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Quantitative gas chromatographic analysis of amino acids on a short glass capillary column
ANALYTICAL
51, 204-219 (1973)
BIOCHEMISTRY
Quantitative
Gas
Acids JiiRGEN Department
Chromatographic
on a Short
Glass
JONSSON, JULIUS
of Biochemistry, University Received
of
Analysis Capillary
EYEM,
AND
Faculty of Pharmacy, The Uppsaln, 751 bl Uppsala,
June
2, 1972;
accepted
July
of Amino
Column JOHN SJiiQUIST Wallenberg Sweden 24,
Laboratory,
1972
A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run. Hydrolyzed peptides have been analyzed. The analyses were performed
with
a precision
varying
viation) depending The amount taken
known
on for
between
sequences of the peptides
chromatography
modification
except
1 and 6% (mean relative
the number of amino acid residues analysis was 20-300 pg. The results for
as S-methylated
cysteine.
and with This
standard
de-
in the peptide. agree with the
the analyses by ion-exchange
amino
acid
can
be analyzed
after
cysteine.
The main purpose of this investigation was to find a suitable method for the quantitative gas chromatographic analysis of peptides. Our requirement was that the procedure should be rapid and applicable to samples no larger than those used in ion-exchange chromatography, in which 100 nmoles per amino acid are analyzed in two separate runs. These analyses are quite time consuming, requiring approximately 5 hr per analysis. With a suitable derivative, gas chromatographic analysis can be completed in half an hour using a single-column system. Recently a high pressure single-column ion-exchange analyzer appeared on the market (Durrum Instr. Corp. Mod D 500). This inst,rument performs a complete analysis in 48 min with a sensitivity of 1 nmole at a signal-tonoise ratio of 3O:l. The reproducibility is said to be 15% at a level of 100 nmoles per amino acid. These data should bc weighed against the very high price of the instrument. The advantages of using a gas chromatograph are the low cost and the great versatility of the system. Another advantage is the great sensitivity that can be obtained, especially when using capillary columns, together with ultrasensitive detectors such as the electron-capture detector (ECD). Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
204 Inc. reserved.
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
205
A number of workers (l-4, 7-9, 13-37) have investigated different kinds of derivatives suitable for gas chromatographic analysis, but they have not succeeded in separating and quantifying all the protein amino acids satisfactorily in a single run. Gehrke and coworkers used a dualcolumn system for analyzing N-trifluoroacetyl (TFA) amino acid n-butyl esters (1) which proved to be useful for even very small amounts of sample. Moss and Lambert (2) investigated the separation of the N-heptafluorobutyric (HFB) amino acid n-propyl esters on an OV 1 column. By using this derivative they succeeded in separating 20 amino acids in a single run and obtained sharp peaks when injecting 15 nmoles per amino acid derivative on a packed column. Pollock (3) investigated the time reduction when N-HFB-n-butyl derivatives were used instead of N-TFA-amino acid n-butyl esters and found it to be 35%. He also proposed the use of an electron-capture detector together with the N-HFB-derivatives in order to increase the sensitivity. Zumwalt et al. (4) were able to detect amounts of N-HFBn-butyl derivatives of cysteine and methionine as low as 2 and 1 pg, respectively, when using an ECD. The possibility of separating the protein amino acids on a single column in a short time and the great sensitivity that can be obtained using N-HFB amino acid n-propyl esters prompted us to select these derivatives for further studies. MATERIALS
AND
METHODS
A. Apparatus a. A Carlo Erba Mod. G 1 gas chromatograph which was equipped with a flame ionization detector and a linear temperature programmer was used. It was modified for glass capillary columns and had an all-glass inlet-splitt’ing device (5). The column used was a 6-m-long, 0.2-mm-i.d. open tubular column with dimethyl siloxane polymer chemically bound to the glass wall1 (6). The properties of the column were as follows: column efficiency, n, and partition ratio, k, for n-octadecane = 24,000 and 13.4, respectively; phase ratio ,f3 = 250; separation number S = 13; polarity (expressed as the retention index I of phenanthrene at 150°C) = 1,723. The detector signal was recorded with a Hitachi Perkin-Elmer recorder (Mod. 65) and integrated with a Hewlett-Packard electronic integrator (Mod. 3371 B) . Nitrogen, purified as previously described (7)) was used as carrier gas. b. For evaporation of solvents, we used a heated aluminium block 1 Prep.ared
by LKB-Produkter
AB,
Stockholm,
Sweden.
206
JBNSSON,
EYEM,
AND
SJijQUIST
fitted with eight stainless-steel needles blowing nitrogen over the samples. c. Ultrasonic Mixer (Millipore) . d. Control amino acid analyses were performed on an automatic ionexchange analyzer (Biochrom BC-200). B. Chromatographic
Conditions
a. Column temperature: Initially 70°C held constant for 4 min, then linearly programmed at a rate of 5”C/min up to 230°C. b. Detector and injection temperature: 210°C. c. Mean carrier-gas velocity: 16 cm/set. d. Splitting ratio: 1:lOO. C. Reagents a. Analytical grade amino acids were obtained from different manufacturers. b. 8 M HCl:n-propanol. Anhydrous HCl was produced by adding coned HCI (p.a., Merck) dropwise to coned H,SO, (p.a., Merck) and passing the HCI gas through a H&SO, drying tower. The gas was bubbled through n-propanol (p.a., Merck) which had been dried over CaSO, and redistilled in an all-glass system. The concentration of WC1 was determined by titration. c. n-Hexane (p.a., Merck) was dried over CaSO* and redistilled in the same manner as n-propanol. d. Heptafluorobutyric anhydride (HFBA) was obtained from Pierce Chemical Co. e. cY-n-Va15-angiotensineamide-II (Ciba) and prolyl-leucyl-glycineamide were kindly provided by Dr. I. SjGholm at this department. D. Treatment
of Sample for Separation Studies
The amino acid derivatives were prepared as described by Moss and Lambert (2) with some modifications. A 500-~1 quantity of amino acid standard solution containing approximately 100 nmoles per amino acid in 0.1 M HCl was pipetted into a l-ml heavy-walled screw-capped glass vial equipped with a Teflon-lined stopper (Reacti-Vial, Pierce Chemical Co.). The solution was evaporated at 100°C under extra pure nitrogen and the sample was stored overnight in a desiccator (over KOH-pellets) . A 500-J amount of 8 M HCl:n-propanol was added to the sample. It was thoroughly shaken by hand and then mixed in the ultrasonic bath until the white precipitate dissolved. Esterification was performed in an oven at 110°C for 10 min. The vessel was immersed in the ultrasonic bath and it,s contents mixed for 30 set while hot. The HCl-propanol was
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
207
evaporated under N, at 100°C. The esterification step was then repeated, but the ultrasonic treatment was omitted. After evaporation, the remaining reagent was removed by azeotropic distillation with 50 ~1 n-hexane at ambient temperature. Acylation of the amino acid esters was then carried out in 50 ~1 HFBA at 150°C for 10 min in a heated aluminium block. After cooling, 2 ~1 of sample mixed with 2 ,ul of fresh HFBA in a syringe was injected into the gas chromatograph for analysis. The retention of each derivative relative to the norleucine derivative (ri8) was calculated. E. Esterification
Studies
Studies were done on a prolonged one-step esterification performed in 8 M HCl n-propanol at 110°C for 30 min. Further, the effect of using 3.5 M HCl:n-propanol for esterification was tested at 110°C for 30 min. Acylation was performed as described under section D. In each study three samples (containing 100 nmoles of each amino acid) were derivatized and analyzed. F. Acylation
Studies
The results of acylation for 10 and 20 min at 150°C were compared. Three replicate samples were derivatized as described in section D. Three others were treated identically with the exception of the acylation procedure, for which the time was doubled. G. Stability
of the Derivatized
Samples
Two samples were derivatized as described in section D and stored at room temperature. The analyses were performed 15 min, 24, 48, and 72 hr after derivatization. H. Determination
of the Relative
Molar
Response
Three replicate samples were derivatized and analyzed as described in section D. The RMR values relative to the norleucine derivative were calculated. RMR = {(area of studied peak) (molar amount of norleucine)} x { ( area of norleucine peak) (molar amount of studied amino acid) }-I. I. Linearity
of the Response
The amount of each amino acid analyzed by the gas chromatographic technique was approximately 25-200 nmoles. This range was chosen in order to permit direct comparison with analyses from the ion-exchange amino acid analyzer. Duplicate samples containing 23, 47, 94, and 188
208
JijNSSON,
EPEM,
AND
SJiiQUIST
nmoles per amino acid were taken through the derivatization and analysis steps according to section D. The amount of the internal standard, norleucine, was constant in all samples, i.e., 92.4 nmoles. J. Application to Peptide Hydrolyzates Peptides were hydrolyzed for 20 hr at 106°C in 2 ml 6 M HCl (Riedel de Ha&, p.a.) containing 27% (v/v) phenol and 200 nmoles of norleucine. The hydrolyzate was taken to dryness on a rotary evaporator at 40°C under reduced pressure. The residue was dissolved in 1 ml of 0.1 M HCl. Two 250-,LL~aliquots were used for gas chromatographic analyses. The remainder of the sample was analyzed on an ion-exchange amino acid analyzer. Eighty micrograms of prolyl-leucyl-glycineamide, 190 pg of cu-L-Va15-angiotensineamide-II and 1.2 mg of insulin were hydrolyzed. RESULTS
The Gas Chromatographic System Figure 1 shows the separation of 19 amino acid derivat’ives, including those of histidine and arginine. ,411 derivatives were sufficiently well
I 0
J
5
10
15 20 MINUTES
25
30
35
FIG. 1. Separation of the N-HFB-amino acid n-propyl ester derivatives of 19 amino acids on a 5.85 m long glass capillary column. Temperature program: 70°C for 4 min, then B”C/min. Full-scale deflection on the recorder represents a sensitivity of the instrument was detector current of 1.3 X 10mil A; 1/8 of th e maximal used. The peak between leucine and isoleucine probably represents allo-isoleucine. The peak behind S-methyl-cysteine represents cysteine (impurity from cystine).
GAS
CHROMATOGRAPHY
OF AMINO
209
ACIDS
separated to make precise qualitative analyses possible; the retentions relative to the norleucine derivative (T,,) are given in Table 1. The most critical amino acids are leucine and isoleucine due to the appearance of a second isoleucine peak emerging between leucine and the main peak of isoleucine. This peak has been observed only when analyzing isoleucine from the standard solution and it probably represents alla-isoleucine. Even when this peak is present the relative retention values for the leucine and isoleucine derivatives are significantly different as indicated by the values of the standard deviations (SD). The double peak appearing between the histidine and lysine derivatives is seen only when cystine is present in the sample. The complete separation of the amino acid derivatives requires only 35 min. The separation could be even clearer if a lower temperature gradient or a longer column were used but this has not seemed necessary for our purposes. The column, when operated under the conditions described in (7)) is stable for at least 3 months. This corresponds to more than 500 analyses.
Relative Retentions, n-Propyl Esters
TABLE 1 Pis, of N-Heptafluorobutyric (Relative to the Norleucine
N-HFBamino acid n-propylester Ala GUY Val Thr Ser Leu Ilel Iler Pro S-Me-Cys CYS Met Asp Phe Glu His LYS ‘Or Arg Cystine
Standard deviation SD 0.499 0.535 0.750 0.837 0.873 0.905 0.913 0.926 1.070 1.137 1.154 1.357 1.391 1.549 1.621 1.653 1.841 1.873 2.039 2.568
0.002 0.002 0.001 0.001 0.001 0.001 0.000 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.003 0.004 0.004 0.004 0.006 0.008
Amino Derivative)
Acid
Relative standard deviation RSD
(%) 0.3 0.4 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.3 0.3
210
JijNSSON,
El-EM,
AND
The Derivatization
SJijQUIST
System
Table 2 lists the results of the different esterification procedures. This study was done because of the somewhat inconvenient repetitive propylation step, in which an extra evaporation is required, as suggested by Coulter and Hann (8). The problems involved in the esterification of the amino acids are: (a) the low solubility of the basic amino acids and cystine in a high concentration of HCl; this results in low recoveries of these amino acid esters (b) the slow reactivity of valine and isoleucine; this demands a high HCl concentration for good yields of the esters. Moreover, histidine and lysine require two periods of 10 min at 100°C for complete esterification (8). The attempted one-step reaction in 8 M HCl:n-propanol failed with respect to the acidic and basic amino acids. The propylation of valine and isoleucine, on the other hand, worked well. In 3.5 M HCI:n-propanol the amino acids dissolved fairly rapidly and as a result the RMR values for the basic amino acids are higher than in the former case. Compared with the two-step esterification procedure in
RMR
Values of the N-HFB Derivative) at Different the 8 M HCI:n-propanol
TABLE 2 Amino Acid n-Propyl Esters (Relative to the Norleucine Esterification Conditions. Expressed as Percentage of Two-Step Esterification Values (RMR %). SD = Standard Deviation Esterification
N-HFB amino acid n-propyl ester Ala Gly Val Thr Ser Leu Ile Pro Met ASP Phe Glu His LYS Tyr Arg
8
M
HCl;
(RMR) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
two s
step SD 4.1 1.9 6.6 3.4 3.8 2.0 3.1 3.2 3.4 3.6 4.3 3.6 0.8 5.9 4.6 5.6
conditions
8 M HCl; (RMR) 105 98 114 95 91 108 117 90 101 88 93 75 61 71 91 73
one step %
SD 1.1 2 0 1.0 4.1 1.3 2.5 4.1 5.8 6.9 3.6 1.7 4.4 6.0 5.0 2.3 6.0
3.5
M
HCl;
(RMR) 107 96 109 95 93 103 103 96 108 95 95 91 86 89 92 90
one step %
SD 2.7 2.2 6.4 3.0 1.7 2.1 2.1 1.9 3.1 2.9 2.8 3.0 3.5 7.0 6.3 7.5
GAS
CHROMATOGRAPHY
OF AMINO
211
ACIDS
8 M HCI:n-propanol, the one-step propylation in the presence of 3.5 M HCl tended to give lower RMR values for the basic amino acids. Therefore, the original two-step propylation procedure was retained. If a onestep procedure is preferable; for example, when analyzing microamounts of amino acids, it should be better to use 3.5 M HCl instead of 8 M HCl. Increasing the acylation time from 10 to 20 min at 150°C had no significant effect on the yields of the amino acid derivatives. An acylation time of 10 min is evidently sufficient at this temperature. The stability of the derivatives with respect to time when stored at room temperature is shown in Table 3. The study extended over a period of 3 days. The results indicate that the derivatives are sufficiently stable during this period.
QuantitativeAnalysis Table 4 gives the RMR values relative to the norleucine derivative. Three replicate samples were derivatized at the lOO-nmole level. Forty picamoles of each derivative were analyzed on the column. The relative TABLE 3 RMR Values of the N-HFB-Amino Acid n-Propyl Esters (Relative to the Norleucine Derivative) When Analyzed Over a Period of 72 hr. Expressed as Percentage of the Values from the First Analysis (RMR %). SD = Standard Deviation N-HFB amino acid n-propyl ester Ala GUY Val Thr Ser Leu Ile Pro Met ASP
Phe Glu His LYS Tyr As
Time between acylation and analysis 15 min (RMR) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
%
24 hr SD
(RMR)
3.1 2.5 2.6 0.9 0.8 0.8 1.8 2.3 2.7 5.2 5.6 7.5 7.3 5.7 7.0 4.5
%
98 94 99 100 100 99 103 99 99 102 101 98 90 92 97 93 -.
. . I.”
48 hr SD 3.4 1.2 3.2 1.6 1.0 2.2 5.0 0.7 2.3 5.8 6.5 10.6 10.8 8.0 9.4 12.0
(RMR) 94 92 97 100 101 100 98 102 100 103 99 101 100 98 101 07
%
72 hr SD 0.6 2.8 0.3 0.5 2.0 1.6 1.0 0.3 2.7 3.4 0.1 4.7 1.8 12.7 7.5 2.5
(RMR)
%
SD
95 92 97 101 101 101 99 102 97 100 100 98 98 93 97
3.5 2.9 2.2 1.1 0.7 0.8 1.2 0.4 2.7 1.1 0.8 3.6 13.7 4.5 2.7
91
2.8
212
RMR
JBNSSON,
EYEM,
AND
TABLE 4 Values of the N-HFB Amino Acid n-Propyl Derivative). Three Replicate Samples Were RSD = Helat.ive Standard IV-HFB amino acid n-propyl est,er Ala Gly Val Thr Ser Leu Ile Pro S-Me-Cys Met Asp Phe Glu His LYS Tyr Ax Cystine
(RMR) 0.56 0.52 0.76 0.95 0.90 1.02 0.75 0.79 0.70 0.73 1.04 1.47 1.20 0.72 0.97 1.58 1.19 0.92
SJiiQUIST
Esters (Relative to the Norleucine Derivatized and Analyzed. Deviation
RSD
(o/c)
4.1 1.9 6.6 3.4 3.8 2.0 3.1 3.2 1.7 3.4 3.6 4.3 3.6 0.8 5.9 4.6 5.6 10
standard deviation (RSD) is less than 5% for most of the amino acid derivatives. Cystine shows the greatest variation (10%). The linearity test is illustrated in Table 5. The amounts analyzed on the column were only 9-75 pmoles per amino acid derivative. Valine, threonine, serine, leucine, isoleucine, aspartic acid, phenylalanine, glutamic acid, histidine, and tyrosine seem to respond linearly in the range studied. The deviations for isoleucine and leucine are related to the separation of the peaks. When larger amounts are injected into the column the leucine value tends to increase at the expense of the isoleucine value. This is most pronounced in the standard analyses because of the interfering peak probably representing allo-isoleucine. The linearity is confirmed by treating leucine and isoleucine as one peak. The deviations from linearity in the lower range shown by some amino acids, are at least partly dependent on factors such as the absolute peak area (at a given amplifier sensit,ivity), the chosen slope sensitivity of the integrator, and the tailing of the particular peak. Figure 2 and Table 6 show the analyses of the hydrolyzed tripeptide, prolyl-leucyl-glycineamide. The molar ratios calculated are in good agreement with the results of ionexchange analysis and the theoret,,cal values. This is also the case for cr-n-Val’-angiotensineamide-II shown in
GAS
Area
CHROMATOGRAPHY
OF
AMINO
213
ACIDS
TABLE 5 Ratios of the AT-HFB Amino Acid n-Propyl Ester Peaks Relative to the Peak the Norleucine Derivative When Derivatized and Analyzed at Different Levels. Expressed in Percentage of the Ratios from t,he Highest Level (AR ‘%). Each Sample Contained 92.4 Nmoles of Norleucine. SD = Standard Deviation
N-HFB amino acid n-propyl ester ~-
Derivatized 23
amounts
of
(nmoles)
47
94
188
_____ (AR)
Ala GUY Val Thr Ser Leu Ile Leu + Ile Pro Met Asp Phe Glu His LYS Tyr Arg Theory
10.2 7.1 12.0 13.0 12.7 9.9 16.0 12.6 10.7 10.4 11.9 12.3 11.4 14.2 9.1 12.4 9.4 12.5
%
SD
(AR)
0.3 1.3 0.0 0.2 0.1 0.1 0.3 0.2 0.4 0.0 0.5 0.6 0.5 4.7 0.2 0.7 0.6 0.0
21.7 18.9 24 8 25.5 25.3 20.7 32.0 25.6 23.7 24.2 24.4 25.0 22.5 24.9 23.0 24.6 24.1 25.0
Fig. 3 and Table 7. The deviation the same in both the ion-exchange The low recoveries of valine may acid is hydrolyzed slowly in 6 M
Molar
%
Ratios, MR, Obtained Prolyl-Leucyl-Glycineamide.
Amino acid
Theory MR
Gly Leu Pro
1 1 1
SD 1.0 0.6 0 .2 0.2 0.9 0 .5 1.6 0.2 0.5 0.6 1.1 0.9 0.4 4.0 0.9 0.5 0.X 0.0
(AR)
%
47.5 45.7 50.6 49.3 50.0 47.8 54.0 50.4 48.3 50.3 49.8 49.4 46.3 46.0 48.4 49.6 48.0 50.0
SD
(AR)
4.0 0.3 3 .9 1.2 1.5 5.1 9.9 1.4 1.8 1.0 1.1 1.3 2.2 12.0 1.7 1.1 4.6 0.0
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
%
SD 1.8 1.5 1.6 1.0 1.8 0.5 2.2 1.2 3.0 7.2 7.6 8.5 9.2 2.1 9.9 10.1 8.4 0.0
from the theoretical value for valine is and the gas chromatographic analyses. be explained by the fact that this amino HCI.
TABLE 6 in t,he Amino Acid Analyses of Hydrolyzed SD = Standard Deviation
Ion-exchange analysis Mll 1.04 1.00 0.96
Gas chrom. analyses (MR)
SD
0.97 1.05 0.97
0.02 0.00 0.01
214
JijNSSON,
EYEM,
AND
SJijQUIST
i? a
IL
J L
I
0
5
I
10 MINUTES
1
I
15
20
FIG. 2. Analysis of hydrolyzed prolyl-leucyl-glycineamide. 8 ng peptide out of 20 pg derivatized. Analysis conditions
The peaks represent aa in Fig. 1.
TABLE 7 Molar Ratios, MR, Obtained in the Amino Acid Analyses of Hydrolyzed cY-n-VaKAngiotensineamide-II. SD = Standard Deviation
Amino acid
Theory MR
Val Pro
2 1 1 1 1 1 1
ASP
Phe His Tyr As
Ion-exchange analysis MR 1.69 1.0 1.06 1.03 0.93 0.93 1.0
Gas chrom. analyses (MR)
SD
1.6 1.2
0.0 0.0
1.1
0.0
1.1 0.8
0.0 0.1
0.9
0.0 0.1
1.1
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
215
MINUTES
FIG. 3. Analysis of hydrolyzed cu-L-VaP-angiotensineamide-II. 20 ng peptide out of 50 pg derivatized. Analysis conditions
The peaks represent as in Fig. 1.
Table 8 and Fig. 4 illustrate the analyses of the hydrolyzate of insulin. The only discrepancies between the two types of analysis and a comparison with the theoretical numbers of amino acid residues in the molecule, arose with valine and cysteine. The same MR value for valine was obtained from the two types of analysis. On the other hand, no agreement was obtained for cysteine between the ion-exchange analysis and gas chromatography. The low valine values in the insulin analyses may be explained in the same way as for angiotensineamide. Furthermore, an impurity was seen in the analyses. According to the ion-exchange analysis, this could be a hexosamine. The mean RMR value of the amino acid derivatives was used for quantifying this peak. DISCUSSION
The injection of an anhydride together with the sample converts the mono-acyl derivative of histidine to the di-acyl derivative. Roach et al. (9) demonstrated this in their GC-analysis of N-TFA histidine n-butyl ester. Moss and Lambert (2) also observed this phenomenon. After acylation in HFBA and ethyl acetate they evaporated the sample at room
216
JijNSSON,
0
5
10
EYEM,
15
AND
SJijQUIST
I
,
20
25
I
30
35
MINUTES FIG. 4. Analysis of hydrolyzed insulin. The amount taken for derivatization was 0.3 mg. The peaks represent 120 ng insulin. The impurity (IMP) is probably a hexosamine. Analysis conditions as in Fig. 1.
temperature, dissolved it in ethyl acetate, and injected an aliquot together with acetic acid anhydride. This resulted in a peak emerging between arginine and cystine. The derivative was probably 3 or 5-Nacetyl-cu-N-HFB histidine. In our analyses the histidine derivative emerges from the column between the glutamic acid and the lysine derivatives when the acylation is performed in HFBA alone and an aliquot is injected with an equal volume of fresh HFBA. The elution sequence of the other amino acid derivatives is in agreement with the one reported by Moss and Lambert. Increasing the volume of fresh HFBA injected did not give better results for histidine. On the other hand, injection of the amino acid derivatives alone gave a smaller histidine peak, and sometimes a peak between arginine and cystine was observed. This could be t’he mono-HFB derivative. In Fig. 1 the peak probably represents the di-HFB histidine derivative. The cysteine peak in Fig. 1 arises from cystine either as an impurity or as a decomposition product. S-methylated cysteine (Sigma Chemical Co.)
GAS
Molar
llatios,
hlli,
CHROMATOGRAPHY
Obtained
OF
TABLE in the Amino SD = Standard Ion
Amino acid
Theory Mli
Ala QY Val Thr Ser Leu Ile Pro Met ASP Phe Glu His LYS ‘I& A% CYS Impurity a Calculated
from
the cystine
AMINO
8 Acid Analyses Deviation exchange analysis MR
217
ACIDS
of IIydrolyxed
Gas chrom. -
Insulin.
analyses .~
(MR)
SD
2.66 4.08 3.63 1.47 2.91 6.05 0.86 1.22 -
2.8 4.3 3.9 1.4 2.9 5.6 1.0 1.2 -
0.2 0.1 0.2 0.0 0.0 0.0 0.1 0.0 -
3.17 2.96 7.05 2.10 1.15 3.99 1.15 4.19 0.65
3.2 3.1 7.2 2.2 1.1 3.8 1.0 7.1a 0.7
0.1 0.1 0.5 0.5 0.1 0.3 0.1 0.4 0.1
values.
was analyzed with a view to the possibility of obtaining higher accuracy for cysteine by modifying it to a more easily analyzed amino acid. This can be done according to Eyem et al. (10). Due to their stability with respect to time, the HFB-derivatives are suitable for analysis on a gas chromatograph supplied with an automatic sample injector. By this method 30 samples could be analyzed in a 24-hr period. This would permit 150 complete analyses in one normal working week, which, compared with what can be accomplished with the ordinary ion-exchange analyzer, is a very high number. At present the amount of each amino acid taken through the whole analysis system is not very low, approximately 50 nmoles. Keeping in mind that this means 20 pmoles on the column, it would seem feasible to scale down the method by means of technical adjustments, such as the use of a splitless injection system (11,12) or a “solvent vent” device similar to that described by Zumvalt et ~2. (4). This would permit the injection of the entire sample into the column which would decrease the required amount of each amino acid in the initial sample to no more than 20 pmoles, provided that the derivatization is reliable at this level.
218
JijNSSON,
EPEM,
AiYD
SJijQUIST
Another way of decreasing the amount needed would be to concentrate the sample by evaporation prior to the injection. This might be somewhat difficult to do without losses under the strict anhydrous conditions required when working with low levels of amino acid derivatives. ACKNOWLEDGMENTS This investigation was supported by grants from the Swedish Medical Research Council. the Knut and Alice Wallenberg Foundation, and the Bank of Sweden Tercentenary Fund. We thank Mr. J. Eriksson for stimulating discussions, Mr. T. Moller for performing ion-exchange amino acid analyses, and Mrs. M. Arwidsson for typing the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
GEHRKE, C. W., Kuo, K., AND ZUMWALT, R. W. (1971) J. Chromatogr. 57, 209. Moss, C. W., AND LAMBERT, M. A. (1971) J. Chromatogr. 60, 137. POLLOCK, G. E. (1967) Anal. Chem. 39, 1194. ZUMWALT, R. W., Kuo, K., AND GEIIRKE, C. W. (1971) J. Chromatogr. 57, 193. EYEM, J., to be published. EYEM, J., to be published. EYEM, J., AND SJGQUIST, J. Anal. Biochem., Submitted for publication. COULTER, J. R., AND HANN, C. S. (1968) J. Chromatogr. 36, 42. ROACH, D., GEHRKE, C. W., AND ZUMWALT, R. W. (1969) J. Chromntogr. 43, 311. EYEM, J., JSNSSON, J., AND SJBQUIST, J., to be published. GROB, K., AND GROB, G. (1969) J. Chromatogr. Sci. 7, 584. GROB, K., AND GROB, G. (1969) J. Chromatogr. fki. 7, 587. JOHNSON, D. E., SCOTT, S. J., AND MEISTER, A. (1961) Anal. Chem. 33, 669. CRUICKSHANK, P. A., AND SHEENAN, J. C. (1964) Anal. Chem. 36, 1191. SMITH, E. D., AND SHEPPARD, H., JR. (1965) Nature London 208, 878. DARBRE, A., AND BLAU, K. (1965) J. Chromatogr. 17, 31. BLAU, K., AND DARBRE, A. (1965) J. Chromatogr. 17, 445. MARCUCCI, F., MUSSINI, E., POY, F., AND GAGLIARDI, P. (1965) J. Chromatogr. 18, 487. LAMKIN, W. M., AND GEHRKE, C. W. (1965) Anal. Chem. 37, 383. GEHRKE, C. W., LAMKIN, W. M., STALLING, D. L., AND SHAHROKHI, F. (1965) Biochem. Biophys. Res. Commun. 19, 328. STALLING, D. L., AND GEHRKE, C. W. (1966) Biochem. Biophys. Res. Commun. 22, 329. GEHRKE, C. W., AND STALLING, D. L. (1967) Separation Sci. 2, 101. STALLING, D. L., GILLE, G., AND GEHRKE, C. W. (1967) Anal. Biochem. 18, 118. STEFANOVIC, M., AND WALKER, B. L. (1967) Anal. Chem. 39, 711. DARBRE, A., AND ISLAM, A. (1968) Biochem. J. 106, 923. JEFFERSON, W. D., JR., AND FTJRST, A. (1968) Anal. Chem. 40, 1911. ROACH, D., AND GEHRKE, C. W. (1969) J. Chromatogr. 43, 303. RQACH, D., AND GEHRKE, C. W. (1969) J. Chromatogr. 44, 269. ZUMWALT, R. W., ROACH, D., AND GEHRKE, C. W. (1970) J. Chromatogr. 53, 171. BERGSTR~~M, K., G~~RTLER, J., AND BLOMSTRAND, R. (1970) Anal. Biochem. 34, 74. LACHOVITZKI, N., AND BJGRKLUND, B. (1970) And. Biochem. 38, 446. GEHRKE, C. W., AND LEIMER, K. (1970) J. Chromatogr. 53, 195.
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
219
33. PELLIZZARI, E. D., BROWN, J. H., TALBOT, P., FARMER, R. W., AND FABRE, L. F., JR. (1971) J. Chromatogr. 55, 281. 34. GEHRHE, C. W., AND LEIMER, K. (1971) J. Chromatogr. 57, 219. 35. BHATTI, T., AND CLAMP, J. R. (1971) Biochim. Biophus. Acta 229, 293. 36. GEHRKE, C. W., ZUMWALT, R. W., AND Kuo, K. (1971) J. Agr. Food Chem. 19, 605. 37. METZ, J., EBERT, W., AND WEICKER, H. (1971) Chromatographiu 4, 259.
51, 204-219 (1973)
BIOCHEMISTRY
Quantitative
Gas
Acids JiiRGEN Department
Chromatographic
on a Short
Glass
JONSSON, JULIUS
of Biochemistry, University Received
of
Analysis Capillary
EYEM,
AND
Faculty of Pharmacy, The Uppsaln, 751 bl Uppsala,
June
2, 1972;
accepted
July
of Amino
Column JOHN SJiiQUIST Wallenberg Sweden 24,
Laboratory,
1972
A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run. Hydrolyzed peptides have been analyzed. The analyses were performed
with
a precision
varying
viation) depending The amount taken
known
on for
between
sequences of the peptides
chromatography
modification
except
1 and 6% (mean relative
the number of amino acid residues analysis was 20-300 pg. The results for
as S-methylated
cysteine.
and with This
standard
de-
in the peptide. agree with the
the analyses by ion-exchange
amino
acid
can
be analyzed
after
cysteine.
The main purpose of this investigation was to find a suitable method for the quantitative gas chromatographic analysis of peptides. Our requirement was that the procedure should be rapid and applicable to samples no larger than those used in ion-exchange chromatography, in which 100 nmoles per amino acid are analyzed in two separate runs. These analyses are quite time consuming, requiring approximately 5 hr per analysis. With a suitable derivative, gas chromatographic analysis can be completed in half an hour using a single-column system. Recently a high pressure single-column ion-exchange analyzer appeared on the market (Durrum Instr. Corp. Mod D 500). This inst,rument performs a complete analysis in 48 min with a sensitivity of 1 nmole at a signal-tonoise ratio of 3O:l. The reproducibility is said to be 15% at a level of 100 nmoles per amino acid. These data should bc weighed against the very high price of the instrument. The advantages of using a gas chromatograph are the low cost and the great versatility of the system. Another advantage is the great sensitivity that can be obtained, especially when using capillary columns, together with ultrasensitive detectors such as the electron-capture detector (ECD). Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
204 Inc. reserved.
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
205
A number of workers (l-4, 7-9, 13-37) have investigated different kinds of derivatives suitable for gas chromatographic analysis, but they have not succeeded in separating and quantifying all the protein amino acids satisfactorily in a single run. Gehrke and coworkers used a dualcolumn system for analyzing N-trifluoroacetyl (TFA) amino acid n-butyl esters (1) which proved to be useful for even very small amounts of sample. Moss and Lambert (2) investigated the separation of the N-heptafluorobutyric (HFB) amino acid n-propyl esters on an OV 1 column. By using this derivative they succeeded in separating 20 amino acids in a single run and obtained sharp peaks when injecting 15 nmoles per amino acid derivative on a packed column. Pollock (3) investigated the time reduction when N-HFB-n-butyl derivatives were used instead of N-TFA-amino acid n-butyl esters and found it to be 35%. He also proposed the use of an electron-capture detector together with the N-HFB-derivatives in order to increase the sensitivity. Zumwalt et al. (4) were able to detect amounts of N-HFBn-butyl derivatives of cysteine and methionine as low as 2 and 1 pg, respectively, when using an ECD. The possibility of separating the protein amino acids on a single column in a short time and the great sensitivity that can be obtained using N-HFB amino acid n-propyl esters prompted us to select these derivatives for further studies. MATERIALS
AND
METHODS
A. Apparatus a. A Carlo Erba Mod. G 1 gas chromatograph which was equipped with a flame ionization detector and a linear temperature programmer was used. It was modified for glass capillary columns and had an all-glass inlet-splitt’ing device (5). The column used was a 6-m-long, 0.2-mm-i.d. open tubular column with dimethyl siloxane polymer chemically bound to the glass wall1 (6). The properties of the column were as follows: column efficiency, n, and partition ratio, k, for n-octadecane = 24,000 and 13.4, respectively; phase ratio ,f3 = 250; separation number S = 13; polarity (expressed as the retention index I of phenanthrene at 150°C) = 1,723. The detector signal was recorded with a Hitachi Perkin-Elmer recorder (Mod. 65) and integrated with a Hewlett-Packard electronic integrator (Mod. 3371 B) . Nitrogen, purified as previously described (7)) was used as carrier gas. b. For evaporation of solvents, we used a heated aluminium block 1 Prep.ared
by LKB-Produkter
AB,
Stockholm,
Sweden.
206
JBNSSON,
EYEM,
AND
SJijQUIST
fitted with eight stainless-steel needles blowing nitrogen over the samples. c. Ultrasonic Mixer (Millipore) . d. Control amino acid analyses were performed on an automatic ionexchange analyzer (Biochrom BC-200). B. Chromatographic
Conditions
a. Column temperature: Initially 70°C held constant for 4 min, then linearly programmed at a rate of 5”C/min up to 230°C. b. Detector and injection temperature: 210°C. c. Mean carrier-gas velocity: 16 cm/set. d. Splitting ratio: 1:lOO. C. Reagents a. Analytical grade amino acids were obtained from different manufacturers. b. 8 M HCl:n-propanol. Anhydrous HCl was produced by adding coned HCI (p.a., Merck) dropwise to coned H,SO, (p.a., Merck) and passing the HCI gas through a H&SO, drying tower. The gas was bubbled through n-propanol (p.a., Merck) which had been dried over CaSO, and redistilled in an all-glass system. The concentration of WC1 was determined by titration. c. n-Hexane (p.a., Merck) was dried over CaSO* and redistilled in the same manner as n-propanol. d. Heptafluorobutyric anhydride (HFBA) was obtained from Pierce Chemical Co. e. cY-n-Va15-angiotensineamide-II (Ciba) and prolyl-leucyl-glycineamide were kindly provided by Dr. I. SjGholm at this department. D. Treatment
of Sample for Separation Studies
The amino acid derivatives were prepared as described by Moss and Lambert (2) with some modifications. A 500-~1 quantity of amino acid standard solution containing approximately 100 nmoles per amino acid in 0.1 M HCl was pipetted into a l-ml heavy-walled screw-capped glass vial equipped with a Teflon-lined stopper (Reacti-Vial, Pierce Chemical Co.). The solution was evaporated at 100°C under extra pure nitrogen and the sample was stored overnight in a desiccator (over KOH-pellets) . A 500-J amount of 8 M HCl:n-propanol was added to the sample. It was thoroughly shaken by hand and then mixed in the ultrasonic bath until the white precipitate dissolved. Esterification was performed in an oven at 110°C for 10 min. The vessel was immersed in the ultrasonic bath and it,s contents mixed for 30 set while hot. The HCl-propanol was
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
207
evaporated under N, at 100°C. The esterification step was then repeated, but the ultrasonic treatment was omitted. After evaporation, the remaining reagent was removed by azeotropic distillation with 50 ~1 n-hexane at ambient temperature. Acylation of the amino acid esters was then carried out in 50 ~1 HFBA at 150°C for 10 min in a heated aluminium block. After cooling, 2 ~1 of sample mixed with 2 ,ul of fresh HFBA in a syringe was injected into the gas chromatograph for analysis. The retention of each derivative relative to the norleucine derivative (ri8) was calculated. E. Esterification
Studies
Studies were done on a prolonged one-step esterification performed in 8 M HCl n-propanol at 110°C for 30 min. Further, the effect of using 3.5 M HCl:n-propanol for esterification was tested at 110°C for 30 min. Acylation was performed as described under section D. In each study three samples (containing 100 nmoles of each amino acid) were derivatized and analyzed. F. Acylation
Studies
The results of acylation for 10 and 20 min at 150°C were compared. Three replicate samples were derivatized as described in section D. Three others were treated identically with the exception of the acylation procedure, for which the time was doubled. G. Stability
of the Derivatized
Samples
Two samples were derivatized as described in section D and stored at room temperature. The analyses were performed 15 min, 24, 48, and 72 hr after derivatization. H. Determination
of the Relative
Molar
Response
Three replicate samples were derivatized and analyzed as described in section D. The RMR values relative to the norleucine derivative were calculated. RMR = {(area of studied peak) (molar amount of norleucine)} x { ( area of norleucine peak) (molar amount of studied amino acid) }-I. I. Linearity
of the Response
The amount of each amino acid analyzed by the gas chromatographic technique was approximately 25-200 nmoles. This range was chosen in order to permit direct comparison with analyses from the ion-exchange amino acid analyzer. Duplicate samples containing 23, 47, 94, and 188
208
JijNSSON,
EPEM,
AND
SJiiQUIST
nmoles per amino acid were taken through the derivatization and analysis steps according to section D. The amount of the internal standard, norleucine, was constant in all samples, i.e., 92.4 nmoles. J. Application to Peptide Hydrolyzates Peptides were hydrolyzed for 20 hr at 106°C in 2 ml 6 M HCl (Riedel de Ha&, p.a.) containing 27% (v/v) phenol and 200 nmoles of norleucine. The hydrolyzate was taken to dryness on a rotary evaporator at 40°C under reduced pressure. The residue was dissolved in 1 ml of 0.1 M HCl. Two 250-,LL~aliquots were used for gas chromatographic analyses. The remainder of the sample was analyzed on an ion-exchange amino acid analyzer. Eighty micrograms of prolyl-leucyl-glycineamide, 190 pg of cu-L-Va15-angiotensineamide-II and 1.2 mg of insulin were hydrolyzed. RESULTS
The Gas Chromatographic System Figure 1 shows the separation of 19 amino acid derivat’ives, including those of histidine and arginine. ,411 derivatives were sufficiently well
I 0
J
5
10
15 20 MINUTES
25
30
35
FIG. 1. Separation of the N-HFB-amino acid n-propyl ester derivatives of 19 amino acids on a 5.85 m long glass capillary column. Temperature program: 70°C for 4 min, then B”C/min. Full-scale deflection on the recorder represents a sensitivity of the instrument was detector current of 1.3 X 10mil A; 1/8 of th e maximal used. The peak between leucine and isoleucine probably represents allo-isoleucine. The peak behind S-methyl-cysteine represents cysteine (impurity from cystine).
GAS
CHROMATOGRAPHY
OF AMINO
209
ACIDS
separated to make precise qualitative analyses possible; the retentions relative to the norleucine derivative (T,,) are given in Table 1. The most critical amino acids are leucine and isoleucine due to the appearance of a second isoleucine peak emerging between leucine and the main peak of isoleucine. This peak has been observed only when analyzing isoleucine from the standard solution and it probably represents alla-isoleucine. Even when this peak is present the relative retention values for the leucine and isoleucine derivatives are significantly different as indicated by the values of the standard deviations (SD). The double peak appearing between the histidine and lysine derivatives is seen only when cystine is present in the sample. The complete separation of the amino acid derivatives requires only 35 min. The separation could be even clearer if a lower temperature gradient or a longer column were used but this has not seemed necessary for our purposes. The column, when operated under the conditions described in (7)) is stable for at least 3 months. This corresponds to more than 500 analyses.
Relative Retentions, n-Propyl Esters
TABLE 1 Pis, of N-Heptafluorobutyric (Relative to the Norleucine
N-HFBamino acid n-propylester Ala GUY Val Thr Ser Leu Ilel Iler Pro S-Me-Cys CYS Met Asp Phe Glu His LYS ‘Or Arg Cystine
Standard deviation SD 0.499 0.535 0.750 0.837 0.873 0.905 0.913 0.926 1.070 1.137 1.154 1.357 1.391 1.549 1.621 1.653 1.841 1.873 2.039 2.568
0.002 0.002 0.001 0.001 0.001 0.001 0.000 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.003 0.004 0.004 0.004 0.006 0.008
Amino Derivative)
Acid
Relative standard deviation RSD
(%) 0.3 0.4 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.3 0.3
210
JijNSSON,
El-EM,
AND
The Derivatization
SJijQUIST
System
Table 2 lists the results of the different esterification procedures. This study was done because of the somewhat inconvenient repetitive propylation step, in which an extra evaporation is required, as suggested by Coulter and Hann (8). The problems involved in the esterification of the amino acids are: (a) the low solubility of the basic amino acids and cystine in a high concentration of HCl; this results in low recoveries of these amino acid esters (b) the slow reactivity of valine and isoleucine; this demands a high HCl concentration for good yields of the esters. Moreover, histidine and lysine require two periods of 10 min at 100°C for complete esterification (8). The attempted one-step reaction in 8 M HCl:n-propanol failed with respect to the acidic and basic amino acids. The propylation of valine and isoleucine, on the other hand, worked well. In 3.5 M HCI:n-propanol the amino acids dissolved fairly rapidly and as a result the RMR values for the basic amino acids are higher than in the former case. Compared with the two-step esterification procedure in
RMR
Values of the N-HFB Derivative) at Different the 8 M HCI:n-propanol
TABLE 2 Amino Acid n-Propyl Esters (Relative to the Norleucine Esterification Conditions. Expressed as Percentage of Two-Step Esterification Values (RMR %). SD = Standard Deviation Esterification
N-HFB amino acid n-propyl ester Ala Gly Val Thr Ser Leu Ile Pro Met ASP Phe Glu His LYS Tyr Arg
8
M
HCl;
(RMR) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
two s
step SD 4.1 1.9 6.6 3.4 3.8 2.0 3.1 3.2 3.4 3.6 4.3 3.6 0.8 5.9 4.6 5.6
conditions
8 M HCl; (RMR) 105 98 114 95 91 108 117 90 101 88 93 75 61 71 91 73
one step %
SD 1.1 2 0 1.0 4.1 1.3 2.5 4.1 5.8 6.9 3.6 1.7 4.4 6.0 5.0 2.3 6.0
3.5
M
HCl;
(RMR) 107 96 109 95 93 103 103 96 108 95 95 91 86 89 92 90
one step %
SD 2.7 2.2 6.4 3.0 1.7 2.1 2.1 1.9 3.1 2.9 2.8 3.0 3.5 7.0 6.3 7.5
GAS
CHROMATOGRAPHY
OF AMINO
211
ACIDS
8 M HCI:n-propanol, the one-step propylation in the presence of 3.5 M HCl tended to give lower RMR values for the basic amino acids. Therefore, the original two-step propylation procedure was retained. If a onestep procedure is preferable; for example, when analyzing microamounts of amino acids, it should be better to use 3.5 M HCl instead of 8 M HCl. Increasing the acylation time from 10 to 20 min at 150°C had no significant effect on the yields of the amino acid derivatives. An acylation time of 10 min is evidently sufficient at this temperature. The stability of the derivatives with respect to time when stored at room temperature is shown in Table 3. The study extended over a period of 3 days. The results indicate that the derivatives are sufficiently stable during this period.
QuantitativeAnalysis Table 4 gives the RMR values relative to the norleucine derivative. Three replicate samples were derivatized at the lOO-nmole level. Forty picamoles of each derivative were analyzed on the column. The relative TABLE 3 RMR Values of the N-HFB-Amino Acid n-Propyl Esters (Relative to the Norleucine Derivative) When Analyzed Over a Period of 72 hr. Expressed as Percentage of the Values from the First Analysis (RMR %). SD = Standard Deviation N-HFB amino acid n-propyl ester Ala GUY Val Thr Ser Leu Ile Pro Met ASP
Phe Glu His LYS Tyr As
Time between acylation and analysis 15 min (RMR) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
%
24 hr SD
(RMR)
3.1 2.5 2.6 0.9 0.8 0.8 1.8 2.3 2.7 5.2 5.6 7.5 7.3 5.7 7.0 4.5
%
98 94 99 100 100 99 103 99 99 102 101 98 90 92 97 93 -.
. . I.”
48 hr SD 3.4 1.2 3.2 1.6 1.0 2.2 5.0 0.7 2.3 5.8 6.5 10.6 10.8 8.0 9.4 12.0
(RMR) 94 92 97 100 101 100 98 102 100 103 99 101 100 98 101 07
%
72 hr SD 0.6 2.8 0.3 0.5 2.0 1.6 1.0 0.3 2.7 3.4 0.1 4.7 1.8 12.7 7.5 2.5
(RMR)
%
SD
95 92 97 101 101 101 99 102 97 100 100 98 98 93 97
3.5 2.9 2.2 1.1 0.7 0.8 1.2 0.4 2.7 1.1 0.8 3.6 13.7 4.5 2.7
91
2.8
212
RMR
JBNSSON,
EYEM,
AND
TABLE 4 Values of the N-HFB Amino Acid n-Propyl Derivative). Three Replicate Samples Were RSD = Helat.ive Standard IV-HFB amino acid n-propyl est,er Ala Gly Val Thr Ser Leu Ile Pro S-Me-Cys Met Asp Phe Glu His LYS Tyr Ax Cystine
(RMR) 0.56 0.52 0.76 0.95 0.90 1.02 0.75 0.79 0.70 0.73 1.04 1.47 1.20 0.72 0.97 1.58 1.19 0.92
SJiiQUIST
Esters (Relative to the Norleucine Derivatized and Analyzed. Deviation
RSD
(o/c)
4.1 1.9 6.6 3.4 3.8 2.0 3.1 3.2 1.7 3.4 3.6 4.3 3.6 0.8 5.9 4.6 5.6 10
standard deviation (RSD) is less than 5% for most of the amino acid derivatives. Cystine shows the greatest variation (10%). The linearity test is illustrated in Table 5. The amounts analyzed on the column were only 9-75 pmoles per amino acid derivative. Valine, threonine, serine, leucine, isoleucine, aspartic acid, phenylalanine, glutamic acid, histidine, and tyrosine seem to respond linearly in the range studied. The deviations for isoleucine and leucine are related to the separation of the peaks. When larger amounts are injected into the column the leucine value tends to increase at the expense of the isoleucine value. This is most pronounced in the standard analyses because of the interfering peak probably representing allo-isoleucine. The linearity is confirmed by treating leucine and isoleucine as one peak. The deviations from linearity in the lower range shown by some amino acids, are at least partly dependent on factors such as the absolute peak area (at a given amplifier sensit,ivity), the chosen slope sensitivity of the integrator, and the tailing of the particular peak. Figure 2 and Table 6 show the analyses of the hydrolyzed tripeptide, prolyl-leucyl-glycineamide. The molar ratios calculated are in good agreement with the results of ionexchange analysis and the theoret,,cal values. This is also the case for cr-n-Val’-angiotensineamide-II shown in
GAS
Area
CHROMATOGRAPHY
OF
AMINO
213
ACIDS
TABLE 5 Ratios of the AT-HFB Amino Acid n-Propyl Ester Peaks Relative to the Peak the Norleucine Derivative When Derivatized and Analyzed at Different Levels. Expressed in Percentage of the Ratios from t,he Highest Level (AR ‘%). Each Sample Contained 92.4 Nmoles of Norleucine. SD = Standard Deviation
N-HFB amino acid n-propyl ester ~-
Derivatized 23
amounts
of
(nmoles)
47
94
188
_____ (AR)
Ala GUY Val Thr Ser Leu Ile Leu + Ile Pro Met Asp Phe Glu His LYS Tyr Arg Theory
10.2 7.1 12.0 13.0 12.7 9.9 16.0 12.6 10.7 10.4 11.9 12.3 11.4 14.2 9.1 12.4 9.4 12.5
%
SD
(AR)
0.3 1.3 0.0 0.2 0.1 0.1 0.3 0.2 0.4 0.0 0.5 0.6 0.5 4.7 0.2 0.7 0.6 0.0
21.7 18.9 24 8 25.5 25.3 20.7 32.0 25.6 23.7 24.2 24.4 25.0 22.5 24.9 23.0 24.6 24.1 25.0
Fig. 3 and Table 7. The deviation the same in both the ion-exchange The low recoveries of valine may acid is hydrolyzed slowly in 6 M
Molar
%
Ratios, MR, Obtained Prolyl-Leucyl-Glycineamide.
Amino acid
Theory MR
Gly Leu Pro
1 1 1
SD 1.0 0.6 0 .2 0.2 0.9 0 .5 1.6 0.2 0.5 0.6 1.1 0.9 0.4 4.0 0.9 0.5 0.X 0.0
(AR)
%
47.5 45.7 50.6 49.3 50.0 47.8 54.0 50.4 48.3 50.3 49.8 49.4 46.3 46.0 48.4 49.6 48.0 50.0
SD
(AR)
4.0 0.3 3 .9 1.2 1.5 5.1 9.9 1.4 1.8 1.0 1.1 1.3 2.2 12.0 1.7 1.1 4.6 0.0
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
%
SD 1.8 1.5 1.6 1.0 1.8 0.5 2.2 1.2 3.0 7.2 7.6 8.5 9.2 2.1 9.9 10.1 8.4 0.0
from the theoretical value for valine is and the gas chromatographic analyses. be explained by the fact that this amino HCI.
TABLE 6 in t,he Amino Acid Analyses of Hydrolyzed SD = Standard Deviation
Ion-exchange analysis Mll 1.04 1.00 0.96
Gas chrom. analyses (MR)
SD
0.97 1.05 0.97
0.02 0.00 0.01
214
JijNSSON,
EYEM,
AND
SJijQUIST
i? a
IL
J L
I
0
5
I
10 MINUTES
1
I
15
20
FIG. 2. Analysis of hydrolyzed prolyl-leucyl-glycineamide. 8 ng peptide out of 20 pg derivatized. Analysis conditions
The peaks represent aa in Fig. 1.
TABLE 7 Molar Ratios, MR, Obtained in the Amino Acid Analyses of Hydrolyzed cY-n-VaKAngiotensineamide-II. SD = Standard Deviation
Amino acid
Theory MR
Val Pro
2 1 1 1 1 1 1
ASP
Phe His Tyr As
Ion-exchange analysis MR 1.69 1.0 1.06 1.03 0.93 0.93 1.0
Gas chrom. analyses (MR)
SD
1.6 1.2
0.0 0.0
1.1
0.0
1.1 0.8
0.0 0.1
0.9
0.0 0.1
1.1
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
215
MINUTES
FIG. 3. Analysis of hydrolyzed cu-L-VaP-angiotensineamide-II. 20 ng peptide out of 50 pg derivatized. Analysis conditions
The peaks represent as in Fig. 1.
Table 8 and Fig. 4 illustrate the analyses of the hydrolyzate of insulin. The only discrepancies between the two types of analysis and a comparison with the theoretical numbers of amino acid residues in the molecule, arose with valine and cysteine. The same MR value for valine was obtained from the two types of analysis. On the other hand, no agreement was obtained for cysteine between the ion-exchange analysis and gas chromatography. The low valine values in the insulin analyses may be explained in the same way as for angiotensineamide. Furthermore, an impurity was seen in the analyses. According to the ion-exchange analysis, this could be a hexosamine. The mean RMR value of the amino acid derivatives was used for quantifying this peak. DISCUSSION
The injection of an anhydride together with the sample converts the mono-acyl derivative of histidine to the di-acyl derivative. Roach et al. (9) demonstrated this in their GC-analysis of N-TFA histidine n-butyl ester. Moss and Lambert (2) also observed this phenomenon. After acylation in HFBA and ethyl acetate they evaporated the sample at room
216
JijNSSON,
0
5
10
EYEM,
15
AND
SJijQUIST
I
,
20
25
I
30
35
MINUTES FIG. 4. Analysis of hydrolyzed insulin. The amount taken for derivatization was 0.3 mg. The peaks represent 120 ng insulin. The impurity (IMP) is probably a hexosamine. Analysis conditions as in Fig. 1.
temperature, dissolved it in ethyl acetate, and injected an aliquot together with acetic acid anhydride. This resulted in a peak emerging between arginine and cystine. The derivative was probably 3 or 5-Nacetyl-cu-N-HFB histidine. In our analyses the histidine derivative emerges from the column between the glutamic acid and the lysine derivatives when the acylation is performed in HFBA alone and an aliquot is injected with an equal volume of fresh HFBA. The elution sequence of the other amino acid derivatives is in agreement with the one reported by Moss and Lambert. Increasing the volume of fresh HFBA injected did not give better results for histidine. On the other hand, injection of the amino acid derivatives alone gave a smaller histidine peak, and sometimes a peak between arginine and cystine was observed. This could be t’he mono-HFB derivative. In Fig. 1 the peak probably represents the di-HFB histidine derivative. The cysteine peak in Fig. 1 arises from cystine either as an impurity or as a decomposition product. S-methylated cysteine (Sigma Chemical Co.)
GAS
Molar
llatios,
hlli,
CHROMATOGRAPHY
Obtained
OF
TABLE in the Amino SD = Standard Ion
Amino acid
Theory Mli
Ala QY Val Thr Ser Leu Ile Pro Met ASP Phe Glu His LYS ‘I& A% CYS Impurity a Calculated
from
the cystine
AMINO
8 Acid Analyses Deviation exchange analysis MR
217
ACIDS
of IIydrolyxed
Gas chrom. -
Insulin.
analyses .~
(MR)
SD
2.66 4.08 3.63 1.47 2.91 6.05 0.86 1.22 -
2.8 4.3 3.9 1.4 2.9 5.6 1.0 1.2 -
0.2 0.1 0.2 0.0 0.0 0.0 0.1 0.0 -
3.17 2.96 7.05 2.10 1.15 3.99 1.15 4.19 0.65
3.2 3.1 7.2 2.2 1.1 3.8 1.0 7.1a 0.7
0.1 0.1 0.5 0.5 0.1 0.3 0.1 0.4 0.1
values.
was analyzed with a view to the possibility of obtaining higher accuracy for cysteine by modifying it to a more easily analyzed amino acid. This can be done according to Eyem et al. (10). Due to their stability with respect to time, the HFB-derivatives are suitable for analysis on a gas chromatograph supplied with an automatic sample injector. By this method 30 samples could be analyzed in a 24-hr period. This would permit 150 complete analyses in one normal working week, which, compared with what can be accomplished with the ordinary ion-exchange analyzer, is a very high number. At present the amount of each amino acid taken through the whole analysis system is not very low, approximately 50 nmoles. Keeping in mind that this means 20 pmoles on the column, it would seem feasible to scale down the method by means of technical adjustments, such as the use of a splitless injection system (11,12) or a “solvent vent” device similar to that described by Zumvalt et ~2. (4). This would permit the injection of the entire sample into the column which would decrease the required amount of each amino acid in the initial sample to no more than 20 pmoles, provided that the derivatization is reliable at this level.
218
JijNSSON,
EPEM,
AiYD
SJijQUIST
Another way of decreasing the amount needed would be to concentrate the sample by evaporation prior to the injection. This might be somewhat difficult to do without losses under the strict anhydrous conditions required when working with low levels of amino acid derivatives. ACKNOWLEDGMENTS This investigation was supported by grants from the Swedish Medical Research Council. the Knut and Alice Wallenberg Foundation, and the Bank of Sweden Tercentenary Fund. We thank Mr. J. Eriksson for stimulating discussions, Mr. T. Moller for performing ion-exchange amino acid analyses, and Mrs. M. Arwidsson for typing the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
GEHRKE, C. W., Kuo, K., AND ZUMWALT, R. W. (1971) J. Chromatogr. 57, 209. Moss, C. W., AND LAMBERT, M. A. (1971) J. Chromatogr. 60, 137. POLLOCK, G. E. (1967) Anal. Chem. 39, 1194. ZUMWALT, R. W., Kuo, K., AND GEIIRKE, C. W. (1971) J. Chromatogr. 57, 193. EYEM, J., to be published. EYEM, J., to be published. EYEM, J., AND SJGQUIST, J. Anal. Biochem., Submitted for publication. COULTER, J. R., AND HANN, C. S. (1968) J. Chromatogr. 36, 42. ROACH, D., GEHRKE, C. W., AND ZUMWALT, R. W. (1969) J. Chromntogr. 43, 311. EYEM, J., JSNSSON, J., AND SJBQUIST, J., to be published. GROB, K., AND GROB, G. (1969) J. Chromatogr. Sci. 7, 584. GROB, K., AND GROB, G. (1969) J. Chromatogr. fki. 7, 587. JOHNSON, D. E., SCOTT, S. J., AND MEISTER, A. (1961) Anal. Chem. 33, 669. CRUICKSHANK, P. A., AND SHEENAN, J. C. (1964) Anal. Chem. 36, 1191. SMITH, E. D., AND SHEPPARD, H., JR. (1965) Nature London 208, 878. DARBRE, A., AND BLAU, K. (1965) J. Chromatogr. 17, 31. BLAU, K., AND DARBRE, A. (1965) J. Chromatogr. 17, 445. MARCUCCI, F., MUSSINI, E., POY, F., AND GAGLIARDI, P. (1965) J. Chromatogr. 18, 487. LAMKIN, W. M., AND GEHRKE, C. W. (1965) Anal. Chem. 37, 383. GEHRKE, C. W., LAMKIN, W. M., STALLING, D. L., AND SHAHROKHI, F. (1965) Biochem. Biophys. Res. Commun. 19, 328. STALLING, D. L., AND GEHRKE, C. W. (1966) Biochem. Biophys. Res. Commun. 22, 329. GEHRKE, C. W., AND STALLING, D. L. (1967) Separation Sci. 2, 101. STALLING, D. L., GILLE, G., AND GEHRKE, C. W. (1967) Anal. Biochem. 18, 118. STEFANOVIC, M., AND WALKER, B. L. (1967) Anal. Chem. 39, 711. DARBRE, A., AND ISLAM, A. (1968) Biochem. J. 106, 923. JEFFERSON, W. D., JR., AND FTJRST, A. (1968) Anal. Chem. 40, 1911. ROACH, D., AND GEHRKE, C. W. (1969) J. Chromatogr. 43, 303. RQACH, D., AND GEHRKE, C. W. (1969) J. Chromatogr. 44, 269. ZUMWALT, R. W., ROACH, D., AND GEHRKE, C. W. (1970) J. Chromatogr. 53, 171. BERGSTR~~M, K., G~~RTLER, J., AND BLOMSTRAND, R. (1970) Anal. Biochem. 34, 74. LACHOVITZKI, N., AND BJGRKLUND, B. (1970) And. Biochem. 38, 446. GEHRKE, C. W., AND LEIMER, K. (1970) J. Chromatogr. 53, 195.
GAS
CHROMATOGRAPHY
OF
AMINO
ACIDS
219
33. PELLIZZARI, E. D., BROWN, J. H., TALBOT, P., FARMER, R. W., AND FABRE, L. F., JR. (1971) J. Chromatogr. 55, 281. 34. GEHRHE, C. W., AND LEIMER, K. (1971) J. Chromatogr. 57, 219. 35. BHATTI, T., AND CLAMP, J. R. (1971) Biochim. Biophus. Acta 229, 293. 36. GEHRKE, C. W., ZUMWALT, R. W., AND Kuo, K. (1971) J. Agr. Food Chem. 19, 605. 37. METZ, J., EBERT, W., AND WEICKER, H. (1971) Chromatographiu 4, 259.