head of the instrument, and rotated for 5 minutes to achieve an even wetness. It was then stopped and 5-pl. aliquots of the mixture to be separated were applied. The rotor was now set a t 400 r.p.m., the developing solution added as a fine stream, and a dire-t current potential of 18 volts per cm. applied. After 20 minutes, a bromophenol blue spot had traveled outward 7 . 5 em. (chromatographic displacement) ; and 2 em. in the direction of the anode [ionographic migration ('7) 1. For separation of amino acids, representative experimental conditions might be illustrated by the fractionation of a solution containing glycine, lysine, and glutamic acid. The conditions were: sample, 0.01M solution of glycine, lysine, and glutamic acid; rotor speed, 400 r.p.m.; flow of rate of developing solvent, 0.6 nil. per minute; buffer and other conditions as above. After 15 minutes, the amino acids had all moved 7.5 cm. from the point of application and mere 1.9 em. apart ( 7 ) . The electrocliroinatogram shown in Figure 5 is a further illustration of the type of separations achieved. It was ohtained from a spot application a t the tn o points indicated by the arrows.
-1 5-pI. aliquot of a solution contain-
ing 0.01 mole per liter each of arginine, histidine, proline, tyrosine, and aspartic acid was applied. The experimental conditions were: phosphate buffer, p H 6.4; ionic strength, 0.008; potential gradicnt, 17 volts per cm.; current, 16 ma.; rotor speed, 316 r.p.m.; temperature, 25" C.; paper, Khatman 3MM, 23 X 46 cm.; flow rate of developing solution, 0.8 ml. per minute; time, 15 minutes. The displacement of the amino acids, in centimeters, from the point of origin to the approximate center of each zone, was: arginine, 8.2; histidine, 8.1; proline, 8.6; tyrosine, 6.6; aspartic acid, 9.0. The distance of separation betn-een the arginine and aspartic acid zones mas 4.5 cm. As the pattern on one half of the electrochromatogram is practically a mirror iniage of that on the other, an authentic sample of one amino acid can be applied to the paper on one side and a mixture on the other. \Then development has been completed, the paper sheet can be folded in half, and the presence or absence of the knon n amino acid in the mixture determined by observing congruence of developed zones. The centrifugal and electrical forces niay be applied independently, in scquence, or simultaneously. It is thus
possible to carry out simple chroniatographic or ionographic as well as electrochromatographic separations. LITERATURE CITED
(1) Clayton, R. A., Strong, F. M., A \ . ~ L . CHEM.26, 1362 (1954).
( 2 ) McDonald, H. J., Bermes, E. IT-., Shepherd, H. G., Chromatog. Methods 2, No. 1, 1 (1957).
(3) McDonald, H. J., Bermes, E. K., Shepherd, H. G., Nuturzc~zssenschafte,L 44, 9 (1957). (1) McDonald, H. J., Bermes, E. IT-., Shepherd, H. G., Proc. Chenl. SOC. (London), 1957, 23. (5) McDonald, H. J., MeKendell, L. V., Naturwissenschajten 44, 616 (1958'1. (6) McDonald, H. J., McKendell, L. V.. Bermes. E. W.,J. Chronzatog. 1, 259 (1958).' ( i )McDonald, H. J., Ribeiro, I,. P., Federation Proc. 17,272 (1958). RECEIVED for review November 17, 1'358. Accepted March 4, 1959. Project snpported in part by a grant-in-aid from the Chicago Heart Association. Leonard J. Banaszak is holder of the New Horizons Fellowship, for 1958-59, supported b>Labline, Inc., Chicago. Luiz P. Ribeiro, postgraduate research fellow 1957-58, acknowledges receipt of a travel grant from the National Research Council Of Brazil. The apparatus described in thls article is manufactured by and avdable from Labline, Inc., Chicago 22, Ill.
Diffusion Analysis of Ions in Gels MARVIN ANTELMAN Hampshire Chemical Corp., Nashua, N. H. SISTER DENISE EBY Saint Joseph College, Emmifsburg, Md. GEORGE 8. KAUFFMAN Fresno State College, Fresno, Calif.
b Methods were needed for qualitative separation of ions and their quantitative estimation by radial diffusion in gels. Mixtures of two and three cations were easily identified after resolution in gelatin and poly(viny1 alcohol) gels. Diffusion distances for cations and anions were noted and compared in gelatin and sodium alginate gels. Individual cations and anions diffuse in accordance with Fick's law, and can be determined b y plotting the variation of radial distance against ionic concentration. Common cations and anions may be quantitatively determined in concentrations between 0.1N and lO.ON, with an This accuracy to approximately 1 technique i s valid for both the cationic and anionic species in a given compound, as each constituent diffuses independently.
yo.
T
objective of this work was to develop further qualitative and quantitative tests for ions by utilizing the property of diffusion in gelatinous media in contradistinction t o solvent flow capillary action and absorption vs. gravity, the properties utilized in paper chromatographic separations. Whereas analytical separations have been accomplished by diffusion alone in gels (f-8),no method has been previously developed for utilizing the property of diffusion (independent of current or other influence) for determining ions quantitatively. This paper describes the partial resolution and detection of some common cations and anions by radial differential diffusion taking place in concentrated gels under the influence of chemical potential gradients, and the behavior of i n d i ~ ~ d u a l i o r i s ~respect ~ i t h to the variHE
ables of time, concentration, and distance when undergoing diffusion, n-hich enables their deteimination. GEL PREPARATION
Thick concentrated gels of gclatin, poly(ving.1 alcohol), and sodium alginate were prepared. Gelatin plates were prepared by softening 16 grams of food grade gelatin in 50 ml. of ccld distilled water, then adding 150 ml. of boiling distilled water instantly to the mass. The hot solution nas poured into Petri dishes to a depth cf 1.5 cm. (watch glasses may also be used). Sodium alginate plates were prepared from Kelcosol (Kelco Co.) in the same manner by dissolving 3 grams of alginate in 100 ml. of hot distilled water. Poly(vinyl alcohol) plates were prepared from Elvanol 72-60 (Du Pont). In this case it was necessary to heat the alcohol (40 grams per 200 ml. of distilled VOL 31, N O . 5, MAY 1 9 5 9
829
water)-water solution for 30 minutes to ensure uniform gelation. Refrigeration hastened gelling.
centric rings. The developing may take from less than a minute to an hour, depending on the reagents concerned. Some combinations of cations and developers failed to give good results. Quantitative. Gel plates were placed on a polar coordinate paper so t h a t t h e periphery of t h e Petri dishes coincided concentrically with the circles on t h e paper. Carefully measured drops (0.05 ml.) of individual ions of known concentration were placed a t the center of the gel plates over the central circle of the polar coordinate paper. This radius, which was constant in all cases, is termed the initial radius, To. Care had to be taken that the drops delivered were perfectly round with no distortion, that the plates were
PROCEDURE
Qualitative. The mixtures t o be resolved (0.lN solutions of cations) were added 0.2 t o 0.4 ml. a t a time from a pipet t o t h e approximate center of t h e plates. After a few minutes had elapsed, noticeable diffusion had taken place. Some gel surfaces were completely dry, while others were wet with excess liquid. Wet surfaces were carefully dried by gently dipping the edge of a filter paper into the liquid. Developing reagents were then added in a sufficient amount to cover the surfaces and develop distinct con-
Table I.
Separation of Cation Pairs in Gelatin by Selective Diffusion
Compounds Diffused
Colors and Ions Inner Outer Purple, Au+++Hg++, Yellow, Hg++ Orange brown, Fe+++, Yellow-green, Cu++
Developer
-..++
(711
C U ( N O ~ )HgClz ~,
Oxine
HAuCla, C U ( N O ~ ) ~Anthranilic acid Cu(NO& Bi(N03)~ Oxine Hg(N03)1,Cu(N03)~ Cyanuric acid Bi( NO3)3, Cu(NO& Oxine Hg(N03)z, Pb(N0a)z KI FeC13, Xi( NO& Oxine Bi(NO3)2,Hg(N03)z K2CrOd Zn( NO3)2, FeC18 FeC13,Hg(N03)2
&Fe( CN), Cyanuric acid
Ni(NO3)2, Hg(?;O3)2 Cyanuric acid t rubeanic acid Mn( S03)2,Hg( N 0 3 ) ~K4Fe(CN)6
+
Ni(XO3)2, Co(N03)Z NaaPOa dimethylgly oxime SnClZ,?\li(N03)2 Rubeanic acid ~
Table
Compounds Diffused
II.
Yellow-green, Cu++, HZ++ Purile brown, Au+++, c u ++ Whitish yellow, Cu++, Bi+++ Orange, Hg++, Cu++ Gray, Bi+++ Cu++ Brown, HgfC, Pb++ Brown, Fe+++,Ni++ Yellow, orange, Bi+++, Hg++ Brown, Zn++, Fe+++ Yellow-brown, Fe+++, Hg++ Yellow, Xi++, Hg++
Yellow, Hg++ Violet, Cu++ Yellow-green, Cu++ Violet, Cu++ Green, Cu++ Yellow, P b + + Green, Ni++ Yellow, Bi+++ Yellow, Z n + + Green, F e + + + Violet, Ni++
Yellow-green, &In++, Hg++ Rust, CO++, Ni++
White, Mn++
Brown, Ni++, Sn++
Violet, S i + +
Pink, Si++
~~
Separations of Three Cations in Gelatin by Selective Diffusion
not jarred, and that the tempeorature remained constant within 1 O or 2 , The radii traveled by the ions after specific time intervals from r o were measured for various concentrations. Measurement was facilitated by rendering invisible ions visible with a suitable developer. This arrested the diffision a t the time interval, necessitating separate ion development for each time interval. Two techniques were used to measure the diffusion radius, r, of an ion. One technique consisted in projecting the image of the ionic zone with an opaque projector onto a screen, and measuring distance r, on the screen with a millimeter ruler. Another method was t o span r with a fine bow divider from a drawing set, then lay the bow divider against the millimeter scale of a Vernier caliper which could be accurately read to millimeters and estimated to tenths of millimeters. The first technique was useful for relative readings and comparison, while the second gave the actual distance traveled by the ion in the gel system. The actual readings were recorded and calibration curves were made for various ions by plotting the distances traveled as ordinates against the logs of the concentrations of the diffused ions for constant time intervals. Time was plotted as abscissa, US. distance traveled for ions, holding concentration constant. Students were then asked to prepare standard series of solutions of various ions, duplicate the above procedure in the same medium for the same time interval, and then construct calibration curves of log concentration us. distance for the time interval. The students were then given unknowns of the ions which they diffused for the same time interval, and asked to evaluate the concentrations of their unknowns by measurement of r and comparison with their calibration curves. Compounds were diffused and r of both the cation constituent and the anion was measured and compared for a single compound.
Colors and Ions Inner zone Middle zone Brown, Fe+++,Hg++, Blue, Fe+++,Ni++ Ni++
Outer zone Violet, Ni++
RA
Red, Mn + +,Cu ++, Xi++
Purple, Xi++,Mn++
White, M n + +
RA
Brown-green, Ni ++, Cu++, Fe+++
Blue-green, Ni++,Cut+ Violet, X i + +
RA&KF
Blue, Mn++,Ni++, Fe+++
Violet, Xi++, M n + +
White, Mn++
RA&KF
Brown-green, &In++, Violet, Cu++,Mn++ Cu++, Hg++
White, Mn++
KF
Brown, Mn++, Hg++, Maroon, Mn++,Cu++ White, Mn++
Qualitative. Many cation combinations were qualitatively resolved in gelatin (Tables I and 11) and Elvanol (Table 111). Considering t h e results obtained here in conjunction with previous results ( I ) , 0.1N solutions of cations when mutually diffused in twos or threes will fall into the following category, in order of greater diffusion distance:
KF
Green, Mn++, Hg++, Blue, Fe + +,Mn + + Fe+++
Mn(I1) > Ni(I1) > Co(I1) > Ag(1) > Cu(I1) > Fe(II1) > Pb(I1)
Developers R.46KF
RESULTS
cu++
White, Mn++
Bi(II1) > Hg(I1) > Au(IJJ) > Hg(1) KF. Potassium ferrocyanide RA. Rubeanic acid
830
ANALYTICAL CHEMISTRY
Quantitative. Figure 1 compares curves obtained by plotting time us.
*
Table 111.
Separation of Cation Pairs b y Selective Diffusion in Elvanol
Colors, Ions, and Diameters of Zones Developer Inner Outer Oxine Cloudy lemon yellow, Cu ++, Transparent yellow, Hg++ Hg++ HAuC14, HgClz Oxine Purple, Au +, Hg + + Transparent yellow, Hg++ Fe( SO&, HgClz Oxine Gold-brown center surrounded Yellow, tinged with bv olive green ring, Fe+++, dull green from gg++ ring around iron zone, Hg++ Fe(Noa)~,Cu(N03)~ Oxine Gold-brown center surrounded Dirty yellow-green, by olive green ring, Fe+++, Cu++ Cu++ Compounds Diffused HgClz, Cu(N03)z
++
+H+
Ti
Table IV. ’5
(15-minute periods) Kelcosol
45
70
T / N E (IYINVThS)
Figure 1. Variation of distance, r , with time, f, for 0.1N solutions of various cations in gelatin
Compound NaC1 KaBr KaI NaF NHaCl NHdBr NH4F KC1 KBr KI KF Table V.
-
Compound
-3
-2
LO9
a
y+
A
A$*
8
Pb*
c.
-I
Figure 2. Log concentration v5. r for group I cations, after 15 minutes’ diffusion in gelatin
distance for 0 . 1 s solutions of various cation nitrates diffusing in gelatin. Figures 2 a n d 3 illustrate how log concentration varies with distance, enabling determination of ions diffusing in a fixed period of time (15 minutes in this case). Constructing such curves for knowris enabled students to determine the concentration of unknorrn solutions of lead nitrate. By diffusing the solutions in gelatin for 15 minutes, and then arresting the diffusion with saturated potassium iodide, measurement of the yellow zone radii, 0.1000, 0.5000, 1,0000, 5.0000, and 10.0000N solutions could be identified with reasonable accuracy. Out of 28 trials, the results were accurate to 10.68%. A plot of time us. distance at various concentrations is illustrated in Figure 4, from which the effect of concentration on the slope of a n r 2’s. t plot may be noted. Halide anion radii were developed with silver nitrate and calcium chloride
Diameters of Diffusion Zones of Anions
BaClz FeCh SlSOI COClZ HgCh
0.LV 12.0 14.6 16 5 6 9 9 7 15 9 9.0 11.0 18.0 17.5 8.2
0.05N 10.9 13.0 10.5 6.2 8.0 14.8
0.01N 8.4 11.9 9.1 5.9 7.7 14.0 7.0 7.2 14.3 15.2 6.9
8.0 9 6
16.8 16.5
7.9
0.005N
Gelatin
0.1N 7.0 15.5 9.0 7.8 7.2 15.0 8.5 7.9 13.5 7.5 7.6
... 10.7 9.0 13.0 6.9 6.3 13.0 13.0
...
0.05N 5.5 14.0 7.0 7.4 6.3 13 0 7.9 7.2 12.5 5 3 7.3
0.01N 4.9 13.2
0.005N
7.1 5.2 12.0 7.1 7.0 12.5 4.5 6.6
6.9 5.0 11.0 5 8 6 5 10.9
4.9 11.9
Diffusion of Compounds as Ions in Gelatin (G) and Kelcosol (K)
Cation Distance, llm. K G 10 7 11 12 11
4 0 5 5
3
10 7 10 9 6
8 5 2 0 5
Anion Distance, Mm. K G
Cation Developer SO4-*
Fe(CN)G-4 Rubeanic acid Fe(CN)8-4 KI
(for fluoride ions) solutions. Table IV compares halide ion radii of sodium, potassium, and ammonium salts and shows that the diffusion of a n anion is affected by the cationic species present. When r is used for determining unknown concentrations of a cation or a n anion in a compound, the anion must always be identical when the cation is being determined, and vice versa-e. g., consistent results will not be obtained if silver nitrate is used for standardization and the unknown is silver cyanide, silver sulfate, etc. Diffusivn radii of 0.1N solutions of individual cation nitrates were measured after 15 minutes in gelatin and found to follow the following sequence in order of greater diffusion distance: Zn(I1) > Hg(1) > Co(I1) > Cr(II1) > Cd(I1) > 4 u ( I I I ) > Ni(I1) Fe(I1) > Hg(I1) > Fe(II1) > Mn(I1) > Ag(1) > Ti(1V) > Cu(1) CU(II) > UOz(I1) > Pb(I1) > hl(II1) Interaction between ions and the diffusion media interferes with normal diffusion. This has been observed with mercury(I), bismuth(III), and
8 5 9 2 9 s 12 4
9 7 6 7 7
Anion Developer
2 0 2 2 0
Ag Ag + +
Ba++ Ag + Ag +
iron(II1) in gelatin. Bismuth(II1) forms white bismuth hydroxide and iron(II1) brown chelate complexes of amino acids. Xevertheless, iron(II1) appears to follow normal diffusion according to Fick’s law. Kelcosol arrests copper(II), copper(I), gold(III), nickel(11), aluminum(III), titaiiium(IV), cadmium(II), and zinc(I1). Some ions diffuse preferentially a t faster rates in some media than in others. A case in point is shown in Figure 5 , which compares the slopes obtained for chlorine (as potassium chloride) after 15 minutes’ diffusion in Kelcosol and in gelatin. Single substances do not diffuse in gels as a compound but as anions and cations, as can be seen from Table V, which compares r (for 0.1N solutions after 15 minutes) of cations and anions in single compounds. DISCUSSION
Qualitative diffusion procedures resolve cations partially. When a cation mixture is placed centrally on a gelatin surface, each component migrates under the influence of the chemical concentrations or potential gradient. The VOL. 31, NO. 5 , MAY 1959
831
zone of each component extends from the starting position to the leading boundary. Each cation contaminates the zone of every more slowly diffusing cation; therefore, only a small fraction of the fastest migrating ion can be separated from others. Figure 6 illustrates this for cations A, B, and C. A component may be retained by a zone in preference to another component if it is heavier, thus diffusing more slowly. It nil1 be retained in preference if it also undergoes a secondary reaction with the diffusion media. This is strictly true only when the cations are of equal concentration in the mixture being resolved, and when they have a common anion. Individual cations follow Fick’s law of diffusion. When time is held con.itant and r and the concentration, c, of the ion in question are varied, solution of Fick’s equation gives:
/ 7t
‘r
-3
DK (t,
- tl)
=
r2
- r1
7L
-I
-2 Lcq
c,
15
Figure 3. Log concentration v5. r for group Ill cations after 15 minutes’ diffusion in gelatin
ous that mutual effects radically alter the diffusion of an individual ion.
where r1 is the diffusion radius traveled for an ion of concentration c1, and r2 is the radius for an ion of concentration cz. K is a constant peculiar to the time interval of the diffusion process and varies inversely with t . D is the diffusion constant. Equation 1 when obeyed by an ion gives straight lines when log c is plotted against r at rarioils time intervals, as is shown in Figures 2, 3, and 5 . Equation 2 represents the solution of Fick’s law when T and t are the variables and c is held constant.
/
9.5
rmr ( t w u n s )
Figure 4. Plots of r v5. t for 0.1 N and 0.001N solutions of silver nitrate diffusing in gelatin
I2 -
Mixtures of cations may be resolved partially by differential diffusion in gels. The concentration of ions may be determined by measuring the distances traveled in a fixed length of time and comparing them m-ith standards. To determine a cation’s or an anion’s concentration by this technique, the same compound must be used to obtain valid results. A single
30
’’x
fl-
w
7 E
,/ KCLCOSOL
A
9-
2 8-
0
r6.
+€LATIN
(2)
K in this case varies directly with c. Einstein has shown that the diffusion constant, D, may be expressed as D = RT/GrvrN
(3)
where R is the gas constant, T is the absolute temperature, N is Avogadro’s number, q is the viscosity of the diffusion media, and T is the mean radius of the ions. A single ion will follow Fick’s law, but not when mixed n-ith other ions of the same charge. Thus studying Figure 2, one would expect when resolving a mixture of 0.1N solutions of mercury(I), lead(II), and silver(1) nitrates that the outer zone would contain mercury(I), the middle, niercury(I) and silver(I), and the inner all three cations. Actually silver(1) is in the outer zone, lead(I1) and silver(1) are in the middle zone, and all three cations are in the inner zone ( 1 ) . It is obvi-
832
ANALYTICAL CHEMISTRY
Figure 6. Schematic drawing of cross contamination of three cations, A, B, and C
on these diffusions rcwived S a C ’ I T I ’ .J:~cwljsoii,Boston University, Boston, certificates from Randbook I’ulilislim, 11:~~s. Inc., for their satisfactory solution of an undergraduate research program: Larry LITERATURE CITED ii. Teter, Fresno Stat,e Collogc~,Fresno, Calif.; Richard ill, ,james 1 ~ . (1) .liltelman, &I., ANAL. CHEM. 26, 1218-19 (1954). ”Id G. ’’-ilkerson, ( 2 ) Harris, F. E., S a s h , L. K., ZhicE., 22, University of Texas, Austin, Tex.; I552 (1950). Gloria nefina, Paula Fetsko, and Con( 3 ) Leupin, O., Iluggli, P., Helv. Chim. Acta 2 2 , 1170-7 (1939). stance St* Joseph ( 4 ) Kicholas, R. E. H., Rimington, C., Biochem. J . 48, 306 (1951). Emmitsburg, N d . ; and Saniuel Hirsch
(5) Strain, H. H., 3Iurphy, G. IV., .IY.~L. CHEM.24, 51 (1952). (6) Veil, S., Compt. rend. 198, 1854 (1934). (7) Zhid., 199, 611 (1934). (8) Zhid., p. 1044. RECEIVEDfor review March 7 . 1058. Accepted December 31, 1958. ?ivieion of Analytical Chemistry, Symposium on Separation Processes through Differential Migration Analysis, 134th Meeting, .4CS, Chicago, Ill., September 1958. Work done under the auspices of the National Cooperative Undergraduate Chemical Research Program (NaCCRP).
Carrier Displacement Chromatography on Ion Exchange Resins DONALD 1. BUCHANAN Radioisotope Service, Veterans Adminisfration Hospital, West Haven, Conn., and Department o f Biochemistry, Yale University, New Haven, Conn.
b Certain volatile bases assume intermediate positions in the order of displacement of amino acids on cation exchange resins and thereby convert partial into complete separations. Acids can do the same on anion exchange resins. A paramount criterion in the search for these selective agents or carriers is the dissociation constant, but aromaticity and size are also considered. Some of these carrier substances may be used in a second way. If a resin is first saturated with an appropriate acid or base, amino acids may separate by differential migration, if displaced into and through the zone of saturated resin. This method of column development has distinct advantages over those using buffers or strong acids. The need for clarification of the terminology and the mechanisms of ion exchange chromatography of arnpholytes are discussed. solutes that bind firmly to an inimobile phase are able to strip conipletely or displace more weakly bound substances, zones “push” one another down a column all a t the same rate, the filial displacing agent acting like a piston ( 3 ) . This is diagrammed in Figure 1, n here thc curves represent concentrations of solute along a column. El en L\ lirii individual affinities for the stationary phase differ greatly (-4 and B ) , pure zones are a h ays separated by a miwd zoiie, the lower limiting size of which is governed by the mechanical imperfections of column operation. It is theoretically impossible to recover any substance completely iii pure form. HEX
Mixed zones tend to be larger when relative affiities differ to a lesser degree ( B and C). Often solutes compete almost equally for the stationary phase (D and E ) and nearly all of one or both substances is mixed x i t h the other. Unlike most types of chromatography, the relative size of a mixed zone does not decrease as less material is chromatographed. Conversely, addition of more material does not increase the absolute sizes of mixed zones beyond certain maxima and the relative quantity of mixed material therefore becomes less. Because of these characteristics displacement chromatography of amino acids has been successful when very large mixtures-e.g., 280 grams of hydrolyzed egg albumin (25)-are separated. Even M hen two solutes differ but slightly in their affinity for the solid phase ( D and E , Figure 1) they may be separated by displacement chromatography if enough of a third substance n i t h a n intermediate affinity is interposed. Such interposed displacers, called “carriers,” have been used in absorption chromatography (9, 12, 15, 17,18,28,34). Recently (5) carriers were used to expand the methodology of Partridge and coworkers (20-27) in the separation by displacement chromatography of mixtures of amino acids on ion exchange resins. The carriers mere nonamphoteric bases and acids, all easily separated from the amino acids by evaporation or ion exchange. The observation of navies ( 7 ) that with bases as me11 as acids the stronger displace the \T ealier on ion exchange columns made the search for carriers
much easier. Although the displacement sequence of a group of substances cannot always be predicted from their dissociation constants, (24, 25), experience is a reliable guide in evaluating the magnitudes of the effects of molecular size and aromaticity in altering the expected displacement order. Some groups of three or more amino acids have dissociation constants and other properties so nearly alike that when the group is chromatograplied by displacement there is little tendency for one to act as a carrier to separate the others. Because of this the hope of finding other carriers to separate thebe groups of very similar amino acids is not great. However, the individual members of such groups are often separable by diff erential migration, if a resin column is first regenerated with the carrier initially used to displace the group and the amino acid mixture is then displaced through the column. I n this case the carrier becomes a competitive developer. Partridge and Brimley (22, 2;) had demonstrated that some amino acids that fail to separate on a sulfonic acid resin may sometimes do so on an anion exchanger. These workers (23) a n d others (10, 19) had also improved some separations by raising temperatures. Cooling may also inzprore a separation (5). These factors were brought t o bear in developing the empiricallJsuccessful schemes (5) for separation of synthetic amino acid mixtures. The present paper illustrates the resolution of a n acid hydrolyzate of protein. VOL. 31, NO. 5, MAY 1959
833
