ANALYTICAL CHEMISTRY
732
Table \-. Comparison of Several Commercial Silicic Acids as Adsorbent for Chromatographic Separation of 2,4-Dinitrophenylamino Acids -
Groups
11. 111, I V
Adsorbent Rlerck 40446‘‘ Rlerck 40665” RIerck 43243‘ Rferck 42316 hfallinckrodt XXE-1 e Mallinckrodt X M H - I Rlallinckrodt XXG-1 llallinckrodt XAP hlallinckrodt R C T - l a Baker A-28ga City Chemical” General Chemicala
2:lh 4:1
2:l 2:1 4:l 2: 1
“I
4:1 2:1 4:1 2:l 2:1 2:l 4:1 2:l 4:1 2:1 4:l
Group I1
B-
B-
BR
B ’ C-
F B-
F
I
C’ A
...
...
.4
A-
D R-
BD C-
y’
F’
B
C
B
C+
...
C
B
...
...
c
C T
...
c+
...
A-
...
Group
Sample DNP-asp, -glu
A...
nL ...
...
e...
...
...
:-
... ... .-. . H ... D R-
B-
Group 111
B R... ...
...
.
.
_______________
Silicic acid 40446, on which the scheme had been worked out, appears to be of intermediate quality, but even the poorest, of the nine samples which were examined extensively gave satisfactory results if 4 to 1 silicic acid-Celite was used for most of t,he chromatograms. It had been anticipated that the t\yo poorePt samples ( l l e r c k 43216 and Baker i1-288) might give unsatisfactory results, as these samples were much more granular than the ot,hers and the rate of flow of solvents through columns of these adsorbents was much greater than for the others. The t,hird poor sample (llallinckrodt, X A P ) also felt somewhat granular arid had a rather fast flow rate, although not so great as the other two poor samples. It may he that after grinding and siftring to lOO-mesh, these samples would also I,c suitable for chromatographic use. T h e results of these tests indicate that it should not 1 ) difficult ~ to find a commercial silicic acid which is suit,ahIefor UPH with the scheme. T h e three samples which were designated by the manufacturer as especially prepared for chromatographic purposes gave the tied results. However, because of the slight variatio:is iri the adsorptive strengths of the various silicic acids, i t will be necessary to determine the development behavior of each of the 2,1-dinitrophenylamino acids with each developer t o be used in t,he method. It should be possible t o determine whether a sample of silicic acid will he suitshle hy running a few exploratory tests. Saniplrs that give poor results for one of the hetter separ%tions, such as Group TI with Z.L\-lO;\-T,, can be e l i m i n a t d rradily. If acceptable results are obtained u-ith this mixture, the tests can he evt,ended t o some of the poorer separations surh as that of 2,1-dinitrophenylaspartic and Z,~-dinitrophrn!-lglutamicacids. If sat,isf;trtory separation3 are ohtnined here, the other portions of the acheme can then be tried. I n some instances more compact zones and better separations can be ohtained by using a weaker developer than that prescribed in the scheme, but a greater volume of developer is required and hence the time required t o run the chromatogram is increased. The degree of improvement attained x i t h the weaker developer often does not warrant t h e additimal time required. T h e sharpness of the zones and t h e interzones become progressively better as the strength of the developer is decreased. Application of Scheme to Study of Lysozyme. The scheme has been successfully employed in determining that, lysine is the t,erminal amino acid residue a t the amino end of the lysozyme niolecule (6) and by a n estension of the method Schroeder has recently found that, the sequence of the first four amino acid
...
B-
...
...
... C
B-
D
... ...
... ...
...
D’
B-
...
DNP-Val,
... ... ... D ... ...
ACKNOWLEDGMENT
-leu
...
B-
... ... ...
...
... B-
... Bt
B’ ... ... ... ... ... ...
B
...
T h e authors wish t o acknowledge indebtedness t o Robert B. Corey and K. A. Schroeder for helpful discussions, and to Lewis Honnen for technical assistance. This investigation was aided by a grant from the Xational Foundation for Infantile Paralysis. LITERkTURE CITED
(1) Biserte, G., and Osteur, R.,
Compt. rend., 230, 1404
(1950). (2) Blackburn, S., Biochem. J.. 45, 579 (1949). (3) Blackburn, S.,and Lowther. A. G.,I b i d . , 48, 126 (1951). (4) Brand, E., Ann. S. 1’. d c n d . Sci., 47, 222 (1948). (5) Consden, R., Gordon, A. H., Martin, A. J. P., and Synge, R. L. RI., Biochem. J . , 41, 59G (1947). (6) Green, F. C., and Schroeder, W.A , , J . A m . Chenz. Soc., 73, 1385 (1961) . ( 7 ) Kent, P. IT., Lawson, G., and Senior, .I..Science, 113, 354 (1951). ( 8 ) LeRosen, .4.L., J . Ani. Chem. Soc., 64, 1906 (1942). (9) SIiddlebrook, W.R., r n t u r e , 164, 501 (1949). (10) Monier. R.. and Penasse, L.. Compl. rend., 230, 1176 (1950). (11) Parti,idge, SI., and Swain, T., S a t i t r e , 166, 272 (1950). (12) Perrone, J. C., Ihid., 167, 513 (1951). (13) Phillips, D. RI. P., and Stephens. ,J. &I.L.. Ibid., 162, 152 (1948). (14) Porter, R. R., and Sanger, F., Riorhem (15) Sanger, F., Ibid., 39, 507 11945). (16) Ibid., 45, 563 (1949). (17) Schroeder, TT. A , . Ann. .\-. 1’. Acnd. Sci.,49, 204 (1948). (18) Schroeder, IT, -I., J . Am. Ciiem. Soc., 74, 281 (1952). (19) Schroeder, TT, A , , Keilin, B., and Leninion, R. M.,Ind. Eng. Chem.. 43, 939 (1951). Malmberg, E. IT., Fong, L. L.. Trueblood. I
