Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride

Article abstract of DOI:10.1007/s00726-010-0650-z

Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz l-Leu-NH₂) and amino acid (l-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C₁₈ column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.

Full text of DOI:10.1007/s00726-010-0650-z

Amino Acids (2011) 40:403–409
DOI 10.1007/s00726-010-0650-z
ORIGINAL ARTICLE
Reversed-phase liquid chromatographic resolution
of diastereomers of protein and non-protein amino acids prepared
with newly synthesized chiral derivatizing reagents based
on cyanuric chloride
•
Ravi Bhushan Charu Agarwal
Received: 18 March 2010 / Accepted: 2 June 2010 / Published online: 18 June 2010
Ó Springer-Verlag 2010
Abstract Two new chiral monochloro-s-triazines (MCT)
were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-
triazine-2-yl)-L-leucine amide and N-(4-chloro-6-piperidi-
taste, aroma, and color among other aspects (Hunt 1985).
The relevance of amino acid enantiomers in wine gained
attention, but gas chromatographic determination of
quantities and pattern of D-amino acids, particularly of
D-proline, has been found not to be correlated with the
storage time of bottled wines (Ali et al. 2010).
nyl-[1,3,5]-triazine-2-yl)-L-leucine) (CDR
1
and 2,
respectively)] by the nucleophilic displacement of chlorine
atoms in s-triazine moiety. One of the Cl atoms was
replaced with piperidine, and the second Cl atom in the
6-piperidinyl derivative was replaced with amino acid
amide (viz L-Leu–NH2) and amino acid (L-Leu). These
reagents were characterized and used as CDRs for chiral
separation of protein and non-protein amino acids, and
were separated on a reversed-phase C18 column. The
reaction conditions were optimized for the synthesis of
diastereomers using one MCT reagent. The separation
method was validated for limit of detection, linearity,
accuracy, precision, and recovery.
Application of different CDRs for indirect enantioreso-
lution is capable of providing better resolution with good
sensitivity as the chromatographic conditions can be easily
optimized. Several reports (Marfey 1984; B’Hymer et al.
2003; Bhushan and Bru¨ckner 2004; Bhushan et al. 2009),
and the literature cited therein, on RP-HPLC enantioreso-
lution of certain protein and non-protein amino acids show
that indirect resolution has been successful. Recently, D- as
well as L-Iva has been detected, and its chiral sequences
were evaluated using Marfey’s reagent in Integramide A
which is a 16-amino acid peptide inhibitor of the enzyme
HIV-1 integrase (De Zotti et al. 2010). Triazine derivatives
of chiral amines and amino acids have been used to prepare
CSP for enantioresolution of chiral amines (Bru¨ckner and
Wachsmann 1996; Lin and Yang 1993; Wachsmann and
Bru¨ckner 1998).
Keywords Cyanuric chloride Á Amino acids Á
Reversed-phase liquid chromatography Á Indirect resolution
Introduction
Taking advantage of trifuctionality of cyanuric chloride
(s-triazine, CC), Bru¨ckner and Wachsmann (2003) syn-
thesized chiral monochloro-s-triazine (MCT) reagents by
introducing L-alanine amide as chiral auxiliary along with
achiral moieties such as methoxy and phenoxy and used
them for chiral separation of a few selected amino acids by
HPLC and reported that the CDRs containing –OCH3
moiety gave better separation and resolution in comparison
to the CDRs containing other achiral moieties. Bhushan
and Kumar (2008) prepared some new dichlorotriazine
(DCT) reagents using L-leucine amide and D-phenylglyci-
namide as chiral auxiliaries and monochlorotriazine (MCT)
reagents using the same two chiral auxiliaries along with
Enantiomers of amino acids are known to have high ste-
reospecificity, different physiological and biological
activities, and differences in metabolic rates as their
binding to different receptor types leads to different bio-
logical response. Amino acids also play an important role
in food industry for their nutritive value and by affecting
R. Bhushan (&) Á C. Agarwal
Department of Chemistry,
Indian Institute of Technology Roorkee,
Roorkee 247667, India
e-mail: rbushfcy@iitr.ernet.in
123

404
R. Bhushan, C. Agarwal
–OCH3 as an achiral moiety; these reagents were used as
CDRs for chiral resolution of protein (except His, Lys, and
Arg)and non-protein amino acids. Literature shows that the
resolution of diastereomers of amino acids prepared with
DCT reagents was better in comparison to those of pre-
pared with MCT reagents (Bhushan and Kumar 2008); the
electronegativity difference of O– (in the –OCH3 of MCT)
and Cl atoms (present in DCT reagents) was considered to
be responsible for the same.
piperidine, L-leucine amide hydrochloride (L-Leu–NH2Á
HCl), L-phenylalanine amide hydrochloride (L-Phe–NH2Á
HCl), D-phenylglycinamide hydrochloride (D-Phg–NH2ÁHCl),
L-leucine (L-Leu), and L-alanine (L-Ala) were obtained from
Sigma-Aldrich (St Louis, MO, USA). All other analytical
grade chemicals and HPLC solvents, MeCN and methanol
(MeOH) used were from E. Merck (Mumbai, India). Purified
water was used throughout all studies.
Taking into account the role of electronegativity of the
atoms of achiral moieties present in the MCT and DCT
reagents, it was considered worthwhile to introduce such
achiral moieties in CC that have atoms of electronegativity
identical to chlorine. Thus, two new monochloro-s-tria-
zines having L-leucine amide and L-leucine as chiral aux-
iliaries and piperidine molecule as an achiral substituent
were synthesized since on the Pauling scale the electro-
negativities are 3.5 for oxygen, 3.04 for nitrogen, and 3.0
for chlorine. These CDRs were characterized and used to
synthesize diastereomers of 18 protein and 8 non-protein
amino acids. The diastereomers were separated by
reversed-phase HPLC. The performance of the two CDRs
was compared. The method was also validated for linearity,
accuracy, and precision. Limit of detection was determined
for all amino acids. To the best of authors’ knowledge, this
is the first report on use of piperidine as an achiral sub-
stituent in CC for preparing CDRs followed by their
application in enantioresolution of a-amino acids.
Synthesis of chiral MCT reagents
Two MCTs were prepared by nucleophilic substitution of
chlorine atoms in cyanuric chloride. One of the chlorine
atoms was substituted by a piperidinyl group and the sec-
ond with chiral auxiliaries (L-Leu–NH2 and L-leucine)
(Fig. 1). MCT reagents were synthesized and characterized
according to methods given in literature (Wachsmann and
Bru¨ckner 1998; Bhushan and Kumar 2008). Chiral purity
of the CDRs so synthesized was established as described
elsewhere (Bhushan and Kumar 2008).
N-(4-Chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine
amide (CDR-1)
Yield: 86%; color: white; mp: 145–150°C; UV [kmax (nm),
in acetone]: 210; IR (KBr): 3,426, 2,365, 1,676, 1,593,
1,565, 1,407, 1,347, 1,308, 1,017, 621, and 460 cm-1
1H NMR (500 MHz, CDCl3) d 0.95–0.99 (dd, 6H, –2CH3),
1.41–1.46 (m, 6H, –3CH2–), 1.59–1.68 (m, 3H, –CH2–CH),
3.74–3.76 (m, 1H, –CH–N), 7.28(s, 1H, –CONH2), 7.72
(s, 1H, –CONH2), 4.12–4.25 (dd, 1H, –NH); analysis:
calculated for C14H23ClN6O: C 51.45%; H 7.09%;
N 25.71%, found: C 51.51%; H 7.05%; N 25.75%.
;
Experimental
Instrumentation and materials
The HPLC system consisting of a 10-mL pump head 1000,
manager 5000 degasser, UV–visible detector 2600, Knauer
manual injection valve and Eurochrom operating software
was from Knauer (Berlin, Germany). Reversed-phase
HPLC was performed on a Lichrospher C18 (250 mm 9
4.6 mm I.D., 5 lm) column from Merck (Darmstadt,
Germany). A pH meter Cyberscan 510 (Singapore) and
Incubator CI-65 (Remi, Mumbai, India) were used. The
Milli-Q system of Millipore (Bedford, MA, USA) was used
to purify double distilled water (18.2 MXcm3). IR spectra
were recorded in KBr pellets on a PerkinElmer 1600 FT
spectrometer (Boardman, OH, USA). Elemental analysis
was carried out using a Vario EL III elementary analyzer.
UV–visible spectra were recorded in acetonitrile (MeCN)
on a Shimadzu UV-1601 spectrophotometer. 1H NMR
spectra were recorded on a Bru¨ker 500 MHz instrument
using CDCl3 as deuterated solvent.
N-(4-Chloro-6-pipredinyl-[1,3,5]-triazine-2-yl)-L-leucine
(CDR-2)
Yield: 79%; color: white; mp: 99–110°C; UV [kmax (nm),
in acetone]: 210; IR (KBr): 3,439, 2,947, 2,360, 2,134,
1,626, 1,585, 1,547, 1,239, 1,117, 1,104, 1,057, 916, 656,
626, 546, 510, and 449 cm-1; 1H NMR (500 MHz, CDCl3)
d 0.94–0.98 (m, 6H, –2CH3), 1.56–1.66 (m, 6H, –3CH2–),
1.71–1.77 (m, 3H, –CH2–CH), 3.70–3.77 (m, 1H, –CH–N),
4.61–4.65 (dd, 1H, –NH); analysis: calculated for
C14H22ClN5O2: C 51.29%; H 6.76%; N 21.36%. found:
C 51.41%; H 6.71%; N 21.40%.
Synthesis of diastereomers of amino acids with MCT
reagents
Derivatization of a-amino acids with MCT reagent was
carried out according to procedure employed by Bhushan
and Kumar (2008). To 30 lL aliquot (3 lmol) of standard
All racemic and chirally pure amino acids were obtained
from Sigma-Aldrich (Bangalore, India). Cyanuric chloride,
123

Reversed-phase liquid-chromatographic resolution
405
Fig. 1 The general scheme for
synthesis of chiral MCT
reagents and the diastereomers
(first letter refers to the
configuration of CDR while the
second refers to that of the
analyte)
solution of DL-alanine (100 mM in 1 M HCl) were added
45 lL NaHCO3 (1 M) and 500 lL aliquots (5 lmol) of
CDR-1 (10 mM in acetone). The mixture was heated for
1 h at 80°C. 10 lL of the resulting solution, containing
diastereomers, was diluted ten times with MeCN, and
20 lL of it was injected onto the column.
Results and discussion
Derivatization of each of the amino acids with each of the
two CDRs was successful in presence of 1 M NaHCO3
providing a pH around 8 in 1 h at 80°C and using twofold
molar excess of CDR that helped preventing kinetic reso-
lution. Thus, 52 diastereomeric pairs were synthesized. The
diastereomers could be stored at 5°C without any decom-
position. The first letter denotes the configuration of CDR
and the second denotes the configuration of analyte in the
diastereomer.
HPLC conditions
Following eluents were used:
eluent A: MeCN (200 mL) ? H2O (800 mL) ? trifluo-
roacetic acid (1 mL),
Separation of diastereomers
eluent B: MeCN (800 mL) ? H2O (200 mL) ? trifluo-
roacetic acid (1 mL),
The chromatographic data for separation of diastereomers
are shown in Tables 1 and 2. Sharp peaks of diastereomers
were obtained as shown in Figs. 2a, b and 3. The L-D
diastereomer eluted with the mobile phase earlier than L-L
diastereomer in each case. MeCN was found to be a better
organic modifier in comparison to methanol as larger
retention times and broader peaks were obtained with the
eluent C: MeOH (900 mL) ? H2O (100 mL) ? trifluo-
roacetic acid (1 mL).
A linear gradient, (I) from 100% A to 100% B and (II)
from 100% C to 100% D, in 45 min was employed. The
separation was carried out at a flow rate of 0.5 mL/min
with UV detection at 230 nm.
123

406
R. Bhushan, C. Agarwal
Table 1 RP-HPLC resolution
of diastereomers of amino acids
prepared with CDR-1 and
CDR-2
No.
Amino acids
CDR-1
CDR-2
kR
kS
4.58
a
RS
4.197
kR
kS
a
RS
1
Alanine
1.85
10.94
14.20
7.55
2.47
1.20
1.11
1.10
1.13
1.12
1.10
1.01
1.11
1.05
1.15
1.04
1.18
1.15
1.17
1.06
1.20
1.37
17.58
11.06
18.79
14.75
15.96
16.98
16.40
19.39
12.80
15.17
16.21
15.36
16.04
18.03
13.79
3.12
18.46
12.79
19.74
15.64
16.70
18.17
16.68
20.09
14.56
15.51
16.38
15.89
17.26
18.77
15.09
3.27
1.05
1.16
1.05
1.06
1.05
1.07
1.02
1.04
1.14
1.02
1.01
1.03
1.08
1.04
1.09
1.05
1.69
2.31
2.20
2.02
0.79
1.75
0.58
1.53
2.73
0.73
0.49
1.08
2.29
1.49
1.82
0.41
2
Valine
13.17
15.84
8.32
1.00
1.89
1.99
0.78
2.06
0.78
0.40
2.07
0.64
4.86
1.00
2.45
1.71
1.61
0.50
1.22
12.08
3
Leucine
4
Isoleucine
Proline
5
6.56
7.45
6
Threonine
Serine
7.41
8.34
7
6.38
7.01
8
Phenylalanine
Tyrosine
15.68
10.05
5.52
15.84
11.18
5.82
9
Chromatographic conditions:
eluents, see experimental;
gradient (I); flow rate
0.5 mL min-1, detection
230 nm
10
11
12
13
14
15
16
17
18
Glutamic acid
Aspartic acid
Asparagine
Cystine
14.77
6.51
17.04
6.79
10.91
6.50
12.93
7.51
NT not tested; k1, k2 retention
factors; a separation factor
Methionine
Tryptophan
Arginine
Histidinea
Lysinea
7.59
8.88
a
Mobile phase involving
6.28
6.63
gradient (II) was successful for
the diastereomers of protein
amino acids 15–17 prepared
with CDR-1
4.60
5.54
NT
NT
12.99
17.78
Table 2 RP-HPLC resolution of diastereomers of non-protein amino acids prepared with CDR-1 and CDR-2
No.
Amino acids
CDR-1
CDR-2
kR
kS
a
RS
kR
kS
a
RS
1
2
3
4
5
6
7
8
2-Amino octanoic acid
2-Amino butyric acid
2-Amino adipic acid
Norvaline
12.45
15.89
15.43
4.91
14.35
16.54
17.28
6.44
1.15
1.04
1.12
1.31
1.04
1.14
1.35
2.27
1.07
2.00
3.85
1.07
2.73
4.26
15.66
16.08
18.22
12.28
11.90
22.79
17.31
18.11
17.23
18.66
14.66
12.59
23.57
17.64
1.16
1.07
1.02
1.19
1.06
1.03
1.02
3.55
1.66
1.11
3.25
0.72
1.62
0.60
Pipecolic acid
15.36
12.80
7.76
15.89
14.56
10.46
2-Phenyl glycine
Isovalinea
Cysteic acid
NR
NR
Chromatographic conditions: eluents, see experimental; gradient (I); flow rate 0.5 mL min-1, detection 230 nm
k1, k2 retention factors; a separation factor, NR no resolution
a
Mobile phase involving gradient (II) was successful for diastereomers of isovaline prepared with CDR-1
latter. The best separation was obtained at 45 min for the
applied gradient. On varying TFA concentration from
0.005 M to 0.03 M, a slight increase in separation factor
was observed.
diastereomers of hydroxyl group containing amino acids
(6 and 7) and sulfur containing amino acids (13 and 14)
prepared with CDR-2 were lower than those of their dia-
stereomers prepared with CDR-1; for aromatic amino acids
(8 and 9) and for acidic amino acids (10 and 11) resolution
values for diastereomers prepared with CDR-2 were higher
than the diastereomers prepared with CDR-1, except the
resolution value of aspartic acid; among the basic amino
acids (15–16), the resolution values were higher for the
diastereomers of tryptophan prepared with CDR-2 than its
diastereomers prepared with CDR-1, while the results were
just opposite for diastereomers of arginine.
The basic mechanism for separation of diastereomers of
amino acids prepared with MR has been explained by
Marfey (1984), Bru¨ckner and Keller-Hoehl (1990), and
Fujii et al. (1997). It was observed from Table 1 that the
resolution values for diastereomers of aliphatic amino acids
(serial numbers 1–5) prepared with CDR-2 were higher in
comparison to those for the diastereomers prepared with
CDR-1, except for alanine; the resolution values for the
123

Reversed-phase liquid-chromatographic resolution
407
Fig. 2 The sections of
chromatograms showing
resolution of diastereomers of
some protein amino acids
prepared with CDR-1.
Chromatographic conditions:
eluent A: MeCN
(200 mL) ? H2O
(800 mL) ? trifluoroacetic acid
(1 mL); eluent B: MeCN
(800 mL) ? H2O
(200 mL) ? trifluoroacetic acid
(1 mL); linear gradient (I) from
100% A to 100% B, in 45 min;
flow rate 0.5 mL min-1
,
detection 230 nm. *Reagent-
related peaks
In case of non-protein amino acids (Table 2), it was
observed that the resolution values for diastereomers of
2-amino octanoic acid and 2-amino butyric acid prepared
with CDR-2 were higher in comparison to those for the
diastereomers of rest of the non-protein amino acids pre-
pared with CDR-2. According to Table 2, it is concluded
that the Rs values for the diastereomers of isovaline (Iva)
prepared with CDR-1 were the highest among the Rs values
of the diastereomers of the rest of the non-protein amino
acids prepared with CDR-1 and 2. The diastereomers of the
same six non-protein amino acids (except Iva) prepared
with (S)-NIFE required higher run and retention times and
showed higher Rs using methanol in the mobile phase
(Bhushan and Agarwal 2010) in comparison to the run
time, retention times, and Rs of their diastereomers pre-
pared with CDR-1 and 2 using acetonitrile (in the present
case) as organic modifier. Use of methanol as organic
modifier in the present case showed good Rs for the dia-
stereomers of the six non-protein amino acids, but it was
not chosen finally as there were longer retention times.
Hydrophobicity of the alkyl side chain of the amino
acids appears to affect the interaction of the diastereomers
123

408
R. Bhushan, C. Agarwal
Fig. 3 The sections of
chromatograms showing
resolution of diastereomers of
non-protein amino acids
prepared with CDR-2.
Chromatographic conditions:
eluent A: MeCN
(200 mL) ? H2O
(800 mL) ? trifluoroacetic acid
(1 mL); eluent B: MeCN
(800 mL) ? H2O
(200 mL) ? trifluoroacetic acid
(1 mL); eluent C: MeOH
(900 mL) ? H2O
(100 mL) ? trifluoroacetic acid
(1 mL); linear gradient, (I) from
100% A to 100% B and (II)
from 100% A to 100% C, in
45 min; flow rate
0.5 mL min-1, detection
230 nm. *Reagent-related peaks
with the ODS material of the column and the separation.
Looking to the alkyl side chain in the structures of analytes,
following observations have been made: with increasing
alkyl group in the side chain the separation factors of the
analytes (diastereomers prepared with CDR-1 and 2)
increased particularly for the following sets: Trp [
Arg [ Asn; Tyr [ Thr [ Ser; Tyr [ Phe; Cys [ Met. For
diastereomers of Ile, Leu, and Ala prepared with CDR-2,
with increasing alkyl group in the side chain the separation
factors of the analytes increased particularly for Ile [
Leu = Ala.
poorly resolved, but its diastereomers prepared with CDR-
2 were well resolved (Rs = 1.53); diastereomers of Asp,
Iva and pipecolic acid prepared with CDR-2 were poorly
resolved, but their diastereomers prepared with CDR-1
were well resolved on reversed-phase column.
Linearity, accuracy, and precision
Varying amounts of derivatives in the range 50–500 pmol
were injected onto column, and linearity studies were
carried out using correlations between the peak areas. The
linearity was acceptable in this range for diastereomers of
D-Ala (R2 = 0.9987) and for diastereomers of L-Ala
(R2 = 0.9994), respectively.
The Rs values for the diastereomers of glutamic acid
prepared with both the CDRs was nearly equal while the Rs
value for the diastereomers of aspartic acid prepared with
CDR-1 is greater than the Rs value for the diastereomers of
the same prepared with CDR-2. It may be due to the polar
interactions between COO- anions present in Asp and NH2
of the L-Leu–NH2 present in CDR-1.
The accuracy and precision studies were carried out by
replicate HPLC analysis (n = 5) of DL-Ala at five different
concentration levels (10, 30, 50, 80, and 100 ng mL-1) and
RSD were less than 2%. The relative standard deviation for
D- and L-Ala varied from 1.20 to 1.46% and 1.25 to 1.80%
for intra-day assay precision and 1.42 to 1.80% and 1.70 to
1.90% for inter-day assay precision. The recovery for
D- and L-Ala varied from 96.8 to 97.6% and 96 to 98.5% for
Among the specific pairs of diastereomers, it was
observed that the diastereomeric pairs of Ser, Pro, Glu, and
Arg prepared with both CDR 1 and 2 were poorly resolved;
the diastereomeric pair of Phe prepared with CDR-1 was
123

Reversed-phase liquid-chromatographic resolution
409
chromatographic separation of diastereomers of amino alcohols,
non-protein amino acids and PenA. Amino Acids. doi:
10.1007/s00726-010-0472-z
intra-day assay and 95.7 to 97% and 95.8 to 97.8% for
inter-day assay. Accuracy of the method was determined
by investigating the standard solutions (100 mM) of L-Ala
spiked with D-Ala in the range 0.1–1.0%. The results
indicate that this method can be applied for detection of
D-Ala in L-Ala up to 0.3% by HPLC.
Bhushan R, Bru¨ckner H (2004) Marfey’s reagent for chiral amino
acid analysis: a review. Amino Acids 27:231–247
Bhushan R, Kumar V (2008) Indirect enantioseparation of a-amino
acids by reversed-phase liquid chromatography using new
chiral derivatizing reagents synthesized from s-triazine chloride.
J Chromatogr A 1201:35–42
Bhushan R, Kumar V, Tanwar S (2009) Chromatographic separation
of enantiomers of non-protein a-amino acids after derivatization
with Marfey’s reagent and its four variants. Amino Acids
36:571–579
Bru¨ckner H, Keller-Hoehl C (1990) HPLC separation of DL-amino
acids derivatized with N2-(5-fluoro-2, 4-dinitrophenyl)-L-amino
acid amides. Chromatographia 30:621–629
Bru¨ckner H, Wachsmann M (1996) Liquid chromatographic separa-
tion of amino acid enantiomers on a silica-bonded chiral
s-triazine column. J Chromatogr 728:447–454
Bru¨ckner H, Wachsmann M (2003) Design of chiral monochloro-s-
triazine reagents for the liquid chromatographic separation of
amino acid enantiomers. J Chromatogr 998:73–82
De Zotti M, Damato F, Formaggio F, Cirsma M, Schievano E,
Mammi S, Kaptein B, Broxterman QB, Felock PJ, Hazuda DJ,
Singh SB, Kirschbaum J, Bru¨ckner H, Toniolo C (2010) Total
synthesis, characterization, and conformational analysis of
the naturally occurring hexadecapeptide integramide A and
a diastereomer. Chem Eur J 16:316–327
Fujii K, Ikai Y, Mayumi T, Oka H, Suzuki M, Harada K (1997) A
nonempirical method using LC/MS for determination of the
absolute configuration of constituent amino acids in a peptide:
elucidation of limitations of Marfey’s method and of its
separation mechanism. Anal Chem 69:3346–3352
Conclusion
Different reactive and detectable groups can be attached
with CC, and new series of CDRs can be prepared that can
be used for improved separation of different chiral phar-
maceutical drugs and amino acids. It was found that sep-
aration and retention times of diastereomers of amino acids
depend upon hydrophobicity of amino acids. The results
obtained from the piperidine-substituted MCT reagents
were comparable to those obtained with other DCT
reagents and were better than the other MCT reagents
having chiral moieties such as methoxy group.
Acknowledgments The authors are thankful to the Ministry of
Human Resources Development, Government of India, New Delhi for
the award of a research assistantship (to C.A.) and to the Alexander
von Humboldt Foundation, Bonn, Germany for donating Knauer
HPLC equipment and to the Council of Scientific and Industrial
Research, New Delhi, India for financial assistance [to R.B.; research
grant No. 01(2334)/09/EMR-II].
Hunt S (1985) In: Barrett GC (ed) Chemistry and Biochemistry of the
amino acids. Chapman and Hall, London, p 55
Lin J-Y, Yang M-H (1993) High-performance liquid chromatographic
separation of enantiomeric amino acids on bis[carbam-
oyl(alkyl)methylamino]-6-chloro-s-triazine-derived chiral sta-
tionary phases. J Chromatogr 644:277–283
Marfey P (1984) Determination of D-amino acids. II. Use of
a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg
Res Comm 49:591–596
Wachsmann M, Bru¨ckner H (1998) Ligand-exchange chromato-
graphic separation of DL-amino acids on aminopropylsilica-
bonded chiral s-triazines. Chromatographia 47:637–642
References
¨
¨
Ali HMS, Patzold R, Bruckner H (2010) Gas chromatographic
determination of amino acid enantiomers in bottled and aged
wines. Amino Acids 38:951–958
B’Hymer C, Montes-Bayon M, Caruso JA (2003) Marfey’s reagent:
past, present, and future uses of 1-fluoro-2, 4-dinitrophenyl-5-L-
alanine amide. J Sep Sci 26:7–19
Bhushan R, Agarwal C (2010) Application of (S)-N-(4-nitrophenoxy-
carbonyl) phenylalanine methoxyethyl ester as a chiral deriv-
atizing reagent for reversed-phase high-performance liquid
123