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Journal of Chromatography A, 1180 (2008) 131137

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 dOrl 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 GCMS 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-uorenylethyloxycarbonyle (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 [812]. An acylation/esterication procedure using alkyl chloroformates [1318] 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 elds, 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 CHClCH2 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

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The diastereomers can then be separated by gas chromatography on a non-chiral column. The present method was developed to provide rapid enantiomeric separation of the 20 proteinogenic amino acids simultaneously. 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 control, derivatization of l amino acid enantiomers has also been performed. A mixture of 20 amino acids, 19 pairs of enantiomers and glycine, was used in the study. 2. Experimental 2.1. Solvents and reagents The 20 amino acids used were purchased from Sigma or Aldrich. Each amino acid enantiomer was dissolved in water to a concentration of 0.1 M for lysine, arginine, alanine, threonine, glycine, proline and cysteine, 0.05 M for serine and valine, 0.02 M for histine, isoleucine, methionine and asparagine, 0.01 M for phenylalanine, leucine and glutamine, 5 103 M for tryptophan, aspartic and glutamic acid and 5 104 M for tyrosine. Standard stock solutions were prepared by mixing the d and l amino acid solutions, each amino acid then having a concentration of 3.12 104 M. Ethanol, pyridine, ethyl chloroformate, trichloromethane, were obtained from Fluka. (S)-(+)-2-chloro-1-propanol 97% was purchased from Aldrich. 2.2. Amino acid derivatization Amino acid derivatization was run following the procedure described by Abe et al. [17]. For each derivatization, 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. 15 L of ethyl chloroformate were added to this solution and the vial was shaken for 10 s. After 5 min, the CO2 that was produced had escaped and 100 L of chloroform were added. The vial was vigorously shaken for 2 min and the derivatives were extracted in the organic layer. Finally, 1 L of the organic layer was injected into the gas chromatography. 2.3. Gas chromatography The Agilent 6890 gas chromatograph system, coupled with a 5973 mass spectrometer as detector, was controlled by MSD ChemStation software. Helium was used as the carrier gas (inlet pressure: 170 kPa). Splitless injection mode was used. Separations were achieved on fused-silica capillary columns: a non-chiral CP-Sil 19 CB column from Varian (30 m 0.25 mm I.D., 0.2 m lm thickness) and a chiral Chirasil-L-Val column from Macherey-Nagel (25 m 0.25 mm I.D., 0.12 m lm thickness).

With the CP-Sil 19 CB column, the injector temperature was set at 230 C and the detector temperature at 250 C. The oven temperature was set at 100 C for 5 min, then programmed to reach 275 C at a rate of 2 C/min. With the Chirasil-L-Val column, both injector and the detector temperatures were set at 200 C. The oven temperature was held at 70 C for 5 min, then programmed to 180 C at a rate of 2 C/min, and nally, held at 180 C for 15 min. 3. Analytical procedures A mixture of the enantiomers of the 20 proteinogenic amino acids was derivatized via several procedures and separated on different columns. Three types of analysis were carried through: (1) analysis on an achiral column with a chiral derivatization procedure, which required the use of a chiral alcohol, (S)-(+)-2-chloro-1-propanol. This reagent allowed the formation of diasteromers, N(O,S)-ethoxycarbonyl 2-chloropropyl ester derivatives, which can be separated on an achiral column. (2) The same analysis with the derivatization of only l amino acids to check that the reaction did not result in any racemization. (3) Analysis of N(O,S)-ethoxycarbonyl ethyl ester derivatives obtained by ethanol/ethylchloroformate derivatization on a chiral column. The derivatization reactions led to acylation of the amino group of the amino acids and esterication of the carboxylic group. The reaction was more complex for the amino acids containing an additional functional group. In the case of serine, threonine, lysine, histidine and tyrosine, either monoand/or bis-acetylated derivatives were obtained, whereas mono- and bis-esteried derivatives were formed from aspartic and glutamic acids. The mono- and bis-substituted derivatives were identied by mass spectrometry. All derivatives prepared in this study were stable, gave signicant peak signals and could easily be identied by mass spectrometry. The selected ion monitoring (SIM) detection mode allowed the selection of the derivatives of interest by neglecting the secondary ones. This detection mode was very sensitive and was used to quantify the species. It allowed also the detection of very tiny amounts of compounds. Thanks to the formation of molecular ion groups, the method provided very clear and very practical chromatograms and automated quantication of the derivatives was carried out when the compounds were well separated. Two derivatives with very similar retention times or co-eluting could also be identied with the SIM detection mode and integrated, albeit manually. The gas chromatograms obtained on an achiral column of N(O,S)-ethoxycarbonyl 2-chloropropyl ester derivatives with the same temperature ramp and with a SIM detection mode are shown in Fig. 1ac. In the rst experiment, l and d amino acids were derivatized and analyzed. Fig. 1b is a detail of Fig. 1a. In the second experiment, run for control, only l amino acids were used (Fig. 1c).

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The third experiment is a chromatogram of N(O,S)-ethoxycarbonyl ethyl esters derivatives detected in scan mode (Fig. 2). The retention times of the amino acid derivatives, their enantiomeric resolution and the mean molecular ions of

chromatogram peaks are reported in Table 1 for the N(O,S)ethoxycarbonyl 2-chloropropyl ester derivatives (corresponding to Fig. 1a and b, and in Table 2 (corresponding to Fig. 2) for the N(O,S)-ethoxycarbonyl ethyl esters derivatives.

Fig. 1. (a) Gas chromatogram of N(O,S)-ethoxycarbonyl 2-chloropropyl ester derivatives of a l and d amino acid mixture. Column Varian CP-Sil 19 CB (30 m 0.25 mm I.D., 0.2 m lm thickness). Column temperature: 100 C, 5 min hold, and then programmed at 2 C/min to 275 C. Carrier gas: helium; column inlet pressure 170 kPa; splitless. Injection: 1 L of the 20 amino acid enantiomer mixture at 9.01 104 M of each amino acid enantiomer. Detection by SIM mass spectrometry. (b) Detail of (a). (c) Gas chromatogram of N(O,S)-ethoxycarbonyl 2-chloropropyl ester derivatives of an 8 l amino acid mixture. Column Varian CP-Sil 19 CB (30 m 0.25 mm I.D., 0.2 m lm thickness). Column temperature: 100 C, 5 min hold, and then programmed at 2 C/min to 275 C. Carrier gas: helium; column inlet pressure 170 kPa; splitless. Injection: 1 L of the 20 l amino acid mixture at 9.01 104 M of each amino acid.

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Fig. 1. (Continued ).

Fig. 2. Gas chromatogram of N(O,S)-ethoxycarbonyl ethylester of a l and d amino acid mixture. Chirasil-L-Val column (25 m 0.25 mm I.D., 0.12 m lm thickness) provided by Macherey-Nagel. Column temperature: 70 C for 3 min, then programmed to 180 C at 5 C/min, 15 min hold at 180 C. Carrier gas: helium; column inlet pressure 79 kPa; splitless. Injection 1 L of 20 amino acid enantiomer mixture at 2.84 102 M of each amino acid enantiomer. Scan detection mode.

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Table 1 Retention time tR , enantiomeric resolution Rs and mean molecular ions of l and d N(O,S)-ethoxycarbonyl 2-chloropropyl ester derivatives analyzed on a CP-Sil 19 CB Derivatives of d,l-Ala Gly d,l-Val d,l-Leu d,l-Ile d,l-Pro d,l-Thr d,l-Ser d,l-Asn d,l-Met d,l-Phe d,l-Cys (bis-acetylated) d,l-Asp (bis-esteried) d,l-Glu (bis-esteried) d,l-Gln d,l-Lys (bis-acetylated) d,l-His (bis-acetylated) d,l-Trp d,l-Tyr (bis-acetylated) ns: not separated. Table 2 Retention time, 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,l-Ala Gly d,l-Val d,l-Pro d,l-Ile d,l-Leu d,l-Asp d,l-Asp (bis-esteried) d,l-Asn d,l-Thr d,l-Ser (bis-acetylated) d,l-Met d,l-Glu (bis-esteried) d,l-Phe d,l-Lys (bis-acetylated) d,l-His d,l-Cys (bis-acetylated) d,l-Tyr (bis-acetylated) ns: not separated. tR (min) 21.1922.00 23.49 26.7327.36 29.88 31.9332.50 33.3634.26 36.6836.90 43.7244.02 43.8044.06 46.7747.21 48.1348.46 49.0949.75 51.8652.46 53.0453.54 62.5863.95 62.67 69.8971.25 69.9271.22 Rs 3.04 2.25 ns 1.78 3.45 1.04 1.26 0.76 0.45 1.4 2.62 2.67 1.82 1.32 ns 1.35 1.27 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) 29.1729.38 31.43 34.0734.28 37.9538.19 38.3338.53 40.6240.84 44.0644.25 45.3045.44 48.6 53.2753.39 57.08 59.59 61.6161.76 67.44 69.99 74.7774.87 75.7 76.45 80.03 Rs 1.54 1.53 1.67 1.41 1.52 1.33 1 ns 0.75 ns ns 0.91 ns ns 0.48 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 121.5 116 102 144 158 158 142 146 132 159 176 192 220 237 251 173 173 254 231 208

129 159

231 208

159 129 204

107

4. Discussion 4.1. Experiment 1 (Fig. 1a and b and Table 1) In Fig. 1a, all amino acids derivatives gave signicant peak signals and 10 of the 19 pairs of amino acid enantiomer derivatives were separated with signicant retention times and enantiomeric resolutions (see Table 1). All d amino acid derivatives had shorter retention times than the corresponding l ones. Better resolution of some derivatives was obtained by using different rates of temperature gradient. This method gave good and reproducible enantiomeric resolutions for alkyl chain amino acids (like alanine, valine, proline, leucine, and isoleucine)

(Fig. 1b). The enantiomeric resolution was satisfactory for serine, threonine, methionine, and aspartic acid, but low for lysine. The other amino acid enantiomer derivatives were not separated. The amino acid enantiomers could be quantied with internal standards when good separations and resolutions were achieved. 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. The bleeding of the column was low and allowed good analysis of the high boiling point derivatives without damaging for the column. The good separations of the different amino acid derivatives (except for isoleucine and leucine) and the low column bleeding allowed the formation of

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ion molecular groups and the use of the SIM detection mode to detect and quantify the compounds described in Table 1. The quantication of all amino acids in one single injection was obtained and was automated with good accuracy. The most important advantage of this method is the separation and identication of 19 amino acids out of the 20 natural amino acids. Arginine was the only one which was not detected. 4.2. Experiment 2 (Fig. 1c) The chromatogram of the l amino acids derivatives (Fig. 1c) run for control showed that no racemization occurred during the derivatization. The mirror image did not exceed 1.6%, a value lower than the degree of purity of 2 chloropropanol. 4.3. Experiment 3 (Fig. 2 and Table 2) As seen on the chromatogram in Fig. 2, it was not possible to form ion molecular groups to identify all of the compounds since too many derivatives were overlapping (Table 2). For example, aspartic acid and asparagine were not well separated and gave overlapping peaks, as did lysine and histidine. Moreover, for the analysis of the basic amino acid derivatives (with high boiling points), the upper temperature limit of the Chirasil-L-Val column had to be used, which caused signicant bleeding and did not allow the detection of tryptophan. The use of the column at high temperature strongly decreased its performance and its lifespan. This phenomenon was not at all observed with the non-chiral CP-Sil 19 CB column, for which the temperature limit was not reached. Nevertheless, the Chirasil-L-Val column allowed the separation of 14 out of 19 enantiomer pairs with generally good enantiomeric resolution. Automation of the calculations could not be carried out with this procedure, because there was too much overlapping of the various derivatives. It should be noted that better resolutions were obtained for bis-substituted compounds compared to the corresponding mono-substituted ones. For example, the enantiomeric resolution of aspartic acid mono ester was 1.04 and 1.26 for the di-ester. 4.4. Mass spectrometry fragmentation The same mass spectrometric fragmentations as those reported by Zampolli et al. [18] were observed for the N(O,S)-ethoxycarbonyl 2-chloropropyl ester and N(O,S)-ethoxycarbonyl ethyl ester derivatives. The main mass peak corresponds to the loss of the acylium ion [CO2 CH2 CHCl CH3 ]+ (m/z = M 121.5) for the N(O,S)ethoxycarbonyl 2-chloropropyl ester derivatives (Table 1) and to the loss of [CO2 CH2 CH3 ]+ (m/z = M 73) for the N(O,S)-ethoxycarbonyl ethyl ester derivatives (Table 2). The loss of acylium ions is characteristic of the cleavage of the C C bond in the -position of the esteried carboxyl group. The other mass peaks could be identied easily: they correspond to the loss of OH+ , 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.

5. Conclusions The derivatization methods described are handy and fast, offering several complementary advantages. The rst 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. Since no column change is necessary, it is very easy to switch from achiral to chiral analysis and to extend the analysis to other molecules, that could be present in more complex mixtures. The separation and the integration of 19 out of 20 amino acids were easily achieved by the formation of molecular ion groups. This method was reproducible and gave satisfactory enantiomeric resolutions for 10 of the 19 enantiomer pairs. The other amino acid derivative pairs were not separated. The percentage of amino acid enantiomers could be quantied with internal standards by the use of the SIM detection mode when good separations and resolutions were obtained. The high boiling point derivatives gave well dened peaks and could be quantied by analysis on the CP-Sil 19 CB column without damage to the column, contrary to the analysis on the Chirasil-L-Val column. This derivatization method was found to be suitable for rapid, single step detection of the presence of amino acid enantiomers in solution, as well as their quantitative determination. However, the best enantiomeric resolutions were always obtained when using derivatization with ethanol and ethylchloroformate on a Chirasil-L-Val column. This method allows the separation of 14 of 19 enantiomeric pairs of the proteinogenic amino acids when associated with the SIM detection mode. The simplicity of these methods makes them readily applicable to many elds, such as foods or paints. Acknowledgements This study was supported by the French Centre National de la Recherche Scientique (CNRS) and by the French Centre National dEtudes Spatiales (CNES). The authors thank Bernard Barbier and Uwe Meierhenrich for their help. References
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