ANALYTICAL CHEMISTRY
502 drive shuts itself off and the bell sounds the signal. The operator can turn off the power line switches or merely set the selector switch to “manual” depending on subsequent procedure. If the recycling procedure is t o be employed, phases are removed as desired and replaced with fresh solvent. Tubes 0 and 49 are connected to complete the solvent cycle and the apparatus is started again by manually advancing the stepping sxT-itches to position No. 1 and then restoring the selector switch to “auto.” The procedure obviously can be repeated as many times as desired. At the completion of the distribution, both phases of each tube are withdrawn together into the tared evaporating dishes shown in Figure 3. The dishes are spun of 0.010-inch stainless steel stock and each has a tab for convenience in handling and numbering. The dishes are 1 inch high, 2l/* inches in outside diameter across the bottom, and 21/2 inches outside diameter across the top. They have a 50-ml. capacity and weigh about 10 grams each. The dishes are arranged in sets of 50 and numbered from 0 to 49 to correspond with the tube numbers of the apparatus. New dishes are weighed and then numbered in order of increasing (or decreasing) weights to facilitate tare weighing. Each set of dishes is contained on a tier rack, the shelves of which are made of perforated stainless steel stock. Each shelf is I 1 inches in diameter and will hold 12 or 13 dishes. The shelves are spaced 1 3 / 4 inches apart on the spindle of a pedestal. The upper end of the spindle has a threaded hole for arcepting a ring which serves as a means of carrying the entire rack and dishes. Each shelf is therefore removable. The dishes on it are arranged in a systematic order for ease in use. Considerable versatility is offered by having two or more complete sets of dishes and racks. Dishes can be cleaned, dried, or tared during the automatic operation of the apparatus. Figure 3 discloses the details of the dishes and rack. A wooden shelflike platform is used t o place the dishes during removal of the solvents from the tubes. The solvents are withdrawn by siphon action through small diameter stainless steel (or glass) tubes, The siphon tube contains a small rubber stopper to seal it to the tube because the siphon action is started by a blowpipe. The latter is attached to the exit of tube 49 and operated by mouth. A small dimple blown on the underside of the equilibration tubes directly opposite the position of the filling tubulations serves as a sump for the siphon and permits complete removal of the phases. Siphoning is started a t tube 0. While the siphon is running in tube 0, dish 1 is placed in position on the shelf in readiness for tube 1. When the siphon stops, it is transferred to tube 1, tube 0 is stoppered, and the siphon is started. Dish 0 is now returned to its rack and dish 2 is placed on the shelf for the next tube. By this procedure the 50 tubes can be siphoned in less than an hour.
As each shelf of the dish rack is filled with dishes, it is placed on the spindle of the rack. Jt-hen all tubes have been emptied, the rack is placed in a chamber where clean warm air is blown through the rack to rvaporat,e the solvents. The time required for this operation, of course, depends on the volatility of the solvents. When high boiling solvents or water is used, the rack is finally placed in a vacuum chamber and given a 2- to 4-hour (or even overnight) evacuation. The dishes are then weighed and curves plotted in the usual manner. The dishes are cleaned by vapor and condensate from boiling solvents. They are placed upside down on their shelves and the entire rack is placed in the washing vessel. This vessel is essentially a still and consists of a boiler and condenser. The cleaning solvent is evaporated by energy from an immersed steam coil and the vapor is effectively and completely condensed a t the top by a flat spiral fin-tube coil. The condenser coil is fixed to the lid of the vessel for ease in removal. The rising vapors and refluxing condensate thoroughly wash the dishes in 1 hour. Because of the tabs on the dishes, they set inclined on the shelf. -411 solvent, therefore, drains completely from the dish surfaces. When washed, the rack is placed in the solvent evaporating chamber for 15 to 20 minutes to remove the residual solvent which may cling to the dishes or rack. The dishes are then ready for taring. To wash the glass tubes of the apparatus, the large sprocket is loosened from the shaft and the actuating arm is raised to avoid contact with the switches. This permits manual movement of the apparatus free from the drive mechanism. Washing is then accomplished in the usual manner. ACKKOW’LEDG3IEh-T
The authors v5sh to acknowledge their appreciation for the advice given by Frederick Krasselt of Consolidated Vacuum Gorp. in the design of the electrical circuit, to Dean Cox for his suggest,ions and assistance in fabricating parts, and to the management of Distillation Products Industries for perniission to publish this paper. LITERATURE CITED
(1) Craig, L. C.. Hausmann, Werner, Ahrens, E. H., and Harfenist, L. CHEX, 2 3 , 1236 (1981). (2) Hickey, F. C . , Ibid., 24, 1993 (1952). (3) Metzch, F. A,, Chenz. 1710. Tech.. 25, 66-72 (1953). (4) Morris, C. J. 0. R., Biochem. J . , 5 5 , 369 (1953). RECEIVED for review October 5 , lY.53. Accepted Kovember 2 3 , 1953. Communication No. 195 from Distillation Products Industries, Division of Eastinan Kodak Co., Rochester, S . Y .
Quantitative Determination of Amino Acids Using Monodimensional Paper Chromatography J. F. ROLAND, JR., and A. M. GROSS Biochemical Research Department, The Armour Laboratories, Chicago,
A
T PRESEST, the determination of the amino acids of a
protein by paper chromatographic methods is an arduous task involving the use of a variety of solvent mixtures, or partial resolution by group?, elutions, and subsequent chromatography (3). Heretofore, a solvcnt mixture capable of resolving a large number of amino acids by monodimensional procedures has not been available. Investigations by Miettinen and Virtanen (IO) have suggested the possibility of achieving desirable amino acid separations by extending the length of resolution time. However, the solvents employed by these investigators required lengthy resolution periods and in preliminary studies the amino acid spots were frequently diffuse and poorly resolved. Recently, hIcFarren ( 9 ) has dewribed the use of several buffered phenols and alcohols for the resolution and quantitation of 18 amino acids. Likewise, Redfield and Guzman Barron (12) have propocred a procedure for the quantitation of amino acids u4ng five different solvent systems and prolonged resolution periods ( 5 to 6 days). Fowden ( 7 , 8) has also proposed a quantita-
111.
tive procedure in which resolutions of the amino acids are accomplished with three different solvent systems and prolonged resolution periods of 5 days. Block f 6 ) has described investigations of amino arid patterns with a great variety of solvent systems using ascending chromatography for 24-hour periods. -4mong these, a solvent mixture composed of 2-butanol and 3oJ, ammonia ( 3 to 1) appeared to have further potentialities for amino acid resolutions if adequate separation times (40 to 48 hours) were employed. The results of investigations along these lines have led t o a comparatively simple and faster procedure for the resolution and quantitative estimation of 10 amino acids. Six other amino acids are resolved in a previously described (6) solvent system. EXPERIMENTAL
Filter Paper. Whatman No. 1 filter paper sheets cut into 8 x 22 inch strips with folded absorbent paper bands stapled to the bottom edge were used in this procedure.
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
503
Previous systems for quantitative amino acid analysis of milligram or smaller portions of peptides and proteins were not entirely satisfactory because of the complexity and the number of systems required for resolution of the amino acids. A simpler and more convenient method has been developed and used routinely for o v e r a year with success. Two monodimensional solvent systenis, 2-butanol-370 ammonia (3 to 1) and 7270 phenol will resolve 16 amino acids sufficiently well for quantitative determination by direct photometry of the ninhydrin spots. -2dditional systems are described for the resolution of histidine and tryptophan. Determinations by this method of the amino acid composition of &lactoglobulin were found to be in agreement with reported values. The method has also been very useful in the identification of microgram quantities of peptides separated by paper chromatography.
Chromatographic Chamber. Circular borosilicate glass jars (12 X 24 inch) are used as developing chambers. Glass photographic development trays, 8.5 X 5.5 inches (Central Scientific Co., Chicago, Ill.), are supported on top of 2-liter glass cylinders. Plate glass sheets (12 X 12 X 0.25 inch) with a 5/8-inch centerbored hole are employed as covers. The glass jar and lid interface should be well sealed wit'h silicone grease. The corner edges of the glass photographic trays should 'oe slightly ground on an emery wheel to permit' placement in the large glass jars. Solvents. Two solvents are employed for separation of the various amino acids with the exception of histidine and tryptophan. SOLVEST 1. 2-But,anol-3% ammonin ( 3 to 1) provides excellent resdution of the following amino acids as individual entities in 45 hours: lysine, arginine, alanine, proline, tyrosine, valine, methionine, isdeucinr, leucine, and phenylalanine (Figure 1). As;,artic-glutamic-cystine, serine-glycine, and histidinethreonine are unresolved complexes in this system. The solvent is prepared by thoroughly agitating 120 ml. of 2-butanol (Matheson Chemical Co., ,Joliet, Ill.) wit'h 40 nil. of 37, ammonium hydroxide ( 5 5 ml. of fresh 15.V ammonium hydroxide diluted to 500 ml.). The solvent is then ready for use. Only peroxidefree 2-liutanol should be eni-
in accordance with those described here. However, solvent system 1 can withstand considerable temperature variations (10' to 12' F.) without distortion of the resolution patterns. Minor adjustments of the ammonia content of the solvent system generally will correct unsatisfactory resolutions. Analytical Preparations. Block ( 9 ) has published many of the details involved in the estimation of amino acid8 and amines on paper chromatograms. I n most instances, the general procedures set forth in his paper were followed. However, certain modifications appeared advantageous. Vacuum-dried samples of all amino acids (chromatographicallj~ pure) are weighed and dissolved in separate volumetric flash to provide a concentration of 5007 of a-amino nitrogen per milliliter in the stock (10% isopropyl alcohol) solutions. Subdilution standards of the appropriate amino acid mixtures are then made to provide a final lower working limit of 0 . 1 0 ~of a-amino nitrogen per 2.5X. For each strip to be iun, four increments of 0.10, 0.20, ~ a-amino nitrogen of anamino acid standard mix0.30, and 0 . 4 of ture containing aspartic acid, glutamic acid, lysine, arginine, serine, glysine, histidine, threonine, alanine, proline, tyrosine, valine, methionine, leucine, and phenj lalanine and two increments of the unknown protein hydrolyzate are routinely micropipeted (Microchemical Specialties Co., 1834 University Ave., Berkeley 3, Calif.) in duplicate on the same filter paper strip. Procedure with Solvent 1. After filter paper strips have been prepared for descending analytical chromatography, they are placed in the chromatographic chambers in the usual fashion and allowed to condition for 2 hours in an atmosphere of the solvent mixture (100 ml.) which has been carefully decanted to the bottom of the glass jar. In addition, a 4-inch band of absorbent paper saturated with the solvent is wrapped around the top of the glass cylinder. (These jars are maintained "conditioned" 3 to 4 weeks without cleaning.) After the proper conditioning interval, the center hole in the glass lid is opened and about 130 ml. of solvent is carefullv introduced into the glass tray with a large hypodermic syringe and needle. The lid hole is again stoppered and the chromatogram alloxed to develop for 40 to 48 hours depending upon the temperature conditions employed. It has been found desirable under conditions where considerable temperature variation occurs to remove and develop one sheet early in the morning phase of the final hours to determine the extent of resolution which has occurred and from this to predict the most appropriate time to remove the remainder of the sheets to he evamined (Figure 1). Procedure with Solvent 2. After filter paper strips are prepared for quantitative chromatographic analysis, they are inserted in the glass jars in stainless steel chromatographic frames and solvent containersl( University Apparatus Co., Berkeley 3
ployed. SOLVEST2. Phenol [Ira]linckrotlt USP XIV) meoared a$ suggested by Block' (2)will readily resolve the following amino acids: cysteic acid, aspartic acid, glutamic acid, serine, glycine, and threonine. llternative m e t h o d s f o r histidine are descrihed in the procedure for solvent 2. llodification of solvent system 1 with ethanol in the following proportions: 2-butanol-ethanol 95-67 ammonium hydroxide (100 to 25 to 35) resolves tryptophan in 24 to 40 hours. The pattern of resolved amino acids with the solvent is as follows (descending): cystine, aspartic acid-glutamic acid, s e r i n e-glycine-histidine, pro1 ine, threonine-alanine, tyrosine, valine-methionine, tryptophan, isoleucine, and phenylalanine-leucine. Resolution Factors, Time, Temperature, and Conditioning. Satisfactory resolution of a large number of amino acids in a single solvent system depends on a variety of factors ( 2 ) . Desirable results have been obtained only when the conditions and equipment are
Table I.
Amino -4cid Analysis of a Simulated Bovine Serum Albumin Hydrolyzate .k
Constituent Lysine
S o . of Assays 6
Standard Error of .\lean 0 17
Difference between Actual Composition and Experimental RIean, OZ 9.21
?
Amino Acid Mean a n d Range,
%
14.00 (13,50-14.50) .irginine , 6.34 (5.61- 7 . 2 1 ) Alanine 10 6.17 (5.80- A . 60) Proline 6 4.73 (4.00- 5 . 5 0 ) Tyrosine 8 4.94 (4.50- 5 . 5 0 ) Valine 5.59 lo (4 70- 6 . 7 0 ) Methionine 9 0.80 (0.57- 0 . 9 8 ) Isoleucine 9 2.57 (2.20- 3 . 3 0 ) Leucine 8 12.20 (11.50-12,70) Phenylalanine 6 6.15 (5.40- 6 . 6 0 ) Aspartic acid 5 11.40 ( 1 0 . 91-1 1.80) Glutamic acid 5 15.30 (14.50-16.60) Serine 6 4.62 (4.20- 5.20) Glycine 7 1.97 (1.70- 2 . 1 0 ) Threonine 7 6.00 (5.40- 6 . 6 0 ) Histidine 4 4.10 (3.80- 4.30) Total 106.70 Difference between Column A a n d B 0.40%
n,
Actual Composition,
% 12.82
0.26
7 45
0 or,
1.28
0.25
0.25
U.42
4 75
0.12
2.37
d.OF
0 20
5.59
5.Q2
0 07
1.24
0.81
5.90
0.14
1.53
2.61
0 14
0.57
12.27
0 22
6.68
6.59
0 21
4.59
10.91
0 35
7.84
16.50
0 20
9.22
4.23
0 06
8.24
1.82
0.15
2.92
5.83
0.12
2.50
4.00
___
106.27
504
ANALYTICAL CHEMISTRY
Calif.). After conditioning for 2 hours, the center hole in the glass lid is opened and the container filled with the solvent mixture and restoppered as described above. As neither solvent 1 nor 2 will resolve histidine as an individual spot, separate sheets resolved in solvent 1 may be set aside and subsequently developed with the Pauly reagent (2) or the hydrolyzate mixture may be resolved in an alternative solvent system such as Z-butanol-
nine. asmrtic "acid, serine-glycine. th;eonine:dutamie
'
a t 0.2% concentration in acetone (14) and containing 1% acetic acid is routinely employed for the development of the resolved ohmmatograms. After resolution, the sheets are dried in the hood a t room temperature ( 7 ) and subsequently dipped in the
a&
A
.9
Lysine Arginine
.8. .7.
Phenylalanine
.4
.3 Proline
2 I
Solvent System No. 1
.I
.2
.3
Conc entration
Figure 2.
4
a "2-N
(rnicroams)
Typical Amino Acid !3 tandard urYe*
Table 11. Comparative Amino AcidI Values of B-Lacto-rlobulina
Arginine Histidine Lysine Tyrosine TryDtoDhan Phenylalanine Cystine~oysteine Methionine Serine
Figure 1. Quantitative Amino Acid Chromatogram
Threonine 1.e"Oi"e Ide"Ci*e Valine Glutamic aoid Aspsrtio acid Glycine Alanine Proline
2.8 1.7 13.0 3.5 2.0t 3.6
2.91 1.63 12.58 3.64 1.94
3.0 3.9 4.9 15.7 5.6 5.6 19.8 11.5 1.6 6.9 5.1
3.22 3.96 4.92 15.50 5.86 5.62 19.08 11.52 1.39 7.09 5.14
...
."-
..
...
..
505
V O L U M E 26, N O . 3, M A R C H 1 9 5 4 This instrument has proved to be very sensitive and the values observed were readily reproducible. The densitometer is adjusted to 0% transmittance density against the edge of each filter paper strip and readjusted as readings are made progreseively up the paper strip. Figure 2 shows curves of the various amino acid standards Then color density units are plotted against micrograms of a-amino nitrogen. RESULTS
Block (3)has published data for thr analysis of samples of brain neurokeratin and also determination of the amino acids present in casein ( 2 ) by the use of paper chromatograms. He has reported an over-all accuracy of & l o % by his procedure.. Stein and Moore have analyzed crystalline bovine serum albumin (BSA) by the use of starch column chromatography ( I S ) . Likewise, Redfield and Guzman Barron ( 1 2 ) have analyzed bovine serum albumin and alro a partial synthetic amino acid mixture by paper chromatographic procedures with excellent results. A similar studywasundertakenat thislaboratory, in which a mixture of 15 amino acids designed to reproduce the composition of amino acids as found in crystalline bovine serum albumin were carried through an acid hydrolytic procedure ( I S ) and analyzed by the paper chromatographic procedure described above. Table I shows the comparative results obtained from this experiment. The individual estimations of the composition of the amino acid mixture are in reasonably good agreement with the actual composition of the synthetic mixture. The assay data and the actual composition for each of the amino acids are presented in Table I. The computation of the standard errors was used as a measuie of the accuracy of the assays. All of the constituents, 15ith the exception of lyqine, glutamic acid, and glycine, were within thc experimental confidence limits ( P = 0.95). The differences between actual composition and the experimental mean, howevcr,
are only -7.84% for the glutamic acid, +9.21% for lysine, and +8.24% for glycine. Analytical data on the amino acid cornposition of crystalline proteins such as p-lactoglobulin (Table 11) and bovine serum albumin when determined by this method have been in good agreement with previously published (12, I S ) values. In general, the procedurrs which have been described provide a less tedious, more rapid, and reasonably accurate approach to the analysis of protein hydrolyzates than has previously been available. ACKNOWLEDGMENT
The authors wish to acknowledge the technical assistance of John RoPevear in the early stages of this work and to express thanks to C. F. Marquardt for the statistical evaluations and also to Jean Hogan and .Janet Dunlevy for photographic apsistanre. LITERATURE CITED
(1) .kcher, R., Fromageot, C., and Justia, l l . , Biochini. et B i o p h y s . Acta, 5 , 81 (1950). ( 2 ) Block. R. J.. A s a ~ CHEY.. . 22. 1327 (1950). (3) Block, R . J., Arch. Biochem. a n d Bibphys.. 3 1 , 266 (1951). (4) Block, R. J . , Proc. SOC.Exptl. Bid. M e d . , 72, 337 (1949).
Block, R . J., Science, 108, 608 (1948). (6) Block, R. J., and Bolling, D., “.-imino .kcid Composition of Proteins and Foods,” 2nd ed., Springfield, Ill., C. C Thomas, (5)
1950.
(7) Fowden. L., Biochetn. J . (Londorc),4 8 , 3 2 7 (1951). (8) I b i d . , 50, 355 (1952). A N A L .C H E W ,24, 650 (1952). (9) JIcFarren, E. F., and llills, J. d., (10) Niettinen, J. K., and T’irtanen, d. I., Acta Chem. Scand., 3, 459 (1949). (11) Patton. il. R., and Chism. P., ..~N.AI.. CHEM.,2 3 , 1683 (1951). (12) Redficld, R. R., and Guzman Barron, E. P.,Arch Biochem. anti BiophZjs., 3 5 , 4 4 3 (1952). (13) Stein, 15‘. H., and JIoore. S., J . B i d . Chem., 178, 7 9 (1949). (14) Toennies, G., and Kolb, J. J., AN.AL.C H E x , 23, 823 (1951).
RECEIVED for review July 3, 1952. Accepted December 28,
1953.
Behavior of the Condensed Phosphates in Ani on-Exc hange Chromat ography JOHN BEUKENKAMP, WILLIAM RIEMAN 111, and SIEGFRIED LINDENBAUM School o f Chemistry, Rutgers University, N e w Brunswick, N. 1. There is need of a rapid and more accurate method for the analysis of mixtures of the lower condensed phosphates, ortho-, pyro-, tri-, trimeta-, and tetrametaphosphates. The successful application of ion-exchange chromatography to numerous difficult analytical separations suggests the possibility of developing an ionexchange procedure for the analysis of mixtures of the polymeric phosphates. A s an approach to this problem, equations were developed to describe the elution graphs of the various phosphoric acids as functions of the pH and concentration of the eluant solutions. Several dozen elutions were performed which indicate the reliability of these equations. This work advances the theory of ion-exchange chromatography and points the way to the development of an accurate procedure for the analysis of mixtures of the polymeric phosphates.
T
HE objectives of this investigation are: (1) to develop equations, based on the plate theory of Martin and Synge ( 6 ) , for describing the elution graphs of polyprotic acids as functions of the concentration of potassium chloride and the pH; (2) to derive an equation by means of whirh it vi11 be possible to calculate the column height required for a given separation; (3) to test the
reliability of these equations by elutions of ortho-, P>TO-, tri-, trimeta-, and tetrametaphosphoric acids; and (4) to use these equations for the development of an ion-exchange procedure for the analysis of a mixture of these acids or their salts. The first, second, and third of these points are described in this paper. The fourth will be reported in a later paper. DERIVATION OF EQUATIONS
Notation. The following notation is used in this paper. a = a parameter in the Gaussian equation, No. 2. -4= chemical s~7mbolfor an acid radical. C = the distribution ratio of an acid-i.e., the total quantity of its anions in the resin of any given plate divided by the total quantity of the acid (both ionized and nonionized) in the solution of the same plate. e = base of the natural system of logarithms. E l , E,, E a , etc. = apparent equilibrium constants for the,exchange of a primary (secondary, tertiary) anion. See Equations 7 to 9. F = fraction of solute eluted between any two given U values. G I R , G ? R , G 3 R = quantity of the primary (secondary, tertiary) anion in the resin phase of any given plate, millimoles. GoS = quantity of nonionized acid in the solution of the same plate, millimoles. GIs, G2S, G3S = quantity of the primary (secondary, tertiary) anion in the solution of the same plate, millimoles.
