condit’ions employed below. This system was chosen to best t,his method. The reaction involves the oxidation of the alcohols in the presence of the coenzyme. nicotinamide adenine dinucleotide (S.\D) : R--CHAOH
I) + NAD e R-CHO + S.iDH2 h
13
(7)
The reactions were run in a pyrophosphate-phosphate buffer, p H = 8.8, and the enzyme solutions were made up and their activity determined by the method of 1-allee and Hoch ( 1 1 ) . The courhe of the reaction was followed by recording the rate of increase of adsorbcnce of S;\DH2 a t 340 mp with a Berkman Xodel 1113 recording spectropho;ometer which was thermostated a t 26.1” C. A-lllsolutions were thermostated a t this temperature prior to mixing. The enzyme solution was rapidly added to the alcohol mixture with a microliter pipet which was forceemptied with a syringe. *is a binary mixture was determined, it was necessary t o measure the initial rates of reaction under two different conditions: solution of 1.25 X 10-b.ll K h D and 8.8 X mg.!ml. of A D H ; and 0.75 x ~ O - ~S.411 J I and 4.4 x mg./ml. of .IDH. Some typical results of the determinations are shown in Table I. Ailthough the accuracy is not, exceptionally good as t,he experimental
procedure was crude [automated methods are generally required to yield accurate results when enzyme-catalyzed reactions are employed ( I ) ] , it is sufficient to demonstrate that the method is valid. The application of this method for the determination of other alcohol mixtures, a-hydroxy acid mixtures [using lactic dehydrogenase (9) 1, and amide-ester mixtures [using trypsin (9)], is being investigated. Also, the optimum conditions for accurate analysis, such as optimum ratios of initial rates of the individual components and optimum values of G and G’ for each component is also being studied and will be reported in detail in the near future. LITERATURE CITED
( 1 ) Blaedel, W. J., Hicks, G. P., “Ad-
vances in Analytical Chemistry and Instrumentation,” C. S . Reilley, ed., Yol. 3, Interscience, New York, in press. ( 2 ) Briggs, G. E., Haldane, J. B., Biochem. J . 19, 338 (1925). ( 3 ) Devlin, T. hl., ANAL.CHEM.31, 977 (1959). ( 4 j Dixon, M., Webb, E. C., “Enzymes,” Academic Press, Kew York, 1960. ( 5 ) Garmon, R. G., Reilley, C. N., ASAL. CHEM.34,600 (1962). ( 6 ) Mark. H. B. Jr., Papa, L. J., Abstracts of 144th Meeting, ACS, Los Angeles, Calif., April 1963, p. 24B. ( 7 ) Michaelis, L., Menton, M. L., Biochem. 2. 49, 333 (1913).
Table I. Determination of Ethanol” in Eth anol-n-Propanol Mixtures
(Alcohol dehydrogenase catalyzed reaction with nicotinamide adenine dinucleotide. Temperature, 26.1” C. Total alcohol concn., 5 X I O - 3 M ) NO. Ethanol in mixtures, % detns. Present Found 2 2 5 2
16 36 50 80
24, 22 31, 35 57-49 73, 77
% n-Propanol can be determined by subtracting yc ethanol found from 1 0 0 ~ o . ( 8 ) Neilands, J. B., “Organic Analysis,” J. Mitchell, ed., Vo1. 4, p. 65, Interscience, Sew York, 1960. ( 9 ) Xeilands, J. B., Stanier, R. Y.,
“Enzyme Chemistry,” 2nd ed., pp. 185-7, Wiley, K e a York, 1958. (10) Papa, L. J., Mark, H. B., Jr., Reilley, C. N., ANAL.CHEM.34, 1443 (1962). ( 1 1 ) Yallee, B. L., Hoch, F. L., Proc. Natl. Acad. Scz. 41, 327 (1955).
H ~ R RB. Y MARK,JR. Department of Chemistry The University of Michigan Ann Arbor, Mich. RECEIVEDfor review March 10, 1964. Accepted April 13, 1964. Acknowledgment is made to the donors of the Petroleum Fund, administered by the ACS, for partial support of this work. Analytical Division, 147th Meeting, ACS, Philadelphia, Pa., April 1964.
Determination of Argon and Oxygen by Gas Chro matogrcphy SIR: To date, there are no rapid and simple methods for the determination of en and argon by gas chromatography. Similar retention times are exhibited by these two gases on all adsorbents at room temperature or above, and t,heir separation can be effwted only under extreme conditions. Vizard and Wynne ( 7 ) reported a n and argon on a 10-meter column of IIolecular Sieve 5A\ oprrated a t room t,emperature, using hyclrogcn carrier; with such a long colunin, 1 owever, retention times were excessively long and sensitivity was Ion.. Lard and Horn ( 3 ) were able to separate oxygen and argon on a 6-foot column of hIolecular Sieve 5 h operated a t loiv temperature ( - i 2 ” C.), using helium carrier gas; for air analyses, however, a second run a t room temperature was necessary to determine nitrogen, since this gas is irreversibly absorbed a t loiv temperatures. Other methods ( I , 6) have been reported in
which oxygen was used as carrier for argon, and argon as carrier for oxygen; sensitivity was very 1)oor, however, due to the similar thermal conductivities of carrier gas and unknown. Krejci, Tesarik, and Janak ( 2 ) have described a procedure employing hydrogen carrier, in which oxygen was converted to water by passage over a palladium catalyst and subsequently removed. This procedure requires two separate runs, one for the purpose of determining the combined 02-Ar peak before separation, the second for the measurement of Xr alone after the oxygen has been removed. An application of this technique has been briefly described in a paper concerned with the determination of dissolved gases in aqueous solutions ( 4 ) . The method described in this report for the separation and determination of oxygen and argon is a modification of the procedure of Krejci. Hydrogen carrier gas, in the presence of a palladium catalyst a t
room temperature, is used to quantitatively convert oxygen to water vapor. The gas mixture is then separated by the usual chromatographic techniques, and each component is independently measured. Only one run is thus required, and the necessity for determining one of the components by difference is eliminated. A block diagram of the system used is shown in Figure 1. The DynatronicChrom-Alyzer-100 employs two hotwire thermal conductivity detectors, the second detector normally being used as a reference detector. I n this work, however, the reference detector was employed as a second measuring detector. A constant current poner supply was used to provide detector cell currents of 350 ma. Hydrogen was used as carrier gas a t a flow rate of 65 cc. per minute. The gas mixture was first passed through a catalyzer, consisting of a 1-inch length of ’/,-inch aluminum tubing filled with 30-60 mesh palladium VOL. 36, NO. 8, JULY 1964
1669
-
EXXAUSP
U
CHROM-AL YZER-IO0
P E R K f N EL MER SAMPLING VALVE OR LIQUID STRIPPING
METER
CHAMBER.' MOL EGUL AR
P A L L AOlUM COLUMN
SIEVE 5A
ON TEFLON CONDUCTIVITY DETECTORS
uTHERMAL
Figure 1.
Block diagram of system
metal. Directly coupled to the catalyzer was a 16-inch ltngth of 3,'16inch Teflon tubing fi1lt.d wit,h 504 Triton X-305 on 30-60 mesh Teflon support' (column number 1), which serves to separate the water vapor from the argon. .1 column temperature of 55' C. was found to give efficient separation. For the analysis of air, a second column is required for the purpose of separating nitrogen and argon. The column used for this purpose consist'ed of 12 feet' of Molecular lot
1
(02)
Response vs. composition of oxygen-argon mix-
Somple size, 250 PI. Chart speed, 2 inches per minute. Peak areas normalized to same instrumental sensitivity range of 1 X. O x y g e n p e a k a r e a actually due to equivalent amount of water vapor
0
Sieve 5-1 followed by 18 t o 20 feet, of inert buffer. The latter was found necessary to allow t,ime for the composite nitrogen-argon peak and the water peak to emerge from the first, detector before the separated nitrogen and argon peaks entered the second detector. The air samples were injected with a standard Perkin-Elmer rotating valve, or with a modified l'erkin-Elmer valve and a glass stripping chamber previously described for the analysis of dissolved gases in solution
v)
(6).
WATER I
N2
116% 5c
l
w
a:
w
>
I
2
3
4
5
TIME (MINUTES) Figure 2. G a s chromatogram obtained on 250-pl. air sample Flow rate 65 cc. per minute, recorder chart speed inch per minute
1670
Figure 3. tures
-TT
I!
C
V O L U M E % OF G A S IN M I X T U R E
ANALYTICAL CHEMISTRY
Figure 2 shows the results obtained when a 250-microliter air sample was analyzed. The composite peak which is first recorded consists of argon and nitrogen which pass t h o u g h column 1 without separation. The water vapor from the catalyzer is separat'ed from the composite on column 1, and follows the composite to be recorded as the second peak. The composite mixture is then resolved in column 2 (Illolecular Sieve 5-1), and the separated gases emerge and are recorded as argon and nitrogen in that order. The wat,er is permanently absorbed by the drying column immediately preceding column 2. It
should be noted that the recorder polarity is switched immediately aft'er the water peak emerges, otherwise the .1r and K2 peaks would be negative. Time required for a complete analysis of the air sample is 5 minutes. To determine that there is a linear relationship between the amount of oxygen in the sample and the measured water peaks produced on the chromatogram, several carefully prepared mixtures of oxygen and argon ranging from looyo O2 to 1OOOjc h r were employed. Each mixture was introduced into the chromatograph by a Perkin-Elmer valve fitted with a 250-microliter sample loop. The results are shown in Figure 3, which also includes the data obtained for argon. Each point represents an average value of 6 replicate samples. The data clearly show a linear response for both gages, and under the conditions of these experiments give calibration factors of 0.36 and 0.50 microliter per. sq. em. of peak area for oxygen and argon, respectively, corresponding to an absolute sensitivity of approximately 10-8 mole for each gas. The accuracy obt'ainable by this method was determined by a series
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 A N D 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 1,400 0,860 0,635 0.490 1 ,700 1.075 0.770
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 5 100 10 0 835 103 15 0 605 105 20 0 450 109 5 1 700 0.50 100 1 050 10 102 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.
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