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.
V O L U M E 2 3 , NO. 1, J A N U A R Y 1 9 5 1
169
complished. I n addition, these accumulated data permit selection of one or more solvents buffered a t one or more pH values for chromatogramming an unknown, thereby identifying an unknown amino acid with greater assurance. '
VALINE LYSINE -A- HISTIDINE -0.ARGININE
I
v . 0 .
60
pH
Figure 1.
Oc BUFFER
Variation of RJ Values of i m i n o . k i d s with pH of Buffer Solvent, ria-cresol
Table 11.
Variation of Rf Values of Amino Acids with pH of Buffer 2.0
Pi1
(Solvent, o-cresol) 6.2
* 0.01 * 0.01
* 0.01
Aspartic acid Glritainic acid
0.01 0.02
Lysine .4rginine Histidine
0.00 * 0 . 0 0 0.02 - 0 . 0 1 0.01 * 0 . 0 1
0.00 0.00 - 0 . 0 0 0.07 - 0 . 0 2
-
n. .00 . ~i 0.00 0.02 * 0.01 0 . 0 2 * 0.01 0 . 0 3 * 0.01 0 05 f O . 0 1 0.11 * 0 . 0 1 0.11 0.01 0 . 2 2 * 0.02" 0 30,* 0 03" 0 42 0 02 0 45 * 0 03 0 39 * O 01 0 39 * O 02 0 5 5 * 0 02" 0 42 - 0 03
0 . 0 1 == n n i 0.03 * 0 . 0 i 0 . 0 4 * 0.00 0.06 * 0.00 0 . 0 9 * 0.01 0 18 * 0 . 0 2 0 . 1 8 == 0 . 0 2 0.33 = 0.02" 0 48 * 0 01" 0 61 * 0 03 0 66 * 0 02" 0 58 - 0 03 0 5.5 a 0 02 0 71 * 0 0 62 * O 02
Cv ,s t.i.n..e. I
Serine Glycine Threonine hlanine Tyrosine Hydroxyproline Valine bcethionlne Tryptophan Norleucine Leucine Isoleucine Phenylalanine Proline
f
f
0.00 0.02
* 0.01 *o.oo
12.0
9.0
0.00 0.00
f
*
0.00 0.00
0.11 * 0 . 0 2 0.27 * 0 . 0 3 0.27 * 0 . 0 1
n nR *
0.03 * 0 . 0 0
* 0.00 * 0.00 * 0.01 * O 01 * 0.02 * 0.02 * 0 01"
0.05 0.06 0.09 0.20 0.18 0.29 0 44 0 66 0 63 0 55 0 52 0 75 0 59
0 03 0 04 * O 03 * O 0'3 * 0 02" * O 03 f f
0.00 0 00
f
0.00 00
*0
0.20 * E o 03 0 . 4 2 *0.0.5 0 . 1 5 * O 01 ..
0.02 0.03 0 03
0.07 0.11 0.06 0.20 0 26 0 54
CI 54
-
* .. .
* o 00 0 00
* 0.00 * 0.01
*0.01 * 0 02 * n 01
*o *0 *0
0 43 - 0 0 40 * O 0 57 * 0 0 50 1 0
021
01 01
01 01 02 01
i m i n o acids separated froin all otlieis sufficient15 for identification - ~-
_ . ~
Table 111.
PH Aspartic acid Glritainic acid Lysine Arginine Histidine Cya t ine Serine Glycine Threonine -4lanine Tyrosine Hydroxyproline Valine Methionine Tryptophan Norleucine Leucine Isoleiicine Phenylalanine Proline a
-
Variation of R / Values of Amino Acids with pH of Buffer (Solvent, p-cresol) 2.0 6.2 9.0 0.04 * 0 01 0 . 0 3 * 0 . 0 1 0 02 * 0 . 0 1 0 . 0 8 * 0 . 0 1 0 07 * 0 . 0 1 0 . 0 3 * 0 . 0 0 0 . 0 3 * 0 . 0 1 0 03 * 0 . 0 1 0 . 0 8 * 0 . 0 2 0 06 * 0.01 0.06 - 0 . 0 1 0.13 - 0 . 0 1 0 . 0 1 * 0 . 0 1 0 . 0 2 ;t 0 01 0 . 0 6 * 0.01 0 . 0 6 * 0 01 0 06 * 0 . 0 1 0 . 0 9 * 0 . 0 1 0 . 1 0 * 0 . 0 1 0 13 = 0 . 0 1 0.15 0 0 2 O 0 . 1 8 * 0.01 0 . 2 0 f O . 0 2 " 0.33 t 0 . 0 1 0 . 2 4 * 0 . 0 2 & 0.31 * 0 02 0 47 * O . O Z Q 0 . 4 9 * 0 . 0 I R 0 . 5 7 * O.OXa 0.63 * 0 . O a a 0 . 6 7 * 0 . 0 3 0 74 = 0 . 0 2 0.72 0 03" 0 70 * 0 . 0 0 0.67 * 0 . 0 3 0 72 * 0 01 0 . 6 6 * 0 03 0 70 * 0 02 0 77 * 0.04'> 0 . 8 2 * 0 01 0 63 * 0.04 0 . 7 2 * 0 01 f
f
12.0
0.02 0.02
* 0.01 f
0.01
0.30 * O.OZa 0 . 4 1 t o 0 63 0 . 6 2 * 0 . 0 4 0 . 8 0 * 0 01 0 . 4 9 f 0 . 0 2 a 0 . 4 9 i.O.04 0 . 0 7 * 0 . 0 1 0 . 0 5 i 0 01 0 OX 0 . 0 1 0 09 * 0 02 0 13 * 0 . 0 1 O , l 3 * 0 . 0 2 0.16 * O . O l 0 . 1 7 * O 02 0 23 0.02a 0 . 2 5 * O.0lo 0 . 4 2 * 0 . 0 2 a 0 . 4 0 * o 01 0 . 3 4 * 0.02° 0 . 3 2 * O . O R U 0 . 5 4 *0.03a 0 . 5 6 f O . 0 2 0.66 0 01 0 . 6 3 * 0 02 0 . 7 0 * 0 01 0 . 8 1 * 0 . 0 2 0 . 8 3 * 0.02" 0 . 8 9 f 0 . 0 1 0 . 7 6 * 0 01 0 . 7 6 += 0 . 0 2 0 . 7 5 * 0 . 0 2 0 76 * 0.0" 0 88 * O . O l a 0 . 8 3 * 0.02 0.76 * 0 . 0 1 0 . 7 6 * 0.02 f
f
f
Amino acids separated f r o m all others sufficiently for identification.
________
170
ANALYTICAL CHEMISTRY Table IV. Variation of R j Values of Amino Acids w i t h pH of Buffer (Solvent, phenol)
PH Aspartic acid Glutamic acid
2.0 0 . 2 1 =t0 . 0 1 0.30*0.01
4.0
6.2
0.14 f 0 . 0 1 0.25+0.02
0.15 * 0.00 0.28*0.01
7.5 9.0 0.10 + 0 . 0 1 ~ 0.12 t 0.01a 0.18+0.01 o.ig*o.oi~
10.0 11.2 0 . 1 1 * 0.01a 0 . 0 8 * 0.02 0.i9io.00~ 0.16to.oi
12.0 0 . 1 0 * 0.015 0.17.to.01~
Lysine Arginine Histidine Cystine Serine Glycine Threonine Alanine Tyrosine Hydroxyproline Valine hlethionine Tryptophan Sorleririne
0 . 1 8 f 0 01a 0 16 =t0 . 0 2 0 . 1 0 =t 0.01'" 0.26 o.0i 0.23 0 20 * 0 . 0 1 0 . 2 0 =t 0.01 0.26 0.25 0.02 0.25 * 0.01 0 . 2 6 * 0.01 0.32 0.35 0.01 0.33 * 0.02 0.37 * 0.02 0.39 0 . 4 3 * 0.03a 0 . 3 8 * 0.01 0 . 4 1 * 0.025 0.45 0.56 0.02 0.58 0.02 0.60 0.02a 0.62 0.54 0.01 0.54 * 0.02 0 . 2 3 + 0 0Za 0.57 0 69 0 . 0 2 Q 0 . 6 9 * 0.0Za 0.68 0.02b 0 . 7 1 * 0.04' 0.71 0 . 7 3 * 0 03" 0 . 7 7 * 0.04 0 . 7 9 * 0.03 0.78 * 0 026 0.81 0 82 * 0 . 0 4 0 . 8 6 * 0.04 0 . 8 5 t 0.04 0.88 * 0.03b 0.86 0.83 * 0.03 0.86 * 0.02 0 . 8 8 + 0.04 0.87 * 0.026 0.87 LeiirinP .~ 0.79 0.02 0.82 * 0 . 0 1 0.83 0.02 0 . 8 3 0.03b 0.83 Isoleucine 0.81 * 0.03 0.81 0.03 0.82 * 0.02 0.83 * 0.03b 0.83 Phenylalanine 0.85 * 0 . 0 5 0.90 * 0.03 0.91 * 0.02 0.89 * 0.02 0.90 Proline 0 . 8 0 0.02 0.82 * 0.03 0 . 8 5 * 0.02 0.86 0.02 0.85 Amino acids separated from all cIthers sufficiently for identification. b Heading occui-red. ~
f
f f
f
f
f
f f
f
-
f
~
f
f
Table V.
f
* 0.01 * 0.01 * 0.015 * 0.01Q f
0.29 0.28 0.34 0.40 0.47 0.61 0.59 0.70 0.79 0.88 0.85 0.83 0.83 0.88 0.84
0 .O P U
* 0.01a * 0.01 * 0.08
f f
0.02 0.02
* 0.02 * 0.01
* 0.02 0.03 * 0.01
f
Variation of R f Values of A m i n o Acids w i t h pH of Buffer
0.01 0.01 0.01= 0.01a
0.01'L 0.01 0.01 0.04 0.04 0.07 0.02 0.02 0.02 0.05 0.03
0.22 to0.10 0 . 18 * 0.026 0 . 2 5 * 0.02atb 0 . 3 4 * 0.02b 0.38 * 0.02'~ 0.60 + O.05b 0 . 5 7 =t 0.0:b 0 . 7 2 * 0.026 0.70 0.02b 0.85 0.03 0 . 8 6 * 0.02b 0.83 0.01b 0.83 0.02b 0.90 * 0.01 0.83 * 0 . 0 1 f f
f f
0.30 0.27 0.32 0.41 0.48 0.62 0.58 0.74 0.78 0.83 0.90 0.87 0.87 0.89 0.88
+
0.02
f
0.01"
* 0.02a * 0.02"
* 0.03a * 0.015 * 0.01" * 0.03 * 0.02 * 0.06 * 0.03 * 0.02 f f f
0.02 0.04 0.02
the side of the chamber and dipped into the solution in the bottom of the chamber. The [I 1-1 Unbuffered 2 0 6.2 9.0 12.0 amino acids were prepared as .hi,urtic acid 0.14 * 0 . 0 1 0.02 0.00 0.10 0 . 0 0 0.08 0.01 0.02 * 0 . 0 2 0.03 0.01 solutions containing 0.1 mg. of Glutaniic acid 0.15 0.02 0.02 * 0 . 0 1 0.12 0.01 0.09 * 0.01 0.02 0.02 0.03 0.02 amino nitrogen per ml. (except in the case of tyrosine and cysLysine 0.06 t 0 . 0 1 0.06 f 0 . 0 1 0.01 fO.00 0.01 t0.00 0.05 f 0 . 0 2 0.04 f 0 . 0 3 Arginine 0.10 1 0 . 0 1 0.11 *o.oi 0.02 t 0 . 0 0 0.02 t 0 . 0 1 0 . 1 0 * o . o z 0.05 f 0 . 0 1 tine) and were applied to the Histidine 0.22 t 0 . 0 2 0.13 f o . 0 1 0.10 1 0 . 0 1 0.09 = t o . o i 0.12 * 0 . 0 3 0.11 f 0 . 0 1 paper as single spots from a 4microliter platinum loop or a 5Cystine 0.08 * o . o i 0.02 * o . o i 0.04 f 0 . 0 0 0.03 t 0 . 0 1 0.02 + o . o i 0.03 *o.o4 Serine 0.19 * o . o i 0.10 - 0 . 0 1 0.09 f 0 . 0 0 0.08 - 0 . 0 1 0.08 +=O.OZ 0.07 *o.o3 microliter pipet, if more accuGlycine 0.17 * o . o i 0 . 0 9 *o.oo 0.08 f 0 . 0 1 0.07 . t o . o i 0 . 0 8 *o.oz 0.06 1 0 . 0 2 rate measurements were desired. Threonine 0.23 t 0 . 0 1 0.12 f 0 . 0 1 0.11 * o . o i 0.10 * o . o i 0.09 1 0 . 0 2 0.08 f 0 . 0 1 Alanine 0 21 f O 01 All amino acid solutions were 0 13 1 0 01 0 10 - 0 00 0 09 * O 00 0 10 f 0 02 0 09 f 0 03 Tvroiine o 56 t o 02 0 54 + n no n 4 7 t n na n 4 5 * n n4 n.. 46 n 44 * O 0 2 b .. + n .. nz .adjusted to a pH of approuiH"ydroxypro1ine 0.23 f O 02 0.14 i0.00 o.iz t o . 0 i 0.05 0.0i 0.10 0.02 mately. 6.2 before chromatoValine 0 31 f 0 . 0 2 b 0 . 2 4 * 0 . 0 O b 0 . 1 9 O.Oib 0 . 1 9 * 0.01b 0 . 1 6 f 0 . 0 2 b gramming. I n the case of hlethionine 0 42 0.02 0.34 * 0.00 0.30 0.00 0.27 * 0.02 0.27 0.03 Tryptophan 0.57 0.02 0.57 * 0.03 0.48 * 0.01 0.58 + 0 . 0 3 b 0 . 6 2 * 0 . 0 2 tyrosine, cystine, and mixtures Notlencino 0 49 0.01 0 . 4 3 *0.00b 0.40 0.02 0 . 3 8 * 0.036 0 . 3 5 0 . 0 2 containing these amino acids, Leucine 0 45 f O 01 0 . 3 8 * 0 . 0 2 b 0.35 0.02b 0 . 3 4 =t 0.02b 0 . 3 2 * 0 . 0 2 the solutions were made more Isoleucine 0.42 * 0 . 0 2 0,34 fO.O1 0.31 f O . 0 1 0.29 f O . 0 2 0.30 1 0 . 0 3 0.28 1 0 . 0 2 Phenylalanine 0.54 * 0.02 0.48 0.OOb 0 . 4 3 0 . 0 1 0 . 4 5 0.02 0.43 * 0.01 0.48 0.03b dilute in order to keep them in Proline 0.24 1 0 . 0 1 0.16 fO.01 0.13 t O . 0 1 0.13 * 0 . 0 1 O,l5 *0.02 0.09 h O . 0 1 solution a t pH 6.2. This made (I Refined 2,4,6-collidine purchased from Koppers Co., Inc., and redistilled t o obtain colorless product. it necessary to spot each such Amino acids separated from all others sufficiently for identification. solution from two to four times in order to build up the desired concentration on the paper. Table VI. Variation of R j Yalues of Amino Acids with pH of Buffer The chromatography chambers consisted of borosilicate (Solvent, 2,4-lutidine) glass jars 21 inches high and 12 PH Unbuffered 2 .o 4.0 6.20 6.2 9.0 inches in diameter (60 X 30 Asliartic acid 0.27 * 0.02 0.09 0.01 0.15 0.01 0.27 0.03 0.14 * 0.01 0.07 0.01 cm.), with a flat ground-glass rim Glutamic acid 0.29 0.03 0.10 0.01 0 . 1 8 * 0.00 0.28 * 0.03 0 . 1 6 i. 0 . 0 1 0.07 * 0.01 at the top. The rim of the Lysjne 0.15 0.02b 0.19 * 0.02 0.08 0.00 0 . 2 3 0.03 0 . 0 6 * 0.02 0.12 0.01 chamber was smeared with stopArginine 0 . 2 1 * 0.02 0.28 0.02 0.06 0.01 0 . 2 5 0.02 0.08 * 0 . 0 2 0.20 f O . 0 1 cock grease and covered with a Histidine 0.36 * 0.01 0.27 0.01 0.17 * 0 . 0 1 0.29 0.02 0.20 * 0.01 0.23 * 0.02 ground-glass plate, in order to Cystine 0.28 * 0.01 0.10 f 0 . 0 2 obtain an airtight seal. The Swine 0.34 * 0 . 0 1 0.15 * 0.01 troughs and stands used to supGlycine 0.30 * 0 . 0 1 0 . 1 5 * 0.01 Threonine 0.38 * 0.02 0.20 * 0 . 0 1 port the troughs, a t a height of Alanine 0.36 * 0 . 0 2 0.19 * 0.02 22 inches from the bottom, Tyrosine 0 . 6 6 * 0.01 0.61 f O . 0 1 were made of stainless steel. Hydroxyiiroline 0.39 f O . 0 2 0 . 2 3 0.02 Valine 0.46 0.01b 0.32 * 0.01b Whatman S o . 1 filter paper was Slethionine 0.55 *0.01 0.49 0.01 o 48 + 0.02: 0 . 4 4 * 0.01 0.46 0.02 cut so that the solvent flow Trypt,ophan 0 . 6 5 * 0.02 0.66 0.01 0.60 * 0.02 0.64 0.02 0.61 *0.02 0 . 7 2 * 0.03b would be parallel with the long Norleucine 0.59 0.01 0 56 * 0 . 0 1 0.61 t o . 0 3 0 . 5 3 * 0.030 0.51 * O . 0 l c 0 . 5 5 A 0 . 0 4 n 57 f n . 0 2 Leucine 0.48 0.02 0.53 * 0.01 0 . 5 1 f 0 030 0 . 4 9 * 0 . 0 2 c 0.51 axis of the rectangular sheets 0.04 Isoleucine 0 56 * 0 . 0 3 0.46 * 0 . 0 2 0.50 fO.01 0 46 1 0 03C 0 . 4 7 f O . 0 3 C 0.03 0.47 and was used in collecting the Phenylalanine 0 62 * 0 . 0 2 0 . 2 8 f a 01 0.55 =t 0 . 0 2 0 59 =t0.03 0.56 1 0 . 0 4 0 . 6 3 1 0.03 data reported here. The room, Proline 0 41 f O . 0 5 0.31 * 0 . 0 3 0.25 0.02 0 31 f 0 . 0 3 0.24 0.02 0.29 f 0 . 0 3 b housing the chromatography " Only paper bnffered. chambers, was controlled generb Amino acids spparated from all others sufficier itly for identification C Heading occurred. ally to 22' + 1" C., in order to prevent condensation on the paper strip and the resulting streaking of the amino acid spots. EXPERIMENTAL PROCEDURE After the chromatogram had been run in the chambers, the papers were dried in an oven a t 90' to 95" C. and then sprayed with The papers were dipped into an approximately 0.066 M buffer 0.1% ninhydrin in aqueous butyl alcohol. I n order t o obtain solution of the desired pH, suspended by one end, and air-dried. color development with ninhydrin, it was found necessary to add The solvent was equilibrated by shaking in a separatory funnel 2% acetic acid ( 5 ) to the 0.1% ninhydrin in aqueous butyl alwith the same buffer. When the two layers had separated, the cohol before spraying chromatograms buffered in the range of solvent-rich layer was placed in the trough and the buffer-rich layer was placed in the bottom of the chamber. The chambers pH 7 5 to 9.0, and from 5 to 7% acetic acid before spraying were all lined by preparing a cylinder, of the same diameter as the chromatograms buffered in the pH range of 9.0 to 12.0. The inside of the chamber, from two sheets of Whatman No. 1 filter color was developed by drying the sprayed chromatograms for paper stapled together. The filter paper cylinder fitted against 10 minutes in an oven a t 90" to 95' C. (Solvent, collidine") 4.0
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f
f f
f
f
V O L U M E 23, NO, 1, J A N U A R Y 1 9 5 1 Table VII.
171
Variations of RI Values of A m i n o Acids w i t h pH of Buffer (Solrent, benzyl butyl alcohol 1 t o 1)
1 .o
PH
2.0
4.0
0.10f0.01 0.14i.0.02
Lysine Arginine Histidine
0.01~0.00 0.02*0.02 0.02+0.01 0 . 0 2 f O . 0 2 0 . 0 2 ~ 0 . 0 1 0.02 f 0 . 0 2
('ybline SGrinF Glycino 'I'hrroniii e Alanine Tyrosine H>-droxyproline Tal i 11c Methionine Trvntouhan
n 07 * n no o:i0 = 0.02 0.16 0 . 0 2 0.47 * 0.04 0.13 0.02 0.45 * 0 . 0 3
0.02 0.06
f f
0.01 0.00
f
0.02 0.04
0.03
=t
* 0.02
0.02 0.0s 0.03 0.12 f 0 . 0 2
0.05
1 f
0.40 * 0.03 0.08 * 0.01 0.40 0.05 0.49 0.04 0.45 0.04 0.67 f 0.02 0.66 0 . 0 2 0 . 6 8 f 0.02 0.65 f 0 . 0 2 0.65 f 0.03 0.60 * 0.02 0 . 6 3 f 0.03 0.58 f 0 . 0 1 0.67 1 0.04 0.65 0.02 0.26 f 0.036 0 . 2 2 * 0.02b f
f f
f
i,entfrom either t h c x hasir (ir neutral amino acids, but do not shon12H 2.0 4.0 6.2 9.0 12.0 any great variation with pH. At pH Anliartic acid 0.00 * 0 . 0 1 0 . 0 1 i. o , o 1 o,oo t 0.00 o 00 * 0 . 0 0 O,OO * 0.00 11.2 two point's are given in Figure 1, for Glutamic acid 0.15 0.03 0.04 0.01 0.00 * 0.01 0.00 0.00 0.00 0.00 both lysine and arginine. Apparently at 0.02 0.00 0.00 0.00 0.00 0.00 0.01 * 0.00 0.01 0.01 this pH lysine and arginine dissociate 01' 0.03 f O . O O 0.00 - 0 . 0 0 0.00 * O . O O 0.02 f O . 0 0 0.03 * 0 . 0 1 ionize to such an extent that they~. may not 0.02 t o , 00 o , O O * 0 , 00 0.00 0.01 0.04 rt 0.01 0,114 0 , 01 0.00 f 0 . 0 0 0.00 1 0 . 0 1 Cyytinv 0.01 = O . O O 0.00 + O . O O o 00 + o . o o appear as a spot, a t all, but rather as a 0.03 t 0 . 0 0 0.08to.01 0.03 t 0 . 0 0 Serine 0.02 f0.00 o 02 + o . 0 1 O , O ~ 0.00 O , O ~+ 0 . 0 1 smear throughout the range of R I values 0 , 0 3 + 0.00 0.03 0.01 Glycine 0.00 0.01 0.05 * O . O O 0.05 t O . 0 1 Threoninr 0.12 f0.01 0.03 * O . O O 0.03 f O . 0 1 Alanine 0.17 i0 . 0 1 0.06 0.01 0.05 f 0.01 0.03 f 0.01 0.05 * 0 . 0 1 condition occurs in phenol 0.18 f O . 0 1 0.18 1 0 . 0 2 Tl-vosinc 0.46 fO.O1 0 , 1 4 1 0 01 0.07 1 0 . 0 1 this pH. 0.04 0.01 0.04 t0.01 IIydroxpliroline 0.08 0.01 0.04 * 0 . 0 0 0.02 0.01 0.17 *o.oi 0.16 t 0 . 0 1 Valine 0.47 1 0 . 0 2 0.16 t 0 . 0 1 0.15 i0.030 Figure 2 presents a method of separating RIethionine 0.47 f O . 0 3 0.19 1 0 . 0 1 0.20 i O . 0 2 " 0.17 t0.02 0.18 f O . 0 4 0.33 f 0.03 0.35 0.03= a mixture of eighteen amino acids by oneTryptophan 0.62 * 0.02b 0.34 * 0 . 0 2 0.33 * 0,040 Korleucine 0 . 7 4 0.03 0.42 0.01 0.42 O . O i a 0 . 4 3 0.020 0.39 0 dimensional chromatography sufficiently Leucine 0.70 t 0 . 0 2 0.38 f O . 0 2 0 36 f O . 0 4 Q 0.36 1 0 . 0 2 " 0.33 f0.03a Isoleurine 0.67 * 0 . 0 2 0.32 * 0.01 0.30 * 0 , 0 3 0 0.30 10.01 0.28 * 0.03" for quantitative determinat,ions. For Phenylalanine 0.66 0.01 0.38 0 . 0 1 0 30 0.01" 0.38 0.03a 0.37 t 0 . 0 " Proline 0.16 * 0.01 0.08 + 0 . 0 1 0 . 0 7 * 0.01 0.09 * 0.01 0.08 1 0 . 0 2 qualitative purposes this can be accomplishcd by spottingseven different sheets of Treading occurred. h A m i n o acids separated from all otliers sufficiently for identification. paper simultaneously, placing them in the respective chambers overnight (16 to 18 hours), and developing t.hem the next day. Tablcs I to I X present the data collected in several solvents at For quantitative purposes it is desirable to allow the solvent to various pH values. The buffer mixtures used to obtain these run off the paper (9) (24 to 48 hours) in order to obtain pH values are given in Table X. They are all standard buffers better separatione. as given by Britton ( 2 ) , modified in some cases to obtain the After submitting this article for publication it was found that desired molarity. lutidine (40 hours) saturat,ed with a pH 6.2 huffer of 0.22 ,If The data obtained for lysine, arginine, histidine, and valine a t various pH values . in the solvent m-cresol are plotted in FigTable 1X. Variation of R / Values of Amino Acids w i t h pH of Buffer ure 1. If a graph is made of all the amino acids a very interesting and useful picture (Solrent. benzyl alcohol) is obtained of the separations achieved a t PH 2.0 4 0 6.2 9.0 12 0 any particular pH. In some cases such Aspartic acid 0.03 0.01 0.00 * 0.00 0.00 * 0 . 0 0 0 00 0.00 0 . 0 0 * 0 00 a graph can be used to predict a pH at Glutamic acid 0.06 0.01 0.02 * 0.00 0 . 0 0 * 0 00 0 . 0 0 * 0.00 0 . 0 1 t 0 01 Table VIII.
(Solvent, butyl alcohol)
f
f
f
f
f
f
f
f
f
;t
f
f
f
f
f
f
f
f
f
f
f
f
'1
~~
~~~
~~~~~~
f
f
xhich a desired separation can be RCcomplished. .4 graph of all the amino acids has not been included in this paper, for the plotting of all the data on a graph the size of this printed page xould present a confusing picture. However, Figure 1 does serve t o show the variation of the Rt values with pH. Lysine, arginine, and histidine have be'en chosen as the most variable of all the amino acids. Valine is more or less representative of the monoamino monocarboxylic acids. Aspartic and glutamic acids follow a pattern some-
Lysine Arginine Histidine
0.00 f O . 0 0 0.01 i o . 0 0 0.01 t 0 . 0 0
Cystine 0.00 fO.00 Serine 0.02 f O . 0 1 Glycine 0.03 f O . 0 1 Threonine 0.03 t 0 . 0 1 Alanine 0 09 t 0 . 0 2 Tyrosine 0 28 t 0 . 0 3 Hydroxyproline 0.07 t O . 0 1 0.26 f O . 0 2 Valine Methionine 0.32 0.03 Tryptophan 0.53 f 0.04 0.48 1 0.01b Norleucine Leucine 0 . 4 3 f 0.01 0 41 f O . 0 1 Isoleucine Phenylalanine 0 . 6 2 1 0.01 Proline 0 . 1 7 * 0.030 a Heading occ urred. b Amino acids separated from all f
0.00 f o . 0 0 0.00 f O . 0 0 0.00 + 0 . 0 0
0.00 1 00 0 0 00 t0.00 0.01 f O . 0 0
0.01 1 0no 0.02 f O . 0 0 0 03 f O . 0 1
0.04 t o . 0 0 0 0 03 1 0 . 0 1 0.02 * 0 . 0 1
0.00 t o . 0 0 0.01 t o . 0 0 0.01 t O . O O 0.02 f O . 0 0 0.03 * 0.00 0.12 f 0.01 0.04 1 0 . 0 1 0.10 * 0.01 0.16 0 . 0 0 0 0.34 0.00 0.28 fO.020 0.23 *0.01 0.20 * 0.01 0.36 0 . 0 1 0.10 *0.01
0 . 0 0 t0.00
0.00 t0.00 0.01 f O . 0 1 0 . 0 1 t0.01 0.01 10.01
0.00 10.00 0.00 * 0 . 0 0 0.01 =0.01 0 01 f 0 00 0 02 = 0 00 0 08 - 0 . 0 0 0 02 * 0 00 0 07 i 0 O l n n 11 i n nia 0 : i j f 0'0ia 0.23 0.01; 0.18 i 0.01 0.15 f 0 . 0 2 Q 0.29 f 0 . 0 2 Q 0.06 0 . 0 1
f
f
f
0.01 t0.00 0 . 0 1 t0.00 0 . 0 2 f0.00 0.03 t 0.00 0.12 =k 0 . 0 1 0.03 f 0.01
0.00 0.01 O.OOb 0.27 f 0.01b 0 23 t 0 01 0.20 f 0 . 0 1 0 38 * 0.01h 0.12 t o . 0 1 0.09 0 16 0.34
f
t i
others sufficiently for identification.
0.03 t 0.00 0.11 f 0.01 0.03 f 0.01 0.10 f 0.01 0.16 t 0.01 0.34 =0.03 0.26 t0.01b 0.22 fO.02 0.20 f 0.01 0.36 O.Ola 0 . 1 2 fO.01 f
f
f
ANALYTICAL CHEMISTRY
172 Table X.
Buffer Mixtures
Molarity and Proportions of Salts 50 ml. of 0.2 M KCl 97.0 ml. of 0.2 M HCl 5 nil. of 0.067 M KC1 10.66 ml. of 0.067 M HCI 2 4 15.42 ml. of 0.067 M NazHPOb 12.92 ml. of 0.067 M citric acid 8 ml. of 0.067 M KHzPO, 2 ml. of 0.067 M NazHPOi 6.2 56 ml. of 0.067 M Na,zHPOI 10 ml. of 0.067 M KHzPOa 7.5 50 ml. of 0.067 M boric acid and KC1 8.55 ml. of 0.067 M NaOH 8.4 50 ml. of 0.067 M boric acid a n d KC1 21.30 ml. of 0.067 NaOH 9.0 50 ml. of 0.067 M boric'acid and KCI 38.82 ml. of 0.067 M N a O H 9.7 10.0 50 ml. of 0.067 M boric acid and KC1 43.90 ml. of 0,067 M S a O H 62.4 10.5 100 ml. of 0.02 N boric acid, KHzPOc, and phenylacetic acid ml. of 0.1 N S a O H 16.50 ml. of 0.067 M N a O H 1 1 . 2 50 ml. of 0.067 M NapHPOi 50 nil. of 0.067 M N a O H 12.0 50 ml. of 0.067 M NazHPOa
PH 1
++
++ +
+ +++
++
+
(paper buffer a t pH 6.2) gave excellent separation of lysine and arginine.
indicated that the relative positions of the amino acids will remain the same and the R , values obtained will not vary by more than the indicated amounts, provided that the operating temperature is not allowed to vary more than * 1' C. Apparently, the greater the temperature variation, the more variable are the R/ values (Table XII). At present a conqtant-temperature room is being considered for housing the chromatographic chambers. In the case of solvents such as collidine or lutidine (Table XII) the Rj values are generally lower a t the higher temperature, whereas with such solvents as phenol little difference is noted. Vapor Saturation. The necessity of having chambers airtight in order to keep them saturated with vapors of the solvent and water is discussed frequently in the literature. In addition, when working with tall chambers it seemed advisable to line the cham-
Certain R/ values noted in Tables I to I X indicate the amino acids separated from all others sufficiently for identification in each solvent a t each pH. Several good separations other than those shown in Figure 2 are tyrosine in p-cresol saturated with buffer of pH 9.0 (paper buffered a t pH 9.0), hydroxyproline in p-cresol saturated with buffer of 12.0 (paper buffered a t pH 12.0), and cystine in water-lutidine (paper buffered a t pH 6.2). Cystine separates from the others in this case, but it usually streaks, owing t o decomposition during the chromatographic run. S o r leucine can be separated sufficiently from all others, a t least for qualitative purposes, in benzyl alcohol saturated with buffer of 9.0 (paper buffered a t pH 9.0). The data accumulated in these tables also permit selection of one or more solvents buffered a t one or more pH values for chromatogramming an unknown-that is, the fact that a given amino acid does not usually have the same R/ values in all solvents and a t all pH values can serve as a useful means of verifyin 4 an identification. ,4n example can be shown from Figure 2. The identification of alanine could be further assured by running an unknown in both phenol (spot 6) and mcresol (spot 7 ) . This information on any amino acid can readily be found in Tables I to I X .
I1 t lIi 9 a t 4 ? I
0
I,
14
I)
l5
DISCUSSION
Various factors that influence the success of this method have been investigated. Variation in R,. It is perhaps debatable whether the variation in the R f values of the amino acids is due to changes in pH, ionic strength, and salt composition of the buffer, or to changes in the water solubility of the solvent due to all three of these factors. The ionic strength ( 4 ) of the buffers was calculated, but no correlation between ionic strength and variation of Rj values could be determined. As a further check, an experiment was conducted using buffers of 0.067, 0.09, 0.143 molarity (Table XI). No significant variations in the Rf values seem evident. Whether the specific salts used in a buffer have any effect on the Rf values is still an open question. In one case when two different buffers of the same pH were tried, very different R, values were obtained. This might have been due to an unfortunate choice of buffer; it was thought that the buffer reacted with the solvent, but this has not been further investigated. The data available a t present seem to indicate that the primary factor controlling the R/ values is pH, because the R/ values fall along a smooth curve, even when various buffers are employed to obtain the pII range from 1 to 12. Heading. Some of the amino acids head in certain solvents, generally between a pH of 6 to 9 (Tables I to I X ) . This is believed to be due to ionization or dissociation of the amino acids in this particular pH range. Whatever the cause, this phenomenon of heading prevents useful separations from being realized. Temperature. Some of the data presented here were collected during the summer months a t higher temperatures than normal, and those who attempt to duplicate these results may not obtain the same absolute R/ values. However, experiments have
0
el7
c
E
F
G
Photocopies of Typical Buffered Chroma tograms A.
Phenol (24 hours) saturated with 12.0 p H buffer, paper buffered a t p H 12.0 1. Aspartic acid ?. Glycine 2. Glutamic acid a . Threonine 6. Alanine 3. Serine B . m-Cresol (40 hours) saturated with 8.4 pH buffer, paper buffered a t p H 8.4
7. Alanine 8. Arginine. Alanine and ar mine arc not always separated so well, unless pH. etc., are carefuqly controlled. 9. Hydroxyproline 12. Valine in. Tvrosine 13. Methionine 11. H k i d i n e C. Benzyl butyl alcohol (40hours) saturated with 8.4 pH buffcr, paper buffered a t p H 8.4 14. Isoleucine 15. Leucine D . o-Cresol (24 hours) saturated with 6.2 pH buffer, paper buffered a t p H 6.2 16. Phenylalanine E . Collidine (24 hours) saturated with 9.0 pH buffer, paper buffered a t p H 9.0 17. Tryptophan F . Benzyl butyl alcohol (12 hours) saturated with 1.0 p H buffer, paper buffered a t pH 1.0 18. Proline G. Water-lutidine (24 hours) 19. Lysine. Usually separates better than this example.
V O L U M E 2 3 , N O . 1, J A N U A R Y 1 9 5 1 Tahle YT.
173
R j Values of Amino Acids as a Function of Molarity of Buffer 7 5/Benzyl Butyl Alcohol 7 . j a 0 067 .If 0 09 M 0 143 M
Aspartic Glutamic Lysine Arginine Histidine
0 00 0 00 0.00
0 00 0 00 0.00 0.01 0.03
0 01 0 01
7 5/m-Cresol 7.5" 0 09 M 0 143-g 0 01 0 00 0 00
0 067
,M
0 01
0 01
0 01
0.02 0.03 0.03 o 03 0.01 0.08 0.09 0.08 0.02 0.26 0.29 0.26 Cystine 0.01 0.02 0.02 0.00 0.01 0 02 0.02 0.03 0.04 0.03 0.03 0 ox Serine Glycine 0.02 0.03 0.03 0 05 0.04 0 04 0.07 0.08 Threonine 0.03 0.04 0.04 0 07 n.12 0.11 Alanine 0.04 0.04 0.03 0 11 0 1.5 0.13 0.25 0.27 0.16 0 27 Tyrosine 0.20 Hydroxyproline 0.03 0.05 0.03 0.18 0 18 0.12 0.15 0.10 0.41 0.39 Valine 0 39 0.19 0.16 0.54 0.53 0 53 Methionine 0.18 0.70 0.36 0.38 0.36 0.72 Trvptophan 0 72 0.74 0.73 0.36 0.35 0.30 0 73 Ndrleiicine Leiicine 0.67 0.30 0.29 0.68 0 67 0.26 Isoleucine 0.26 0.27 0.65 0.64 0 64 0.23 Phenylalanine 0.40 0.39 0.38 0.78 0.81 0 81 Proline 0.09 0.10 0.63 0.62 0 62 0.08 Sotation iised for convenience, 7.8/benayl butyl alcohol indicates t h a t benzyl butyl alcohol satiirated with buffer of p H 7.5 was used in chromatography trough. Second 7.6 indicateP that paper mas buffered at p H 7.5.
0.00 0.03
~-
Table X11.
R: \-allies of .Amino Acids as a Function of Temperature 2 0 'Phenol 2.0 L-niined Chamber 1 5 . 5 O C. 21.0'' C. 2 6 . 5 O C.
Asrmrtir Glutainic Lysine -4rginine Histidine Cystine Serine Glycine Threonine Alanine Tyrosine Hydroxyproline Valine Tryptophan Methionine Norlriicine Leiicine Isoleucine Phenylalanine Proline
0.18 0.28 0.21 n.3.i 0.27
0.17 0.27 0 .. 2328 0.31
0.18 0.28
....
.... 0.20
.... 0.22
0.20 0.22
....
n
21
, . .
n.30
o
52
....
.... .. .
0.72
n';x
....
.. .
0 . 24 30 0.27
0.2.;
. .. ....
0.50
....
.
.,
0.72
....
0.7.:
0.78
0.73
0.86 0.80
0.81 0.79
0.7'1 0.80
....
. .
Q.O/Collidine 9.0 Lined ChambTr 2 1 . 5 ' * 1' C. 27.5O * 3 O C. 0.03 * 0.01 0.03 * O.O0
0.02 0.02
o 01 * O.O1
:;::::$ ::::2;:"0 0 . 1 6 t0.01
0.03 n.10 n . 11
* 0.01
+
0.01
* 0.01
:::; * n :,::* 0":;
0.55 0.01 0.13 *0.02 2.5 + 0 . 0 1
0.43 0.02 0 . 3 9 * 0.01 0.35 * n . o i n , 2n * oo. ,oo2i o.,j1
0.12 1 0 . 0 1 0.02 0.07
0.08
01 0 02
i. 0 . 0 2
:::!:;:; i-
0.48
* 0.03
0.20
*o
0.40 0.35
* 0 02 * 0.03 * 0.04
0.11 * O 01
0.31 0o ., 4l 56
_________
bers ( 1 0 ) with a suitable material that, would dip into the liquid at the bottom and become saturated with the solvent-saturated water, so that the chamher would be kept saturated with the vapors of hoth, throughout its ent'ire height. Four different chanihrrs werv prepared : one contained water in the bottom, another o-cresol-saturated water (u-ater-rich), another watersaturated o-cresol (solrent-rich), and the fourth w : i ~k f t dry. The troughs in all thc chamhers n-ere filled with wattTr-satnrated n-crcsol and the paper was buffered . a t pH 6.2. Table XIII. Table XIII the data in these four chambers are presented with the dat'a previously collected in a chamber set, up in the convent,ional manner-namel~-. unlined with water-sat,urated o-cresol in the trough and o-cresol-saturated water (water-rich) in the bottom of the chamber. T,argc $T;;;$ca;itd differences in the Xj values of t,rgptophan and Lysine phenylalanine occurred under these varied condiArginine tions. The small differences noted between the Histidine R, values obtained in the chamber containing water and that containing thc water-rich layer probablJGlycine Threonine can lie explained by the fact that the chamber conAlanine tainitrg water in t,he bottom apparent]?. became ~$$!~pro~ine saturated by diffusion from the vapors of the chamValine Xlethionine bey until it. wa8 essentially water-rich; tho chamber Tryptophan containing water took longer to come to equilibrium and t o give reproducible Ri values th*,n did Isoleucine Phenylalanine the chamber containing the water-rich layer. BeProline cause the partial pressures of each component are
:z2p
*0
2 :::: 02
* nn , 02 n2
the same in two liquid layers a t equilibrium, i t might seem surprising that such different R, values were obtained when the solvent-rich layer was used in the bottom of the chamber. The explanation seems to be that the paper used for the chromatogram adsorbs water more readily than it does solvent. Thus, the cresol solution is no longer saturated with water. Because a deficiency of water exists in the chamber, the equilibrium is upset and the R , values are lower. Collection of data from the chamber which was left dry was discontinued after the second day, for it rvas obvious that a similar, but somewhat more exaggerated, condition existed. The R, values of tryptophan and phenylalanine are greater and more reproducible in the lined water-rich chamber than in the similar conventional but unlined chamher. All the data collected in Tahle XI11 were ohtained by buffering the paper only. It is preferable to buffer both the solvent and the paper. Buffering. Buffering the solvent only, by equilihration in a separatory funnel, did not prove SUPcewful, as heading and pink fronts developed, aoften occurs on an unbuffered chromatogram when other substances are not added to the chamber or thc solvent. Buffering only the paper gave reproducible R , values and usually eliminated the previously mentioned difficulties. However, when the paper only is buffered, a large pH gradient is observed from the top to the bottom of the paper as determined by indicators. Higher molarity buffers were used to buffer the paper and less of a pFI gradient was observed, but with some solvents wat,erlogging occurred. Consden, Gordon, and Martin (6) observed a similar condition in hydrolyzates, or mixhres, in which t,he ratio of soluble inorganic salts to amino acids was high: this could be eliminated by impregnating the
paper wit,h salt and using the solvent equilibrated with saturated salt solution. Thus, waterlogging was eliminated by buffering both the paper and the solvent and at the same time less of a pF1 gradient was observed from the top to the bottom of the paper. pH of Solution Chromatogrammed. Another annoying condition was encountered when mixtures of amino acids were chromatogrammed. When lysine, arginine, and histidine were chromatogrammed singly, reproducible R , values were obtained, but when
Rj \'slues of .Amino Acids as a Function of Vapor Content of Chamher Unlined Dry
....
....
.... .... .... ....
n
0.5
0.10 0 07 0.17 0.29 0.35 0.29 0.47 0.43 0.42 0.46 0.50
Chamber Lined Lined Bottom of Chamber Solvent-rich Water-rich water
~~~
Lined
. .... .. .. ..., .... , . , . ,
0.00
0.01
0.00 * 0.00 0.02 1 0 . 0 1 0 . 0 6 * 0.01
n no * 0 . 0 0 n 03 . t n . o i
0.04 * 0.00 0.07 t 0 . 0 1 0.06 * o . o i 0.10 + o . o i n 08 * n . n i 0.19 O ,l4 * *0 O .. 0O I3 0.36 1 n . m
0.17 0.31 0.46 0.55 * n . m 0.62 0.60 1 0 . 0 5 n 63 0.56 + n . o ? n , s 7 0.50 - 0 . 0 4 0.53 0.66 + 0 . 0 1 0 . 7 4 0.63 1 0 . 0 2 0.59
0.46 * n . n 3
.. .. .... . ..
0 . 0 0 10.01
;t00.. 0 2 1 2 10.02
h0.02
10.03
+n.oi +o.oo
*0.02 *0.02 10.01
.., . . . ..
Unlined Kater-rich 0.02 0.05
0 00 0.00 0 06
* 0.01 * 0.04 *o.oo * 0.01
* 0.02
0.00 * 0.01 0.03 k 0 . 0 1 0.04 * 0.01 0 . 0 6 ' ~ t t . o l 0 . 0 7 ==o.oi 0.09 * o . o i 0.10 1 0 . 0 2
... ....
** 0n . 0012
0 . 1 85 **0 0. 0. 022 0.32 * o . o 4 n 43 *o.o3 0.46 * o . o 7 0.60 * o . o 3 0.53 *0.02 0.50 * 0 . 0 3 0 . 7 4 1 0 . 0 4 0 64 * 0 . 0 6 0.62 t 0 . 0 2 0.60 1 0 . 0 3
0.18 0.32 0.47 0.62
+o.o3
1 0 02 10.04 0 . 6 3 1 n 08 0.58 * 0 . 0 2 0.54 1 0 . 0 1
-
~~
.__ -
-
174
ANALYTICAL CHEMISTRY
these amino acids were present in mixtures, often they could not be located. As an example, tyrosine alone has an R/ value of 0.29 in m-cresol saturated with 9.0 p H buffer (paper buffered a t pH 9.0) and histidine an R / of 0.35. When these two amino acids were chromatogrammed together, histidine turned up a t Rt 0.14 and tyrosine a t R/ 0.26. This particular combination gave a clue, for when the tyrosine solution was prepared, it was acidified to get the tyrosine completely into solution. $pparently the acidity of the tyrosine solution was sufficient to override the buffered solutions and the buffered paper, so that when these amino acids were spotted together and chromatogrammed, a serious shift in R/ values resulted, particularly histidine. Subsequent experiments showed that when the solution or mixture of amino acids was first adjusted to a pH between 5.5 and 7.5 (1, S), the R, values of the amino acids in mixtures were reproducible. Unfortunately, this imposes another problem which has never been completely solved; in this pH range cystine is nearly quantitatively precipitated and tyrosine is rather insoluble. The only solution a t present seems to be to work with more dilute solutions and to build up a concentration on the paper by repeated spotting before chroniatograniming. SUMMARY
Each of twenty amino acids may be separated from all others by employing several one-dimensional buffered chromatograms. The paper chromatograms are buffered by dipping the paper into buffer of the desired pH and molarity and air-drying, and by equilibrating the solvent with the same buffer rather than with water. Different, but more reproducible, Rfvalues are obtained if all chambers are lined with a filter paper which dips into the solvent-saturated buffer in the bottom of the chamber. All solutions must be adjusted to a pH between 5.5 and 7.5 before spotting in order to prevent heading of some of the amino
acids in mixtures and to obtain reproducible R/ values for lysine, arginine, and histidine. The temperature in the room housing the chromatographic chambers must be thermostatically controlled to * 1O C.to prevent condensation on the paper and the resulting streaking and elongation of spots. Particularly with solvents such as collidine, the more the temperature varies, the more the R/ values of t,he amino acids vary. ACKNOWLEDGMEYT
The author is grateful to Gotfred Haugaard and R. A. Sullivan of the Sational Dairy Research Laboratory, Inc., for many helpful suggestions and criticisms, and to James A. Mills of this same laboratory, who did many of the routine experiments. LITER4TURE CITED
(1) Aronoff, S., Science, 110, 590 (1949). (2) Britton, H. T. S., “Hydrogen Ions,” New York, D. Van Nos-
trand Co., 1942. 13) . . Bull, H. B.. Hahn. J. W., and Baptist, V. R., J. Ant. Chenz. Soc.. 71,550 (1949). (4) Clark, TV. bl., “Topics in Physical Chemistry,” Baltimore, hld., Williams and Wilkins Co., 1948. (5) Consden, R., and Gordon, A. H., N a t u r e , 162, 180 (1948). (6) Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J . , 38, 224 (1944). (7) Haugaard, G., and Kroner, T. B., * J . Am. Chem. Soc., 70, 2135 (1948). ( 8 ) Karnovsky, M. L., and Johnson, M. J., ANAL.CHEW,21, 1125 (1949). (9) Miettinen, J. K., and Virtanen, A. I., Acta Chem. Scand., 3, 469 (1949). (10) Winsten, W.A , , Science, 107, 605 (1948). RECEIVEDApril 25, 1950. Presented before the Division of Biological CHEMICALSOCIETY, Chemistry a t the 117th Meeting of the AMERICAN Philadelphia, Pa.
Determination of Trivalent and Tetravalent Manganese W. S. FYFE C’niversity of Otago, Dunedin, New Zealand With acetylacetone, the manganic ion forms a stable complex which has no oxidizing action on acidified potassium iodide. In the presence of acetylacetone, manganese tetrachloride loses chlorine and forms the stabilized trihalide. The chlorine evolved in this reduction chlorinates the acetylacetone. This chlorination can be reversed in the presence of acidified potassium iodide in which case the iodine is liberated. Thus, by estimating the iodine liberated i t is possible to determine the tetrahalide in the presence of the trihalide.
A
C E T n A 4 C E T O x Eis selective in stabilizing the ferric and manganic ions (3). When manganese dioxide was dissolved in hydrochloric acid in the presence of acetylacetone, no chlorine was evolved; when treated mith potassium iodide the solution liberated iodine as follows:
+ 1/2Cl2 = KCI + ‘/Jz
MnC14 = iLInCl8 ‘/&I2
+ KI
The reactions involved in the solution of manganese dioxide in the presence of acetylacetone are: Mn02
+ 4HC1 = 1InCl4 + 2H20
+
21vInC14 z(CH,COCH&OCH,) 2MnC1,(CH&OCH~COCH3),-1
(3)
=
+ CH,COCHClCOCH, + HCl
(1)
(4) and with acidified potassium iodide:
(2)
The rate a t which this liberation of iodine occurs is identical with the rate a t which chlorinated acetylacetone reacts with potassium iodide, and is affected by both iodide and hydrogcn ion concentrations (6).
CH3COCHCICOCH3
+ 2HI
=
CHaCOCHzCOCH,
+ HCl + I1
(5)
Equation 5 involves a dechlorination; the chlorine evolved liberates iodine, which combines with potassium iodide to form polyiodides. These are not efficient iodinating agents. As the
