modified scheme has been devised which should overcome this difficulty. In addition, the new sequence of operations yields the same information with one less operation, which will save time. The new scheme is shown in Figure 7. It haa not yet been tested thoroughly, although each component operation is known to be satisfactory. Future Work. While the scheme and its constituent techniques are believed, a t present, to be practical research tools, there are still directions in which fruitful work can be done. The area of greatest potential usefulness, application to high-boiling
mixtures, is yet to be properly explored. In this connection, operation with capillary columns appears mpecialy attractive. Trapping of the column effluent is not possible in this case, but trapping and operating on the portion of injected sample which issues from the sample splitter appears feasible.
(3) Emmett, P.H. Divieion of Petroleum Chemiitp A&, 136th Meeting, Boston, pril 1969. (4) Keulemans, A. I. M., Voge, H. H., J . Phys. chem. 63,416-80 (1969). (5) Kokea, R. J., Tobm, H. Jr., Emmett P. H., J . Am. C h a . &. 77, 686d
LITERATURE ClTED
(8) Whitman, B. T., Nature 182, 391
N., Cie linski, E. W., Coates, $. J., J . &romalog. 3, BO (1960). (2) Coulson D. M., ANAL. CHEM.31, 906 (1959).
RECEIVED for review November 17, 1960. Accepted January 30, 1961. Division of Analytical Chemistry, 137th Meeting, ACS, Cleveland, Ohio, April 1960.
(1956). (6) Martin, R. L., ANAL. CHEM. 32, 330-86 (1960).
n, C. J. Coleman, H. J., ‘ 7 b 2 m r C . .. Rsll.. k. T.. Zbid..~.32. 424 (igwj.
(1958).
(1) Brenner
Analysis of Gas-Liquid Chromatog rams by a Punched Card Technique ROBERT K. TANDY, FRANK T. LINDGREN, WALTER H. MARTIN, and ROBERT D. WILLS Donner laboratory of Medical Physics and the Lawrence Radiation laboratory, University of California, Berkeley, Calif.
b A
method is described whereby gas-liquid chromatograms may be analyzed using a punched card technique. Although the application presented involves analysis of fatty acid methyl esters in which a beta particle ionization detector is used, with minor revisions this method has potential applications to all gas-liquid chromatographic work. The advantages of this technique are: elimination of nearly all manual arithmetic calculations, equivalent or greater accuracy to existing manual techniques, and ease of data manipulation and storage.
D
the last decade one of the most promising and exciting developments for the analysis of volatile organic compounds of biological interest has been the technique of gasliquid chromatography ( 1 ) . An important application of this technique is in the study of long-chain fatty acids and their relationship to both normal and abnormal lipide metabolism. However, one of the technical difficulties encountered in this and other applications of gas-liquid chromatography (GLC) is the complicated nature of the data. I n the fatty acid studies, for example, there are about 100 biologically occurring fatty acids. In a typical gas-liquid chromatographic analysis approximately 25 or more of these fatty acids are frequently resolved on each chromatogram. Tabulation and comparison of such extensive data are very tedious manual tasks. Usually, the amount of each fatty acid component has been quantitated by measuring URINO
under each peak on the chromatogram is calculated by multiplying the product of the elution time and pcak height by a first order correction function based upon the peak height. Thus, for a given chromatogram, the mass of each component is calculated together with the total mass of all chromatographic components present. For convenience, the mass per cent of each component aa well as its retention time (relative to methyl stearate) is also calculated. An additional calculation correcting each chromatographic component on the basis of its relative retention time completes the program.
the area under each chromatographic peak through integration, planimetry, and triangulation (2). If carried out manually, these methods are laborious and unfortunately subject to frequent human error. To avoid the above-mentioned technical difficulties, the authors have developed a technique for analysis of gss-liquid chromatograms using punched cards together with an a p propriate computer (IBM 650). In essence, each chromatogram consisting of a complicated sequence of peaks is reduced to a small deck of IBM punched cards (one for each chromatographic component). The basic date placed on these input IBM punched cards consist of the elution time and peak height of each chromatographic component. From these data, a measure of the area
Figure 1. Relationship between triangulated peak width and retention time
-E
EXPERIMENTAL
For a given fatty acid methyl ester component on a gas-liquid chromato-
4
z
g 52
Y
i ll-
# Chromatogrophic run 1406
0
I
0
20
I
I
40
I
I
I
60
I
80
I
I
100
RETENTION TIME, MINUTES
VOL 33, NO. 6, MAY 1961
665
gram, the peak width (at the baee line by triangulation) is roughly proportional to the elution time. F'igure 1 shows this relationship for a duplicate analysis of chm~ l calculated l
7 - 1
I
I
I
1
I
I
1
1.1
@ 0 Q @ 8
Ed g
Methyl orothidonoto Mothyl oleoto Mothyl Ilnoleotr Mothy1 rteoroto Mothyl polmitote
1.0
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e
8
0.9
0.1
PEAK HEIGHT, INCHES,AT 1 VOLT
Figure 2. helght
g
1.1
E::
-
1.1
-
1.0
-
8
R n t correction function involving peak
0.9 I 0.2
0.1
I
I 0.4
I
I 1
I I I I I 0.b 0.8 1.0
I 3
1 4
1
RELATIVE RETENTION TIME
Figure 3. Second correction function involving relative time
FULL SCALE (DEFLECTION) 10" * 3.12 x IO-' cmps (33.3x)
t
2
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1"lsEnw31~ U
Figure 4.
1406
666
ANALYTICAL CHEMISTRY
',
(33.3x) ( a t . ) p
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a
7
Duplicate analysis of chromatographic run
6
NO.
matographic run No. 1406. Anotlicr relationship is that, for a given component, the peak Iiright is approximately 85% the triangulated height. Thua, for any component, a measure of the triangulated area (which is approximately 95% of the absolute peek area) can be obtaiiied from the product of the peak height and the elution time. This value is, of course, only a first approximation to component mass, because for a given component using our beta particle ionization detector (3) peak area (or peak height) is not exactly linear with component mass but is actually somewhat sigmoidal. Using a standard calibration mixture, Figure 2 shows the first order correction factors, based only on peak height, that are necessary to correct each component. This function is a stepped interval approximation using calibration data of the five principal fatty acid methyl ester components. The sample
c I
niiwws for each component covrr the range usually encountered in GLC of the blood lipides and are the order of from 5 to 50 pg. This first order function primarily corrects for the nonlinearity of all calibration curvcs, irrespective of differences in detector response for different fatty acid methyl ester components. Sample masses were measured both by injection of different meascred volumes of a standard calibration mixture and by successive dilution of such a calibration mixture. Variations in absolute retention time for a given component do not introduce appreciable errors. Thus, under our operating conditions (S), the retention time observed for methyl stearate decreases from approximately 18 to IO minutes for the early and late phases, respectively, of the usable life span of a column. If desired, an additional second order correction can be made. This correc-
OUTPUT DECK
INPUT D E C K SEl X
f
PH
SEI a
AMP1
Figure 5.
tion funrt,ion is illuRtrated in Figure 3 and is obtained from calihation data of methyl ester componentsof from 1O:o to 20:4. Ihch point shown is the mean value of three correction factors determined ovcr the usual mam rtlnge (1 to 50 pg.) cncountered for each of these components. The first correction interval starts a t zero time and the laat one (which includes 20:4) ends a t D9.9 (stearate time units). Additional calibration data, particularly of componenta of the CS series, will allow further refinement in this correction function. Thus, this second function correcta primarily for different detector responses to each methyl ester chromatographic component. A typical gas-liquid chromatogram is shown in Figure 4. This methyl ester sample is a total serum p h w pholipide fraction from a 21-yearsld normal female. Detaila of GLC tachnique and apparatus used for all gas
1
PH
AMP1
MI
Rtll
MIX M I %
MI
Input and output deck for ret No. 1406 VOL 33, NO. 6, MAY 1961
661
set
I
1406 1406 1406 1406 1406
1406 1406 1406 1406 1406 1406 1406 1406 1406
t
PH
2.11 9.07
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9.65
7.14
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21.30
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24.93
1406 1406 1406 1406 1406 1406 1406
29.35 33.33 37.09 41.81 47040 51001
57.27 66.94 74.88 78666
07
REL 1
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001
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87
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018
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20432 2.761 30073 3.464
.
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.
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.
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.
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Figure 6.
ANAlYnCAL CHEMISTRY
b
.
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33 2.54 8.78 52 087 25 33 33 25
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.
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12.92 79 1.22 35 47 47 35
46 2.08 3.24 140.32
IBM printout sheet for gas chromatographic set No. 1406
chromatographic work presented here are described in detail elsewhere (3). Liquid phase is polydiethylene glycol, succinate. Column temperature is 195" C., argon carrier gas flow rate is 80 ml. per minute, and detector temperature approximately 195' C. For this run two recorders are used, one operating continuously a t lox, and the other at 1X until the 20:4 component has been resolved and thereafter at 33.3X. Chart speed is 15 inchea per hour and retention time for methyl stearate is 12.07 minutes, as measured from the negative deflection of the solvent front. The actual manual work necessary to read a chromatogram such as run 1406 (shown in Figure 4) consists in placing an appropriate base line on each chromatogram, noting the recorder input amplification factor used, and measuring the retention time and peak height for each component. The base line, obtained from a solvent injection of the same volume, recorded at the same amplification is conveniently traced on the chromatogram over a translucent fluorescent light box. These input data are transcribed to standard 80-column 7a/* x 31/4 inch IBM cards using a Model 026 card punch machine and a Model 056 verifier. Figure 5 illustrates the usage of each column for both input and output data in the reading procedure of run NO. 1406. For each component, the input data are punched on the columns 1 through 19 of a single IBM card. Columns 1 through 5 contain the chromato668
Ml
lotoo 10.00 10.00 I0.00 1.00 I0.00
026
8.88 10003 12.0713050
1406
1406
6
AMPL
graphic number, 6 to 10 the elution time of the component, 11 to 14 the peak height, and 16 to 19 the amplification factor with reference to a 1-volt signal giving a 10-inch (full-scale) recorder deflection. Before the computer operation, each input deck of IBM cards is arranged numerically by set (run) number and each card within a deck is arranged in sequence by retention time. In this order, the cards are ready for the program. Following computation there results for each input card a corresponding output card which hea in addition to the input data the following: first order corrected mass, retention time (relative to methyl stearate), first order masa per cent, second order mass per cent, and second order corrected mass. A final output card contains the set number as well as the sums of both the first and second order masses. Although an IBM 650 electronic computer is used here, any standard digital computer may be used for this operation with slight revisions in the actual program details. The programmed computations p r e ceed in the following manner for each set. First, the peak height is divided by the amplification factor. Then this normalized peak height is multiplied by an appropriate factor determined from the first correction function (Figure 2). This corrected peak height is multiplied by the elution time. The resulting product is stored and as each card is processed the first order component mass is summed. Also, the retention time of each component is
divided by the stearate retention time which is identified by an X-punched card. The first order calculation is completed by dividing each component mass by the total mass of each set giving a first order mass per cent value for each component. The accuracy of this first order determination of the maases of each principal chromatographic component resolved and ita mass per cent of the total maas resolved is approximately 5 to 10%. This accuracy is limited primarily because of ditrerences in response to dfierent fatty acids by our beta particle ionization detector. For some applications a second order correction may be desired or needed. The program for the second order correction multiplies each first order component mass by a correction factor based upon relative elution time. Thus, this function (see Figure 3) essentially corrects for differences in detector response to different fatty acid methyl esters. This is accomplished by a table look-up program similar to the one used to determine the appropriate factors for a given normalieed peak height (used in the first order correction). Finally, after the masa of each component has been corrected and the total mass for each set has been summed, the second order masa per cent value for each component is calculated. For set 1406, Figure 6 shows both the first and second order corrected output data. To obtain this type of data p r i n t out each output deck (obtained from the computer) is processed through a
Model 407 printer. The total computer time necessary t o process a single chromatographic run consisting of an input deck of 28 IBM cards (such as set 1406) is approximately 30 seconds. Table I shows the results of both these first and second order corrections for a standard calibration mixture over the sample maas range of 0.9 to 232 p g . Although quantitation of trace components ae well as inadequately separated components still presents difficulties, all principal GLC components are quantitated to within appro$mately 6%. To attempt further cofrections would probably not yield additional accuracy since resistors used for different amplifications, recorder response, and over-all calibration are not easily controlled much beyond this level of accuracy. Also, the elide-wire width of the recorder imposes an ultimate limitation on accuracy. However, the relative accuracy of comparing the composition of two methyl ester eamples is somewhat greater, approximately 2% for the principal components. DISCUSSION AND CONCLUSIONS
The principal advantage of this punched card technique is that it takes most of the manual drudgery out of chromatographic data proccssing. However, base line pLzcement and
limited manual work are still required. Although further automation in the reading of gasliquid chromatograms is possible, certain considerations a t this time do not favor such extension of this technique. For instance, R principal difficulty in determining the neceseary input data is that of placing an appropriate base line on each chromatogram. This is particularly true of the early base line contour if solvent injection is employed. Judgment and experience are necessary to do this because uncontrollable drifts in the base line frequently occur. Reasons for these drifts include amplifier instability, intermittent gas leaks, alterations in gas flow rates, and liquid phase bleed, as well as temperature fluctuations of apparatus components. Most of these factors operate independently of each other; however, factors such as temperature and liquid phase bleed are closely related. For analysis of gas-liquid chromatograms in general, as well as the specific determinations of the input data for our punched card reading technique, the use of blank chart paper is suggested Experience has shown that graphed chart paper has several disadvantages. First, the critical placement of the base line on the chromatogram is frequently made difficult by the presence of adjacent horizontal lines. Similar dif-.
ficulty is rncountercd when measuring cach peak height because both peak extremity and base line may be close to distracting horizontal lines. Also, the vertical lines tend to interfere with the mcasurcment of the retention time of each chromatograph component. On the other hand, a definite advantage in using blank chart paper is that the actual chromatograms may be photographed easily for reproduction. ACKNOWLEDGMENT
The authors thank the editors of the American Journal of Clinical Nutrition for permission to reproduce Figure 4, from “Fatty Acid Distributions in Serum Lipoproteins,” by F. T. Lindgren, A. V. Nichols, and R. D. Wills [Am. J . Clin. Nutrition 9, 13 (1961)l. LITERATURE CITED
James, A. T., Martin, A. J. P., Biochm. J . 50, 679 (1952). (2) James, A. T., Wheatly, V. R., Zbid., (1)
63, 269 (1956). (3) U ham, F. T., Lindgren, F. T., ~IcEols,A. v.,Univereity of California
Lawrence Radiation Laboratory Rept.
No.9039 (1960).
RECEIVED for review November 9, 1960. Accepted Februa 6 1961. Work SUP ported in part b %arch Grant H-1882 (C5) from the Xational Heart Institute, Public Health &Mace,Bethesda, Md., and b the U. S. Atomic Energy Commission, daahington, D. C.
Gas-Liquid Chromatography of Amino Acid Derivatives DONALD E. JOHNSON, SARA 10 SCOTT, and ALTON MEISTER Deportment of Biochemistry, Tufts University School of Medicine, Boston, Mass.
b Gas-liquid chromatography of a number of N-acetylamino acid n-amylesters has been carried out on 2- to 8-foot columns packed with Chromosorb W coated with 0.5 to 5% polyethylene glycol. A standard procedure for preparation of the derivatives, and columns satisfactory for the separation of 35 amino acids including 1 8 of the common protein amino acids, have been developed. Preparation of the derivatives and chromatography take about 2 hours, and quantities of amino acid of the order of lo-’@ mole can be detected. Preliminary quantitative studies indicate a relatively high conversion of amino acid to derivative.
M
of the recent advances in the chemistry and biochemistry of amino acids, peptides, and proteins may be attributed to the development of chromatographic procedures. Tne elegant quantitative ion exchange proANY
cedures of Moore and Stein (6),now available on an automatic basis (7), have been of particular value, and make possible a complete amino acid analysis of a mixture containing approximately lo-’ mole of each amino acid within 18 to 24 hours. The present work was undertaken in an effort to evaluate the applicability of gas-liquid chromatography to amino acid analysis. If analyses could be carried out on very small amounts of amino acids, studies on proteins available only in small quantity would be possible. Gas chromatography might be useful as an adjunct to paper and ion exchange chromatography. Furthermore, if analysis time could be decreased appreciably, investigations requiring a large number of analyses might become experimentally feasible. Several previous attempts to employ gas-liquid chromatography for amino acids have been recorded in the literature. Hunter, Dimick, and Corse (3)
chromatographed the aldehydes derived from leucine, valine, and isoleucine by reaction with ninhydrin, and Zlatkis, Oro, and Kimball (IO) extended this approach to four additional aliphatic amino acids (norleucine, norvaline, a-aminobutyric acid, and alanine). Bier and Teitelbaum (2) explored the usefulness of several amines obtained from amino acids by decarboxylation. Bayer, Reuther, and Born ( 1 ) were successful in separating the methyl esters of several amino acids by gas phasa chromatography. Youngs (9) chromatographed the N-acetyl-n-butyl estera of glycine, alanine, valine, leucine, isoleucine, and proline on a column of firebrick coated with hydrogenated vegetable oil; although leucine and isoleucine were not separated, quantitative analyses of hydrolyzates of gelatir! f G r the other four amino acids agreec! closely with data reported previousIg in the literature. Earlier, Weygand an< Geiger (8) had prepared several KVOL 33, NO. 6. MAY 1961
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