. 403–408. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 5, 2004, pp. 451–457.
ˇ
Original Russian Text Copyright © 2004 by Guranda, Shapovalova, Svedas.
A New N-Acyl Derivative of (S)-Cysteine
for Quantitative Determination of Enantiomers
of Amino Compounds by HPLC
with a Precolumn Modification with o-Phthalaldehyde
^
1
D. T. Guranda, I. V. Shapovalova, and V. K. Svedas
Department of Bioengineering and Bioinformatics and Belozerskii Institute of Physicochemical Biology,
Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
Received August 25, 2003; in final form, October 29, 2003
Abstract—N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enanti-
omers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was com-
pared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctional-
ized amines and amino alcohols. After the NAC-modification, the resulting diastereomeric isoindoles were dif-
ficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols.
The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition,
allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the
amino component and the thiol reagent favored the diastereomer separation. This method was used for deter-
mination of the absolute concentration of individual enantiomers of amines in the course of stereoselective
enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis
based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of peni-
cillin acylase.
Key words: amines, chemoselective acylation, chiral analysis, penicillin acylase from Alcaligenes faecalis,
thiol reagent
1
INTRODUCTION
N-acyl derivatives of cysteine [2, 6, 11–14], thiosugars
[7, 15], and 3-mercapto-2-methylpropionic acid [5]. A
comparative analysis demonstrated that the structure of
mercapto compound used for the modification of pri-
mary amino groups mainly determines both the diaste-
reoselectivity of separation [2, 5–7, 16] and the stability
of resulting isoindoles [5, 17]. Interpretation of the
experimental results with the application of molecular
The reversed phase HPLC with a precolumn modi-
fication of amino groups by a reagent composed of
Ó-phthalaldehyde and optically active thiol-containing
compound is one of the well-known methods of indi-
rect determination of enantiomers of amino com-
2
pounds. This approach is based on a preliminary mod-
ification of the primary amino groups by the chiral modeling suggests that the mechanism of chiral dis-
reagent and subsequent separation of the resulting dia- crimination of diastereomeric isoindoles is based on the
different conformational rigidity of two diastereomers
determined by intramolecular interactions [16]. Elec-
trostatic interactions, hydrogen bonds, and π-interac-
tions play the key role in this process [5, 6, 8, 16, 18].
stereomeric indoles on an achiral stationary phase. This
method is used for enantiomeric analysis of a wide
number of amino compounds, including α-amino acids
[1, 2], their esters and amides [3–5], amino alcohols [5–
7], arylalkylamines [8], α-alkyl-α-amino acids [3–5,
9], and nucleoamino acids [10]. For the first time, the
possibility of stereomer separation was demonstrated
when NAC was used as an SH-reagent [1, 2]. Further,
other chiral compounds were also applied for the for-
mation of diastereomeric adducts with OPA: various
Despite numerous attempts of applying this
approach to chiral analysis of a wide number of amino
compounds, the method of OPA precolumn modifica-
tion is now mainly used for the determination of amino
acid enantiomers [19, 20]. This limitation is connected
with the fact that diastereomers of the NAC-modified
nonfunctionalized amines and amino alcohols are
poorly separated [6, 14] unlike the effective fraction-
ation of the corresponding amino acid derivatives.
1
Corresponding author; phone/fax: +7 (095) 939-2355; e-mail:
vytas@belozersky.msu.ru
2
Abbreviations: Alcaligenes PA, penicillin acylase from Alcali-
genes faecalis; NAC, N-acetyl-(S)-cysteine; NPPC, N-phenyl-
acetyl-(R)-phenylglycyl-(S)-cysteine; OPA, o-phthalaldehyde;
and rpHPLC, reversed phase HPLC.
In this study, we demonstrated the possibility of
analyzing stereomers of amino alcohols and nonfunc-
1068-1620/04/3005-0403 © 2004 MAIK “Nauka /Interperiodica”

404
GURANDA et al.
tionalized primary amines by HPLC with the precol-
umn OPA modification and with the use of NPPC, a
new optically active thiol reagent. This method was
successfully used both for the determination of enanti-
omeric excess and the absolute concentration of amine
stereomers in various chemical preparations and for
monitoring the stereoselective enzymatic processes
without fractionation of the analyzed compounds and
other components of the reaction system.
O
HN H
H3C
O
O
*
C
HN
COOH
H
HN
COOH
*
H
*
HS
HS
RESULTS AND DISCUSSION
NAC
NPPC
Separation of Diastereomeric Isoindoles
Scheme 1. Chiral SH-reagents for the determination of opti-
cal purity of primary amines: traditional NAC, N-acetyl-(S)-
cysteine, and NPPC, a new N-phenylacetyl-(R)-phenylgly-
cyl-(S)-cysteine.
NPPC, a new thiol reagent was proposed for the
determination of enantiomers of primary amines by the
rpHPLC with the precolumn OPA modification. The
enantiomerically pure NPPC was synthesized by a
chemoenzymatic method based on the regioselective
acylation of (S)-cysteine amino group catalyzed by
Alcaligenes PA [21, 22]. The proposed mercaptan and
NAC, the classic SH-reagent (Scheme 1), were used for
the separation of stereomers of a number of aliphatic
and aromatic amines and amino alcohols (Scheme 2).
The treatment of primary amines with a mixture of
OPA and optically active thiol results in two diastereo-
meric isoindole adducts (Scheme 3) with a characteris-
tic light absorption maximum at 340 nm. The diastere-
omers were analyzed by rpHPLC on a C-18 column in
isocratic regime using a spectrophotometric detector.
NH2
*
NH2
*
OH
R
R
CH3
(I) 2-Naphtyl–
(II) Ph–CH2–CH2–
(III) Bzl–
(XI) p-Cl–Ph–CH2–
(XII) Ph–CH2–
(XIII) Ph–
(IV) Ph–
(V) p-Cl–Ph–
(XIV) CH3–CH2–
(XV) CH3–
(VI) (CH3)2CH–(CH2)3–
(VII) CH3–(CH2)4–
(VIII) CH3–(CH2)2–
(IX) (CH3)2CH–
(X) (CH3)2CH(OH)–(CH2)3–
NH2
(XVI)
*
Bzl
CF3
Evidently, the parameters of chromatographic sepa-
ration are determined by the structure and ionization of
adducts resulting under the conditions of analysis. Pre-
liminary experiments demonstrated that the values of
capacity coefficient (k'), selectivity coefficient (α, and
resolution factor (R) of the isoindole derivatives were
improved when pH of mobile phase was shifted from
an acidic to neutral value and acetonitrile concentration
decreased. This observation is in a good agreement
with the published results [10, 16] and is probably due
to the dissociation of cysteine carboxyl group. There-
fore, we further used a buffer with pH 6.8 as a mobile
phase.
Scheme 2. Structures of the amines studied.
R
O
CHO
CHO
HN
COOH
H
+
*
HS
R1
As follows from the table, the separation efficiency
of the formed diastereomers (α and R parameters)
depends on both the nature of amine component itself
and the structure of SH-reagent. The resolution selec-
tivity of amino alcohols is significantly higher than that
of nonfuctionalized amines, which is explained by the
intramolecular hydrogen bond formed between the cys-
teine carboxyl and the alcohol group [16]. A similar
nature of side radicals of the amino component and the
thiol reagent (in this case, N-acyl moiety of cysteine)
favors the diastereomeric resolution. For example, ali-
phatic amino compounds [amines (VI)–(VIII) and
amino alcohols (X), (XIV), and (XV)] are separated
more efficiently with the use of classic reagent NAC,
whereas the selectivity of NPPC derivatives is higher
for arylamino compounds [amines (I), (II), and (V) and
*
H2N
R2
H
R
O
R
O
HN
COOH
H
HN
COOH
H
*
*
+
S
S
R1
R1
R2
N
R2
*
*
N
H
H
Scheme 3. Formation of diastereomeric isoindole adducts
in the reaction of chiral primary amines with OPA and opti-
cally active thiol.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

A NEW N-ACYL DERIVATIVE OF (S)-CYSTEINE
405
The determination of stereomers of amino compounds by HPLC with the precolumn OPA modification using NAC and NPPC
as optically active SH-reagents
NAC
NPPC
CompoundA
B
αC
RD
EC, %E
αC
RD
EC, %E
B
'
k1
'
k1
(I)
83.5
55.9
24.1
16.9
48.1
52.2
69.9
42.6
36.4
24.5
90.1
31.1
21.5
30.2
13.2
34.1
1.00
1.06
1.05
1.00
1.04
1.13
1.06
1.05
1.02
1.23
1.00
1.00
1.07
1.23
1.19
1.07
0
20
20
20
20
20
20
20
15
15
15
15
15
15
10
10
20
113
1.07
1.10
1.00
1.02
1.06
1.09
1.04
1.00
1.05
1.00
1.03
1.13
1.18
1.17
1.15
1.08
1.41
1.65
0
35
35
35
35
35
40
35
30
30
30
30
30
30
30
30
35
(II)
0.67
1.02
0
95
(III)
(IV)
(V)
47.6
38.1
83.1
18.4
0.41
0.97
0.44
0.45
0
0.95
0.80
1.02
0.92
0.36
4.15
0
(VI)
(VII)
(VIII)
(IX)
(X)
111
53.2
45.9
23.8
62.2
26.1
23.2
10.3
0.58
0
(XI)
(XII)
(XIII)
(XIV)
(XV)
(XVI)
A
0.42
2.35
2.96
2.72
2.00
1.41
0
1.41
3.86
3.03
1.39
6.28
62.7
B
C
D
'
'
'
'
'
'
'
'
1
Note: The corresponding structure is given in scheme 2; k1 = t1 – t , k = t2 – t ; α = k1 /k2 ; R = (t2 – t )/(W + W ), where t
0
0
0
1
2
2
E
'
'
is a dead time, t1 and t2 are the retention times of isomers, and W and W are the half-widths of the peak areas of isomers. Eluent
1
2
composition is a content of acetonitrile (by volume).
amino alcohols (XII) and (XIII)]. The most pro- The (R)-isomer of ε-amino alcohol is eluted before the
nounced is the difference in the separation efficiency corresponding (S)-isomer in the case of both reagents.
when the selectivities of NPPC and NAC isoindoles of
aromatic amino compounds (I), (IV), (XI), and (XII)
The Determination of Absolute Concentrations
of Enantiomers: An Analysis of Reaction Mixture
were compared with those of aliphatic structures (VIII)
and (X).
The interaction of OPA and thiol-containing com-
Thus, the NAC isoindole adducts are characterized
pounds with amino acids is well studied and used for
by a low diastereoselectivity of absorption, and only
the quantitative spectrophotometric determination of
some aliphatic amino alcohols can satisfactorily be sep-
primary amino groups [23–25]. We could expect that
arated. The use of NPPC improves the analysis of ste-
the application of HPLC with precolumn OPA modifi-
reomers of amino alcohols and provides for the deter-
cation would offer the possibility of determination of
mination of enantiomers of nonfunctionalized amines.
not only the relative composition or enantiomeric
A more efficient resolution of the NPPC-diastereo-
excess, but also the absolute concentration of stere-
meric isoindoles can probably be explained by the real-
omers of amino compounds as well. It is also of inter-
ization of additional intramolecular interactions with
est, because this method could be used for the quantita-
phenylacetyl and phenylglycine residues of the modi-
tive analysis of primary amines and their stereomers in
fier.
multicomponent systems due to a characteristic absorp-
The elution order of diastereomeric forms was
determined after the modification of individual enanti-
omers. For both chiral reagents, (S)-enantiomers of β-
amino alcohols and (R)-enantiomers of nonfunctional-
ized amines (the HO-group changes the attribution of
configuration according to the Cahn–Ingold–Prelog
nomenclature) are eluted first.
tion maximum of isoindoles at 340 nm.
The dependence of maximum optical density of the
formed chromophore adduct on the OPA concentration
and concentration of a sulfhydryl reagent in the reac-
tion mixture was studied in order to determine the con-
ditions of the most efficient modification of amino
groups. The absolute concentration of primary amino
A similar elution order was observed for the stere- groups can be determined with the use of practically
omers of isoindole derivatives of α-amino acids [2, 6]. equimolar amount of OPA and three to fourfold excess
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

406
GURANDA et al.
A
2.0
(a)
(b)
2
1
1.5
1.0
0.5
2
1
0
5
10
15
20 0
2
4
6
8
10
[NPPC]/phenylalaninol
[OPA]/phenylalaninol
Fig. 1. The dependence of maximal value of optical absorption at 340 nm on the concentration of (a) the thiol reagent and (b) OPA
in 0.4 M borate buffer (pH 9.6): (a, curve 1) 0.1 mM phenylalaninol, (a, curve 2) 0.25 mM phenylalaninol, (black symbols) 0.3 mM
OPA or (open symbols) 3.0 mM OPA; (b, curve 1) 0.1 M phenylalaninol, (b, curve 2) 0.25 mM phenylalaninol, and 3 mM NPPC.
of NAC or 8–10-fold excess of NPPC (Fig. 1). The hydrolysis of N-phenylacetyl derivative (XII) moni-
modification of all the studied compounds [except for tored by an ordinary HPLC (detection at 210 nm) and
amine (XVI)] took 10–15 min at an approximately by chiral analysis at 340 nm with the precolumn modi-
100 µM reagent concentration. The formation of chro- fication. Note that both analyses were carried out on the
mophore adduct from amine (XVI) proceeded for 3 h same chromatographic column.
possibly due to a low nucleophilicity of its amino
group. The molar absorption coefficient of the resulting
isoindoles is the same for both stereomers (ε340 6000 ±
Thus, the use of the new NPPC chiral thiol com-
pound improved the HPLC analysis of stereomers of
amino alcohols and allowed the enantiomeric analysis
of nonfunctionalized amines with the use of the OPA
precolumn modification. This method is characterized
by a good resolution, sensitivity, and reproducibility of
signal and can be applied to the quantitative determina-
tion of enantiomers of primary amines in both individ-
ual preparations and reaction mixtures.
100 M–1 cm–1) and does not depend on the nature of N-
acyl moiety of SH-reagent.
This method is useful for the qualitative analysis of
chiral amines and amino alcohols due to a high stability
of the formed isoindole adducts. The absorption inten-
sity of the samples diluted with eluent remained
unchanged within 4–5 h, which is the necessary prereq-
uisite for the qualitative analysis. The quantitative
HPLC analysis of stereomers was carried out on the
basis of peak areas, which were identical for two dia-
stereomeric forms after modification of racemates of
the examined amines. An analysis of standard solutions
of individual enantiomers confirmed the absence of
racemization in the course of modification of amino
groups. The detection limit was 3–5 pmol. The linear
dependence of detected signal on the concentration of
determined substance for (IV), (XII),), and (XIV) was
observed within the concentration range of enantiomers
of amines from 20 pmol to 20 nmol (data not given),
which indicates ample opportunities of the method.
EXPERIMENTAL
OPA (Koch Light, UK); NAC and phenylacetyl
chloride (Sigma, United States); (R)- and (S)-phenyl-
glycinol, and phenylacetic acid (Aldrich Chemie, Ger-
many); (S)-cysteine, (R)-, (S)- and (±)-1-phenylethy-
lamine, (R)- and (S)-phenylalaninol, (±)-2-aminopen-
tane, (R)- and (S)-1-(2-naphtyl)ethylamine, (R)- and
(S)-2-amino-1-propanol, (±)-2-amino-4-phenylbutane,
(±)-2-amino-6-methylheptane, and (±)-6-amino-2-
methyl-2-heptanol hydrochloride (Fluka, Switzerland);
phenylmethylsulfonyl fluoride, methanol and sodium
dodecyl sulfate (Merck, Germany); (±)-1-(4-chlorophe-
nyl)ethylamine, (±)-2-heptylamine, (±)-2-aminobu-
We used our method for the spectrophotometric tanol, (±)-p-chlorphenylalaninol, and (±)-2-amino-3-
monitoring of the absolute concentrations of enanti- methylbutane (Acros, Belgium); phenylacetamide
omers of amino compounds in the course of two stere- (Reakhim, Russia); and acetonitrile (Kriokhrom, Rus-
oselective enzymatic reactions: an enzymatic acylation sia) were used in this study. (±)-1-Benzyl-2,2,2-trifluo-
of racemates of amines and amino alcohols and Alkali- roethylamine was a kind gift of Professor V.A. Solo-
genes PA-catalyzed hydrolysis of N-acyl derivatives of shonok (Institute of Oil Chemistry and Bioorganic
amines (Fig. 2). Other components of the reaction mix- Chemistry, Kiev, Ukraine). N-Phenylacetyl derivatives
tures did not hinder the analysis. A good agreement was of amino compounds were synthesized as previously
observed between the results of the stereoselective described [21]. (R)- and (S)-mandelic acid and its
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

A NEW N-ACYL DERIVATIVE OF (S)-CYSTEINE
407
c, å
0.3
Optical purity
1.0
(a)
(b)
0.8
0.6
0.4
0.2
0.2
0.1
0
40
80 120 160 200
0
0.2
0.4
0.6
Time, min
Conversion degree
Fig. 2. Acylation of (±)-phenylalaninol with (R)-mandelic acid amide catalyzed by Alcaligenes PA. (a) Integral curves of changes
in the component concentrations of the reaction mixture and (b) the dependence of enantiomeric excess on the conversion degree:
(᭹), (R)-mandelic acid amide; (᭿), (R)-mandelic acid; (᭡), (R)-mandelyl-(S)-phenylalaninol; (᭢), (R)-mandelyl-(R)-phenylalaninol;
(᭜), (±)-phenylalaniol; (᭝), (S)-phenylalaninol; (᭞) (R)-phenylalaninol. The black symbols designate the results of ordinary HPLC
at 210 nm, and the open symbols indicate the data of chiral analysis by HPLC at 340 nm with the precolumn OPA modification.
The solid line and the hatched line in Fig. 2b represent the calculated curves obtained by mathematic modeling of the enzymatic
reaction with stereo selectivity of 50.
amides, (R)-phenylglycine, (R)-phenylglycinamide, phenylglycine amide (0.3 M) to the amino group of (S)-
and an Alcaligenes PA preparation were presented by cysteine (0.2 M) with the use of Alcaligenes PA as a
the DSM company (the Netherlands). The concentra- catalyst at 25°C and pH 10 of aqueous medium. At the
tion of active sites of penicillin acylase was determined moment of maximal accumulation of the product, the
according to [26].
reaction mixture was ajusted to pH 12; the unreacted
(R)-phenylglycine amide was extracted with ethyl ace-
tate, and the water phase containing N-(R)-phenylgly-
cyl-(S)-cysteine was used an the next stage without any
additional purification.
HPLC-analysis. Chromatographic system con-
sisted of a Waters M6000 module of eluent supply, a
Reodyne 7125 injector with the injection loop of 50-µl
volume, a Nucleosil C-18 Chrompack column (Varian,
250 × 4 mm, 5 µm), and a Waters M481 LC detector.
At the second stage, the amino group of N-(R)-phe-
The chromatograms were registered on a Multikhrom nylglycyl-(S)-cysteine was acylated by phenylacetyl
program-apparatus complex (Ampersand, Russia). chloride in an aqueous acetone solution at pH 7.5–8.0
Flow rate was 1 ml/min. A mixture of 6 mM phosphate and at 0–5°ë in an ice bath. After the end of reaction,
buffer and acetonitrile was used as a mobile phase. acetone was evaporated on a rotary evaporator and the
Isoindole diastereomers were analyzed at 340 nm with product was extracted with ethyl acetate from the acidic
the use of a 10–40 vol % gradient of acetonitrile in solution (pH 3) and recrystallized from aqueous etha-
6 mM phosphate buffer (pH 6.8). An HPLC analysis of nol. The detailed procedure of the synthesis and physi-
chemical components was carried out at 210 nm using cochemical characteristics of NPPC will be given in our
6 mM phosphate buffer (pH 3.0) containing 30% (vol) next publications.
acetonitrile and sodium dodecyl sulfate (0.7 g/l) as a
Precolumn OPA modification of amino groups.
mobile phase. The following retention times were
Primary amino groups were modified as follows: a
observed: phenylglycine, 5.5 min; N-(R)-phenylglycyl-
methanolic solution of SH-reagent (50 µl) containing
(S)-cysteine, 11 min; phenylglycine amide, 20 min;
thiol (40 mM NAC or 100 mM NPPC) and OPA
phenylacetamide, 4.4 min; and phenylacetic acid,
(20 mM) was added to a 0.5 mM solution of amine
6.8 min.
(900 µl) in 0.4 M borate buffer (pH 9.6). The reaction
The kinetics of indole adduct formation was contin- mixture was stirred for 15 min, diluted with the chro-
uously monitored at 340 nm on a Shimadzu UV-1601 matographic eluent, centrifuged for 3 min at 1200 rpm,
spectrophotometer at 25°C in 0.4 M borate buffer and analyzed. The OPA solution and the thiol solution
(pH 9.6).
were stored for a month at +5°ë in a dark container.
Synthesis of N-phenylacetyl-(R)-phenylglycyl-
Quantitative analysis of the reaction mixture. A
(S)-cysteine. NPPC was prepared by chemoenzymatic stereoselective enzymatic reactions of acylation of a
synthesis based on a regioselective acylation of amino racemic amine and a hydrolysis of racemates of the N-
group of (S)-cysteine by the action of penicillin acylase. acylated amino compounds catalyzed by Alcaligenes
At the first stage of the synthesis, N-(R)-phenylglycyl- PA were carried out in a thermostatic cell of a 719 S
(S)-cysteine was prepared by the acyl transfer from (R)- Titrino pH-stat (Metrohm) at 25°ë, pH 10 or pH 7.5,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

408
GURANDA et al.
11. Bruckner, H., Wittner, R., and Godel, H., J. Chromatogr.,
respectively. For the quantitative registration of
changes in the component concentrations, an aliquot
1989, vol. 476, pp. 73–82.
(50–100 µl) was taken from the reaction mixture, 12. Euerby, M.R., Partridge, L.Z., and Gibbons, W.A.,
J. Chromatogr., 1989, vol. 483, pp. 239–252.
diluted with the eluent (1 ml, pH 3.0) for the complete
solution of the reaction components and the stop of
enzymatic reaction. One portion of the obtained solu-
tion was analyzed by HPLC at 210 nm, and another
13. Bruckner, H., Haasmann, S., Langer, M., Westhauser, T.,
Wittner, R., and Godel, H., J. Chromatogr. A, 1994,
vol. 666, pp. 259–273.
portion was subjected to the OPA modification (for the 14. Guranda, D.T., Kudryavtsev, P.A., Khimiuk, A.J., and
Svedas, V.K, Abstracts of Papers, Int. Symp. 100 Years of
Chromatography, Moscow, Russia, 2003, pp. 388–389.
determination of concentrations of individual enanti-
omers of amino compounds) and analyzed on the same
chromatographic column at 340 nm.
15. Jegorov, A., Triska, T., Trnka, T., and Cerny, M., J. Chro-
matogr., 1988, vol. 434, pp. 417–422.
16. Duchateau, A.L.L., Boesten, J.M.M., and Cous-
ACKNOWLEDGMENTS
sens, B.B., Chirality, 1995, vol. 7, pp. 547–555.
This study was supported by the Russian Founda-
tion for Basic Research, project no. 03-04-48472 and
Federal Grant-in-Aid Program (TsP) Integration,
project no. 33384.
17. Jacobs, W.A., Leburg, M.W., and Madaj, E.J., Anal. Bio-
chem., 1986, vol. 156, pp. 334–340.
18. Lindner, W., in Indirect Separation of Enantiomers by
Liquid Chromatography, Zief, M. and Crane, L.J., Eds.,
Chromatographic Chiral Separations, Chromatographic
Science Series, vol. 40, NewYork: Marcel Dekker, 1988,
p. 116.
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