ence of complex-forming reagents in the background electrolytic solution (9, I d ) . RESULTS
As a test of the effectiveness of separations in tapered paper on a polystyrene foam support, separations are reported with platinum wire electrodes attached to the paper. Because most of the ions were colorless, their paths through the paper could not be seen unless the migration was discontinued and the paper treated with special reagents. As these reagents discolored the paper or were effective in ultraviolet light, clear photographs have not been obtained. To demonstrate the degree of separation of the ions during the separation, drawings which indicate the location and width of the zones have been made ( I d ) ; the regions occupied by the ions are indicated in black. The continuous separation of silver and copper ions in the presence of a complexing agent in small tapered sheets of paper, supported on a polystyrene block 16 inches square and 2 inches thick, is illustrated by Figure 2. Similar separations were obtained with sheets 0.05 and 0.10 inch thick. A continuous separation of cobaltous and ferric ions in a large sheet of tapered paper 0.03 inch thick supported on a polystyrene block 2 feet square is shown in Figure 3. Similar results were obtained in paper 0.05 inch thick. A continuous separation of silver, cadmium, bismuth, and aluminum ions in large tapered paper sheets 0.03 inch thick is portrayed by Figure 4. Similar separations were achieved in paper 0.05 inch thick. Mixtures of various ions have also been separated in large tapered paper sheets like those shown in Figures 3 and 4. I n 0.1M acetic acid plus 0.05M malonic acid, and a t an applied potential of only 50 volts, ferric and manganous ions,
each 0.005M, and magnesium and ferric ions, each 0.005M, separated much like cobaltous and ferric ions (Figure 3) (paths followed by the ions located with yellow ammonium sulfide and with 8quinolinol in ultraviolet light). With 0.05M citric acid in 0.3M ammonia and with an applied potential of only 50 volts, thallium (located with hydrogen sulfide) and aluminum (located with 8-quinolinol in ultraviolet light) were separated completely in the large paper sheets. With a potential of 150 volts, the thallous ion zone was scarcely displaced; the aluminum ion zone moved about halfway to the anode. With 0.05M malonic acid in 0.05M ammonia and a n applied potential of 160 volts, thallium, arsenic, and aluminum ions were readily separated. The thallous ions migrated nearly to the cathode; the arsenious ions did not migrate from the center of the paper; and the aluminum ions migrated all the way to the cathode. The polystyrene foam support has been used with paper sheets shorter than those shown in Figures 3 and 4 but about the same width of base. These shorter sheets have the advantage of faster flow rate. Because the zones were narrower a t the base, the separations were nearly as effective as in the longer, tapered sheets. The foam support has also been used with square sheets of soft paper. With small square sheets of paper and conditions like those of Figure 2, the distance of the separation was not so great as with the tapered paper sheets. ACKNOWLEDGMENT
Some of the background electrolytic solutions described in this report-Figures 3 and 4, for example-were selected by Harlan D. Frame, Jr., from his studies of one-way paper electrochromatography. The effectiveness of the con-
tinuous separations depends, in large measure, upon the selective migration of the ions in these solutions and upon the suitable electrical conductance. LITERATURE CITED
(1) Block, R. J., Durrum, E. L., Zweig, G., “Manual of Paper Chro-
matography and Paper Electrophoresis,” Academic Press, New York. 1955. Durrum, E. L., Chem. Eng. News 27, 601 (1949); J . Am. C h m . SOC.72, 2943 (1950). Grassmann, W., “Tagung der Physiologen und physiologischen Chemiker,” Gottingen, 1949; Naturwissenschaften 38, 200 (1951). Hobart, M. H., Rose, C. F. M., J . Clin. Pathol. 8, 338 (1955). Lederer, M., “Introduction t o Paper E1ectroph;resis and Related Methods, Elsevier, New York, 7 9.5.5.
( 6 ) McDonald, H. J., “Ionography,
Electrophoresis in Stabilized Media,” Year Book Publishers, Chicago, 1955. (7) PuEar, Z., Croat. Chem. Acfa 28. 195 .. 11956).
(8) Sato, T . - R . , Kisieleski, TV. E., Norris, TV. P., Strain, H. H., ANAL.CHEM.25, 438 (1953). (9) Sato, T. R., Norris, W. P., Strain, H. H.. Ibid.. 24. 776 (1952). (10) Strain, H. H.,‘ Cirnegie Inst: Wash., Yenr Rook .. . ... 48. . - 87 11949). (11) Strain, H. H., ’Sato, T. R., ANAL. CHEM.28, 687 (1956). (12) Strain. H. H.. Sullivan. J. C.. Ibid., 23. ’816 (1951). (13) Svensson, H., Brattsten, I., Arkiu Kemi Mineral.Geol. 1, 401 (1949). (14) Wolstenholme, G.,I E. W., Millar. E. C. P., eds., Ciba Foundation Symposium on Paper Electrophoresis,” Little, Brown & Co., Boston, 1956. (15) Rood, S. E., Strain, H. H., ANAL CHEhl. 26, 260 (1954). RECEIVEDfor review May 29, 1957. Accepted August 26, 1957. Division of Analytical Chemistry, 131st Meeting, ACS, Miami, Fla., April 1957. Based on work performed under the auspices of the U. S. Atomic Energy Commission. Presented in part at Instruments and Measurements Conference, Stockholm. Sept. 19, 1956. \--
- - I
Ion Exchange Paper in Rapid Separation and Identification of Basic Amino Acids Arginine, Histidine, and Lysine from Casein Hydrolyzates MURRAY M. TUCKERMAN Department o f Chemistry, Rensselaer Polytechnic Institute, Troy,
b A newly available sulfonic acidtype cation exchange resin paper allows separation of arginine, histidine, and lysine from casein hydrolyzates in 90 minutes. A pH 5.2 acetate buffer i s used as the developing
N. Y., and Sterling- Winthrop Research Institute, Rensselaer, N. Y.
solvent in conventional descending chromatography technique,
C
MATERIALS with ion exchange functions have been prepared in sheet form by chemical treat-
ELLGLOSIC
ment of cotton fabric (4, 5) or paper (6, ‘?‘), by impregnation of paper with pulverized resin (8),and by incorporation of the resin with a slurry of paper fibers before the sheet is formed (a). The utility of these materials has VOL. 30, NO. 2, FEBRUARY 1958
231
penerally been shown by t'lie resolution of ternary inorganic mixtures. Lautsch, Xaiiecke, and Broser ( 7 ) , hoxever, report R , values for a number of amino acids on carboxylated paper at' various pH values. Partridge and coworkers (10-15) showed the eff ecbireness of displacemcnt development, of aiiiino acids on ion exchange columns. Step\~iseelut,ioii from Don-ex40 ( S a - ) was reported by Moore and Stein (9) in 1951 t o give successful separation of almost all common amino acids. This mcthod iTas adapted for preparative purposes by Hire. LIoore, and Stein (3) the following year. This larger sc*alty fractionation involvrs prc~liniiiiary scyarat'ion 011 Don-ex40 (SH,+) int'o the basic amino ac.ids.-arginine, histidine, and l>-sine.-.n-liich appear in n.t~ll-separatec1 bands; tyrosine plus phenylalanine, the aromatic amino :irid; and the ncutral plus acidic amino a d s . This preliminary separation is followed by further resolution of the last two groups, to give complete scparation of tlie common amino acids.
0
-1
ARGININE
~
#LYSINE
I
= I
HISTIDINE
0
PHENYLALANINE
0
TYROSINE 0
L
1
LEUCINE
ISOLEUCNE
VALINE
0
~
~
ILPININE MIXTURE
Figure 1. location of zones of individual amino acids and mixture developed a t 25" C. with pH 5.2, acetate buffer
Table I.
Standards Developed at
25" C.
Cm. from Point of Application
Arginine
EXPERIMENTAL
I n December 1956 an experimental ion exchange paper was made available by the Rohm & Haas Co., Philadelphia, Pa. I t contains about 2870 by n-eight of IR-120 ( S a + ) type cation exchange resin. The company states that this paper has an exchange capacity of about 3 peq. per square centimeter. Four microliters of 0.0lJf solutions of various aiiiino acids and of an amino acid mixture n-ere spotted across a sheet of tlie ion exchange paper and developed a t 25" C. by descending chromatography with various buffer mixtures; the solvent front was allorn-ed t o progress 22 cm. beyond the point of sample application. The sheets air-dried. sprayed with ninhydrin (1.2.3-triketoliydrindene)reagent (1.00 gram of ninhydrin in 400 grams of absolute alcohol denatured with O.5yc benzene, 150 grams of dichlorodifluoromethane. and 600 grams of dichlorotetrafluoroethane, packed in aerosol spray cans). and examined after 1 hour. A schematic diagram of the sheet on which a pH 5 . 2 acetate buffer 0.2M in sodium acetate was used for development is shown in Figure 1. This buffer IYas chosen for sharpness of separation of the basic amino acids, buffering close to the optimum p H for the ninhydrin reaction, and ease of preparation. The buffer is a mixture of 0.2M acetic acid and 0.4M sodium acetate. Approximately 6 pl. of a standard solution 0.01M in each amino acid \yere transferred t o a strip of ion exchange paper 3 em. wide by 30.5 cni. long by means of a Spinco-Durrum clectrophoresis apparatus sample striper (Beckman Instruments, Inc.. Belmont, Calif., Part 300-806), to form a streak across the width of the paper about
232
ANALYTICAL CHEMISTRY
1.8 1 8
3.3 3 3 34
8 2
15 5
1.8 AV.
100 I?, Std. dev.
Histidine
04
13
6 cni. from one end. The strip n a s developed by conventional descending chromatography in a chamber saturated with mater vapor, a t 25" C., using the p H 5.2 acetate buffer described above. After the solvent front had progressed 22 em. beyond the point of sample application (about 90 minutes), the strip was removed from the chamber. air-dried a t 25" C., sprayed with ninhydrin reagent, and allowed to develop a t 25" C. for 1 hour, then a t 100" C. for 10 minutes. As the color formed by ninhydrin is not stable, a permanent record was obtained by reading the strip in the Spinco Analytrol recording, integrating photometer. Each band appears to approximate a normal Gaussian distribution. Average R/ values based on half the band width at half the band height and their standard deT-iation calculated by the method of Dean and Dixon (1) are listed in Table I. A simple alarm system conserves the time in which the strip is being developed, without the risk of having the developing buffer run off the paper, thus making impossible the determination of R/ values. Two enameled magnet-wire leads were forced into opposite edges of the paper strip about 3 mm. above the desired end point and connected t o a Cenco electronic relay (Central Scientific Co.. Chicago, Ill., Catalog S o . 99783)
Lysine
Phenylalanine
Leucine
5.2 5 2 5 2 23 6
13.; 13.3 13 4
19.8
1 1
BO 8
1 5
19.6
19 5 88 5 1 6
in the normally open position. The other side of the relay was connected to a bell-buzzer through the usual stepdown transformer. As the solvent front passed between the two leads. the resistance dropped from about 300 megohms to about 50,000 ohms, thus tripping the relay. The above method of separation and identification n-as applied to Parenamine (Winthrop-Stearns, Inc., XenYork 18, Lot S127NE), a modified casein hydrolyzate having the stated per cent composition: lysine 0.35, leucine 0.36, threonine 0.14, isoleucine 0.27, methionine 0.27, tryptophan 0.08. phenylalanine 0.17, valine 0.28. arginine 0.19, and histidine 0.12. R, values found (Table 11) show the presence of arginine, histidine, lysine, aromatic amino acids, and neutral and acidic amino acids. DISCUSSION
Ion exchange paper offers the follon ing advantages over ion exchange columns: smaller sample. shorter time for separations, and elimination of fraction collecting and fraction analysis by direct staining. The separation of amino acids n-as chosen deliberately
LITERATURE CITED
Table II.
Band
I 1.9 1.7 1.8 8.2
AV.
100
Parenamine Developed at
25" C.
Cm. from Point of Application I1 I11 IV 5.1 12.9 3.4 4.9 12.9 3.3 12.9 3.4 5.0 58.5 22.7 15.5
as a rigorous test of the ability of the paper t o separate closely related substances and because separation and identification of amino acids in complex mixtures normally require several working days. The three basic amino acids are readily identified. The order of separation, however, does not follow the order of p K for the amino group nor the sequence found b y Moore and Stein (9) but rather the sequence obtained by Hirs, Noore, and Stein (S), in \vhich lysine has a greater R, value than histidine. As there is almost complete overlap of tyrosine and phenylalanine, the aromatic group can be identified, but the components
V 19.2 19.6 19.4 88.4
cannot be distinguished. Leucine has the smallest Rf value in the neutral and acidic amino acid group, so that R, values greater than that for leucine identify this third group.
Dean, R . B., Dixon, K . J., ANAL. CHEX 23, 636 (1951). Hale, D. K., Chem. R. I n d . (Londoii) 1955, 1147. Him. C. H.. &Toore.s..Stein. \v. H.. J.'BioZ. Chem. 195,669 (1952). ' Hoffpauir, C. L., Guthrie, J. D., Ibid., 178, 207 (1949). Hoffpauir, C. L., Guthrie, J. D., Tezttle Research J . 20, 617 (1950). Kember. K . F.. Kells. R. A . , LVatw-e Lautsch,
w:>LTaneclie,
G., Broser,
W,, 2. Suturforsch. 8b, 232 (1953).
Lederer. ll,, ,4nal. C h ~ m .Ictu 12, 122 (1955). Noore, S., Stein, W. H.. J . Biol. Chem. 192, 663 (1951).
ACKNOWLEDGMENT
The author xishes to express his gratitude for advice and encouragement to Frederick C. Xachod, SterlingM3nthrop Research Institute, and Robert A. Osteryoung, Rensselaer Polytechnic Institute. Thanks are also expressed to Dita Froelich for her careful preparation of the figure.
Partridge, S. M. Ibid.. 48. 313 (1951). r-r, I aL L L l U l j C ,
U.
_*I., Y
.
' L " . L L J ,
AL.
L
.)
Pepper, K. IT., Ibid., 46, 334 (1950). (15) Partridge, S. M., \Irestall, R. G., Ibid., 44, 418 (1949). RECEIVEDfor revien- 1 I a y 22, 1957. Accepted October 16, 1957,
Potentiometric Titration of Halide Mixtures A. J. MARTIN Polychemicals Department, Experimental Station,
b
To satisfy the need for accurate analyses of halide mixtures in mole ratios as high as 50 to 1, potentiometric titration with silver nitrate in dilute nitric acid was studied. The end point detection is based on the intersection of straight lines. The inaccuracies in individual halide analyses resulting from mixed salt formation are reproducible and predictable, and thus subject to correction. The correction factors for intersection-point data are calculated by statistical analysis using the Doolittle solution of partial regressions. The corrected inflection-point analyses show a standard deviation about 1.5 times greater than that of intersection-point data. Limits of detection of one halide in the presence of large amounts of another are determined for this method.
P
titration O f iodide, bromide, chloride, and some mixtures of these halides with silver nitrate was first reported in 1893 by Behrend ( I ) . I n 1920 Liebich pin-pointed the two problems which h a w prevented wide application of the titration. He reported that halide mixtures could be resolved only if the mole ratios were OTEKTIOMETRIC
E. 1.
du Pont de Nemours & Co., Inc., Wilmington, Del.
greater than 1 to 10 (4, 5, 9). At mole ratios less than 1 t o 10, the end points were difficult t o locate precisely. He observed that direct titration to inflection points gave positive errors for iodide and bromide, and a negative error for chloride (IO). These effects were attributed t o mixed salt formation-Le., the formation of solid solutions of the silver halides (7, I S , I S ) . Many improvements and modifications have been made to the direct titration of halide mixtures (2, 6,6, 9,11, la), but none has eliminated the main problem, that of inaccuracy in individual halide determinations in halide miutures. Levy described the use of an additive correction factor in microtitrations ( 8 ) , but the author found additive correction inapplicable to macrolerel titrations.
loned less than 0.005 mmole of chloride to diffuse through its tip in 4 hours. Materials. Standard solutions of potassium iodide, potassium bromide, and sodium chloride n ere made u p by weight from the pure dried salts t o be exactly 0.1OOO.Y. The silver nitrate solution was prepared from reagent grade silver nitrate and the concentration ma5 adjusted to 0.1.Y. The solution was accurately standardized by the Volhard titration of pure sodium chloride. Procedure. Known amounts of t h e halide solutions were pipetted into a 400-ml. beaker and diluted t o 150 nil. with distilled 1T-ater. S e x t . approximately 1.5J1 nitric acid solution was added dropnise until a p H of about 1 was attained with indicating paper. Too high a concentration of nitric acid was avoided as it might oxidize the iodide ion to iodine.
EXPERIMENTAL
Apparatus. The potentiometric d a t a were obtained using a Leeds and Northrup &lode1 7655 hydrogen ion potentiometer or a Beckman Model H-2 p H meter operated on the p H scale. The silver indicator electrode was a wire, 1.5 mm. in diameter, held in a rubber stopper and soldered to a n electrode cable. A Beckman No. 4970 calomel electrode served as a reference electrode. The calomel electrode al-
RESULTS AND CONCLUSIONS
Curve I of Figure 1 shows the iodide and bromide inflection points obtained when a solution containing 0.5 mmole each of iodide and bromide and 5.0 mmoles of chloride mas titrated. Curves I11 and IV show the first derivative curves of the two inflection points. (These two curves have different units on the ordinate to bring the heights to VOL. 30, NO. 2, FEBRUARY 1958
233
