Chamber Size and R, Values of Amino Acids R. A. CLAYTON’ Department o f Biochemistry, George Washington University School o f Medicine, Washington 5,
Because of the extreme importance of R , values as physical constants, it is unfortunate that they are not more reproducible. This study was undertaken in an attempt to determine the effect of chamber size on the Rt values of amino acids. The term, “critical volume,” has been introduced to define the volume of solvent necessary to saturate a specific chamber under a given set of experimental conditions. I t has been found that when the amount of developing solvent used exceeds the critical volume, the R , values will be constant and at a minimum; below the critical volume the values will be at a constant maximum. Sotation of these volumes in future communications would greatly aid in the standardization of paper chromatography.
D. C.
layer was then used. Because on standing this solvent system propagates a fourth component, butyl acetate, the solvent v a s always prepared immediately before use. I n order t o study the influence of chamber size on Rj values, three papers were developed simultaneously in different-sized chambers of volumes of 37, 14, and 8 liters, hereafter referred to as chambers -4,B , and C. respectively. The distance from the surface of the developing solvent to the base line was kept constant. All experimental work was carried out in a constant temperature room at 25“ i l oc . After development, the air-dried chromatograms were sprayed with a butyl alcohol solution of ninhydrin (0.125%) in the usual manner. Over 100 chromatograms were run (13 amino acids pei paper) in an attempt t o clarify the influence of chamber size on Ri values. RESULTS AND DISCUSSIOZ
T
H E importance of paper chromatography as a research tool is unquestioned. I n many instances, the R j value has become as important a physical constant as the melting point. I t is unfortunate, therefore, t h a t it is frequently ex1 remely difficult, if not impossible, to reproduce published R , values with a given solvent system. This difficulty is generally circumvented by a comparison of the Rj value of a standard sample with the R j of the compound in question. Cnfortunately, such an approach is not aln-ays possible. S o attempt will be made in this communication to survey the literature completely with respect to variables in paper chromatography. Strain’s review article on chromatography ( 8 ) , as well as the articles by Cassidy ( 2 ) , Kon-kabany and Cassidy ( d ) , and Block, Le Strange, and Zweig ( 1 ) serve well in this capacity. These authors discuss the influence of such factors as t’ype of paper, pH, purity of solvents, and temperature. Landua, Fuerst, and Alvapara ( 5 ) reported on the effect of the p H of the sample on the observed R , values of amino acids. McFarren ( 6 J , working with buffered developing solvents, showed the variation of R, values of amino acids with the p H of the developing solvent. Underwood and Rockland ( 7 , 9) reported on the effect of the aforementioned factors on small scale paper chromatography. However, even though all these variables be rigidly controlled, R , values, with a given solvent system, have been found to var?- as much as 507, from one laboratory to another. A marked deviation in hi values of amino acids with varying chsmber sizes was first reported by Clayton and Strong (3). The present work was undertaken in a n attempt to elucidate further the relationships between chamber and solvent volumes and R j values. The common n-butyl alcohol -acetic acid-water system a-as chosen for this study.
T o report over 1300 R , values would, in this author’s opinion, be inadvisable. 111 an attempt to present these data in a clear and concise manner, the results obtained with five representative amino acids under varying conditions of chamber size and solvent volume have been reported. The effect of chamber size on R , values is typified by the data in Table I. From these data. it appears t h a t when a given volume of solvent (150 ml.) is used in each chamber, the R , values of the amino acids increase as thp volume of the chamber increases. These increases were fairly constant with the 13 amino acids studied, varying from 20 t o 27%. The effect of varying the solverit volume n-ith a given chamher was studied nest. The results of such a study as carried out n-ith chamber d are shown in Table 11. These data show that the R j values are constant and at a masimum n-hen 300 ml. of solvent (or less) is used, and are a t constant minima n-hen more than 375 ml. of solvent is used. This volume of solvent, above which the Rr value is a t a minimum, and below which it is at s masimum, has been termed the critical volume. \Then such a study was made with chamber B , no discrete critical volume was found. K h e n less than 75 ml. of developing solvent i w s used, Rj values were a t constant maxima, in good agreement n-ith the maxima obtained in chamber A . Above 175 ml. of developing solvent, the values were a t the same constant minima, as observed in the larger chamber. Hoivever, brtn-een 75 and 175 ml., the R , values were approsimately midwaJbetween the maxima and minima. At first it v a s thought that
Table I.
Effect of Chamber \-ohme on Rj \-slues (150 nil. of developing solvent)
A (37 liters)
EXPERIMENTIL
Preparation of Samples. The following amino acids were used in this study: L-tyrosine, obmethionine, DL-leucine, DLtrj-ptophan, bcysteine, bcystine, L-arginine, m-alanine, Lhistidine, nL-valine, L-glutamic acid, nL-aspartic acid, and glycine. Approximately 20 mg. of each amino acid was dissolved in 3.0 meq. of hydrochloric acid and diluted to 10 ml. with water. Operating Procedure. Whatman No. 1 paper was used as supplied by the manufacturer. Two to 4 y of each amino acid a-as applied t o the paper a t the base line a t point,s 1 em. apart. Ascending chromatograms were developed for 12 to 16 hours with a solvent system which rTas prepared by equilibrating 300 ml. of 7Z-bUtyl alcohol, 300 ml. of viater, and 72 ml. of glacial acetic acid in a separatory funnel. After thorough shaking, the twophase system was allowed to stand for 15 minutes; the upper
Table 11.
Ri x 100 B (14 liters)
Effect of Solvent Volume on Rr Values in Different Chambers Chamber B
Chamber d M I . of Solvent 150 Leucine Tryptophan hletliionine Valine Tjrosine
Present address, Research LaboIatory, T h e .kmerican Tobacco CO., Richmond 24, Va. 1
904
81 61 62 64 50
C ( 8 liters)
300
375
400
700
Ri X 100 80 61 63
65 50
68 54 51 51 44
66 53
67
50
__
49
50
79 60 62
51
51
b3
44
44
50
54
1\11, of Solvent 7 5 100 150 175
78 62 65 62 51
R j X 100 73 71 5; 5: 54
56 48
65 53
JJ
49 52
48
43
200 66 53 29 50 43
V O L U M E 28, NO. 5, M A Y 1 9 5 6
905
Table 111. Constancy of R j Values with Respect to Critical Volumes Above Critical Volume -4 (375 nil.) B (175 ml.)
C Rf
Tyrosine
41
43
x
Below Critical Voiuni? A (350 ml.) B ( 5 0 rnl.) 100
41
3C
'OT
25t
1CHAMBER A (37L.1
VOLUME OF SOLVENT (MI3 Figure 1. Comparison of R, values of leucine in three different-sized chambers with varying amounts of developing solvent Maximum Hi,81; minimum, 65
these difierences w e i ~not significant, but, repeated investigation shov-ed other\vise. These d a t a are also presented in Table 11. \l-hen the effect of varying the solvent volume was studied with the smallest chamber, it v a s found t h a t the critical solvent volume for this %liter chamber was so small that it n-as, for all piwtical purposes, a l x a y s excceded. Thus, n h e n as little as 20 ml. of solvent \vas used, the R , values for the 13 amino acids studied were a t t h e same constant minima as obtained in t h r two larger chambers. Figure I shows the variation in R , value of the amino acid leucine in the three chambers with different amounts of developing solvrnt. T h e maximum and minimum valucs olitainrd in t h e three chambers are in good agreement, but t h r witical solvent volume differs markedly according t o t h e volume of the chamber. If these variations iri I Z j vrilucs are due t o critical volumes alone, then one should be able t o observe constant values in any chamber, wgnrdlrss of its volume, if the critical volume of that chamber is known. The constancy of R , values Tvith respect t o ciitical volumps is shon-n in Table 111. I n this table. the XI values obtained in the t h r w different chambers are compared when the volume of polvent in each chamber is either a h o w or ~ C I O Mthe critical volumc of t h a t chamber. Pre-equilibration and Chamber Volumes. The effect of preequilibration on chamber size phenomena was investigated. With descending paper chromatography, probably the moat eoninion equilibration technique is t o suspend the papers in a sealed chamber above a given volume of solvent for 2 t o 4 hours, and then t o introduce additional solvent into the trough. Because the results of such a study would depend on the amounts of developing solvent used-i.e., whether i t was above or below the critical volume of the chamber-this technique could not be used in connection with this work. Instead, the paper \vas suspended
above the developing solvent in a sealed chamber for 3 h o u i ~ , after which i t was lowered into the developing fluid. Development time x a s varied from T to 13 hours ivith no significant Rj differences with a given volume of solvent. T h e effect of preequilibration was studied in the three chambers u-hen the amount of developing solvent was above and belon the critical volumes of the chambers. When the amount of solvent \vas above the critical volumes of the three chambers, there were no sigiiificaiit differences between the values obtained n-ith or ivithout pi'requilibration. Of the 13 amino acids tested, only t ~ ~ o x i r :ind ie tryptophan shoived a variation n-ith pt,r-c~quilihi~ation.The R , values of these two amino acids increased at)out 870 ivheii I ) I ( ' equilibrated. S o significant rhange n-as o l x r ~ ~ v c twith l the othei. amino acids tested. Liken-ise, when the amount of solvent used \v:i+ lc~s?than the critical volume of chamber '4, pre-cquilihratiori 1i:itl no rft't,c,t on the R , values. H o w v e r , rvith chamber H there \v decrease in R , values for leucine, methiorkc. a i ~ dvaline under these conditions. Slight decrrases w i ' e also olwerved for ti,yptophan and tyrosine, but it is questionable Ivhether thcee chaiigrs are significant. These d a t a are presented in Table I\-. I t is difficult t o rationalize these results. I t i? known t h a t butyl acetate forms under the experimental conditions n-ithin :t short time, aiid it might be argued t h a t the introduction of rrltttively large amounts of this fourth romponent a t the beginning of the developing period would he expected t o alter obseivtl values. It also seems tenable t h a t such an alteration c.oulcl result in variations with some amino acids and not with others. However, one must also explain the lack of variation in R j values with and n-ithout pre-equilibration in chambers d and ('>and indeed, the absence of it pre-equilibration effect in chamber H when the volume of solvent n-as above the critical volunie of that chamber. I t is felt that these pie-equilihratioii studies only rmphasize the importance of chamber size because the variations in R , valucs caused by these differences in chamher volumes cannot be circunivented b y pre-equilibration. THEORY
\Yith most xmter-soluble compounds, the R , values iricrease as the xmter content of the developing solvent is increawd. In this system, butyl alcohol is much more volatile than n-ater, and unequal vaporization of the components of the solvent s!.stem results. Because the composition of the vapor and liquid phases a t equilibrium is dependent on the partial pressures ~ i i d mole fraction of each component, factors which n-ould favor IFmoval of t h e mnst volatile component would r r d t in :I tic,veloping liquid richer in n-ater. K h e n a given volunie of solvent is used, the larger the chamber, the greater the vaporization of the butyl alcohol, and the richer the liquid phase becomes v-ith respect t o water. Below t h e critical volume, unrqual vapoi,ization of the solvent components results in a developing liriuitl richer in water, and maximum R f values are observed. The constancy of R , values belotv t h e critical solvent volume must he ascribed t o t h e partition coefficients of the amino acid hrt\\-rc>n the mobile and stationary phases, and t o the maximum :iniount of Tmter n-hich can be held by the paper under the pwvniling t'xperimental conditions.
Table IY. Effect of Pre-equilibration on R f \-slues i n Chamber B (50 ml. of solvent)
3-Hour Equilibration
K O
Eauilibration Rj
Leucine Tryptophan Methionine Valine Tyrosine
X 100
79
68 56
83
52 52
60 62
50
47
ANALYTICAL CHEMISTRY
906 SUMMARY
T h e relationship between chamber volume and solvent volume has been investigated. A new term, critical volume, has been introduced which defines the volume of solvent necessary t o saturate a specific chamber under a given set of experimental coriditions. T h e R , values of amino acids and presumably of other water-soluble compounds (preliminary work with car boh\ drates gave results similar t o those reported here) vary with a given solvent system according t o the volume of the chamber, the volume of developing solvent, and the volatility of the organic solvent. ( I n preliminary work only slight variations in the R; values of amino acids were observed \Then a less volatile org:tnic solvent-i.e., phenol-water-was used.) These variations a l e not eliminated by pre-equilibration. It is felt t h a t the most repi oducible chromatograms can be obtained when small chambers are used because the amount of solvent generally eweeds the critical volume of the chamber. I n v i e x of this evidence,
the difficulty in reproducing R i values from one laboratory t o another might be greatly mitigated if authors report chamber and solvent volumes as part of the description of evperimental coiiditions. LITERATURE CITED
(1) Block, R. J., Le Strange, R., Zweig, G., “Paper Chromatography, A Laboratory 11anuai.” Academic Press, Ken- York, 1957. ( 2 ) Casaidy, H. G., AYAL.CHEY.24, 1415 (1952).
(3) Clayton, R. A , , Strong, F. AI., Ibid., 26, 1362 (1954). (4) Kowkabany. G. S.. Cassidy, H . G., Ibid., 22,817 (1950). (5) Landua, A. J., Fuerst. R., dwapara, J., Ibid., 23, 162 (1951). (6) hIcFarren, E. F., Ibid., 23, 168 (1951). (7) Rockland, L. B., Underwood, J. C., Ibid.. 26, 1557 (1954). (8) Strain, H. H., Ibid., 23, 25 (1951). (9) Underwood, J. C., Rockland, L. B.. Ibid., 26, lS53 (1951). RECEIVED for review September 27, 1935. Accepted March 8, 1956. Dirision of Biological Chemistry, 1’27th AIeeting, hCS. Cincinnati, Ohio, lRIarch 1955.
Radiotracer Studies of Analytical Methods for Styrenated Oil Acids and Esters E. G. BOBALEK, J. R. BRADFORD’, FRED LEUTNERQ,and ROBERT AKIYAMA o f Technology, Cleveland 6, Ohio
Case Institute
TIost analytical niethods for sty renated paint vehicles fail to separate neutral po1)styrene and the oil-acidst>rene copolymer. Armitage and Kut’s method for such fractionation, which depends on differential solubilit? of the copolymer’s calcium soaps and neutral polyst! rene in wet ethjl acetate, was investigated using I adiotracer techniques in polymers prepared from stj rene tagged on the alpha-carbon with carbon-lt. This procedure does not alwajs work. Apparently no method yet exists for determining polymeric species without supplementary anal) sis of the separated fractions. Fractionation data suggest that exten&\c copolymerization of styrene and fatty acids can occur, but that its extent taries with conditions of resin synthesis.
S
T l X E S E drying oil reaction products have been used com-
mercially in paint vehicles for more than 10 years, but the precise chemical nature of these “copolymer oils” is yet unknown. Process conditions of manufacture greatly alter their paint formulation properties, and exact reproducibility of polymerization recipes is a serious problem. Studies of these factors have been hindered by the lack of precise analytical methods for fractionating and estimating the amount of homopolymer and copolymer in tlie product oils. I-arious fractionation schemes have been proposed. Kappelnieier’s ( 4 ) extensive studies led him t o conclude t h a t very little of the drying oil acid escapes reaction with styrene, and probably very little neutral polystyrene is formed-i.e., polystJ-rene unreact’ed with fatty acid. On the other hand, Armitage and K u t ( 1 ) and others ( 6 ) claim to have found a fractionation technique, which, a t least for the reaction product of p-eleostearic acid and styrene, s h o w the presence of considerable quantities of nearly neutral polystyrene. If the latter claim is correct, the .lrmitage-Kut fractionation procedure might be a useful supplement to the Iiappelmeier method of analysis of stgrenated oils and alkyds. This would be trueif neutral polystyrene and the acid copolymer can always he separated in commercial polymers, and if the Armitage-I
