ANALYTICAL CHEMISTRY
162 11 t o 14. Excellent agreement in sensitivity measurements was obtained between Equation 6 and experimental data for both orifices. ACKNOWLEDGMENT
The assistance of Jesse P. Nehrulst and Robert E. Bader of the Emil Greiner Co. in performing some of the laboratory experiments, the help of Charles F. Saladino in preparing the figures, and the permission of the Emil Greiner Co. t o publish this information are gratefully acknowledged. LITERATURE CITED
(1) Arbogast, J. F., personal communication, Aug. 26, 1949. (2) Cook, N. C., personal communication, NOT, 8. 1947. (3) Gilmont, R.,ASAL. C H E X . , 20,89 (1948).
Gilmont, R., ISD.ENG.CHEM.,ANAL. ED., 18, 633 (1946). Goodwin, R. D., J . Chem. Education, 24, 511 (1947). Greiner Go., Emil, Xew York, N. Y., Bull. C.M. 97 (1949). Ibid., I.C.M. 96 (1947). Greiner Co., Emil, New York, K.Y., Catalog G 15070 (1950). Perry, "Chemical Engineers' Handbook," 2nd ed., p. 847, S e n York, RIcGraw-Hill Book Co., 1941. Plambeck, L., Jr., Pedlow, G. W.,Jr., and Bartholomew, W ,H., personal communication, April 7, 1947. Ritzer, J., et al., The Brinewell, 3, S o . 14, 1 (1946). Spadaro, J. J., et al., IND.EXG.CHEV., A N ~ L ED., . 18, 214 (1946). Spillane, L. J., and Goodwin, R. D., J . Chein. Education, 25, 78 (1946). RECEIVED October 18, 1949. Presented before the Division of Physicai and Inorganic Chemistry a t the 116th Lleetinp of the A\IERTCN CHEIIICAL S o c ~ r :Y.,~.itlontir City, S . .I.
Paper Chromatography of Amino Acids E f e c t of p H of Sample 11.1.
ALTON J. LANDUA, ROBERT FUERST, AND JORGE AWAPARA D . Anderson Hospital for Cancer Research, Uniuersity of Texas, Houston, Tex.
It was found that many amino acid solutions could be made to yield good chromatograms by adjustment of the pH of the sample before placing the sample on paper. This was especially true for acid protein hydrolyzates from which most of the acid had been removed by repeated evaporation. The pH of the sample was found to affect the spread of a spot and also its position on the final chromatogram. Similar results were obtained for individual amino acids and for mixtures. The data may be used to determine what pH of the sample will result in the most compact spots and most advantageous positions on a final chromatogram for a desired separation, or may serve as a basis for further interpretation and theoretical speculation on the mechanisms of these separations.
P
APER chromatography has been widely used for the separation of amino acids ( 3 ) . One of the most important factors affecting the quality of a chromatogram is the pH of the solution t o be analyzed. pH effects were recognized by Consden, Gordon, and Martin ( 4 ) , investigated briefly by Bull, Hahn, and Baptist (2), mentioned by Miettinen and Virtanen ( 5 ) ,and considered in some detail for lysine by Aronoff ( 1 ) . Hydrochloric acid protein hydrolyzates, repeatedly evaporated t o dryness, fail t o give good chromatograms by two-dimensional chromatography, when phenol is used first and 2,4-lutidine second as solvent, unless the pH of the sample is further adjusted before application t o the paper. Adjustment of samples from other sources also results in good one- and two-dimensional chromatograms, using other solvents and combinations of solvents. The present investigation was undertaken to determine the effect of sample pH alone on the final chromatogram, and also whether such data could be used t o advantage in resolutions of amino acids and some related substances.
Serial dilutions of 0.2 N hydrochloric acid and 0.2 N sodium hydroxide from 2 X 10-1 N to 2 X 10-6 N were made up and equal volumes of each dilution and the amino acid solution were mixed. One sample consisted of equal volumes of glass-distilled water and the amino acid solution. The pH's of the resulting solutions were measured with a Beckman pH meter, Model G. Then 0.02 nil. of each sample, equivalent to 2 micrograms of amino nitrogen, was placed on Whatman No. 4 filter paper in such a way that the solvent ascended (6) in the short or slow direction; one sheet was used for each compound in each chromatographic solvent. The range of the distance traveled by solvents for different chromatograms was 26.3 * 3.8 em. in phenol, 26.0 * 3.5 cm. in lutidine, and 27.6 * 3.7 cm. in butanol. The solvent boundary was marked with a paper punch. Chromatograms were dried in an oven a t 100" C. for 15 to 20 minutes, sprayed with 0.057, ninhydrin in water-saturated 1butanol, redried for 15 to 20 minutes at 100" C., and then placed on an incandescent illuminator. Here the top of the spot, the bottom of the spot, and the center of most intense color were marked. These distances from the starting point were then measured and Rr was calculated by:
R/ = EXPERIMENTAL PROCEDURE
Solutions of amino acids and related substances were made up to contain 200 micrograms of primary amino nitrogen per milliliter of glass-distilled water, the same concentration of secondary amino nitrogen for proline and hydroxyproline, and the same concentration of primary a-amino nitrogen for substances also containing other types of nitrogen in their molecules. The chromatographic solvents included phenol, 2,4-lutidine, and 1-butanol. All were C.P. grade and were saturated with glass-distilled water a t the temperature of the experiment, the range of which was 21.2" * 2.3 C. Lutidine and butanol were used without purification, but the phenol was distilled from zinc dust, that portion boiling from 177 to 181' C. being used.
distance to point in question distance of travel of solvent boundary
These values were plotted against the pH of the original applied solution. Several solutions containing from four to eight compounds, each having 2 micrograms of amino nitrogen, were then run by one-dimensional chromatography and by two-dimensional chromatography, using different solvents, combinations of solvents, and various pH's. DISCUSSION
It was decided that a graphical presentation of the results would illustrate the effect of pH of the applied solution more
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1
163
Solvent, Phenol
Solvent, Phenol
Rjf 0.7
.--
a.,ar-a-t....--a.--a----a
0.5
DL
0.6
- A i AN/N€
Solvent, Phenol
1
\ -
0.5
GLYCYL G LYCl N E 1
1
'L
0.3 0.2
I
I
1
I
I
at 0.8 a7 0.6
I
I
0.8
1
/I
0.5
0.4 nr
D L- L EUCINE
a7
L
0.1
L
I
-
ARG/N/N€
- GL U TAMIC AClo
I
1
1 O.7
-
L-LEUCYLGLYCYf GLYCINE I
I
0.2
1'
L
I
- ASPARAGINE
1
I 0.5 1
1
A
0.4
ai 0.0
-
-
DL ASPAA TIC AC/D I
2
4
6
PH
8
t o 1 2
Ok3k
1
/ P
I
GLYCINE 4
6
e
IO
12'
PH Figure 1. R/ us. pH
Lower curve represents bottom of spot, center curve center of color, and upper curve top of spot
ANALYTICAL CHEMISTRY
164 Solvent, Phenol
Solvent, 2,CLutidine
Solvent, 2.4-Lutidine
DL-ORNITHINE 0.7
L
- ARGININE
DL-THREONINE
0.3
I
I
&O
L
_--,-------.-*e
0.1
DL- VAL IN E
0.6
2
4
6
I
8
,
,
i o 1 2
- ASPARTIC ACID
DL-ASPARTIC AC/D 2
4
6
6
PH
PH Figure 2.
i o 1 2
G L YC YLG L YcINz 2
4
6
PH
Rj us. pH
Lower curve represents bottom of spot, center curve center of color, and upper curve top of spot
8
1 0 1 2
16s
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1 Solvent, 1-Butanol
Solvent, 2,CLutidine
Solvent, 2,4-Lutidine
8
O-O
0.3
1
DL-ORNf THINE
I
1
1
ast \ HISTAMINE
0.41
DL-PHENYLALANINL
I
02
1
L
HYDROXY L
,
- PROLINE
QO
- PROLfNE
1DL- * - A M INO BUTYRICACID I
I
I
'"1
DL-SERINE
o*tl
1-0.1
1-
. a2F
1
DL- LEUCIN€
L
e..---
0.0
:\-
-
ARGININE
I
I
I
-------__
---..
-e-----
l-o.~} 1- CYSTEINE
I
L-L EUC YLG LYC YL GLYC/NE
I
I
1
I
I
J
I
I
,
0.3 0.4. -
0.2
DL-THR E 0NINE I
Dl- ME THIONINE
I
0.6
A5
t L
I
h
.'I
a4
I
- TRYPTOPHAN
'
DL-VALINE 2
4
1-0.1 8
6
1 0 1 2
R,
us.
L
- GLUTAMIC
--. ,ACIQ
I
L- GLUTATHfONE 2
4
6
PH
PU Figure 3.
-0.1
pH
Lower curve represents b,ottom of spot, center curve center of color, and upper curve top of spot
8
1 0 1 2
166
ANALYTICAL CHEMISTRY Solvenf, 1-Butanol
Solvent, l-Butanol
one chromatogram that no color was developed far samples, tho pH's of which were around the isoelectric point. Variability in
Solvent, 1-Butanol
Chromatogram of I(+)-Cysteine Hydrochloride at Various pH's
Figure 6 .
Chromatogrsphhic solvent. water-saturated 1-butsnol
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1
Figure
167
6. Chromatogram of DLOmithine
Chromatographic solvent, water-saturated 1-butanol
Hydrochloride at Various pH's
150
2.72 3W
357
369
?70
1205
PH
Figure 7.
Chromatogram of L ( + ) - G l u t a m i c Various pH's
Acid
ut
Chmmatocraphic sohent, water-saturated ZC-lutidlno
R, and area of the spot mound tho isoelectricpoint can be seen for most of the substances studied. The effect of certain functional groups is most e l e d y illustrated by a comparison of the graphs for ethanolamine and thc basic amino acids in phenol a t high pH's, where the ionization of the basic group is suppressed. An effect may also be noted for the extra carboxyl group of aspartic and glutamic acids a t low DH. It is also interesting to compare glutamic acid and glutsmine, and aspartic acid and asparagine. One-dimensional chromatograms of solutions containing scparable components of equal concentration showed no significant departure from the curves of Figure 1. The same holds true for two-dimensiona] chromatograms in the first solvent. Measurements in the direction of the second solvent were unreliable be-
2
T;
+-i z 2,'t- LUTIDINE
\ Fieure 9.
Two-Dimensional Chromatograms
~
1. DL-Serine
2. 3. 4.
Glylycin. DL-o-Alanine L (-)-Tryptophsn
5. 6. DL-Methionine DL-Valinc 7. DL-Phenylalanine 8. DL-leucine of ,-ami;o
cnoh
Chromatographic saturated 2.4-lufidinc solvents, second wster-saturated 1-butanol firat, watcr-
upPeI, 6.84:
~ ~ m2.99 r ,
ANALYTICAL CHEMISTRY
168 cause of the formation of a brown line, which preliminary investigations show to be different from the “pink front” of Consden, Gordon, and Martin (4). This brown line slows the solvent, causes an irregular solvent boundary, and contains a wetted area above. It gives a positive aldehyde reaction with Schiff’s reagent. All the amino acids appear below. These effects are minimized but not caused t o disappear by a combination of oven and air drying between solvents. Mainly for this reason, absolute R , values in the direction of the second solvent could not be checked, and data concerning the effect of p H of the applied solution on the chromatogram in the direction of the second solvent were inconclusive. Therefore, the data in Figures 1 t o 4 should be used with extreme caution in two-dimensional chromatograms. For purposes of identification it is still best. t o use mixed chromatograms with known substances, or determination of position relative t o a known substance such as a-alanine, which is relatively insensitive to pH changes. However, the unknown solution should be adjusted to a pH favorable for separation in the first solvent, and the first solvent should be the one in which the amino acids are more sensitive t o pH changes. For instance, if it is suspected that the basic amino acids are present, the solution should be adjusted to pH 10+ and run in phenol first and Iutidine second. The effect of inorganic salts on chromatograms ( 4 ) cannot be minimized. The authors’ investigations show that this factor is best treated as a separate variable. For this reason buffers were not used in the present investigation. Bbove a certain undeter-
mined concentration, the amino acids in the solution also have an effect on each other. Figure 9 represents two-dimensional chromatograms of a solution containing equimolar concentrations of valine, methionine, tr-yptophan, leucine, phenylalanine, serine, glycine, and a-alanine with butanol first and lutidine second as solvents. The pH of the applied solution of the upper chromatogram was 6.84 and of that of the lower was 2.99. Although all these substances are relatively inert toward pH changes, it will be seen that the two chromatograms are different in separation and area of the resulting spots. Serine and glycine separate from each other and from a-alanine a t pH 6.84, when phenol is used first and lutidine second as solvents, but the remaining five do not separate from each other completely. Chromatograms of substances more sensitive to pH show similar but more extreme effects. pH effects for solvents chemically related to the three used here are roughly parallel. The cresols are similar to phenol, the collidines to lutidine, and the alcohols to butanol. LITERATURE CITED
(1) (2)
Aronoff,S., Science, 110, 590 (1949). Bull, H. B., Hahn, J. W., and Baptist, Lr. H., J . A m . Chern.
Soc.,
71, 550 (1949).
( 3 ) Consden, R., Nature, 162, 359 (1948). (4) Consden, R., Gordon, A. H.. and hlartin, A . J . P., Biochem. J . , 38, 224 (1944). ( 5 ) Miettinen, J. K., and Virtanen, 8 . I., Scta Chem. Scand., 3, 45964 (1949). (6) Williams, R. J., and Kirby, H., Science, 107, 481 (1948).
RECEIVED March 10,1950.
Buffered Filter Paper Chromatography of the Amino Acids EARL F. MCFARKEN National Dairy Research Laboratories, Inc., Oakdale, Long Island, .Y. I-
I
S A recent paper by Karnovsky and Johnson (8)the utilization of paper impregnated with buffer for the chromatography of penicillin broths is discussed, and the Rjvalues of the penicillins are shoii n to vary with the p H of the buffer. In the work reported here a similar method is used in chromatograniming the amino acids, and the Rivalues are shown to vary in a manner similar to those of the penicillins. Haugaard and Kroner ( 7 )were the first to apply buffered paper to the chromatographv of amino aci ’s. They also used an applied voltage, but this bas not been done here. However, it was felt that the buffered paper technique was an improvement and should be developed further, other solvents being incorporated and pH valurs on both side8 of the isoelectric point of the monoamino monocarboxylic acids being investigated. By employing several solvents buffered at a chosen pH betwren 1.0 and 12 0, it i i poqsible to separate each amino acid from all others by oncdimensional chromatography. At the same time, some of thr, difficulties of paper chroniatographv as previously wed, particularly two-dimensional, have been overcome-namely, maiiipulation of large sheets, poorly defined spots, irreproducihle R values, and inseparability of qome of the amino acids. \ considerable amount of data is presented here, but it is felt justifiable because it is believed that the data are repioduciblc. The irreproducibility of R j values has been a fault of chromatography in the past, and has been a result of the failure to study and specify carefully all thr conditions of which the values are a function. Because of this fact, many of the data in the literature have been useless to other R-orkers in this field. R , values serve as one means of identification, if they are reprotlucible. Thus, it is necessarr to have accurate data available
In this particular case, accurate data are also needed, so that others may investigate further some of the solvents a t pH values not reported here without having to repeat work already ac-
~
I n the past it has not been possible to resolve all of the twenty common naturally occurring amino acids in a mixture into individual spots on either a onedimensional or twro-dimensional chromatogram. Two-dimensional chromatography gives improved separation, but produces poorly defined spots and irreproducible Rf values. I n the present described method, it has been found possible to separate by one-dimensional chromatograph3 each amino acid from all others in a mixture by employing several solvents buffered at a selected pH. The separation of each amino acid into individual well defined spots makes possible the identification of each amino acid in an unknown mixture without having to resort to special reagents to confirm the identity of one or more of the acids. Ninhydrin can be used as the sole developing reagent, and the amino acids can be identified with assurance by referring onl) to their Rr values or relative positions. The improved separation will also enable quantitative determination of amino acids by direct photometq, elution, or similar methods with greater accuracy.
