Joww~ of Chromarog-raphy, 390 (1987) 434438

Elsevicr scicncc Publishers B.V., Amsterdam - Printed in The Netherlands

CHROM. 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 mix-
tures and (ii) to compare the efficiency of the suggested method to the classical pro-
cedure’ 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. stain-
less-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) (Su-
peko, 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, re-
spectively) 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

NOTES 435

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 acyl-
ation 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 mix-

tures and characteristic representatives of amino acids provided the following results.
(I) As to the optimum esterification time, with 1 mol thionyl chloride con-
taining 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 es-
terification 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, indepen-
dently of the esterification agent used, are the same. (ii) The advantages of our es-
terification 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 es-
terification of lo-15 mg of amino acids. In contrast, the classical prescription12 re-
quires 1.5 cm3 hydrochloric acid-n-butanol for 1 mg amino acid.
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 NOTES 437

TABLE II
REPRODUCIBILITY OF THE DETERMINATION OF DIFFERENT AMOUNTS OF AMINO ACIDS AS
THEIR N-TRIFLUOROACETYL n-BUTYL (A) AND ISOBUTYL (B) ESTERS
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 A B
acid
Injected Peak Mean R.S.D R.S.D. Peak Mean R.S.D. R.S.D.
amowlt area per /%i area pr (%I
(Mb) 1 MT 1H

Alan& 44.52 361 359 4.1 1.1 393 389 11.9 3.0
22.26 361 399
11.13 354 376
Glycine 44.48 184 194 8.1 4.7 301 299 2.1 0.7
22.24 19% 300
11.12 201 297
Vnline 45.12 4cm 399 9.5 2.4 422 419 3.6 0.9
22.56 408 420
11.28 389 415
Leucine 41.60 304 310 5.1 1.7 419 420 3.1 0.7
20.80 311 417
10.40 314 423
Cysteine 40.28 313 309 4.6 1.5 180 190 8.4 4.4
20.14 304 194
10.07 310 195
Hydroxy- 10.00 458 472 14.0 3.0 388 391 4.4 1.1
praline 20.00 486 389
10.00 471 3%
Proline 67.24 379 379 9.5 2.5 647 639 7.5 1.2
33.62 388 639
16.81 369 632
Methionine 29.Y 229 240 14.0 5.8 253 250 4.2 1.7
14.78 236 245
7.39 256 251
Asprrtic 74.64 386 386 1.6 0.4 600 610 9.0 1.5
acid 37,32 385 615
18.66 388 616
Phenyl- 7444 356 352 15.8 4.5 616 630 13.7 2.2
alaine 37.22 335 645
18.61 366 628
Tyro&!C 36.20 376 376 3.5 0.9
18,lO 373
9.05 380
Glutamic 110.32 375 364 10.5 2.9 583 591 6.9 1.2
acid 55.16 354 595
27.58 364 595

(Continued on p. 438)
 438 NOTES

TABLE II (conrirrucrl)

Amino A B
acid
hjecred Peak Mem R.S.D. R.S.D. Peak Mean R.S.D. R.S.D.
l*)(owrt amgcr (%) areu pr (%/oi
lrsl lM Ipcg

LysiYK 59.80 241 232 9.6 4.1 23,6 240 4.0 1.7
29.90 234 244
14.95 222 240
Arginir 51.64 II8 116 4.7 4.0 118 116 5.3 4.5
25.112 111 110
12.91 120 120
Tryptophin 44.04 221 225 7.8 3.5 201 207 5.5 2.6
22.02 234 210
11.01 220 211
Hiti 36.20 108 207 11.5 5.5 177 171 13.6 7.9
30.72 2iM 180
15.36 220 155

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 de-
termine the main constituents of natural matrices by gas chromatography.

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