Determination of Chemical and Enantiomeric Purity of α-Amino Acids and their Methyl Esters as N-Fluorenylmethoxycarbonyl Derivatives Using Amylose-derived Chiral Stationary Phases

Article abstract of DOI:10.1002/bkcs.11694

Liquid chromatographic enantiomer separation and simultaneous determination of chemical and enantiomeric purity of α-amino acids and their methyl esters as N-fluorenylmethoxycarbonyl (FMOC) derivatives was performed on three covalently bonded type chiral stationary phases (CSPs) derived from amylose derivatives. The enantiomer separation of α-amino acid esters as N-FMOC derivatives was better than that of the corresponding acids, especially for CSP 1 and 2. Chemical impurities as the corresponding racemic acids present in several commercially available racemic amino acid methyl esters were observed to be 0.49–17.50%. Enantiomeric impurities of several commercially available L-amino acid methyl esters were found to be 0.03–0.58%, whereas chemical impurities as the corresponding racemic acids present in the same analytes were found to be 0.13–13.62%. This developed analytical method will be useful for the determination of chemical and enantiomeric purity of α-amino acids and/or esters as N-FMOC derivatives using amylose-derived CSPs.

Full text of DOI:10.1002/bkcs.11694

Article
DOI: 10.1002/bkcs.11694
BULLETIN OF THE
M. F. Islam et al.
KOREAN CHEMICAL SOCIETY
Determination of Chemical and Enantiomeric Purity of α-Amino Acids
and their Methyl Esters as N-Fluorenylmethoxycarbonyl Derivatives
Using Amylose-derived Chiral Stationary Phases
†,
Md. Fokhrul Islam,†,§ Suraj Adhikari,†,§ Man-Jeong Paik,‡, and Wonjae Lee
*
*
†College of Pharmacy, Chosun University, Gwangju 501-759, South Korea. *E-mail: wlee@chosun.ac.kr
‡College of Pharmacy, Sunchon National University, Suncheon 540-950, South Korea.
*E-mail: paik815@sunchon.ac.kr
Received January 2, 2019, Accepted January 26, 2019
Liquid chromatographic enantiomer separation and simultaneous determination of chemical and enantio-
meric purity of α-amino acids and their methyl esters as N-fluorenylmethoxycarbonyl (FMOC) derivatives
was performed on three covalently bonded type chiral stationary phases (CSPs) derived from amylose
derivatives. The enantiomer separation of α-amino acid esters as N-FMOC derivatives was better than that
of the corresponding acids, especially for CSP 1 and 2. Chemical impurities as the corresponding racemic
acids present in several commercially available racemic amino acid methyl esters were observed to be
0.49–17.50%. Enantiomeric impurities of several commercially available L-amino acid methyl esters were
found to be 0.03–0.58%, whereas chemical impurities as the corresponding racemic acids present in the
same analytes were found to be 0.13–13.62%. This developed analytical method will be useful for the
determination of chemical and enantiomeric purity of α-amino acids and/or esters as N-FMOC derivatives
using amylose-derived CSPs.
Keywords: Amino acid, Chiral stationary phase, Enantiomer separation, N-Fluorenylmethoxycarbonyl
derivative
Introduction
enantiomers.12,16 Also, immobilized polysaccharide-based
CSPs are a new generation of chiral chromatographic mate-
rials that combine the remarkable enantioselective perfor-
mance of the polysaccharide derivatives with solvent
versatility and column robustness in enantiomeric
resolution.17–19 Recently, three covalently bonded amylose
phenylcarbamate-derived CSPs (Chiralpak ID, IE, and IF)
were developed and have been shown in some
applications.17,19–21 In this study, a simple and convenient
analytical method is developed for the enantiomeric resolu-
tion of α-amino acids and their methyl esters as N-FMOC
derivatives and is applied for determination of their enan-
tiomeric and/or chemical purities. The normal phase chiral
HPLC is performed on three covalently bonded amylose
phenylcarbamate-derived CSPs under simultaneous ultravi-
olet (UV) and fluorescence detection. The application of
newly developed immobilized CSPs for the determination
of the enantiomeric and/or chemical purities of several
commercially available α-amino acid methyl esters is
described in this study.
Pharmaceutical research for chiral drugs is mostly inclined
towards the development of selective stereoisomer and test-
ing of the individual stereoisomers.1 It is because of the fact
that two enantiomers of a chiral drug may behave differ-
ently in chiral environments of living organisms.2,3 Amino
acids are extensively used as essential building blocks for
chiral drugs and their enantiomerically pure isomers are
employed as a useful chiral drug intermediates and nutri-
tional supplements.2 Therefore, in pharmaceutical industry,
the development of effective chiral analytical methods to
determine enantiomeric purity and to obtain enantiomeri-
cally pure chiral drugs or their intermediates is of high
interest.4,5 The fluorenylmethoxycarbonyl (FMOC) group
for α-amino acids can be used as a derivatizing agent for
enantiomer separation and may provide advantages with
respect to selectivity and sensitivity during fluorescence
detection.6,7 Among several techniques for enantiomeric
separation, chiral high-performance liquid chromatography
(HPLC) using polysaccharide-derived chiral stationary
phases (CSPs) have been the most powerful technique.3,8–15
In particular, amylose tris phenylcarbamate represents a
major group of chiral selectors used in the preparation of
CSPs for the liquid chromatographic separation of
Experimental
Chemicals and Reagents. HPLC-grade 2-propanol, hex-
ane, ethyl acetate, and tetrahydrofuran were purchased from
J.T. Baker (Phillipsburg, NJ, USA). HPLC-grade acetoni-
trile, toluene, and dichloromethane were purchased from
§These authors have equally contributed on this work.
Bull. Korean Chem. Soc. 2019
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Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
Fisher Scientific (Hampton, NH, USA) and Merck
(Kenilworth, NJ, USA). Trifluoroacetic acid (TFA) was
obtained from Acros Organics (Geel, Belgium). The deriva-
tizing agent, FMOC chloride, was procured from the Fluka
Company (Buchs, Switzerland). All racemic amino acids
were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Racemic and/or L-amino acid methyl esters of norleucine,
norvaline, phenylalanine, serine, leucine, and valine were
obtained from Chem-Impex International (Bensenville, IL,
USA), Advanced ChemTech (Louisville, KY, USA), Acros
Organics (Geel, Belgium), Bachem (Bubendorf, Switzer-
land), and Alfa-Aesar (Haverhill, MA, USA). The other
racemic and L-amino acid methyl esters were purchased
from Sigma-Aldrich. Two commercially available FMOC
D- and L-phenylglycine, were purchased from the Fluka
Company (Buchs, Switzerland). The N-FMOC derivatized
racemic and L-amino acids and methyl esters were prepared
according to conventional methods.22 Racemic or L-
α-amino acid (5 mmol) was dissolved in 10% aqueous
sodium carbonate solution (12.5 mmol). Dioxane (7.5 mL)
was then added and the mixture was stirred in an ice-bath.
After that, 9-FMOC chloride (5 mmol) was added slowly
and stirred at room temperature for 5 h. Now, the reaction
mixture was poured into water and extracted with ether.
The aqueous solution obtained was acidified with c-HCl in
an ice-bath. Finally, the resulting N-FMOC α-amino acid
was filtered and dried under vacuum. In order to prepare
racemic or L-FMOC α-amino acid methyl ester, the corre-
sponding FMOC α-amino acid (1 mmol) synthesized in the
previous step was dissolved in 5 mL of anhydrous metha-
nol with N,N0-dicyclohexylcarbodiimide (1.1 mmol). The
mixture was stirred at room temperature for 12 h, filtered
and dried under vacuum to get N-FMOC α-amino acid
methyl ester.
injector, and an HP 1046A programmable fluorescence
detector. The chromatographic analysis was performed
under simultaneous UV 262 nm and fluorescence [excita-
tion (Ex.) 264 and emission (Em.) 312 nm] detection. All
enantiomeric separations of α-amino acids and methyl esters
as N-FMOC derivatives were performed using isocratic
mobile phases [10 or 20% 2-propanol/hexane (v/v) with or
without 0.1% TFA] at an ambient temperature (approximately
25 ꢀC) with a flow rate of 1 mL/min. Three covalently
bonded CSPs, CSP 1 (Chiralpak ID), CSP 2 (Chiralpak IE),
and CSP 3 (Chiralpak IF) (250 mm × 4.6 mm, I.D., 5 μm)
having chiral selectors of amylose tris (3-chlorophenylcarba-
mate), amylose tris (3,5-dichlorophenylcarbamate), and amy-
lose tris (3-chloro-4-methylphenylcarbamate), respectively,
were purchased from the Daicel Company (Tokyo, Japan).
Results and Discussion
Enantiomeric Separation of α-Amino acids and Methyl
Esters as N-FMOC Derivatives. The enantiomeric sepa-
ration results of nine α-amino acids and methyl esters as N-
FMOC derivatives with normal phase eluents on three
newly developed immobilized CSPs are summarized in
Tables 1–3. The conventional mobile phase system (2-pro-
panol/hexane) was used for the entire enantiomeric
separation process. The chromatographic parameters of
enantioseparation and retention factor were significantly
influenced by the nature of the mobile phase, the used CSP,
and the analyte type.12,18 Based on the overall results from
the nine analytes, the most successful chiral CSP was CSP
1 having amylose tris (3-chlorophenylcarbamate) as a chiral
selector except for two analytes of amino acids (entries
2 and 9, Table 1). Conversely, CSP 3 with amylose tris
(3-chloro-4-methylphenylcarbamate) showed the lowest
enantioselection and resolution of the investigated analytes,
as shown in Table 3. In general, the enantioseparation
results obtained from CSP 2 were lower than those
Liquid Chromatography. Analysis was performed using
an HP series 1100 HPLC system (Palo Alto, CA, USA),
consisting of a G1310A iso pump, an automatic sample
Table 1. Separation of the enantiomers of α-amino acids and methyl esters as N-FMOC derivatives on CSP 1.
FMOC amino acid FMOC amino acid methyl ester
Entry
Analyte
α
k01
Rs
Conf.
α
k01
Rs
Conf.
1
2
3
4
5
6
7
8
9
Alanine
Leucine
1.37
1.00
1.75
1.12
1.07
1.19
1.32
1.31
1.07
2.67
2.22
5.21
2.19
2.49
3.33
5.80
6.06a
2.20
5.18
—
L
—
L
L
L
L
L
L
D
3.68
1.74
4.03
2.85
2.32
1.36
1.65
2.14
1.61
5.03
3.22
5.84a
3.72
4.06
6.16
7.94
5.09
3.42
20.99
6.50
15.48
12.50
13.02
5.43
7.36
11.19
6.08
L
L
L
L
L
L
L
L
L
Methionine
Norleucine
Norvaline
Phenylalanine
Phenylglycine
Serine
9.56
1.50
1.21
3.21
4.52
4.11
0.83
Valine
Mobile phase: 10% 2-propanol/hexane (v/v) with 0.1% TFA for FMOC amino acids and 10% 2-propanol/hexane (v/v) for FMOC amino acid
methyl esters, flow rate: 1 mL/min, UV: 262 nm, fluorescence detection: Ex. 264 nm, Em. 312 nm, k01: retention factor of the first eluted
enantiomer, α: separation factor, Rs: resolution factor, Conf.: the absolute configuration of the second eluted enantiomer.
a20% 2-propanol/hexane (v/v) with or without 0.1% TFA.
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Article Enantiomer Separation of α-Amino Acids and Esters as N-FMOC Derivatives KOREAN CHEMICAL SOCIETY
Table 2. Separation of the enantiomers of α-amino acids and methyl esters as N-FMOC derivatives on CSP 2.
FMOC amino acid
FMOC amino acid methyl ester
Entry
Analyte
α
k01
Rs
Conf.
α
k01
Rs
Conf.
1
2
3
4
5
6
7
8
9
Alanine
Leucine
1.15
1.17
1.05
1.09
1.07
1.11
1.02
1.31
1.15
3.53
3.13
5.86
3.08
3.45
5.02
6.92
6.78
3.77
2.15
1.90
0.79
1.02
0.96
1.41
0.23
3.21
1.85
D
D
D
D
D
D
D
L
1.14
1.08
2.03
1.09
1.16
2.48
1.54
1.38
1.15
6.34
5.00
10.49
5.09
5.56
6.10
7.75
12.63
4.23
2.01
1.09
12.24
1.18
2.21
10.81
6.08
5.16
1.88
D
D
D
D
D
D
D
L
Methionine
Norleucine
Norvaline
Phenylalanine
Phenylglycine
Serine
Valine
D
D
Mobile phase: 10% 2-propanol/hexane (v/v) with 0.1% TFA for FMOC amino acids and 10% 2-propanol/hexane (v/v) for FMOC amino acid
methyl esters, flow rate: 1 mL/min, UV: 262 nm, fluorescence detection: Ex. 264 nm, Em. 312 nm, k01: retention factor of the first eluted
enantiomer, α: separation factor, Rs: resolution factor, Conf.: the absolute configuration of the second eluted enantiomer.
obtained from CSP 1, although most of the analytes were
baseline separated with good resolution and separation fac-
tor. Additionally, as shown in Tables 1 and 2, the enantiose-
paration of α-amino acid methyl esters as N-FMOC
derivatives was much greater than that of the corresponding
α-amino acids. It is considered that the ester moieties of
amino acid methyl esters might involve and favor the chiral
recognition mechanism on CSP 1 and 2. The L-isomers of
all investigated analytes except for valine (entry 9) in
Table 1 were preferentially retained on CSP 1. An inversion
of the elution order between amino acid and methyl ester
(valine, entry 9) indicates competition of different enantio-
meric separation mechanisms. Unlike on CSP 1 in Table 1,
the D-isomers of all investigated analytes were preferentially
retained on CSP 2 in Table 2, except for serine and its
methyl ester (entry 8). The 3,5-dichloro moiety on the phe-
nylcarbamate chiral selector of CSP 2 might involve in the
different chiral recognition interactions during enantiodiscri-
mination. But, such a different elution order for these ana-
lytes depending upon the used CSPs is difficult to
rationalize and predict. Rather, the enantiomer elution order
must be established by systematic screening of combinations
of CSP and mobile phase conditions. The reporting of such
results might be of great interest to analysts and might be a
significant step in understanding the chiral recognition
mechanisms on polysaccharide-derived CSPs.
Effect of Different Mobile Phases on Enantiomer Sepa-
ration. Table 4 shows the comparative mobile phase
results of chromatographic enantiomeric separation of five
α-amino acids as N-FMOC derivatives between the conven-
tional alcoholic mobile phase system (2-propanol/hexane)
and the non-alcoholic mobile phase system (dichloro-
methane, ethyl acetate, or tetrahydrofuran/hexane) on CSP
1. The separation factors and elution orders on CSP 1 are
dependent on the properties of the mobile phase (alcoholic
or non-alcoholic mobile phase).18,19,23 In general, 10%
2-propanol/hexane (v/v) with 0.1% TFA as a conventional
type mobile phase afforded the greatest enantioselectivities
with high resolution factors. Presumably, hydrogen bonding
interactions in the alcoholic solvent system might be signif-
icantly involved in enantiodiscrimination between the ana-
lyte and chiral selector of the CSP. Among the other non-
Table 3. Separation of the enantiomers of α-amino acids and methyl esters as N-FMOC derivatives on CSP 3.
FMOC amino acid FMOC amino acid methyl ester
Entry
Analyte
α
k01
Rs
Conf.
α
k01
Rs
Conf.
1
2
3
4
5
6
7
8
9
Alanine
Leucine
1.05
1.11
1.27
1.07
1.10
1.08
1.24
1.00
1.00
3.12
2.31
4.74
2.54
2.66
4.22
5.59
5.82
3.19
0.71
1.49
3.54
0.96
1.40
1.10
3.60
—
L
L
L
L
L
L
L
—
1.05
1.30
1.07
1.21
1.14
1.47
1.04
1.33
1.00
5.26
3.58
10.62
3.85
4.69
6.70
7.21
9.31
4.64
0.53
1.74
0.67
1.47
1.00
3.00
0.32
2.74
—
L
L
D
L
D
D
D
L
Methionine
Norleucine
Norvaline
Phenylalanine
Phenylglycine
Serine
Valine
—
—
—
Mobile phase: 10% 2-propanol/hexane (v/v) with 0.1% TFA for FMOC amino acids and 10% 2-propanol/hexane (v/v) for FMOC amino acid
methyl esters, flow rate: 1 mL/min, UV: 262 nm, fluorescence detection: Ex. 264 nm, Em. 312 nm, k01: retention factor of the first eluted
enantiomer, α: separation factor, Rs: resolution factor, Conf.: the absolute configuration of the second eluted enantiomer.
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BULLETIN OF THE
Article Enantiomer Separation of α-Amino Acids and Esters as N-FMOC Derivatives KOREAN CHEMICAL SOCIETY
Table 4. Effect of the mobile phase on the enantiomeric separation of amino acids as N-FMOC derivatives on CSP 1.
Mobile 10% 2-propanol/hexane
40% dichloromethane/hexane 20% ethyl acetate/hexane
(v/v) with 0.1% TFA (v/v) with 0.1% TFA
15% tetrahydrofuran/hexane
(v/v) with 0.1% TFA
phase
(v/v) with 0.1% TFA
k01
1.37 2.67 5.18
Analyte
α
Rs
Conf.
α
k01 k01
1.07 4.42 1.05
Rs
Conf.
α
Rs
Conf.
α
k01
Rs
Conf.
Ala
Leu
Phe
PG
L
—
1.11 5.23 1.26
1.04 3.59 1.08
1.09 5.09 1.37
1.17 8.31 2.34
1.12 3.85 2.25
L
L
L
L
L
D
—
L
1.00 4.28
1.00 3.22
1.00 5.83
1.00 6.87
—
—
—
—
—
—
—
—
D
1.00 2.22
—
1.00 2.91
—
1.19 3.33 3.21
1.32 5.80 4.52
1.07 2.20 0.83
L
L
D
1.05 4.30 0.87
1.11 5.15 1.49
1.03 2.95 0.38
L
L
Val
1.07 2.74 1.07
Flow rate: 1 mL/min, TFA: trifluoroacetic acid, k01: retention factor of the first eluted enantiomer, α: separation factor, Rs: resolution factor, Conf.:
the absolute configuration of the second eluted enantiomer, UV: 262 nm, fluorescence detection: Ex. 264 nm, Em. 312 nm.
alcoholic mobile phases used, the highest enantioselectiv-
ities were shown by 40% dichloromethane/hexane (v/v)
with 0.1% TFA (α = 1.04–1.17, Rs = 1.08–2.34). The 15%
tetrahydrofuran/hexane (v/v) with 0.1% TFA as the mobile
phase showed the lowest enantioselection and most of the
analytes under consideration were unresolved. Also, the use
of different mobile phases does not always provide the con-
sistent elution order of the investigated analytes. For exam-
ple, the reversal of elution order of alanine was observed in
the case of 20% ethyl acetate/hexane (v/v) with 0.1%
TFA (entry 1, Table 4). The binding energy change due
to solvation effect of the analyte or dipole–dipole inter-
action by the polar aprotic solvent of ethyl acetate may
result in its reversal of elution order.23,24 However, as
competitive and different chiral recognition mechanisms
between the chiral selector and each enantiomer might
also occur during enantiodiscrimination process in dif-
ferent mobile phase systems, it is difficult to explain
clearly about such a reversal in elution order. It should
be mentioned that the advantage of the mobile phase
compatibility of covalently bonded CSPs may be useful
for applications of enantiomer separation, especially pre-
parative enantiomer separation.19
Simultaneous Determination of Chemical and/or Enan-
tiomeric Purities. As a typical application, the developed
analytical method in the present study was employed for
the simultaneous determination of chemical and enantio-
meric purities of commercially available α-amino acids
methyl esters as N-FMOC derivatives on CSP 1. Amino
acid esters are prepared from the corresponding amino
acids as starting materials and, therefore, a convenient
analytical method for the determination of chemical
and enantiomeric purities is important. In Table 5, the
chemical purities of racemic amino acid methyl esters as
N-FMOC derivatives determined using CSP 1 are pre-
sented. The chemical impurities (the corresponding race-
mic amino acids) present in the racemic amino acid
methyl esters were found to be 0.49–17.50%. Racemic
norvaline methyl ester (Chem-Impex International) had
the highest amount of racemic norvaline impurity
(17.50%), whereas phenylglycine methyl ester (Sigma-
Aldrich) showed the lowest racemic phenylglycine
impurity (0.49%). Figure 1 shows the typical chromato-
grams of the resolution of racemic methionine methyl
ester (Sigma-Aldrich), racemic norleucine methyl ester
(Chem-Impex International), and racemic valine methyl
Table 5. Chemical purities of commercially available racemic amino acid methyl esters as N-FMOC derivatives on CSP 1.
Chemical puritya
Entry
Analyte
Company
(racemic amino acid: racemic methyl ester)
1
2
3
4
5
6
7
8
9
Alanine
Leucine
Sigma-Aldrich
Bachem
4.06: 95.94
0.54: 99.46
11.56: 88.44
7.62: 92.38
17.50: 82.50
0.56 : 99.44
0.49: 99.51
0.79: 99.21
0.51: 99.49
Methionineb
Norleucine
Norvaline
Phenylalanine
Phenylglycine
Serineb
Sigma-Aldrich
Chem-Impex International
Chem-Impex International
Advanced ChemTech
Sigma-Aldrich
Acros
Valine
Alfa-Aesar
Mobile phase: 10% 2-propanol/hexane (v/v) with 0.1% TFA, flow rate: 1 mL/min, UV: 262 nm, fluorescence detection: Ex. 264 nm, Em. 312 nm.
aAverage value of six times determination.
b20% 2-propanol/hexane (v/v) with 0.1% TFA.
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BULLETIN OF THE
Article Enantiomer Separation of α-Amino Acids and Esters as N-FMOC Derivatives KOREAN CHEMICAL SOCIETY
commercially available L-amino acid methyl esters on
CSP 1. The chemical impurities of L-amino acid methyl
esters represent the ratio of racemic amino acids and
racemic methyl esters. The enantiomeric impurities of L-
amino acid methyl esters are the corresponding D-amino
acid methyl esters. As shown in Table 6, the range of
chemical and enantiomeric impurities obtained for com-
mercially available L-amino acid methyl esters was
found to be 0.13–13.62% and 0.03–0.58%, respectively.
Interestingly, it was observed that the chemical impuri-
ties of these analytes are pretty higher than the enantio-
meric impurities. Figure
2
shows the typical
chromatograms for the resolution of commercially avail-
able racemic and L-alanine methyl ester as N-FMOC
derivatives including the total chiral impurities. For the
chemical purity of racemic alanine methyl ester (Sigma-
Aldrich), the ratio of racemic alanine: racemic its methyl
ester was 4.06: 95.94 (Figure 2, middle, entry 1, Table 5).
On the other hand, the four peak ratios for the chiral
purity of L-alanine methyl ester (Sigma-Aldrich) were
D-acid: L-acid: D-methyl ester: L-methyl ester =
0.20:13.42:0.50:85.88 (Figure 2, bottom). For the
broad applicability of our developed method, we also
determined the enantiomeric ratios of commercially
available FMOC D- and L-phenylglycine analytes on
CSP 1. The enantiomeric impurities for commercially
available FMOC D- and L-phenylglycine (Fluka com-
pany) were found to be 0.35 and 0.60%, respectively,
and are shown in Table 7. The chromatograms of enan-
tiomer separation of these analytes for the determina-
tion of enantiomeric purities under simultaneous UV
and fluorescence detection on CSP 1 are shown in
Figure 3.
Figure 1. Chromatograms of the resolution of (a) racemic methio-
nine methyl ester (Sigma-Aldrich) (amino acid: methyl ester =
11.56: 88.44), (b) racemic norleucine methyl ester (Chem-Impex
international) (amino acid: methyl ester = 7.62: 92.38), and
(c) racemic valine methyl ester (Alfa-Aesar) (amino acid: methyl
ester = 0.51: 99.49) as N-FMOC derivatives on CSP 1 using 10%
2-propanol/hexane (v/v) with 0.1% TFA as a mobile phase.
ester (Alfa-Aesar) as N-FMOC derivatives on CSP
1. Similarly, the chemical and enantiomeric purities of
commercially available L-amino acid methyl esters as
N-FMOC derivatives on CSP 1 were determined.
Table 6 shows the results of all chiral impurities of
Table 6. All chiral ratios including chemical and enantiomeric purities of commercially available L-amino acid methyl esters as N-FMOC
derivatives on CSP 1.
Enantiomeric
Chemical puritya
puritya
Total chiral puritya
racemic acid:
racemic methyl ester
D-methyl ester:
L-methyl ester
D-acid: L-acid: D-methyl
ester: L-methyl ester
Entry Analyte
Company
1
2
3
4
5
6
7
8
9
Alanine
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Chem-Impex International
Chem-Impex International
13.62:86.38
0.13:99.87
0.60:99.40
13.03:86.97
8.60:91.40
0.58:99.42
1.94:98.06
0.62:99.38
0.26:99.74
0.58:99.42
0.05:99.95
0.05:99.95
0.07:99.93
0.28:99.72
0.03:99.97
0.26:99.74
0.05:99.95
0.05:99.95
0.20:13.42:0.50:85.88
0.02:0.11:0.05:99.82
0.01:0.59:0.05:99.35
0.04:12.99:0.06:86.91
0.14:8.46:0.26:91.14
0.03:0.55:0.03:99.39
0.03:1.91:0.25:97.81
0.07:0.55:0.05:99.33
0.04:0.22:0.05:99.69
Leucineb
Methioninec
Norleucine
Norvaline
Phenylalanine Advanced ChemTech
Phenylglycine Sigma-Aldrich
Serinec
Valine
Acros
Sigma-Aldrich
Mobile phase: 10% 2-propanol/hexane (v/v) with 0.1% TFA, flow rate: 1 mL/min, UV: 262 nm, fluorescence detection: Ex. 264 nm,
Em. 312 nm.
aAverage value of six times determination.
bCSP 2 was used.
c20% 2-propanol/hexane (v/v) with 0.1% TFA.
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BULLETIN OF THE
Article Enantiomer Separation of α-Amino Acids and Esters as N-FMOC Derivatives KOREAN CHEMICAL SOCIETY
Figure 3. Chromatograms of the enantiomer resolution of com-
mercially available (a) FMOC D-phenlyglycine (Fluka) (D:
L = 99.65: 0.35) and (b) FMOC L-phenylalanine (Fluka) (D:
L = 0.60: 99.40) under simultaneous UV (top) and fluorescence
(FL) (bottom) detection on CSP 1. See chromatographic condi-
tions in Table 7.
was applied to determine the chemical and/or enantiomeric
Figure 2. Chromatograms of the enantiomer resolution of racemic
purities of several commercially available racemic and L-
alanine (top), racemic alanine methyl ester (Sigma-Aldrich) (race-
amino acid methyl esters. The present study is expected to
mic amino acid: racemic methyl ester = 4.06: 95.94) (middle), and
be useful for the determination of the chiral purity of
L-alanine methyl ester (Sigma-Aldrich) (D-acid: L-acid: D-methyl
ester: L-methyl ester = 0.20: 13.42: 0.50: 85.88) (bottom) as N-
FMOC derivatives on CSP 1 using 10% 2-propanol/hexane (v/v)
with 0.1% TFA as a mobile phase.
α-amino acids and their related compounds as N-FMOC
derivatives in pharmaceuticals.
Acknowledgement. This work was supported by the
National Research Foundation of Korea (NRF) grant
funded by the Ministry of Science, ICT & Future Planning
(2015R1A4A1041219).
Table 7. Enantiomeric ratios of commercially available FMOC D-
and L-phenylglycine on CSP 1.
Entry
Analyte
Company
D:La
References
1
2
FMOC D-phenylglycine
FMOC L-phenylglycine
Fluka
Fluka
99.65:0.35
0.60:99.40
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a
Average value of six times determination.
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In conclusion, a simultaneous UV and fluorescence HPLC
analysis for the enantiomeric separation and determination
of chemical and/or enantiomeric purities of α-amino acids
and their methyl esters as N-FMOC derivatives on three
covalently bonded type CSPs derived from amylose phe-
nylcarbamates was performed. Several mobile phases were
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on enantiomer separation. In general, the performance of
CSP 1 was the greatest among three CSPs and the degree
of enantiomer separation of α-amino acid esters was higher
than that of the corresponding acids. This analytical method
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6

BULLETIN OF THE
Article Enantiomer Separation of α-Amino Acids and Esters as N-FMOC Derivatives KOREAN CHEMICAL SOCIETY
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