Determination of Lysine, Arginine, and Histidine by High Temperature Paper Chromatography STANlMlR M. SlBALlC and NADA V. RADU Deportment of Biochemistry, Institute of Hygiene, Belgrade, Yugoslovia
b Basic amino ocids were determined in cosein hydrolyzote b y horizontal paper chromatography after adsorption on ion exchange resin ond subsequent elution. The separation was made on filter paper in 3 hours at 40' C. with phenol saturated b y phosphate buffer o t pH 6.8. Separated amino acids were dyed b y ninhydrin, spots were eluted b y a mixture of ethyl alcohol and woter, and the dye was read at 570 mp.
METH?DS
for a quiqk separation of armno acids by h g h temperature (5, 8) and ion exchange (7) paper chromatography have been described. The experience of H i m a (5),and pf Thompson (9)who determined amino acids in vegetable materials and protein hydrolyzates, led to the development of the method described herein. As a preliminary, basic amino acids were separated from other amino acids from 100 mg. of casein hydrolyzate by adsorption of Dowex 50-X4 resin in the ammonium form, followed by elution with 3N " O H . Basic amino acids were adsorbed also on Zeocarb 225 as well as on Amberlite IR-120 resins. Development of the chromatograms of basic amino acids according t o Thompson's procedure (9) required 84 hours; this was reduced to 31/* hours by applying increased temperature. Chromatographic Chamber. For the development of t h e chromatograms, a glass-paned chamber 61 X 15.5 X 30 em., whose edges are sealed by an alloy of water glass and talcum powder, is used. A glass frame, 68 X 38 X 4 em., is glued t o the top edges of the chamber; a matching glass pane with two openings 0.5 em. in diameter a n d 4 om. from the shorter edges is leaned against the frame. Below each hole there is a glass trough, 28.5 X 3 X 3 cm., fastened 10 em. from the bottom of the chamber. Two glass rods, used to support the filter paper, are b e d parallel to each trough at a distance of 9 cm. The first rod is 8 em. from the bottdm of the chamber and the second is 6 em. The chamber is placed in a large incubator whose temperature is 40" C. Filter Paper. Whatman No. 1 chromatographic paper, 60 X 60 cm.,
Y
P
Figure 1. Horizontal poper chromatogram of lysine, orginine, and histidine after 3 hours of development with phenol pH 6.8, 40' C. S. Standard, 16 pg. of lysine, 8 pg. of orginine, and 6 fig. of histidine in 0.01 ml. H. Hydrolyzate, 200 ~ g of, hydrolyzed casein in 0.01 ml.
is halved and buffered. When t h e paper dries, at room temperature, it is cut into 24 X 30 em. sheets. Reagents. Phosphate buffer, p H 6.8 (200 ml. of 0.067M Na2HPOI plus 250 ml. of 0.067M KH,P06). Phenol-Solvent Mixture. Commercial phenol is distilled and mixed with an equal volume of buffer. The lower phase is used for the development of the chromatogram, and the upper for saturation of the chamber. Ninhydrin, 0.5 gram, is dissolved in 98 ml. of acetone and 2 ml. of glacial acetic acid. Glass Columns for Ion Exchange Resin. The columns are made of glass buret tubes (65 X 0.8 em.) with capillary tubes (8 X 0.2 em.) attached a t the bottom. At the bottom of the columns glass wool is placed, 3 em. high. Then they are filled with 45 em. of resin in the ammonium form (SI, and topped with 2 cm. of glass beads. The speed of the liquid flowing through the resin is 12 ml. per hour. This column is sufficient for adsorption of basic amino acids from 100 to 200 mg. of casein hydrolyzate. Sample Preparation. Approximately 200 mg. of casein are hydrolyzed with 40 ml. of 6 N HC1 for 20 hours in a sealed tube. After removal of the acid, t h e residue is dissolved in 10 ml. of water and nitrogen is determined in an aliquot (1 ml. of hydrolyzate). From the remainder of the hydrolyzate an amount is weighed which contains 16 mg. of nitrogen; it is diluted to about 50 ml., its p H is brought t o 7.0 with 2N ",OH, and then it is passed through the column. Acidic and neutral amino
acids are rinsed with deionized water until a negative reaction on ninhydrin is reached. After completion of rinsing, basic amino acids are eluted with 130 ml. of 3N ",OH. The eluate is dried at below 50" C. and the residue is dissolved in 5 ml. of 10% isopropyl alcohol (a). Standard Solution of Basic Amino Acids (General Biochemical, Inc., Chagrin Falls, Ohio). Lysine (16 mg.) arginine (8 mg.), and histidine (6 mg.) are dissolved in approximately 30 ml. of water, the p H is brought to 7.0 with 2N ",OH, and the solution is diluted t o about 50 ml., and passed through another column of resin, of the size used previously. After elution, rinsing, and evaporation, the residue is dissolved in 10 ml. of 10% isopropyl alcohol. Procedure. On the buffered paper, 8.5 em. from t h e narrower edges, four pencilled lines are drawn.4 cm. in length and 1.6 cm. apart (Figure 1). T o these lines 0.01 ml. of standard solution and hydrolyzate are applied in turn by micropipet. At a distance of 7 em. from t h e edge, the sheet is folded at a right angle and placed over a rod io the trough. Another sheet is prepared in the same way and put in the other trough. On the bottom of the chamber are two Petri dishes containing water saturated with phenol. The chamber is covered with a lid and left a t 40" C. for '/n hour and then, through the holes in the lid, 60 ml. of buffered phenol are added to each trough, the holes are closed, and the chromatograms are developed for 3 hours. After that the papers are removed, left at room temperature for VOL. 33, NO. 9, AUGUST 1961
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24 hours, pulled thrwgh the solution of ninhydrin, and again left in darkness at room temperature for 12 hours. The colored spots are cut out, weighed to the nearest milligram (9), cut in strips, and placed in test tubes. For lysine and arginine 10 ml. of 50% ethyl alcohol are added and for histidine 5 ml. of 50% ethyl alcohol are added. The tubes are shaken for 30 minutes, the contents are filtered, and the color intensity is read at 570 mp. Absorbance for hydrolyzate is divided by the absorbance for standard, and the factor obtained is multiplied by the amount of standard amino acids applied on paper. From these data the percentage of individual basic amino acids in the hydrolyzate is calculated (3).
Table I. lysine, Arginine, and Histidine in Casein as Determined by Horizontal Paper Chromatography
(Results expressed against 16 grams of nitrogen) Chromatogram Lysine Arginine Histidine 1 2 3 4 5 6 7 8 9
Av. Std. dev. (6)
8.72 8.74 8.66 8.40 8.63 8.69 8.55 8.83 8.55
8.67
3.69 3.70 3.98 3.82 3.80 3.81 3.78 3.73 3.75 3.78
3.10 3.04 3.30 3.04 3.04 3.04 3.08 3.09 3.20 3.10
0.139
0.087
0.089
n
.
on caseinic hydrolyzate are shown in Table I. The standard deviation for these amino acids shows that these determinations might be repeated with good accuracy. These values are in good agreement with literature values (1, 41
LITERATURE CITED
(1) Bergdoll, ?*I. S., Doty, D. M., IND. EKC.CHEM.,A K A L . ED. 18, 600 (1946). (2) Block, R. J., “Paper Chroniatog-
raphy,” Academic Press, New York,
1952. (3) Fischer, F. G., Dorfel, H., Biochem. 2. 324, 544 (1953). 14) Guirard. B. hl.. Snell. E. E.. Williams. R.J., Proc. Soc.‘ Exptl. Biol: Med. 61; 158 (1946). (5) Himes, J. B., Metcalfe, L. D., ANAL. CHEM.31, 1192 (1959). (6) Roberts, H. R., Kolor, 111. G., Ibid., 29, 1800 (1957). (7) Ibid.. 31. 565 (1959). ? S i Rob&,”. R:, Kolor, M. G., Nature 183, 460 (1959). (9) Thompson, F. J., Morris, J. C., Gering, k‘. R., ANAL.CHEM.,31, 1028, 1031 (1959). (10) U.S.Dept. Agr., Washington, D. C., “Amino Acid Content of Foods,” 1957. \
RESULTS AND DISCUSSIONS
In Figure 1 is shown a chromatograni obtained after the solution of basic amino acids (standard solution and casein hydrolyzate after elution from Dowex 50-X4 resin) was chromatographed for 3l/* hours at 40’ C. by paper and phenol, both buffered at p H 6,8. The results obtained from the determination of the three amino acids
hydrin was much better if carried out at room temperature than a t any other increased temperature. Blanks were not taken into consideration, as the determinations of standard amino acids on the same paper were available. Only the size and weight of the spots should be taken into consideration, to be as accurate as possible.
lo).
The development of color with nin-
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RECEIVEDfor review May 11, 1960. Accepted February 27, 1961.
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bpurious Kecovery Tests in Tocopherol Determinations V. H. BOOTH Dunn Nutritional laboratory, Milton Road, Cambridge, England
b Fat solvents contained small amounts of reducing substances that simulated tocopherols by reacting with the ferric chloride-bipyridyl reagent. In proving new methods these substances enable 100% recovery to be reported, even though some tocopherol has really been lost. The substances were separable from tocopherols by paper chromatography, but were not always separable by column chromatography. Reducing substances were also found in solvents after they had percolated through columns of various adsorbents.
I
N DISCUSSIONS on the determination of tocopherol, while attention is given to removing interfering substances from the material to be analyzed, little has been written about artifacts introduced into the system by the reagents. Writings on determination include a comprehensive review by Dicks (4), general articles by Lehman (11) and by the Analytical Methods Committee ( I ) , and paragraphs on the Emmerie-Engel test or other particular aspects by Kjolhede @), Edis-
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ANALYTICAL CHEMISTRY
bury, Gillow, and Taylor (6), and Diplock, Green, Edwin, and Bunyan (6). .Most authors give directions for purifying ethyl or alcohol diethyl ether but not always other solvents. For example, Lehman (11) says the absorbance in the control can be reduced by distilling ethyl alcohol. Quaife and Dju ( 1 2 ) say “petroleum ether must be purified owing to impurities which give an Emmerie and Engel reaction,” and refer to Werner: The paper by Werner ( 1 4 ) , however, is on etherpresumably diethyl-not petroleum. Hobson-Frohock (8) observed a n effect of water on the color developed in the Emmerie-Engel reaction. Rindi (IS) reduced error by making a complete blank run in every test. Some authors circumvent reducing reactions, and measure absorbance a t 292 m,u; Lambertsen and Braekkan (10) did this and corrected for irrelevant absorbance by a geometric procedure. These references provide scanty information on reducing substances present in solvents, and on tocopherol simulators eluted from ndsorbents. In this paper experiments are de-
scribed that show how such artifacts may interfere with accuracy, so that apparently complete recoveries have little meaning unless the recovered tocopherol is properly characterized.
BlPYRlDYL COLOR TEST
In most methods for the determination of tocopherols the lipides are extracted, saponified, and chromatographed, and a modified EmmerieEngel test is done on the residue. In this test tocopherol reduces ferric salt to ferrous, which reacts with 2,2’bipyridyl (2,2’-bipyridine) to give a red color. The modification used in the present work was that of the Analytical Methods Committee (1). Standard procedure included a blank test for subtracting from each reading. With 3.5 ml. of 0.072% bipyridyl 0.5 ml. of 0.2% FeCI,.6H20, both in ethyl alcohol (f), and optical path of 1 cm., this blank absorbance \$as usually about 0.060 after 2 minutes in the dark. With more dilute reagents the blank was lower.
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