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Journal of Chromatography A, 1180 (2008) 131–137

Separation of amino acid enantiomers VIA chiral derivatization and non-chiral gas chromatography
Maryl` ne Bertrand a,b,∗ , Annie Chabin a,b , Andr´ Brack a,b , Frances Westall a,b e e
a

Centre de Biophysique Mol´ culaire, CNRS, rue Charles Sadron, 45071 Orl´ ans Cedex 2, France e e b Universit´ d’Orl´ ans, 6, avenue du Parc Floral, 45067 Orl´ ans Cedex 2, France e e e

Received 19 October 2006; received in revised form 22 November 2007; accepted 3 December 2007 Available online 8 December 2007

Abstract Two GC–MS methods for the enantioselective separation of the 20 proteinogenic amino acids are compared. Ethyl chloroformate and 2chloropropanol were used to derivatize amino acid enantiomers. The diastereomers formed were separated on a non-chiral column by capillary gas chromatography. The separation performances were compared to those obtained when using non-chiral derivatization on a chiral column. © 2007 Elsevier B.V. All rights reserved.
Keywords: Non-chiral capillary GC; Derivatization; Ethyl chloroformate; 2-Chloropropanol; Amino acid diastereomers

1. Introduction Several processes can be used to separate amino acid enantiomers [1]: liquid chromatography (HPLC) [2], gas chromatography (GC) [3,4,15] on chiral columns, or chiral capillary electrophoresis [5]. The most commonly used methods consist in precolumn derivatization of amino acids by reversed-phase chromatography [6]. For example, Einarsson and Josefsson [7] used the (+)-9-fluorenylethyloxycarbonyle (FLEC) as a chiral reagent for the separation of amino acid enantiomers in liquid chromatography. These methods offer chromatographic reproducibility with subpicomole sensitivity and are commonly used in HPLC but not in GC. Nevertheless, capillary gas chromatography is ideal for the analysis of enantiomers because of its inherent high resolution, short analysis time and coupling capabilities with mass spectrometry. Methods of enantiomer separation on chiral stationary phases by gas chromatography were reviewed by Schurig [3]. Chiral separation of amino acid mixtures on a non-chiral column is a widely used method. In order to be separated by gas chromatography, the amino acids need to be derivatized

to become volatile. Many derivatization methods have been reported [8–12]. An acylation/esterification procedure using alkyl chloroformates [13–18] is the most convenient method of derivatization, offering the following advantages: minimal handling of the sample (no heating and no evaporation), the use of an aqueous medium for the reaction, and inexpensive reagents. This method has been used in many fields, such as the analysis of wines [19], food matrices [20] or the binding media in paint [21,22]. Within the context of understanding the origin and behaviour of important prebiotic molecules transported to the early Earth in extraterrestrial materials (meteorites, micrometeorites), we are particularly interested in developing analytical tools for the separation of enantiomers in connection with experiments dedicated to the study of the stability of amino acids under space conditions. We have developed a method based on the use of ethyl chloroformate and 2-chloropropanol to convert the amino acid enantiomers into diastereomers N(O,S)-ethoxycarbonyl 2chloropropyl ester derivatives according to the reaction: H2 N − ∗ CHR − COOH− − →EtOCO − NH − ∗ CHR −−
pyridine EtOCOCl

∗ Corresponding author at: Centre de Biophysique Mol´ culaire, CNRS, rue e Charles Sadron, 45071 Orl´ ans Cedex 2, France. Tel.: +33 2 38255558; e fax: +33 2 38631517. E-mail address: bertrand@cnrs-orleans.fr (M. Bertrand).

−CO − OCOEt− − − − − − EtOCO − NH − ∗ CHR −−−−−→
pyridine

CH3 −∗ CHCl−CH2 OH ∗

−CO − O − CH2 − CHCl − CH3

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.12.004

0. leucine and glutamine. The oven temperature was set at 100 ◦ C for 5 min.D. All derivatives prepared in this study were stable. 2. threonine. then programmed to reach 275 ◦ C at a rate of 2 ◦ C/min. 1a–c. gave significant peak signals and could easily be identified by mass spectrometry. This detection mode was very sensitive and was used to quantify the species.and bis-esterified derivatives were formed from aspartic and glutamic acids. Separations were achieved on fused-silica capillary columns: a non-chiral CP-Sil 19 CB column from Varian (30 m × 0.D. The mono. In the first experiment. Finally. With the Chirasil-L-Val column. proline and cysteine. 15 L of ethyl chloroformate were added to this solution and the vial was shaken for 10 s. (S)-(+)-2-chloro-1-propanol. N(O. .. 5 × 10−3 M for tryptophan. Each amino acid enantiomer was dissolved in water to a concentration of 0. Helium was used as the carrier gas (inlet pressure: 170 kPa).05 M for serine and valine.12 × 10−4 M. The derivatization reactions led to acylation of the amino group of the amino acids and esterification of the carboxylic group. Amino acid derivatization Amino acid derivatization was run following the procedure described by Abe et al. was used in the study. 0. aspartic and glutamic acid and 5 × 10−4 M for tyrosine. histidine and tyrosine.12 m film thickness). derivatization of l amino acid enantiomers has also been performed. only l amino acids were used (Fig. whereas mono. Chromatogr. each amino acid then having a concentration of 3.2. 100 L of standard solution was put into a 1 mL micro reaction vessel from Supelco and derivatized with 34 L of 2-chloropropanol (or 23 L of ethanol) and 13 L of pyridine.and bis-substituted derivatives were identified by mass spectrometry. alanine. which required the use of a chiral alcohol.25 mm I. was controlled by MSD ChemStation software. It allowed also the detection of very tiny amounts of compounds.2 m film thickness) and a chiral Chirasil-L-Val column from Macherey-Nagel (25 m × 0. trichloromethane. (S)-(+)-2-chloro-1-propanol 97% was purchased from Aldrich. In the second experiment. Bertrand et al. and finally. Ethanol. albeit manually. The results obtained using 2-chloropropanol as a derivatization agent on a non-chiral column are presented for the enantiomers of the proteinogenic amino acids and compared to those obtained with ethanol on a chiral column. For each derivatization. ethyl chloroformate. 2. Standard stock solutions were prepared by mixing the d and l amino acid solutions. Three types of analysis were carried through: (1) analysis on an achiral column with a chiral derivatization procedure. isoleucine.01 M for phenylalanine. l and d amino acids were derivatized and analyzed. This reagent allowed the formation of diasteromers. Solvents and reagents The 20 amino acids used were purchased from Sigma or Aldrich. With the CP-Sil 19 CB column. coupled with a 5973 mass spectrometer as detector. run for control. The oven temperature was held at 70 ◦ C for 5 min. threonine.132 M. pyridine.1. arginine. 1b is a detail of Fig. [17]. Experimental 2. were obtained from Fluka. 19 pairs of enantiomers and glycine. methionine and asparagine. 0. the method provided very clear and very practical chromatograms and automated quantification of the derivatives was carried out when the compounds were well separated. 3. 1a.1 M for lysine. 1 L of the organic layer was injected into the gas chromatography.02 M for histine. 0. The gas chromatograms obtained on an achiral column of N(O. In the case of serine. 2.S)-ethoxycarbonyl 2-chloropropyl ester derivatives with the same temperature ramp and with a SIM detection mode are shown in Fig.25 mm I. Splitless injection mode was used. For control. A mixture of 20 amino acids. (2) The same analysis with the derivatization of only l amino acids to check that the reaction did not result in any racemization. which can be separated on an achiral column. (3) Analysis of N(O. After 5 min. The selected ion monitoring (SIM) detection mode allowed the selection of the derivatives of interest by neglecting the secondary ones.. A 1180 (2008) 131–137 The diastereomers can then be separated by gas chromatography on a non-chiral column. The reaction was more complex for the amino acids containing an additional functional group. both injector and the detector temperatures were set at 200 ◦ C. The present method was developed to provide rapid enantiomeric separation of the 20 proteinogenic amino acids simultaneously.3. either monoand/or bis-acetylated derivatives were obtained. Gas chromatography The Agilent 6890 gas chromatograph system. the CO2 that was produced had escaped and 100 L of chloroform were added. Thanks to the formation of molecular ion groups. / J. held at 180 ◦ C for 15 min. lysine. Two derivatives with very similar retention times or co-eluting could also be identified with the SIM detection mode and integrated. 1c). The vial was vigorously shaken for 2 min and the derivatives were extracted in the organic layer. then programmed to 180 ◦ C at a rate of 2 ◦ C/min. 0. Analytical procedures A mixture of the enantiomers of the 20 proteinogenic amino acids was derivatized via several procedures and separated on different columns. the injector temperature was set at 230 ◦ C and the detector temperature at 250 ◦ C. Fig. glycine.S)-ethoxycarbonyl 2-chloropropyl ester derivatives.S)-ethoxycarbonyl ethyl ester derivatives obtained by ethanol/ethylchloroformate derivatization on a chiral column.

2) for the N(O. A 1180 (2008) 131–137 133 The third experiment is a chromatogram of N(O. 2).. / J. (b) Detail of (a). Carrier gas: helium. Detection by SIM mass spectrometry.25 mm I. 0. Fig. Carrier gas: helium..25 mm I. column inlet pressure 170 kPa.S)-ethoxycarbonyl ethyl esters derivatives detected in scan mode (Fig. and then programmed at 2 ◦ C/min to 275 ◦ C. (c) Gas chromatogram of N(O. The retention times of the amino acid derivatives. (a) Gas chromatogram of N(O.S)-ethoxycarbonyl 2-chloropropyl ester derivatives of an 8 l amino acid mixture. 0.2 m film thickness). splitless.2 m film thickness).01 × 10−4 M of each amino acid enantiomer.S)ethoxycarbonyl 2-chloropropyl ester derivatives (corresponding to Fig.S)-ethoxycarbonyl ethyl esters derivatives. 5 min hold.01 × 10−4 M of each amino acid.S)-ethoxycarbonyl 2-chloropropyl ester derivatives of a l and d amino acid mixture.D. Column Varian CP-Sil 19 CB (30 m × 0. and in Table 2 (corresponding to Fig. . 5 min hold. Injection: 1 L of the 20 l amino acid mixture at 9.M. 1a and b. their enantiomeric resolution and the mean molecular ions of chromatogram peaks are reported in Table 1 for the N(O. Bertrand et al. splitless. Column Varian CP-Sil 19 CB (30 m × 0. Chromatogr. column inlet pressure 170 kPa. Injection: 1 L of the 20 amino acid enantiomer mixture at 9. and then programmed at 2 ◦ C/min to 275 ◦ C.D. Column temperature: 100 ◦ C. Column temperature: 100 ◦ C. 1.

(Continued ). Fig.134 M. Bertrand et al.D. Gas chromatogram of N(O.S)-ethoxycarbonyl ethylester of a l and d amino acid mixture. Scan detection mode. 2. Carrier gas: helium. A 1180 (2008) 131–137 Fig. then programmed to 180 ◦ C at 5 ◦ C/min. 0.12 m film thickness) provided by Macherey-Nagel. Column temperature: 70 ◦ C for 3 min. Chirasil-L-Val column (25 m × 0. 1.25 mm I. splitless. .. / J. 15 min hold at 180 ◦ C. Injection 1 L of 20 amino acid enantiomer mixture at 2. Chromatogr. column inlet pressure 79 kPa.84 × 10−2 M of each amino acid enantiomer.

l-Asp d.58–63. All d amino acid derivatives had shorter retention times than the corresponding l ones.46 53. The good separations of the different amino acid derivatives (except for isoleucine and leucine) and the low column bleeding allowed the formation of .78 3.l-Lys (bis-acetylated) d.61–61.l-Asp (bis-esterified) d.13–48.95–38.09–49. The use of the high performance CP-Sil 19 CB column led to very narrow peaks and good resolution mainly for alkyl amino acid derivatives.44 69.91 ns ns 0. Discussion 4. A 1180 (2008) 131–137 135 Table 1 Retention time tR .l-Ile d.27 Molecular ions 116 102 144 142 158 158 160 188 141 146 114 249 202 192 156 182 220 280 M 189 175 217 215 231 231 233 261 232 219 277 249 275 265 318 255 293 353 M − 73 116 102 144 142 158 158 160 188 159 146 204 176 202 192 245 182 220 280 tR (min) 29. threonine.26 36.45 1.27–53.84 44.95 62.l-Leu d. but low for lysine.07–34.54 – 1. / J.l-Phe d.21 48.25 69.06–44. The enantiomeric resolution was satisfactory for serine.26 0.S)-ethoxycarbonyl 2-chloropropyl ester derivatives analyzed on a CP-Sil 19 CB Derivatives of d. Table 2 Retention time.l-Met d.44 48.76 0.33 1 ns 0.54 62. 1a.03 Rs 1.99 74.l-Cys (bis-acetylated) d.02 43.l-Ser (bis-acetylated) d.82 1. Bertrand et al.4 2.08 59.59 61.68–36.45 80.l-Ser d.67 1.75 51.36–34.l-Tyr (bis-acetylated) ns: not separated. proline. leucine.90 43.19–22.04 1.53 1.33–38.l-Phe d.l-Thr d.86–52.l-Lys (bis-acetylated) d. valine.l-Asn d. and aspartic acid.77–74.l-Cys (bis-acetylated) d.50 33. enantiomeric resolution Rs and mean molecular ions of l and d N(O.l-Pro d.l-Gln d. This method gave good and reproducible enantiomeric resolutions for alkyl chain amino acids (like alanine.04–53.67 1.41 1.25 ns 1.5 116 102 144 158 158 142 146 132 159 176 192 220 237 251 173 173 254 231 208 129 159 231 208 159 129 204 107 4. enantiomeric resolution Rs and mean molecular ions of l and d N(O.S)-ethoxycarbonyl ethyl ester derivatives analyzed on a Chirasil-L-Val Derivatives of d.62 2.35 1.l-His (bis-acetylated) d.28 37.l-Pro d. tR (min) 21.M.62–40. Experiment 1 (Fig.75 ns ns 0.l-Thr d. The amino acid enantiomers could be quantified with internal standards when good separations and resolutions were achieved.l-Ile d. 1b).1.l-Trp d.17–29.93–32. methionine.48 ns ns ns Molecular ions 116 102 144 158 158 142 146 132 141 176 192 220 237 251 173 156 254 130 107 M 238 224 266 280 280 264 268 254 281 298 314 342 358 372 295 295 376 353 330 M − 121.72–44.53 40.45 1.80–44. all amino acids derivatives gave significant peak signals and 10 of the 19 pairs of amino acid enantiomer derivatives were separated with significant retention times and enantiomeric resolutions (see Table 1).l-Glu (bis-esterified) d.00 23.l-Val d.19 38.l-Asn d.l-Met d.43 34.73–27.92–71.l-Tyr (bis-acetylated) ns: not separated.6 53.7 76.l-Ala Gly d. Chromatogr.l-Ala Gly d.06 46.l-Asp (bis-esterified) d.89–71.38 31.l-His d.87 75.l-Leu d.46 49.52 1. The other amino acid enantiomer derivatives were not separated.32 ns 1.88 31.67 69.36 29.22 Rs 3.04 – 2.30–45. The bleeding of the column was low and allowed good analysis of the high boiling point derivatives without damaging for the column.25 45.l-Glu (bis-esterified) d. Better resolution of some derivatives was obtained by using different rates of temperature gradient.49 26. 1a and b and Table 1) In Fig.l-Val d. and isoleucine) (Fig.77–47.39 57.76 67.

Nakao. as well as their quantitative determination. This method allows the separation of 14 of 19 enantiomeric pairs of the proteinogenic amino acids when associated with the SIM detection mode. A. 70 (1998) 890. 4. J. the Chirasil-L-Val column allowed the separation of 14 out of 19 enantiomer pairs with generally good enantiomeric resolution. This phenomenon was not at all observed with the non-chiral CP-Sil 19 CB column. Y. Sandra. M. H.26 for the di-ester. Kim. Einarsson. Experiment 2 (Fig. [11] C. For example. T. A. Chem.4. [14] P.3. Oh.-R. Josefsson. for which the temperature limit was not reached. Valleix. Chromatogr. 2 and Table 2) As seen on the chromatogram in Fig. Automation of the calculations could not be carried out with this procedure. J. B 717 (1998) 57. 70 (1987) 151. The use of the column at high temperature strongly decreased its performance and its lifespan. the enantiomeric resolution of aspartic acid mono ester was 1. J. Terabe. Acknowledgements This study was supported by the French “Centre National de la Recherche Scientifique (CNRS)” and by the French “Centre National d’Etudes Spatiales (CNES)”. 4. it was not possible to form ion molecular groups to identify all of the compounds since too many derivatives were overlapping (Table 2). Chromatogr. J. Simek. Abe. Mass spectrometry fragmentation The same mass spectrometric fragmentations as those reported by Zampolli et al. The other mass peaks could be identified easily: they correspond to the loss of OH+ . The first method allows simultaneous analysis of a mixture of amino acid enantiomers without requiring a chiral column change by using (S)-(+)-2-chloro-propanol as a derivatization agent. [10] S. The mirror image did not exceed 1. However. aspartic acid and asparagine were not well separated and gave overlapping peaks. Moreover. The quantification of all amino acids in one single injection was obtained and was automated with good accuracy. This derivatization method was found to be suitable for rapid. such as foods or paints. Husek. [9] I. K. J. J. J. Assoc. 25 (2002) 661. Chromatogr. Duncan.M. The simplicity of these methods makes them readily applicable to many fields. / J. A 1071 (2005) 59. Dreux.136 M. The other amino acid derivative pairs were not separated. Anal. 59 (1987) 1191. Skacani. Sci. Kim. as did lysine and histidine. J. Since no column change is necessary. J. [13] P.W. D.-H. A 1180 (2008) 131–137 ion molecular groups and the use of the SIM detection mode to detect and quantify the compounds described in Table 1. Chromatogr. S. it is very easy to switch from achiral to chiral analysis and to extend the analysis to other molecules. [8] M. Off. The loss of acylium ions is characteristic of the cleavage of the C C bond in the -position of the esterified carboxyl group. H. a value lower than the degree of purity of 2 chloropropanol. Wan. Sep. Anal. Chem. B.L. offering several complementary advantages. A 669 (1994) 125. Petritis. [12] K. FEBS Lett. MacKenzie. single step detection of the presence of amino acid enantiomers in solution. u [7] S. [5] H. Nishi. 4. Chromatogr. Nakahara. Minami. Westhauser. 2. that could be present in more complex mixtures. [3] V. P.04 and 1. References [1] K. [18] were observed for the N(O.2. 1c) run for control showed that no racemization occurred during the derivatization.6%. I. Woo. D.-H.G. The high boiling point derivatives gave well defined peaks and could be quantified by analysis on the CP-Sil 19 CB column without damage to the column. P. Amino Acids 24 (2003) 43. Elfakir. Krupcik. or H2 O for the derivatives containing an additional functional group and to the loss of [HO C6 H4 CH2 ]+ (m/z = 107) for the tyrosine derivatives. [4] I. Poljak.S)-ethoxycarbonyl ethyl ester derivatives. J. Schurig.S)-ethoxycarbonyl 2-chloropropyl ester and N(O. T. [6] H. D. 5. Armstrong. Arginine was the only one which was not detected. which caused significant bleeding and did not allow the detection of tryptophan. J. Conclusions The derivatization methods described are handy and fast. Husek. Nevertheless. Experiment 3 (Fig.5) for the N(O. Chromatogr. LC–GC 19 (2001) 986.W. Brownson. It should be noted that better resolutions were obtained for bis-substituted compounds compared to the corresponding mono-substituted ones. T. for the analysis of the basic amino acid derivatives (with high boiling points). A 906 (2001) 275. Blomberg. L. Mabry. 1c) The chromatogram of the l amino acids derivatives (Fig. A 913 (2001) 331. Chem. Br¨ ckner. The authors thank Bernard Barbier and Uwe Meierhenrich for their help. Spanik. Bertrand et al. contrary to the analysis on the Chirasil-L-Val column.L. .S. C.S)ethoxycarbonyl 2-chloropropyl ester derivatives (Table 1) and to the loss of [CO2 CH2 CH3 ]+ (m/z = M − 73) for the N(O. the upper temperature limit of the Chirasil-L-Val column had to be used. [2] P. The percentage of amino acid enantiomers could be quantified with internal standards by the use of the SIM detection mode when good separations and resolutions were obtained. Chromatogr. For example.J. B 665 (1995) 15. The main mass peak corresponds to the loss of the acylium ion [CO2 CH2 CHCl CH3 ]+ (m/z = M − 121. Chromatogr.S)-ethoxycarbonyl ethyl ester derivatives (Table 2). A 875 (2000) 43. 280 (1991) 354. The separation and the integration of 19 out of 20 amino acids were easily achieved by the formation of molecular ion groups. the best enantiomeric resolutions were always obtained when using derivatization with ethanol and ethylchloroformate on a Chirasil-L-Val column. This method was reproducible and gave satisfactory enantiomeric resolutions for 10 of the 19 enantiomer pairs. because there was too much overlapping of the various derivatives. The most important advantage of this method is the separation and identification of 19 amino acids out of the 20 natural amino acids. Husek. Anal. Lee.

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