limits for duplicate samples a t the 99% confidence level are found by the formulaz i -% to b e 3 =t0.23. N is
d3
the number of samples = 2, t is obtained from the t distribution tables a t the 99% confidence level and at 68 degrees of freedom, and s = 0.12. CONCLUSIONS
I n the range 93 t o 100% anhydride, neither the accuracy nor the precision of the method is dependent on the anhydride concentration. Also, in this range, the over-all standard deviation for the method is 0.16 with 16 degrees
of freedom and there is no significant difference in results between 2- or 8hour warm-up times. The instrument calibration was found not to change linearly over a 3-week period. The precisions of the standard aniline, triethylamine, and spectrophotometric methods are compared and the spectrophotometric method with a standard deviation of 0.16 is found to be more precise than the other two. LITERATURE CITED
(1) Davies, 0. L., “Design and Analysis of Industrial Experiments,” Chap. 7 ,
Hafner, New York, 1956.
(2) Dixon, W. J., Massey, F. J., “Intro-
duction to Statistical Analysis,” McGraw-Hill, New York, 1951. (3) Goddu, R. F., Le Blanc, N. F., Wright, C. M., ANAL.CHEM.27, 1251 (1955). (4) Holman, R. T., Edmondson, P. R., Zbid.. 28. 1533 f 1956). (5) Lihig: F. J:, Mandel, J., Peterson, J. M., Zbid., 1102 (1954). (6) McClure, J. H., Roder, T. M., Kinsey, R. H., Zbid., 27, 1599 (1955). (71 Nicolas. L.. Burel, R., Chim. anal. 33,
RECEIVEDfor review March 17, 1959. Accepted November 16, 1959. Work presented a t the Southeastern Regional Meeting, ACS, Gainesville, Fla., December 1958.
Direct Amino Acid Analysis by Gas Chromatography ALBERT ZLATKIS, JOHN F. ORO, and A. P. KIMBALL Deportment of Chemistry, University of Houston, Houston, l e x .
b Mixtures of amino acids that yield volatile aldehydes can b e rapidly analyzed by injecting a small sample of an aqueous solution of amino acids into a specially designed reactor-gas chromatographic unit. The amino acids are oxidized to aldehydes in a microreactor which is a part of a continuous flow system. The aldehydes a r e chromatographically separated, catalytically cracked, and analyzed in a thermal conductivity cell. The complete analysis of a mixture of seven amino acids can b e performed in less than 1 hour.
T
utilization of procedures whereby amino acids may be converted to volatile compounds offers a convenient method for their analysis by gas-liquid chromatography (14). Hunter, Dimick, and Corse (8) converted leucine, isoleucine, and valine into the corresponding aldehydes by oxidation with ninhydrin. These were collected in cold traps and then sampled for introduction into the gas chromatographic apparatus in a closed system. Bayer, Reuther, and Born (I, 2) esterified several amino acids with methanolic hydrochloric acid, extracted the esters with ether, and then chromatographed the ether extract. Similar techniques based on the chromatography of aldehydes, amines, hydroxy acid esters, and N-acylated amino acid esters have been reported more recently by Bier and Teitelbaum (3, 4,Liberti (8), and Youngs (12). The approach presented in this paper makes use of a special reactor-gas HE
162 *
ANALYTICAL CHEMISTRY
chroniatographic unit. The method is simple and rapid because it requires one single operation. An amino acid solution is injected into a continuously flowing system where oxidation, separation, reduction, drying, and analysis take place in a relatively short time. A heated niicrorcactor containing ninhydrin effects the oxidation of the amino acids to aldehydes. Separation of the latter takes place in a chromatographic column. After leaving the column, the aldehydes are cracked to methane and water over a nickel catalyst. The water is then removed by a drying column and the analysis is made by a thermal conductivity cell. This procedure has been applied to mixtures of amino acids, and in general it may be used for any amino acid which can be oxidized to a volatile aldehyde. APPARATUS AND REAGENTS
A schematic diagram of the system is shown in Figure 1. Reactor A consists of a 6 X l/4 inch glass U-tube containing 30% ninhydrin (Nutritional Biochemicals Corp.) on C-22 firebrick (Johns-Manville Co.) , a graded diatomaceous earth. The tube was heated with a heating tape and the temperature kept a t 140’ C. The chromatographic column, 10 foot X inch copper tubing, contains 30- t o 60-mesh aqua regia-treated C-22 firebrick ( I S ) coated with 10% of an equal mixture of ethylene and propylene carbonates. Reactor B was a 12 X ’/4 inch glass U-tube containing a nickel-kieselguhr catalyst of 30 to 60 mesh. The drying column was a 1 foot X inch glass U-tube filled with a Molecular Sieve of 10 t o 30 mesh.
This reactor-chromatographic train was tied into a Perkin-Elmer Vapor Fractometer, Llodel 154B, through its gas-handling system. PROCEDURE
d ninhydrin-amino acid solution was prepared by mixing 1 part of a saturated aqueous solution of ninhydrin with 1 part of a 0.28Jf solution of amino acids and kept in an ice bath until ready for sampling. Fifteen microliters of this solution were injected into reactor A with a Beckman liquid sampler. Reactors A and B were at 140’ and 4 2 5 O C., respectively; the detector cell, chromatographic column, and drying column were at 25’ C. Hydrogen mas used as the carrier gas at a flow rate of 100 ml. per minute. The analysis of amino acids by the reactor-gas chromatographic technique takes place in a flowing stream through a series of steps.
Oxidation of Amino Acids. The amino acids are oxidized with ninhydrin t o volatile aldehydes and carbon dioxide. T o ensure a complete reaction, a n excess of ninhydrin is used by keeping the reagent in the injecting solution and having the firebrick coated with 30% ninhydrin. A temperature of 140’ C. in reactor A ensures a n almost instantaneous oxidation. Lower temperatures generally result in incomplete reactions. The ninhydrin-firebrick material in reactor A is effective for about five sample injections. Mixtures of amino acids such as alanine, a-amino-*butyric acid, valine, norvaline, leucine, isoleucine, and nor-
I
I
cc2
34,
5u
LO
30
20
T , U E . VIVII'ES
Figure 2. acids
I
Chromatogram of a mixture of seven amino
I I
'
-
Figure 1. Schematic diagram of reactor-gas graphic unit
chromato-
the amino acids by oxidative decarboxylation. The water which is present in the amino acid solutions progresses very slowly through the chromatographic column and does not interfere in the analysis. Reduction and Hydrocracking. As each aldehyde emerges from the chromatographic column, i t passes into reactor B over a bed of nickelkieselguhr catalyst a t 425' C., where it is cracked and reduced t o methane and water. The purpose of reactor B is twofold. Primarily, i t solves the problem created by the presence of water in the amino acids solutions or any aqueous solution to be analyzed by gas chromatography. As there is no convenient way to dry aldehydes following a chromatographic separation, a new approach obviated this difficulty by catalytically hydrocracking the aldehydes to methane and water and then drying the methane (16): catalyst_*
leucine which yield volatile aldehydes upon oxidation with ninhydrin have been analyzed by this method. The analysis of other amino acids which also yield volatile aldehydes such as glycine, phenylalanine, and methionine has not been completely investigated. Glycine gives rise to formaldehyde, which polymerizes readily under the conditions used (6). The aldehydes obtained from phenylalanine and methionine have a low volatility ( 7 ) and their analysis would require higher operating temperatures and special stationary phases such as the one used for the separation of methional (11). Chromatographic Separation. The aldehydes produced in reactor A flow into the chromatographic column, where they are separated. The selection of the packing material, flow rate, and temperature ensures efficient resolution in a relatively short time. I n particular, the isomeric aldehydes 3-methylbutanal and z-methylbutanal, corresponding to leucine and isoleucine, respectively, were resolved in 33 minutes. An additional advantage of the use of the ethylene carbonate-propylene carbonate as a stationary phase is that acetaldehyde is completely resolved from the carbon dioxide derived from
CnHznilCHO
+ ( a + 2)Hz 425". (n+i)CH4 + H2O
Secondly, it increases the sensitivity of the method, as only methane is being detected by the thermal conductivity _______
Table 1.
~~
_ _ _ _ _ _ _ ~ -
cell, and the cell may be operated a t room temperature where it is more sensitive. Furthermore, no calibrations are necessary for the individual aldehydes because of differences in their thermal conductivities. However, the carbon factor must be observed in final calculations, because different aldehydes produce quantities of methane in accordance with their carbon number as shom-n in the above equation. The carbon dioxide produced in the oxidation reactor is converted to methane and mater when it passes through reactor B. It appears as the first peak on chromatograms involving amino acids. The nickel catalyst of reactor B shows no signs of losing its activity after having been subjected to over 50 samples. At low temperatures of the catalyst (300" C.) considerable tailing of the chromatogram peaks is evident. Drying. Because a relatively large amount of water is present from both the original solution as well as the hydrocracking step, it is necessary to desiccate the methane as it flows to the detector. A drying agent such as Drierite, or better yet, a Molecular Sieve is used. By using a Molecular Sieve, i t is possible to show t h a t all the carbon dioxide had been converted to methane in the hydrocracking reactor as carbon dioside is irreversibly adsorbed by the Molecular Sieve while methane passes through. The drying column must be replaced after five runs
~ ~ _ _ _ _ _ ~ _ _ _ _ _ _ _
~~
Analysis of Known Mixtures of Amino Acids Ifmoles per Liter
Run Amino Acid Mixture A Alanine a-Amino-n-butyric acid Valine X'orvaline Leucine Isoleucine Sorleucine
Total hfixture B Alanine Norvaline Total Alanine normalized.
Present
1
2
3
40 40 41 40 41 41 40
40 40 43 40 42 42 -. 35 282
40 39 40 42 39 41 40 281
40 42 41 42 39 40 39 283
283 55 45
100
55
47 102
Found"
5
6
40 39 41 41 42 41 37 281
40 40 41
40 39 43 40 43 39 36 280
40 40 42 40 41 41 37 281
...
55 f 0 45 f 1 100 f 1
-
-
-
55
46 101
55
44 99
45 100
Average f std. dev.
4
55
37
43 43 35 279
...
... ...
... -
...
VOL. 32, NO. 2, FEBRUARY 1960
f0 f1 f1 =t 2
f2
f L =t 2
f1
163
RESULTS AND DISCUSSION
The reactor-gas chromatographic technique was applied to known mixtures of amino acids (Figure 2). The amino acids m-ere qualitatively identified by the retention time of the corresponding aldehydes. Knonn mixtures of aldehydes, passed through the system under identical conditions, gave analogous chromatograms. The quantitative determination of the aniino acids is based on measurement of tlic peak areas, correction for the number of carbons of each aldehyde, and normalization. Becauw each peak is really due to methane, no calibrations are necessary. Individual aldehydes passed through the system give peak areas which are a linear function of their concentration. The results of the analysis of tTo synthetic mixtures of nniino acids, are illustrated in Table I. The error for the values of each amino acid, with the exception of norleucine, is less than 5%. This is of the same order of magnitude as the error involved in measuring the areas of the respective peaks by the weight method. Also, when the values for total amino acid are considered, the error becomes even smaller, suggesting a cancellation of experimental random errors. To demonstrate that the amino acids had reacted completely with ninhydrin, 15 pl. of water or a saturated aqueous ninhydrin solution mere injected into the reactor after each amino acid run. The chromatograms showed no evidence of unreacted amino acids a t the operating temperature of 140" C. At lower temperatures (100' C.) small amounts of amino acids remained unreacted. -In analysis of a casein hydrolyzate
solution by this method showed four main peaks on the chromatogram corresponding to alanine, valine, leucine, and isoleucine in the approximate relative composition to be expected from casein. The other amin? acids present in the misture did not' produce any other det'ectnble yolatile products with the exception of carlion dioxide. The carbon dioxide pcnk. although considerably enlarged. n'ns sufficiently separated from the alanine p : i k to nllon- the measurement of this amino acid. As the amino acid norvaline (also norleucine and a-amino-n-butyric acid) is not normally present in proteins, it should be possible to use it as a marker for the analysis of t'he other amino acids. This n-odd involve diluting a small amount of norvaline solution with the protein liydrolyzat,e solution and injecting the mixture into the chromatographic system. Knowing the exact molarity of the norvaline solution and the dilution factor, the absolute concentration of each one of t'he aliphatic amino acids present' in a protein hydrolyzate could be calculated. This has the added adi-antage that there is no need t'o measure the sample size of the mixture of amino acids to be injected if t'he response of the norvaline solution has been carefully determined beforehand. One microgram of an amino acid can be det'ected using this technique. With the application of nen- clet'ection systems (9, 10): it should be possible to increase the senaiti\-it>-so that as little as 0.001 y TTill be detected. The approach presented here is direct, sensitive, and rapid for analyzing amino acids which produce volatile aldehydes on oxitlation with ninhydrin. I n its
present form the method can be useful in certain specific applications, such as the analysis of the per cent composition of strictly aliphatic amino acids of proteins, and determination of the leucineisoleucine ratios of certain proteins or peptides. The method can also bc important in the confirmation of results obtained by radio tracer techniqucs on the incorporation of analogs of certain aliphatic amino acids into proteins. LITERATURE CITED
(1) B a y , E., "Gas Chromatography 1958, D. H. Desty, ed., p. 333, Academic Press, S e w York, 1958. (2) Bayer, E., Reuther, K. H., Born, F., Angezo. Chem. 69, 640 (1957). (3) Bier, &I., Teitelbaum, P., Ann. K.Y . Acad. Sci. 72. 641 11959). 14) . . Teitelbhum, 'P., Federation (4) Bier. Bier, M M., Proc. ' h o c . 17, 191 (1958). (5) Bradford, B. IT., Harvey, D., Chalkley, D. E., J . Znst. Petrol. 41, fin ioE5) 80 ((1955). (6) Hunter, I. R., Dimick, K. P., Corse, (6)"IJ. \TI.. C h e w & Ind. (London) 1956.294. ( 7 ) Huiiter, I. R., Potter, E.' F., ANAL. CHEM. 30, 293 (1958). (8) Liberti, A., "Gas Chromatography 1958,'' D. H. Desty, ed., p. 341, Academic Press, Kew York, 1958. (9) Lovelock, J. E., J . Chromatog. 1, 35 (1958). (10) McSVilliam, I. G., Dewar, R. -4., Nature 181, 760 (1958). (11) Or6, J., Guidry, C. L., Zlatkis, h.,Food Research 24, 240 (1959). (1'21 YoUll@, c. G., A N A L . CHEhf. 31, 1019 (1059). (13) Zlntkis, A , , Ling, S., Knufman, H. R., Ibid., 31, 945 (1959). (14) Zlatkis, .I,,Orb, J., Ibid., 30, 1156 (1958). (15) Zlatkis, A,, Ridgway, J. A,, Nature 182, 130 (1958). RECEIVEDfor review July 13, 1959. Accepted October 30, 1959. Work supported in part with a grant from the Robert .I,Kelch Foundation to J.F.O.
Gas- Liq uid Chroma t o g ra phy of Pyr id ines Using a New Solid Support ANDREW W. DECORA and GERALD
U. DINNEEN
Laramie Petroleum Research Center, Bureau of Mines, U.lS. Department of the Inferior, lararnie, Wyo.
b A solid support has been developed for the gas-liquid chromatography of pyridines, superior to Chromosorb or Celite 545; symmetric peaks were obtained when nonpolar substrates were used. The solid support was prepared from a commercial detergent by heating it and then extracting it with petroleum ether. The porous residue was used as a liquid substrate carrier. The new solid support was used to study the selectivity of several nonpolar and slightly polar liquid 164
ANALYTICAL CHEMISTRY
substrates. Decided differences in the selectivities of the liquid substrates permit a wide choice in the use of these substrates for separations of pyridines. The study on the selectivity of the liquid substrates was used to select two columns employed in series to separate a test mixture of 14 pyridines.
0
phase of the Bureau of Mines investigation on the characterization of shale oil is determination of the ?;E
pyridines produced in retorting oil shale. Attempts to apply conventional gas-liquid chromatographic techniques using Chromosorb and Celite 545 to the analysis of shale-oil tar bases were hampered by asymmetry of the peaks obtained. Symmetric peaks are obtained when strongly polar liquid substrates are used on either Chromosorb or Celite. However, a solid support which gives symmetric peaks only when strongly polar
