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Esterification of amino acids with thionyl chloride acidified butanols for their gas chromatographic analysis
Joww~ of Chromarog-raphy, 390 (1987) 434438 Elsevicr scicncc
CHROM.
Publishers
B.V., Amsterdam
-
Printed in The Netherlands
19 273
Nate
Estnifkation of amino acids with thionyl chloride acidified butanols for their pas chrornatographic analysis I. MOLNAR-PERL*.
M. PINTfZR-SZAKkS
and V. FABIAN-VONSIK
Institrrie of Inorganic and Analytical Chemisrry. L. Etitvds University, Muzeum krt. 4/B, 1088 Budapest t Hungary / (First ra+ved
September
22nd. 1986; revised manuscript
received November
17th, 1986)
The advantages in the preparation of amino acid methyl esters of using thionyl chloride-methanol, instead of HCI-methanol (methanol saturated by different amounts of hydrogen chloride gas), have been detailed’- 9. Recently the efficiency of n-propyl ester&cation of amino acids was reportedlO. To our knowledge, there are no literature data on the utilization of thionyl chloride -butanols for the esterification of amino acids. Our work was intended (i) to provide a suitable derivatization procedure for the rapid butyl esterification of amino acids with a good reproducibility in order to clarify the optimum conditions using thionyl chlorideebutanols as esterification mixtures and (ii) to compare the efficiency of the suggested method to the classical procedure’ l. MATERIALS
AND
METHODS
Raagents All reagents and standard amino acids were of analytical purity, obtained from Reanal (Budapest, Hungary).
Apparatus A Chromatron G.C.H.F. 18.3 gas chromatograph (VEB Chromatron, Berlin, G.D.R.) equipped with a flame ionization detector and a 3 m x 3 mm I.D. stainless-steel column was used. Nitrogen was the carrier gas at a flow-rate of 60 cm3/min. The column packing consisted of 3% SP-2250 on Supefcoport (80-100 mesh) (Supeko, Bclkfonte, PA, U.S.A.). The column temperature was increased from 100 to 3lo’C at 12 ‘Cimin. The temperatures of the injector and detector were 260 and 350X respectively. Ester@=ation A 1-2 cm3 volume of the 1 M hydrochloric acid stock solution of the amino acids (each 1.55 mg/cm3) was pipetted into a 25-cm3 vessel (the ground joint of which can bc fitted to a vacuum distillation device and to a reflux condenser, respectively) and evaporated to dryness under vacuum. To the residue, I cm3 of n- or 0021~%73’X7’W3.50
IV
1987 Elsevier Science Publishers
B.V
435
NOTES
isobutanol was added containing 1 M of thionyl chloride. The reflux condenser was then fitted and the apparatus placed in a water-bath. Esterification took place at 100°C for 60 min. After cooling to room temperature, the solution was evaporated under vacuum, in a water-bath at 60°C to a syrupy substance. The residue was quantitatively transferred with 5 x 0.1 cm3 dichloromethane into a 2-cm3 Pierce Reacti-Vial. A 0.5-cm3 volume of trifluoroacetic anhydride was added and the acylation was carried out for 10 min at IXX. A stock solution of 1 cm3 was prepared, 5-10 ~1 aliquots of which were injected into the gas chromatograph. RESULTS
AND DISCUSSION
Our model esterifications performed with the thionyl chloride-butanol mixtures and characteristic representatives of amino acids provided the following results. (I) As to the optimum esterification time, with 1 mol thionyl chloride containing n- or isobutanol, respectively, to be on the safe side, we used 60 min (Tables I and II). n-Butyl esterifications were complete within 10 min, isobutyl esterification within 30 min, with the following exceptions: n-butyl esterification of valine, leucine and tryptophan and isobutyl esterification of histidine became quantitative after 40 min. Note: the prolonged reaction time necessary for the quantitative derivatization of valine, kucine, tryptophan and histidine is independent of the quality of the esterification agent applied. (2) In order to select the most advantageous derivatization conditions, Le., the optimum molar ratios of thionyl chloride to butanols (Table I), the concentration of thionyl chloride was varied between 0.5 and 4.0 M (using n-butanol) and 0.5 and 1.5 M (in the case of isobutanol), respectively. The n-butyl esterifications are quantitative in the concentration range of 0.75540 M. Nevertheless, the concentration of thionyl chloride recommended is in the range of 0.75-1.5 M, since (i) when using a thionyl chloride concentration higher than 1.5 M the methionine was eluted in two peaks, and (ii) the stock solution of the derivatized amino acids became yellowish, probably due to side reactions (Table I, A). In the thionyl chlorideeisobutanol system, thionyl chloride concentrations of 0.75 and 1.O M can be utilized for analyses, since (i) in the case of 0.5 M the esterification of valine was not quantitative (Table I, B) and (ii) interfering peaks were detected at >2 M. (3) The most advantageous volumes of the 1 M thionyl chloride containing butanols were also investigated. In both cases, using the thionyl chloride-n-butanol or -isobutanol mixtures, 0.5-2.0 cm3 can be applied. We propose the use of 1 cm3. (4) The reproducibility of the determination of different amounts of amino acids esterified by the suggested method is summarized in Table II. (5) Comparing the peak area responses and the analytical reproducibilities of the N-trifluoroacetyl, n- or isobutyl esters prepared by the suggested method with those obtained by the classical method” (Table I), the following conclusions could be drawn. (i) The peak area responses and the analytical reproducibilities, independently of the esterification agent used, are the same. (ii) The advantages of our esterification method are realized in the direct preparation of the reactant and in the fact that l-2 cm3 of the thionyl chloride-butanol mixture result in quantitative esterification of lo-15 mg of amino acids. In contrast, the classical prescription12 requires 1.5 cm3 hydrochloric acid-n-butanol for 1 mg amino acid.
8’1 b’f E6l 261 161 661 161
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8
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(9)
S‘P S'OI b8f
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6.X L'9Z POE
P’E f’8 VII
8'f 8'fI l9E 69E WE LLE 09f 6Sf (Ml)
(x/,)‘as .a3 P'b 6'0[ 6bZ +lSZ rlfZ SSZ 6SZ IS2 (SO[)
ozz
I’S Z’I I
b'f 9'bl PEP PSB SIP 6fb 9Zb 9fb (f81)
S'P E'S1 Lff 9ZE IEE WE Eff Iff (181)
P'b 6'EI ZIE ZIE POE 9ff POE WE (161)
Z'b 8'8 OIZ PIZ L61 91z 8lZ SO5 EPZI)
E'b h'b1 LEE EEE EZC SZE SSE sot‘ (981)
b'b b'9l LLC 9Lt pop SLE 99f Z9f (891)
O'b 5'01 PSZ 592 9bZ 9SZ 9SZ 9tz (LEI)
E'b I'fI ZOE ZIE 162 ZIE 185 9OE (OfI)
X'S f'f 611 611 811 811 Sll PZL (XL )
612 9lZ ozz 90Z LEZ (261)
NOTES
437
TABLE II REPRODUCIBILITY OF THE DETERMINATION OF DIFFERENT AMOUNTS THEIR N-TRIFLUOROACETYL n-BUTYL (A) AND ISOBUTYL (B) ESTERS
OF AMINO ACIDS AS
Esters were prepared with 1 cm3 of butanol containing 1 M thionyl chloride: esterification time 60 min. All peak areas listed reprcuent the mean of at least three measurements. A fnirro acid
B
A
Injected amowlt
Peak area per
(Mb)
1 MT
Mean
R.S.D
R.S.D. /%i
Peak area pr
Mean
R.S.D.
R.S.D. (%I
1H
Alan&
44.52 22.26 11.13
361 361 354
359
4.1
1.1
393 399 376
389
11.9
3.0
Glycine
44.48 22.24 11.12
184 19% 201
194
8.1
4.7
301 300 297
299
2.1
0.7
Vnline
45.12 22.56 11.28
4cm 408 389
399
9.5
2.4
422 420 415
419
3.6
0.9
Leucine
41.60 20.80 10.40
304 311 314
310
5.1
1.7
419 417 423
420
3.1
0.7
Cysteine
40.28 20.14 10.07
313 304 310
309
4.6
1.5
180 194 195
190
8.4
4.4
Hydroxy-
10.00
14.0
3.0
388 389 3%
391
4.4
1.1
20.00 10.00
458 486 471
472
praline Proline
67.24 33.62 16.81
379 388 369
379
9.5
2.5
647 639 632
639
7.5
1.2
Methionine
29.Y 14.78 7.39
229 236 256
240
14.0
5.8
253 245 251
250
4.2
1.7
Asprrtic acid
74.64 37,32 18.66
386 385 388
386
1.6
0.4
600 615 616
610
9.0
1.5
Phenylalaine
7444 37.22 18.61
356 335 366
352
15.8
4.5
616 645 628
630
13.7
2.2
Tyro&!C
36.20 18,lO 9.05
376 373 380
376
3.5
0.9
Glutamic acid
110.32 55.16 27.58
375 354 364
364
10.5
2.9
583 595 595
591
6.9
1.2
(Continued
on p. 438)
438
NOTES
TABLE II (conrirrucrl) Amino
A
B
acid hjecred l*)(owrt lrsl
Peak
Mem
R.S.D. R.S.D. (%)
amgcr lM
Peak areu pr Ipcg
Mean
R.S.D.
R.S.D. (%/oi
LysiYK
59.80 29.90 14.95
241 234 222
232
9.6
4.1
23,6 244 240
240
4.0
1.7
Arginir
51.64 25.112 12.91
II8 111 120
116
4.7
4.0
118 110 120
116
5.3
4.5
Tryptophin
44.04
225
7.8
3.5
201 210 211
207
5.5
2.6
11.01
221 234 220
36.20 30.72 15.36
108 2iM 220
207
11.5
5.5
177 180 155
171
13.6
7.9
Hiti
22.02
This work in addition to our earlier studies forms part of our comprehensive research concerning the development of new and fast analytical methods13-1 * to determine the main constituents of natural matrices by gas chromatography. REFERENCES
9 IO I1 12 13 14 15 16 17 18
S. Makiaumi and H. A. Saroff, J. Gus Chrcw~togr., 3 (1965) 21-27. P. 8. Hyen and W. Black, Ccm. J. Bbchem.. 43 (1965) 949-952. J. F. Cavins and M. Friedman, BiochemWry, 6 (1967) 3766-3770. R. G. Ackman, S. N. H-r, M. Kates, A. K. Scn Gupta, G. Eglinton and I. Maclean, J. Chromutogr., u (1969) 256-261. E. kllum, V. A. Close, W. Patton, W. Pereira and B. Halpem, AnuI. &o&cm., 31 (1969) 227-234. M. Ga, Alrd. Chcm., 39 (1%7) 1677-1679. C. A. Atkins and D. T. Canvin, CUR. J. Biochem., 49 (1971) 949-952. R. M. ClpfioIi. W. E. seifert, Jr, and D. E. Sutherland, Biockewr. Biaplys. Res. comnun., 55 (1973) 67-72. H. Falter, K. Jayaaimhulu and R. A. Day, Anal. Biochem., 67 (1975) 359-371. S. P. Caqana Filbo and G. Goissis, J. Chromarogr., 236 (1982) 197-m. K. Blau and G. S. King (Editors), Handbook of Derivatives for Chrarsalography, Heyden & Son, London, 1978, Ch. 2, pp. 3e90. D. Roach and C. W. Gchrke, J. Chromatogr., 44 (1969) 26e278. I. Mol&-Rrl, M. Pint&r-Szakitcs, A. KBvig6 and J. Pet&zy, J. Chrmtogr., 295 (1984) 433-443. I. Molnir-&rl, M. PintbSzakBcs, A. KGvigb and J. Petrirczy, CM&y&. Rex, 138 (1985) 83-89. 1. Molntr-Perl. M. Pintir-Stakbs, A. K’Svig& and J. Fetr&xy, Food Chem., 16 (1985) 163-174. 1. M&r-Pwl, M. Pint&r-Szakkcs, A. K&h&, J. Petr&xy, U. P. Kralovinszky and J. Jakab, Food Ckem., 20 (1966) 21-38. 1. Mob&-M. M. Pin&r-Szakbs, R. Wittrnann, AI. Reutter and K. Eichner, J. Chromatogr., 361 (1986) 31 l-320. 1. Molnir-krl and M. Pint&-Szak&, 2. Guam.-Unters.-F., 183 (1986) M-25,
CHROM.
Publishers
B.V., Amsterdam
-
Printed in The Netherlands
19 273
Nate
Estnifkation of amino acids with thionyl chloride acidified butanols for their pas chrornatographic analysis I. MOLNAR-PERL*.
M. PINTfZR-SZAKkS
and V. FABIAN-VONSIK
Institrrie of Inorganic and Analytical Chemisrry. L. Etitvds University, Muzeum krt. 4/B, 1088 Budapest t Hungary / (First ra+ved
September
22nd. 1986; revised manuscript
received November
17th, 1986)
The advantages in the preparation of amino acid methyl esters of using thionyl chloride-methanol, instead of HCI-methanol (methanol saturated by different amounts of hydrogen chloride gas), have been detailed’- 9. Recently the efficiency of n-propyl ester&cation of amino acids was reportedlO. To our knowledge, there are no literature data on the utilization of thionyl chloride -butanols for the esterification of amino acids. Our work was intended (i) to provide a suitable derivatization procedure for the rapid butyl esterification of amino acids with a good reproducibility in order to clarify the optimum conditions using thionyl chlorideebutanols as esterification mixtures and (ii) to compare the efficiency of the suggested method to the classical procedure’ l. MATERIALS
AND
METHODS
Raagents All reagents and standard amino acids were of analytical purity, obtained from Reanal (Budapest, Hungary).
Apparatus A Chromatron G.C.H.F. 18.3 gas chromatograph (VEB Chromatron, Berlin, G.D.R.) equipped with a flame ionization detector and a 3 m x 3 mm I.D. stainless-steel column was used. Nitrogen was the carrier gas at a flow-rate of 60 cm3/min. The column packing consisted of 3% SP-2250 on Supefcoport (80-100 mesh) (Supeko, Bclkfonte, PA, U.S.A.). The column temperature was increased from 100 to 3lo’C at 12 ‘Cimin. The temperatures of the injector and detector were 260 and 350X respectively. Ester@=ation A 1-2 cm3 volume of the 1 M hydrochloric acid stock solution of the amino acids (each 1.55 mg/cm3) was pipetted into a 25-cm3 vessel (the ground joint of which can bc fitted to a vacuum distillation device and to a reflux condenser, respectively) and evaporated to dryness under vacuum. To the residue, I cm3 of n- or 0021~%73’X7’W3.50
IV
1987 Elsevier Science Publishers
B.V
435
NOTES
isobutanol was added containing 1 M of thionyl chloride. The reflux condenser was then fitted and the apparatus placed in a water-bath. Esterification took place at 100°C for 60 min. After cooling to room temperature, the solution was evaporated under vacuum, in a water-bath at 60°C to a syrupy substance. The residue was quantitatively transferred with 5 x 0.1 cm3 dichloromethane into a 2-cm3 Pierce Reacti-Vial. A 0.5-cm3 volume of trifluoroacetic anhydride was added and the acylation was carried out for 10 min at IXX. A stock solution of 1 cm3 was prepared, 5-10 ~1 aliquots of which were injected into the gas chromatograph. RESULTS
AND DISCUSSION
Our model esterifications performed with the thionyl chloride-butanol mixtures and characteristic representatives of amino acids provided the following results. (I) As to the optimum esterification time, with 1 mol thionyl chloride containing n- or isobutanol, respectively, to be on the safe side, we used 60 min (Tables I and II). n-Butyl esterifications were complete within 10 min, isobutyl esterification within 30 min, with the following exceptions: n-butyl esterification of valine, leucine and tryptophan and isobutyl esterification of histidine became quantitative after 40 min. Note: the prolonged reaction time necessary for the quantitative derivatization of valine, kucine, tryptophan and histidine is independent of the quality of the esterification agent applied. (2) In order to select the most advantageous derivatization conditions, Le., the optimum molar ratios of thionyl chloride to butanols (Table I), the concentration of thionyl chloride was varied between 0.5 and 4.0 M (using n-butanol) and 0.5 and 1.5 M (in the case of isobutanol), respectively. The n-butyl esterifications are quantitative in the concentration range of 0.75540 M. Nevertheless, the concentration of thionyl chloride recommended is in the range of 0.75-1.5 M, since (i) when using a thionyl chloride concentration higher than 1.5 M the methionine was eluted in two peaks, and (ii) the stock solution of the derivatized amino acids became yellowish, probably due to side reactions (Table I, A). In the thionyl chlorideeisobutanol system, thionyl chloride concentrations of 0.75 and 1.O M can be utilized for analyses, since (i) in the case of 0.5 M the esterification of valine was not quantitative (Table I, B) and (ii) interfering peaks were detected at >2 M. (3) The most advantageous volumes of the 1 M thionyl chloride containing butanols were also investigated. In both cases, using the thionyl chloride-n-butanol or -isobutanol mixtures, 0.5-2.0 cm3 can be applied. We propose the use of 1 cm3. (4) The reproducibility of the determination of different amounts of amino acids esterified by the suggested method is summarized in Table II. (5) Comparing the peak area responses and the analytical reproducibilities of the N-trifluoroacetyl, n- or isobutyl esters prepared by the suggested method with those obtained by the classical method” (Table I), the following conclusions could be drawn. (i) The peak area responses and the analytical reproducibilities, independently of the esterification agent used, are the same. (ii) The advantages of our esterification method are realized in the direct preparation of the reactant and in the fact that l-2 cm3 of the thionyl chloride-butanol mixture result in quantitative esterification of lo-15 mg of amino acids. In contrast, the classical prescription12 requires 1.5 cm3 hydrochloric acid-n-butanol for 1 mg amino acid.
8’1 b’f E6l 261 161 661 161
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(4)
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8
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f'f 1'91 IOf
1'1 E'b 88f Z8E 88E Z6E 06f
Z'f I’b Ofl SZI IEI Sfl OEI 6't 6'PI EXP
(9)
S‘P S'OI b8f
(vi) ‘a’s .a3 uv4
S'b 9'OZ SfZ
S’Z 5’8 8ff
S‘l E‘S fSb
9'9 Z'fl 002
O'b i'bi ZSf
6'S x.zz t8E
I'S Z'bl 19f
L’E Z.I[ m.
8’5 Z’E LLZ
O'f O'S1 66b 10s Sib PIS 805 969 (965)
6.X L'9Z POE
P’E f’8 VII
8'f 8'fI l9E 69E WE LLE 09f 6Sf (Ml)
(x/,)‘as .a3 P'b 6'0[ 6bZ +lSZ rlfZ SSZ 6SZ IS2 (SO[)
ozz
I’S Z’I I
b'f 9'bl PEP PSB SIP 6fb 9Zb 9fb (f81)
S'P E'S1 Lff 9ZE IEE WE Eff Iff (181)
P'b 6'EI ZIE ZIE POE 9ff POE WE (161)
Z'b 8'8 OIZ PIZ L61 91z 8lZ SO5 EPZI)
E'b h'b1 LEE EEE EZC SZE SSE sot‘ (981)
b'b b'9l LLC 9Lt pop SLE 99f Z9f (891)
O'b 5'01 PSZ 592 9bZ 9SZ 9SZ 9tz (LEI)
E'b I'fI ZOE ZIE 162 ZIE 185 9OE (OfI)
X'S f'f 611 611 811 811 Sll PZL (XL )
612 9lZ ozz 90Z LEZ (261)
NOTES
437
TABLE II REPRODUCIBILITY OF THE DETERMINATION OF DIFFERENT AMOUNTS THEIR N-TRIFLUOROACETYL n-BUTYL (A) AND ISOBUTYL (B) ESTERS
OF AMINO ACIDS AS
Esters were prepared with 1 cm3 of butanol containing 1 M thionyl chloride: esterification time 60 min. All peak areas listed reprcuent the mean of at least three measurements. A fnirro acid
B
A
Injected amowlt
Peak area per
(Mb)
1 MT
Mean
R.S.D
R.S.D. /%i
Peak area pr
Mean
R.S.D.
R.S.D. (%I
1H
Alan&
44.52 22.26 11.13
361 361 354
359
4.1
1.1
393 399 376
389
11.9
3.0
Glycine
44.48 22.24 11.12
184 19% 201
194
8.1
4.7
301 300 297
299
2.1
0.7
Vnline
45.12 22.56 11.28
4cm 408 389
399
9.5
2.4
422 420 415
419
3.6
0.9
Leucine
41.60 20.80 10.40
304 311 314
310
5.1
1.7
419 417 423
420
3.1
0.7
Cysteine
40.28 20.14 10.07
313 304 310
309
4.6
1.5
180 194 195
190
8.4
4.4
Hydroxy-
10.00
14.0
3.0
388 389 3%
391
4.4
1.1
20.00 10.00
458 486 471
472
praline Proline
67.24 33.62 16.81
379 388 369
379
9.5
2.5
647 639 632
639
7.5
1.2
Methionine
29.Y 14.78 7.39
229 236 256
240
14.0
5.8
253 245 251
250
4.2
1.7
Asprrtic acid
74.64 37,32 18.66
386 385 388
386
1.6
0.4
600 615 616
610
9.0
1.5
Phenylalaine
7444 37.22 18.61
356 335 366
352
15.8
4.5
616 645 628
630
13.7
2.2
Tyro&!C
36.20 18,lO 9.05
376 373 380
376
3.5
0.9
Glutamic acid
110.32 55.16 27.58
375 354 364
364
10.5
2.9
583 595 595
591
6.9
1.2
(Continued
on p. 438)
438
NOTES
TABLE II (conrirrucrl) Amino
A
B
acid hjecred l*)(owrt lrsl
Peak
Mem
R.S.D. R.S.D. (%)
amgcr lM
Peak areu pr Ipcg
Mean
R.S.D.
R.S.D. (%/oi
LysiYK
59.80 29.90 14.95
241 234 222
232
9.6
4.1
23,6 244 240
240
4.0
1.7
Arginir
51.64 25.112 12.91
II8 111 120
116
4.7
4.0
118 110 120
116
5.3
4.5
Tryptophin
44.04
225
7.8
3.5
201 210 211
207
5.5
2.6
11.01
221 234 220
36.20 30.72 15.36
108 2iM 220
207
11.5
5.5
177 180 155
171
13.6
7.9
Hiti
22.02
This work in addition to our earlier studies forms part of our comprehensive research concerning the development of new and fast analytical methods13-1 * to determine the main constituents of natural matrices by gas chromatography. REFERENCES
9 IO I1 12 13 14 15 16 17 18
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