of air analyses. Twenty replicate samples (250 microliters each) gave a n average value of 21.1y0 for oxygen, (thLoretica1 20.9%) and o.94yO for argon (theoretical 0.9301,). These values were calculated by multiplying measured peak areas by the calibration factors above. The standard error of the average for this series of samples was O.5y0;using Student's t-test as a criterion, the differences between experimental and theoretical values for both gases were not significant at the 95% level. This method has a decided advantage over existing methods in that it is rapid, requires only a single sample run,
and shows a high degree of sensitivity and accuracy. I t should be useful in the analysis of respiratory gases, where the oxygen-argon ratio does not remain constant. Similarly, in the determination of dissolved air in polluted waters the method should be proved useful, since the oxygen-argon ratio may vary considerably in such media.
p. 255, Academic Press, New York,
1961. (3) Lard, E. W., Horn, R. C . , ANAL. CHEM.32. 878 f 1960). (4) Swinnerton,-J. W,'Linnenborn, V. J., Cheek, C. H., Ibzd., 34, 483 (1962). ( 5 ) Ibid., p. 1509. (6) Szonntag, E. L., Steward, J. R., Symposium on Gas Chromatography, Third Delaware Regional Meeting, ACS, February 1960. (7) Vizard, G. S., Wynne, A,, Chem. Ind. (London) 1959, 196.
LITERATURE CITED
(1) Greene, S. A., Advances in Gas Chromatography, ACS Symposium, New York. Seotember 1957. D. D 105. (2) Krejci,&M., Tesarik; 'K., F a k , J., in "Gas Chromatography, H. J. Noebels, R. F. Wall, N. Brenner, eds.,
J. W. SWINNERTON V. J. LINNENBOM C. H. CHEEK U. S. Naval Research Laboratory Radiation Division Washington, D. C. 20390
Taurine as Reference Standard in Chromatography of Plant Material Extracts SIR: Protein hydrolyzates and plant materials containing aspartic acid are conveniently analyzed for amino acids with fraction collector column chromatography. Aspartic acid provides an easily identifiable reference position for determining buffer change and comparison with the standard curve. Certain free amino acid extracts of abnormal or otherwise depleted extracts may not contain this built in reference point. The addition of aspartic acid routinely to all samples would solve the problem only when it was certainly absent in the test estract. I t would be desirable to use an auxiliary reference amino acid for analysis of extracts from virus infected or growth regulator treated plants for example. The added reference materials should be normally foreign to plants, react predictably with ninhydrin, and preferably elute early in the run of the first buffer. EXPERIMENTAL
The improved Moore, Spackman, and Stein ( 5 ) fraction collector column chromatography method for the separation of neutral and acidic amino acids was employed. Analyt'ical runs were made at 50' C. and the change from pH 3.25 to pH 4.25 buffer was made a t 2.15 times the aspartic acid peak. Paper chromatography was conducted on Whatman No. 1 chromatographic grade sheets 18lIa X 22';* inches using 88% phenol :water (4,'l v./v.) adjust3d to p H 5.5 t'o 5.8 with NaOH or with 0.1 yo 8-hydroxyquinoline in 'the long direction '(6) and butanol-acetic acid-water (100/22,'50, v,/v./v.) in the short direction. Single direction determinations were in the latter solvent system. h n a l ais of effluent fractions from the c o u m n was accomplished by a variation of the Rosen method ( 7 ) .
r-
One-half milliliter of cyanide-acetate buffer (0.0002M NaCN in pH 5.3 acetate buffer) and l/z ml. of 3y0 ninhydrin dissolved in peroxide-free, redistilled (119' to 123' C.) methyl cellosolve were added to each 2-ml. fraction. RESULTS AND DISCUSSION
The best quality taurine available from one biological supply house cantained appreciable quantities of another ninhydrin positive material. On paper this impurity had nearly the same position as glycine in one- and two-dimensional runs. Taurine from another source was apparently pure on paper, but eluates from the column showed a small amount of glycine impurity. Since the naturally derived taurine was chromatographically impure, synthetic taurine was made (3, 4). The final product contained 25.50y0 sulfur and 11.47% nitrogen, nearly the same as the theoretical values of 25.62% and ll.19yo, respectively. A sample containing 0.8 p J 1 of taurine was added to the analytical column with several chromatographically pure amino acids in known concentration. Its elution peak was about 70 ml. which is the representative position of the chief component in commercial taurine in relation to aspartic acid a t 113 ml. Decomposition of synthetic taurine in a Gallenkamp Melting Point apparatus (328' C.) was characteristic of purified natural taurine. Usefulness of synthetic taurine as an internal standard depends also upon predictability of its reaction to standard amino acid tests. Synthetic taurine conforms to Beer's law in the Rosen ( 7 ) test a t several concentrations using isopropyl alcohol-water as a diluent. The color yield of several lots of synthetic and natural taurine at 0.1 and
0.2 p M per tube nearly agreed with Rosen's value of 100% of leucine. Leucine equivalents of three times recrystallized synthetic taurine a t 0.10 to 0.40 p M per tube and a t various dilutions fell between 98 and 109% in these determinations (Table I). Ethyl alcohol was substituted for isopropyl
Table 1.
Leucine Equivalents of Synthetic Taurine
Amino acid Der Dilut;be,o ent, ml. P M 0.10 5 10 0.15 5
Syn. taurine O.D., meanb 0,390 0.245 0.560 0.335 0.715 0.420 0.920 0.570 1.175 0.670 1.300 0.740 0.540 0.410
Equiv. of
Leucine leuO.D., cine, mean 70 0 385 101 106 0 230 0 535 105 10 0 310 108 0.20 100 0 715 10 0 420 100 5 0 885 104 0.25 10 0 530 107 1 150 0.30 5 102 10 0 650 102 1 300 5 0.35 100 10 0 755 98 15c 0 540 100 20 0 420 98 1 400 0.40 5 1,400 100 10 0 835 0,860 103 15 0,635 0 605 105 20 0.490 0 450 109 5 1 700 0.50 1 ,700 100 1.075 1 050 10 102 0.770 15 0 780 99 20 0 555 0.560 99 1 930 0.60 5 1,900 100 1 325 1.325 10 100 0.905 -100 0 905 15 0 715 20 0.700 98 0 2-ml. aliquots per tube before dilution. * Means of 2 readings a t 570 mw. c Higher dilutions used to reiuce 0 . D . below 0.800.
VOL. 36, NO. 8, JULY 1964
1671
alcohol in several additional determinations and gave nearly identical values. The Rosen ( 7 ) ninhydrin reagent is relatively stable. When it is used in the fraction collector method, it greatly reduces the need for checking reagent stability with an internal standard. Taurine used as an internal standard serves principally as a position marker in this method. Beta-2-thienyh~alanine (8),for instance, would not assist in determining buffer change since it (,lutes mitiwny in the second buffer and has less utility in the fract,ion collector method. 130th D-L-ortho phosphoscrine and taurine have some of the desired characteristics; however, the former elutes close to cysteic acid and is morc likrlly to be found in plants than taurine. Taurine elutes in an area where overlapping is not likely to occur (9). The compound is isolated from the muscle of certain shell fish and froin iiiaminalinn wastes. I t appcars to be relatively uncommon in plants. Red algae ( 2 ) and young peas ( I ) reportedly contain taurine. I n the latter case it was claimed t,o be an inter-
mediary in conversion of inorganic sulfur supplied as Xa2S3404. I n our laboratory column chromatography of free amino acid extracts of primary leaves of 10 bo 21-day old plants of Vigna sinensis var. Black and of %day old plants of Piswti sativum var. Alderman revealed no taurine (unpublished). Taurine may thus be eit,herinconspicuous or absent in legumes unless supplied with abundant inorganic sulfur. Contamination of plant extracts with tsurine present in animal origin fertilizers is unlikely with current plant growing practices. Synthesis of taurine is relatively easy and purity of a long term supply can be assured. I t has the desired properties of ninhydrin reaction and mobility stated for the natural product. As a reference point it will not replace aspartic acid or other internal standards for all column chromatography. I t can be helpful, however, to plant pathologists and plant physiologists interested in analysis of certain abnormal plants where a fraction collector system is used.
LITERATURE CITED
(1) Bisnas, B. B., Sen, S. P., Sei. Cult.
(Calcutla) 22, 697 (1957). (2) 'Lindberg, B., Acta Chem. Scand. 9, 1323 (1955). (3) Marvel, C. S., Bailey, C. F., "Organic Synthesis," Tol. 11, p. 558 Wiley, New York, 1943. (4) Marvel, C. S., Sparberg, M. S.,Ibid., p. 563. (5) Moore, S., S ackman, D. H., Stein, W.H., ASAL.$HEM. 30,1185 (1958). (6) Porter, C. A., Margolis, D., Sharp, P., Contrib. Boyce Thompson Insl. 18, 466 (1957). (7) Rosen, H., Arrh. Riochern. Biophys. 6 7 , 10 (1957). (8) Siegel, F. L., Roach, Mary K., ANAL. CHEY.33. 1628 i1961). (9) Zacharius, R. if.,Talley, E. A,, Ibid., 34, 1551 (1962). J. G . KAR.4s H. M. SELL C. L. HAMNER D. J. DEZEELT Departments of Botany and Plant Pathology, Horticulture, and Biochemistry Michigan State University East Lansing, Mich. RESEARCH supported in part by Xational Institutes of Health, Grants E-4581 and AI-04581-02. Published with the approval of the Director, Michigan Agricultural Experiment Station as Journal Article No. 3267.
Qualitative Analysis by Gas Chromatography Choice of a Retention Data System SIR: In a paper by Merritt and Walsh (18, 19), a system of retention data was suggested which they called Isothermal Retention Volume Ratio (IRVR) and Programmed Temperature Retention Volume Differences (PTRVD). In discussing their results and comparing them to existing systems they presented a very short and drastic rebuttal of the retention index (RI) system described by Kovats and Wehrli (14, 24) and completely ignored the Rx,g system described by Evans and Smith ( 7 ) . However, these two systems were the subject of a 1or.g discussion at' the Gas Chromatography Symposium held in 1962 in Hamburg (237, and the first one (RI) is currently used by a great number of workers (1, 6. 8, 10, 12, 13, 15, 16, 23> 26, 26). A lot of papers which appeared recently used ret'ention indices for identification purposes. Kaiser (13) used them to present a lot of retention data in his book. This shows t,hat RJ may be computed from relative retention voluirres if data on n-alkanes are given. Su-oboda (23) discussed the use of RI. Zulaica and Guiochon (25, 26) uscd them to identify diesters used as plasticizers in poly vinyl chloride and showcd that t,he indices of homologous comlioucds increase in a constant amount. of about 100, when one carbon is added to a normal chain. Kovats uses them to identify the numerous components of tangerine essential oil. Baron 1672
ANALYTICAL CHEMISTRY
and Maume ( I ) showed that the use of R I increment or A I-Le., the difference between the R I of a compound on polar and apolar liquid phases-may be used to distinguish radial and equatorial groups in cyclohexane derivatives. Loewenguth (16) used R I to identify C6-Cs olefins without needing the pure compounds: tentative identifications were ascertained by infrared spectromy 011 trapped compounds. I t is ieved that the choice by these authors of the R I system over any other existing system before the IRVR, was made because it is really a good system. I t may first be pointed out that the R I system is not limited to the use of -4piezon L and Emulphor 0. As first suggested by Kovats ( 1 4 ) this system was used with advantage with more than 20 different phases and a lot of retention data given in the literature as retention volumes (absolute or relative) may easily be recomputed as retention indices. I t would be erroneous to think that the R I system is so limited: in this case it would be far less useful. For identification purposes R I increments may be used as long as the R I themselves. These increments are the difference between the R I of one compound on two different, phases, whatever these phases. Of course the two phases used by Kovats and Wehrli are to be preferred because of the amount of data these authors collected and be-
cause the rules they gave to compute the A I a priori may be used easily (24). However these rules are still valid with a lot of other pairs of stationary phases and presumably with almost all possible pairs : the individual increments pertaining to the different adhering zones have only to be computed from previous measurements. The measurement of RI (as that of R,,g factor) may be made in several different ways depending on the complexity of the mixture. In easy cases, as the measurement of R I of pure compounds, it is possible to inject a mixture of the sample and the convenient nalkanes. Then the computation of the R I may be made directly from the logarithm of retention distances of the peaks, measured from the air peak. In more difficult cases it is possible either to inject in successive runs the sample and a mixture of the convenient nalkanes if the apparatus used is sufficiently stable or to allow for variations of retention distances by ,,introducing one n-alkane (or more) in the sample, the choice of the alkane(s) being made to have no interferences between it (them) and any compound of the sample. If no variation in the retention distance of this (or these) n-alkane is meawred, the computation of all the RI may be made from the logarithm of retention distances. In the other case retention volumes must be computed
