Quantitative Evaluation of Paper Chromatograms of Condensed Phosphate Mixtures Using Modified Solvents and a Densitometer D. N. BERNHART and W. B. CHESS Research laborafories, Vicfor Chemical Works, Chicago Heights, 111.
b Mixtures of condensed phosphates are clearly and rapidly separated b y a paper chromatographic technique employing a modified acid solvent. Quantitative evaluation of chromatograms i s based on hydrolysis and color development of each component, followed b y measurement of the resulting color intensities on the paper with a densitometer.
I
paper chroniatography has proved to be the most effective method of clearly separating mixtures of condensed phosphates (1, 2, 6,6). Quantitative evaluation of these chromatograms is, however, lengthy and tedious. I t involves the cutting out of each band, elution with ammonium hydroxide, hydrolysis Tvith acid, and color development with molybdate, and requires such a large sample that clear separations are difficult whcn more than four components are present in a sample. The possibility of using a densitometer was investigated and a good procedure developed. N THE PAST DECADE
REQUIREMENTS FOR PROCEDURE
Four specific details were necessary for development of the procedure. Selection and Adaptation of a Densitometer. The Spinco dnalytrol has been used for evaluating chromatograms of biological materials; therefore i t should be of value for evaluating t h e blue phosphomolybdate bands. The stock blue filters were replaced with a matched pair of red filters. The B-2 cam furnished with t h e instrument gave very poor results with phosphomolybdate bands. A B-4 cam which has a straight absorbance characteristic functioned very well. However, the B-5 cam recently available, slightly modified in the high absorbance range, made the instrument somewhat easier to adjust and gave results equal to those obtained with the B-4 cam. Color Development. Complete development of t h e reduced phosphomolybdate complex on t h e paper strip t o its maximum color intensity is of utmost importance. The spraying and drying technique reported by
1026 *
ANALYTICAL CHEMISTRY
Karl-Kroupa ( 6 ) was folloived, but irradiation by ultraviolet light was not a sufficiently drastic treatment to reduce the phos1:homolybdate complex quantitatively. A second spraying, using stannous chloride as reducing agent, n-as tried to reduce the complex more drastically. It was necessary to regulate the strength of stannous chloride and acidity of this spray carefully, to obtain complete color development, not get a blue background on the paper, and limit sample size so that each fraction Ivould not contain more than 50 y of phosphorus pentoxide. Complete Hydrolysis of Condensed Phosphates t o Orthophosphate on Paper. Prolonged drying after spraying n i t h t h e acidic molybdate solution is not feasible, as the perchloric acid in the paper promotes charring. Trichloroacetic acid is used in t h e solvent; enough of this acid remains on t h e paper t o obtain complete hvdrolysis if t h e paper is left in the 80" to 90' C. drying oven for at least 30 minutes. By this technique quantitative recoveries were obtained even on the higher polymeric phosphates. Modification of Solvents. It was considered \I-orthnhile t o attempt t o modify Ebel's acid solvents (2) to give clearer and more rapid separation than previouqly reported. hlistures of acetic acid and various chlorine-substituted acetic acids with acetone and with acetone-methyl ethyl ketone mixtures n ere tested. Ebel's original solvents with acetone substituted for isopropyl alcohol were best. By using the modified solvent it is possible to separate orthophosphate, pyrophosphate, tripolyphosphate, and trimetaphosphate clearly from each other and from the higher condensed phosphates in 3 hours a t 10' C. The separation of tripolyphosphate from trimetaphosphate is so marked that a basic solvent is not needed for separation of the cyclic phosphates as reported by Karl-Kroupa (6). The run a t 10' C. prevents hydrolysis. Figure 1 shows a typical four-, five-, six-, and seven-component separation a t 10' C. for 3 hours. Tetraphosphate falls between tripolyphosphate and trimetaphosphate. (The tetraphosphate was added to the mixture in the form of the guanadinium salt.) For polyphosphoric acids and glassy phosphates, a solvent richer in water
was used with an overnight run on a longer paper. Ten components (including tri- and tetrametaphosphates) were separated in 16 hours. REAGENTS AND MATERIALS
A11 reagents are reagent grade and all water used is distilled. Solvents. Prepare solvents A and B fresh daily. SOLVENTA. Mix 25 nil. of 20% trichloroacetic acid solution [dissolve 100 grams of trichloroacetic acid in water, add 7 ml. of ammonium hydroxide (28% KH3), and dilute to 500 nil. with ~ a t e r ]10 , ml. of water, and 65 ml. of acetone. SOLVESTB. hIix 25 ml. of 207, trichloroacetic acid solution, 17 nil. of water, and 58 ml. of acetone. Acid Molybdate Solution ( 3 ) . Dissolve 10 grams of ammonium molyb4H,O] in 200 ml. date [(SH4)6110702 of water, add 10 ml. of hydrochloric acid (37%) and 50 ml. of perchloric acid (72%), and dilute to 1 liter with water. Stannous Chloride Solution. Prepare a stock solution veekly, containing 8 grams of stannous chloride (SnC122H20) in 2 ml. of hydrochloric acid (37%). Prepare the spray solution every day by diluting 5 drops of the stock solution to 100 nil. with 0.2LV hydrochloric acid. Filter Paper. Carl Schleicher and Schuell Co., paper 589, orange ribbon, is obtained in 20 X 20 inch sheets and cut into 10 X 10 inch sheets. PROCEDURE
Sample Preparation. Soluble sodium phosphates ranging from 50 t o 70% phosphorus pentoxide. Dilute 0.25 gram t o 100 ml. with water. Detergents. Dilute 0.5 gram to 100 ml. with water. Polyphosphoric acid. Add about 0.20 gram to 50 ml. of a chilled 20% solution of ammonium acetate. Dilute to 100 ml. with water. Preparation of Paper. Draw a starting line 1 inch from t h e bottom of a 10 X 10 inch sheet of filter paper perpendicular t o t h e grain. With a pencil, section off four 1-inch zones, 11/* inches from each end of t h e paper and 1 inch from each other on t h e starting line. Using a micro-
pipet, apply 20 pl. of the sample solutions as evenly as possible across each of the 1-inch zones. Allow the paper t o dry, staple it in a cylindrical position, and place it in a cylindrical jar (6 inches in diamet.er and 12 inches tall) containing 100 ml. of solvent. Cover the jar with a sheet of polyethylene and secure n-ith a rubber band. Overnight runs may be made by using a tnllcr jar and longer sheet of papcr. Time of Run. For commercial sodium triphosphates, detergents, and other compounds with five or less components (ortho-, pyro-, tripoly-, trimeta-, hypoly-) use solvent A for 2 hours a t room temperature or 3 hours at about 10” C. For samples with more components (glassy phosphates and polyphosphoric acid) use solvent B for IG hours a t IO” C. Development of Chromatograms. Remove the paper from the jar and d r y in a n 80‘ t o 90’ C. oven for a t least 45 minutes. Then spray on both sides with acidic molybdate solution and d r y in t h e 80’ t o 90’ C. oven for 5 t o 7 minutes. Spray both sides of the paper with t h e stannous chloride spray solution and dry for 5 minutes in t h e 80’ t o 9O0 C. oven. Carry both oven treatments only to the point where the paper ceases to be damp. Quantitative Interpretation. Cut out the 1-inch strips of paper containing the hands of each sample and run them through t h e Spinco Analytrol a t a setting t o give maximum curve peaks without flattening. Count the inteeration “ b h s ” for each comnonent (ortho, pyro, etc.) and total. The number of blips for each component divided by the total number of blips and multiplied hy 100 is equivalent to the relative per cent of each component. By obtaining the total phosphorus pentoxide of the sample. absolute results may he calculated.
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I
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Figure 1 .
Typical chromatographic seporotion of phosphates in descending order 1. 2. 3. 4.
ortho, Ortho, Ortho, Ortho,
pym. pyro, pyro, pyro,
M p d y , trimeta tripoly, trimeto, high polymeric tripdy, trimeta. tetrameto, high polymeric lripoly, tetrapoiy, trimeto, letrameta, high polymwic
I~~~ ~
~~
~
~
Table I. Synthetic Mixtures of Pyro- and Triphosphates Pyrophosphate, % Added Recovered 16.0 1G.G 12.0 12.2 10.0 9.7 7.7 8.0 6.2 G.0 3.7 4 0 3.2 2.0
RESULTS
The accuracy of the method aas tested on synthetic mixtures containing pyro- and triphosphates (Table I). The tetrasodium pyrophosphatc was prepared from the commercial grade material by three recrystallizations and by heating the resulting hydrate to 400’ C., which converted it to the anhydrous form. The triphosphate N’BS prepared by continuous recrystallization, using acetone until no pyro- band was visible by chromatography, and the phosphorus pentoxide and loss were theoretical for the hexahydrate product. It v a s not converted to the anhydrous form. Table I1 i3hows results on various detergents compared with the elution method. Table I11 shows results of three typical single determinations on polyphosphoric acid samples, canipared with results obtained by Huhti and Gartaganis (4) on acids with the same phosphorus pentoxide content.
~~
A
B C
D E
Triphosphat,e, % Added Recovered 84.0 83.4 88.0 87.8 90.0 89.3 92.0 92.3 94.0 93.8 96.0 9fi.3 96.8 08.0
Doviatian, % .. Abs. -0.8 -0.2 -0.7 +0.3 -0.2 +0.3 -1.2
Table 11. Relative Per Cent of Phosphates in Various Detergents Orthophosphate Pyrophosphate Triphosphate Elution Analytrol Elution Annlytrol Elution Analytrol 2.5 21.0 20.6 2.4 76.5 77.0 3.8 24.2 3.5 24.0 72.0 72.5 6.4 27.6 6.5 27.6 60.0 65.9 1.0 48.0 47.6 51.0 51.2 1.2 12.6 13.5 8R.G 83 0 3.8 3.5 Table Ill.
Composition of Strong Phosphoric Acid 85.0% P,OL 86.2% PsOs Analytrol Huhti ( 4 ) Analytrol Huhti ( 4 ) Analytrol 3.5 2.3 3.0 1.5 2.1 11.0 6.0 3.0 3.2 7.0 7. 1.. 7 7.7 11.0 3.3 2.8 14.0 11.0 5.2 12.0 6.0 11.3 10.5 5.3 4.7 9.5 5.5 10.0 9.6 9.0 5.0 7.7 8.6 3.5 3.2 9.0 3.3 6.0 7.9 7.6 4.0 3.8 2.8 3.3 6.0 6.0 21.7 6F.2 29.4 66.0 30.9
84.2% P*O,
Hnhti ( 4 ) 3.6 10.6 11.6 13.1 Tetra. 12.2 Pents 9.8 Heis Hepta 8.2 5.9 Oeta 4.9 Nona 20.2 Higher Ortho Pyro Tri
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VOL. 31, NO. 6, JUNE 1959
1027
SUMMARY
This is a rapid method with sufficient precision t o give reliable results on large numbers of research and control samples of condensed phosphates and phosphate detergents. The modified solvents and use of the densitometer each contribute to the saving of considerable time over previously published methods. One operator can evaluate 20 samples per day with time during running, drying, etc., for other work. If i t is desired to use an elution tech-
nique for evaluation (1, 4-7), use of the modified solvents will decrease the time needed for separation and eliminate the necessity for a two-solvent, two-dimensional separation. ACKNOWLEDGMENT
The authors express their gratitude to P\larie LTndermood for assisting in development of the method. LITERATURE CITED
(1) Crowther, Joan, ANAL. CHEW 26, 1383 (1954).
(2) Ebel, J. P., Bull. SOC.chim. France 20,991,998 (1953). (3) Hanes, C. S., Isherwood, F. .4,, Nature 164, 1107 (1949). (4) Huht,i, A. Id., Gartaganis, P. A.., Can. J . Chem. 34,785-97 (1956). 15) Karl-Krout~a. Editha. ANAL. CHEM. ' 28,1091-7 (i956). ' (6) Smith, M. J., Ibid., 31, 1023 (1959). ( 7 ) Westman, A. E. R., CronTther, J., J . -4m Ceram. s O C * 37, 420 (1954).
RECEIVEDfor review May 9, 1958. Accepted November 28, 1958. Division of Analytical Chemistrv, 133rd Meeting, ACS, San Francisco, Calif., April 1958.
Purification of Plant Amino Acids for Paper Chromatography JOHN F. THOMPSON, CLAYTON 1. MORRIS, and ROSE K. GERING U. S. Plant, Soil and Nutrition laboratory, Agricultural Research Service, U. S. Departmenf o f Agriculfure, Ifhaca, N. Y.
b In a method for the purification of amino acids by use of ion exchange resins, basic amino acids are retained on Dowex 50 on the ammonium form and other amino acids are retained on the hydrogen form. All amino acids are eluted with ammonia. The hydrolysis of labile substances is avoided by carrying out procedures at 0" to 6" C. Some amines may also b e obtained in a separate fraction, and satisfactory recoveries were obtained with standards, protein hydrolyzates, and plant extracts.
S
rater-soluble materials such as salts and sugars interfere n i t h the satisfactory separation of amino acids by paper chromatography (4)(Figure 1). Purification procedures which have been utilized include solvent extraction ( I ) , electrolytic ( 5 ) , and ion exchange resin methods (3, 13, 15, 16). Solvent extraction methods cause a breakdown of acid-labile compounds, losses of amino acids in the removal of lipides, and incomplete recovery of amino acids through the multiplicity of operations ( g ) . Electrolytic methods result in losses of amino acids (18) and the breakdown of arginine (18, 19) and glutamine (9). They also are cumbersome ( I ) . and do not successfully remove organic acids and nonionic substances. Disadvantages of published procedures for the purification of amino acids with ion exchange resins include the incomplete recovery of amino acids (3, 15, I7), incomplete separation of salts ( I S ) , use of reagents which cause hydrolysis of amides ( I S ) , loss of basic OME
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ANALYTICAL CHEMISTRY
amino acids ( 6 ) , and esterification of glutamic acid (16). The procedures described utilize a sulfonic acid ion exchange resin t o remore interfering substances from amino acids so as to avoid the breakdown of labile substances and accomplish the separation of the basic amino acids and some amines from the other amino acids. EXPERIMENTAL
Materials and Reagents. Deionized SS7ater. Water previously passed through a commercial water purifier is p u t through a column made u p of 2 Darts of Amberlite IRA400 a n d 1 part of IR-120. Ion Exchange Resin. Dowex 50-X4 1200 to 400 m&h) (The Dow Chemical Co.) is soaked in water overnight and stirred \vith a n equal volume of water in a cylinder. The fines are removed by decanting the supernatant after 30 minutes and repeating the process twice. Hydrogen Form. Donex 50-X4 is heated for 16 hours a t 100" C. n-ith 2 volumes of 1N sodium hydroxide, then poured into a column, and drained. (A convenient column is made by attaching a coarse sintered-glass funnel, 5 em. in diameter, to a glass tube a t least 40 cm. long.) After the column of resin is washed with deionized Tvater to remove the excess sodium hydroxide, it i.: treated with five column volumes of 6LV hydrochloric acid. The hydrochloric acid is removed with deionized n-ater until the effluent is free of chloride ions. Smmonium Form. Doxex 50-X4 in the hydrogen form is treated, in a column, with 10 volumes of 2N ammonium hvdroxide made with deionized water. The resin column is mashed with boiled deionized r a t e r (10 to 20 column
volumes) until the effluent reaches a p H of 8 to 9. If not used within a week, i t should be thoroughly washed just before use to remove dissolved resin. This resin should be preserved n-ith chloroform to prevent the growth of microorganisms. Glass Columns for Dowex 50. Columns are made of glass tubes (20 X 0.9 em.) with capillary tubes (10 X 0.2 em.) attached at the bottom. The top is flared to approximately a 60" taper and 3 to 4 em. in diameter. -4small plug of glass wool is placed in the column and the hydrogen or ammonium form of resin is added to a depth of 7 em. The t x o columns are arranged so that the column of ammonium resin is directly over that of the acid resin. ,4n aqueous solution having approximately 500 y of amino nitrogen, by ninhydrin analysis ( I $ ) , is adjusted to pH 7.0 =t0.1 with ammonium hydroxide or hydrochloric acid and cooled to 0" to 6" C. The resin columns are similarly cooled and subsequent operations are performed a t this temperature. The sample solution is poured on the column of ammonium resin and the effluent passes directly on the column of acid resin. The resins are then rinsed n i t h four successive 10-ml. portions of cold deionized water. If the samples contain sufficient amounts of polyphenolic substances t o interfere with separation of amino acids, the columns are further rashed with 80% alcohol (about 100 ml.) until the effluent gives no blue color with a 0.1JIsolution of ferric chloride. The effluent solution contains anions and nonionic materials. The two columns are separated and eluted individually. The column of Dowex 50 in ammonium form retains the basic amino acids and the amines. The basic amino acids and some amines are eluted with 80 ml. of 2N ammonium hydroxide.
