Quantitative Determination of Amino Acids by Circular Paper Chromatography KAMBHAMPATI KRISHNAMURTHY and MAHADEVA SWAMINATHAN Central Food Technological Research Institute,
M ysore,
A procedure for the separation of 16 amino acids from a mixture, on a series of circular paper chromatograms by using different solvent mixtures is described. The solvent mixtures used are phenol-I-butanol-acetic acid saturated with water, benzyl alcohol-tert-amyl alcohol saturated with water, 1-butanol-acetic acid saturated w-ith water, rn-cresol saturated with pH 8.4 buffer, and phenol saturated with pH 12 buffer. The chromatograms of the unknown amino acid mixture and known mixture of standard amino acids are developed side by side on the same paper. For the quantitative determination of the amino acids the chromatograms are treated with ninhydrin and the colored bands due to each amino acid in the standard and unknown chromatograms are cut. The color is extracted with 5 ml. of 75% alcohol and estimated in a Klett-Summerson photoelectric colorimeter using 560 mp filter. The method yields highly reproducible results in the determination of the amino acid content of casein, in agreement with those reported by other workers.
India
ninhydrin according to the method described by Thompson et al. ( 2 1 ) and Giri etal. (10). Preliminary accounts of the present investigation have already appeared (19-14). EXPERIMENTAL
Apparatus. The apparatus used for the development of chromatograms is shown in Figure 1 and is briefly described below. A circular glass trough 30 cm. in diameter and 15 em. in height inverted over a glass plate served as a chromatographic chamber. A circular glass stand (26 cm. in diameter) with four legs (each 5 em. in height) served as the support for the filter circle. The developing solvent was kept in a small Petri dish (10 ml. in volume) at the center over a raised platform so that the edge of the Petri dish was on the same plane as the circular glass ring. Three Petri dishes containing the components of the solvent mixture were placed a t the bottom of the cha-ber to keep the atmosphere saturated with respect to the solvents and water.
I
ri
S
ISCE the appeaIance of the now classical publication of
Consden, Gordon, and Martin ( 3 ) on partition paper chromatography, several variations of the technique have been described by many workers (1). One of these is due to Rutter ( f g ) , who employed circular filter paper on which the components separated out in concentric rings instead of the usual spots obtained ip the ascending or descending technique with filter paper sheets ( 1 , s ) . The circular paper chromatographic technique has been applied by different workers to the separation of amino acids (&If, 18, 80). It has been reported to possess certain advantages over the ascending or descending one-dimensional technique, the most important of these being: speed, sharpness of separations, and simplicitj- and compactness of apparatus; but it suffers from the same disadvantages as the one-dimensional technique in that it has not been found possible so far to separate and identify the different amino acids using any one solvent misture (8, 20). A method for the separation of the overlapping amino acids by circular paper chromatographic technique has recently been described by Giri and Rao ( 8 ) , but this involves a complicated procedure of cutting out, eluting and spotting the overlapping amino acids, and again ruriiiing a chromatogram with suitable solvents. Recently McFarren ( 1 5 ) published a neiy modification of one-dimensional technique for the separation and identification of the different amino acid. from a mixture, by employing filter paper and solvents buffered a t selerted p H values. McFarren and Mills (16) have successfully applied the above procedure for the quantitative determination of amino acids in lactoglobulin. The present investigation mas undertaken nith the object of developing a circular paper chromatographic procedure, similar in principle to the buffered paper technique of SlcFarren ( I S ) , in viex of the numerous advantages possessed by the former method over the ascending or descending techniques on filter paper sheets (20). As a result of this investigation, procedures have been developed for the separation of 16 commonly occurring amino acids by circular paper chromatographic technique using different solvent mixtures (see Table I ) . The separated amino acids have been quantitatively determined by reaction with
Figure 1. Apparatus for development of circular paper chromatograms A . Circular glass trough B . Filter circle C. Circular glass stand D . Petri dish to hold developing solvent E . Wick F. Inverted beaker GI. G?. and Ga. Petri dishes to hold components of solvent mixture Hi Glass plate
Table I. Summary of Quantitative Paper Chromatographic Procedure
Solvent I-Butanol-acetic acidwater (1O:lO:5O)4
pH of Buffer For For saturating buffering solvent filter circle
..
Phenol-l-butanolacetic acid-aater (20 :20:8:40)a
..
Benzyl alcohol-tertamyl alcohol ( 1 : 1 ) ' z saturated with water Phenol
..
m-Cresol
a
1396
Ratio by volume.
12.0
8.1
Amino Acids Separated .. Tyrosine, proline, alanine, arginine, and cystine 2.0 Tyrosine, alanine, glutamic acid, threonine. lysine, cystine, and histidine N o buffer Phenylalanine, leucine, or 8 . 4 isoleucine, and methionine 12,O Tlir,eonine, alanine, glycine, serine, aspartic acid, and glutamic acid 8.4 Phenylalanine, methionine, valine, histidine, tyrosine, arginine, and alanine
V O L U M E 27, N O . 9, S E P T E M B E R 1 9 5 5
1397
Tahle 11. Relative Distances Traveled by Amino Acids in Different Solvents PhenolBenzyl l-Buthnol- Aloohol?w 1Acetic toilPhenol Cresol ButanolAcidAmyl Saturated Saturated Acetic Water Alcohol- with p H with pH AeidAmino (20:20: water 12.0 8.4 water Acids 8:40)" ( l : l : 2 ) s Buffer Buffer (40:10:50)11 1. Aspartic 0.51 0.00 0.256 0.00 0.46 2. Glutamio 0.648 0.00 0.358 0.06 0.51 3. Arginine 0.51 0.14 1.00 0.42b 0.41b 4. Histidine 0.48b 0.24 0.85 0.58~ 0.36 5. c y s t i n e 0.38b 0.14 0.50 0.12 0.30b 6. Lysine 0 . w 0.11 0.99 0.22 0.34 7. serine 0.52 0.12 0.47b 0.15 0.47 8. Glycine 0.53 0.14 0.546 0.21 0.47 9. Threonine 0.696 0.24 0.656 0.27 0.54 10. Alanine 0.76a 0.25 u.74b o.36h 0.616 11. Proline 0.96 0.30 1.00 0.94 0.64i 12. Tyrosine 0.836 0.57 0.82 0.501 0.70b 13. Tryptophan 0.97 0.96 0.87 0.88 14. Methionine 0.96 0:7Ob 0.96 0.85k 0.84 15. Valine 0.96 0.57 0.91 0.71b 0.82 16. Phenylalanine 0.98 1.00b 1.00 1.oob 0.94 17. Leucine 1.00 0.87b 0.99 0.90 0.98 18. Isoleucine 1.00 0.916 0.99 0.91 1 no Ratio by volume. b Amino acids separated from &I1 others sufficiently for identification and Quantitative determination. ~
~~
Hydrolysis of Proteins. the hydrolysis of protein m m accomplished with 6N hydrochloric acid (25 ml. per gram of the samvle) hv autoclavine a t 15-nound n r e m m for R.h m r a The ...acid- was removed by repeated. evapo;ation a t reduced pressure. The residue was taken up in hot water and made UD to a known ~~
~
~~
~~.... ~ -
.
Buffers. T6e buffer solutions used in these investieations were
"I"...
Solvent Mixtures.
All the solvents used for preparing the
Figure 2. C:ircular paper ehromatogram showing separat i o n of oy Sti,ne, lysine, g l u t a m i e acid, threonine, alanine, and t,.irosine Solvent.
M. C.
CY. LY. Hi. Ar
Phenol-1-butsnol-aeetic addwater (20:20: 8: 40). circle buffwed at pH 2.0.
M i x t u r e of known amino aoida Aeid hydrolyzate of casein
Cystine Lysine
Histidine Arsinine
GI". Glutamio Th. Threonine AI. Alanine Gly. Gly+ Ty.'
Tyrosine
Filter
Figure 3. Circular paper c h r o m a t o g r a m showing separat i o n of phenylalanine, leucine, isoleucine, and m e t h i o n i n e Solvent. Benzyl elmhol, tert-amyl alcohol (1: 1) saturated with water. M. M i x t u r e of known amino acids Ph. Pheoylslanine Ca. Aeid hydrolyzate of casein v. Valine ~ 11. lcolcueine Ty. Tyrosine L. Leucine Pr. Proline M e . Mothienine
solvent mixtures were of analytical reagent quahty and were distilled before use. Phenol-1-butanol-acetic acid-water. Twenty volumes of phenol were mixed with 20 volumes of I-butanol, 8 volumes of acetic acid, and 40 volumes of water. The mixture was shaken for 10 minutes and allowed to separate. The top layer consisting of a mixture of solvents was separated and used in conjunction with filter papers buffered a t pH 2.0. Benzyl alcohol-tert-amyl alcohol-water, A mixture of 50 volumes of benzyl alcohol and 50 volumes of tert-amyl alcohol was saturated with water, by shaking with 100 volumes of water for 10 minutes, and the two layers were then allowed to separate. The tap layer was used as solvent in conjunction with either unbuffered filter paper or filter paper buffered a t p H 8.4. Phenol saturated with p H 12.0 buffer was prepared according to McFarren (15). This solvent was used in conjunction with filter circles buffered a t p H 12.0. m-Cresol saturated with p H 8.4 buffer was prepared according to MeFarren (15). This solvent was used in conjunction with filter papers buffered at pH 8.4. 1-Butanol-acetic acid-water. This solvent mixture was prepared according to Partridge (17) by shaking 40 volumes of 1butanol with 10 volumes of acetic acid and 50 volumes of water. After settling, the top layer was used as the solvent. Procedure for Separation and Determination of Amino Acids. On the circumference of a circle (about 4 cm. in diameter) drawn from the center of a circular filter paper (Whatman No. 1) of 28 em. in diameter, six equidistant points were marked. The test sample and known quantities of standard amino acid solutions were spotted a t alternate points. Usually in the case of the test solution, a sample containing 20 y of amino nitrogen was spotted to get good chromatograms. In the case of known amino acids, 4 to 8 y of each amino acid was used. Spotting of the solutions was done with a calibrated micropipet in a thin streak (1 cm. in length) along the line of the circumference of the circle on either side of the marked points. I n the case of dilute standard amino acid or unknown solutions, it is necessary t o build up the required concentration on paper by repeated spotting in 5.~1. quantities with drying in between in a current of warm air. A wick rolled from a strip of filter paper (2 X 3 cm.) and cut a t one end in the form of a brush, similar to that described by Giri et al. (9),was fixed t o a hole a t the center of the filter paper. The filter paper was placed on the glass stand with the brushlike end of the wick dipping in the solvent in the Petri dish inside the chromatmraphic chamber. The chromatogram was irrigated for 8 to 12 hours until the solvent front reached the edge of the
ANALYTICAL CHEMISTRY
1398 paper, the time taken being controlled by the size of the nick. The filter paper was taken out, dried in a current of warm air, andirrigatedagainintheahovemitnner. After thechromatogram had been irrigated two or three times, the filter paper uv.6 dried and sprayed with 0.5% ninhydrin in acetone containing 5% acetic acid. The chromatogram was heated a t 60" C. for 15 minutes to develop the color of the amino acids. For quantitative determination of the amino acids, the ninhydrin stained hands corresponding to the standard and unknown of each amino acid were cut, rolled, and placed in separate test tubes containing 5 ml. of 75% slcohol. The color was eluted by gently agitating the contents of the test tube for 5 minutes. The color of the extract was measured in a Klett-Summerson colari neter using a 560 mp filter. The quantity of the amino acid in the test sample was estimated from the calibration curve drawn for each of the amino acids. It is desirable to draw the calibration curve for the amino acids by running the mixture of different amino acids of known concentrations on the same paper, side by side, with the test saanples.
Table IV.
Amino Acid Composition of Casein
Calculated to 16 grams of nitrogen present I m i n o Add Results Aspartic Glutarnio Arginine Lysine, Histidine cystine Seri"C Glycine Threonine T"r0Si"e
.&nine Methionine valine Phenylalanine Leucine Isoleuelne
6.4 25.1 4.9 8.6 3.7 0.6 5.5 2.4 4.9
Block
($1
7.0 23.0 4.2 8.6
3.?
6.1
3.1 3.4 6.8 6.1 10.7 7.0
RESULTS
Table "1.
.Determination of Certain Amino Acids i n -JJsingDifferent Solvent Mixtures
c r a m s of Nitrogen) 0.62 0.60 4.9 3.0
Solvent henol-1-buthnol-aeeti~ acid -rater .Butanol-aoefie acid-water .Butanol-acetic acid-water ,-Cresol saturated with pH 5.4 buffer henolsaturated with pH 12.0 buffer i 2 i Phenol-1-butanol-soetic aeld-w*ter ~
~
..
~~
5."
4.8
5.4 buffer .0 buffer -water
(1) I-Butanol-acetic aeid-l-yhter (2) Phenol-1-bolanol-aoetle acid-water /./
-
P.""",
_et.._e+eA
"ith
.,"*
d
The composition of the different solvent mixtures used for the Beparation of various amino acids is presented in Table I. Table I1 gives the relative distance traveled by different amino acids with the various solvent mixtures.
3.1 3.0 3.2 3.0 6.1 6.0 3.3
in Figures 2 to G.
Figure 4. Circular paper chromatogram showing separation of aspartic acid, glutamic acid, serine, glycine, threonine, and alanine Solrent. Phenol-asturated with pH 12.0 buffer. Filter circle buffered at pH 12.0. M. Mixture of known amino acids Se. Serine H. Acid hydrolyzate of protein Gly. Glycine Asp. Glu.
Aspartio acid Glufamio acid
Th. Ai.
Threonine Alaninc
Table 111 gives the content of certain amino acids in casein determined after separating them on the circular paper chromatograms hy different solvent systems. There is good agreement lmt,ween the values obtained. The figures obtained far the content of 1G amino acids in casein by the present procedure together with the values reported in the literature (Z) are presented in Table IV DISCUSSION
The results obtained in the present investigation show t h a t 16 amino acids could he quantitatively determined b y the procedure outlined in this paper. The advantages of the present
V O L U M E 27, NO. 9, S E P T E M B E R 1 9 5 5
1399 using as solvent a mixture of lnl-amyl alcohol and benzyl alcohol saturated with water. Work is now in progress on the analysis of some food proteins using the procedure described in this paper. ACKNOWLEDGMENT
The authors wish to thank V. Subrahmanym for his keen interest and helpful suggestions. LITERATURE CITED
Figure 6. Circular paper c h r o m a t o g r a m showing separat i o n of phenylalanine, methionine, valine, histidine, tyrosine, arginine, a n d a l a n i n e Solvent.
m-Cresol saturated w i t h OH 8.4 buffer. Fi1-r oimle bufferrd et OH 8.4.
C. A d d hydrolyzate of aascio M. Mixture of known amino acids Me. Methionine
s.
Serine
Ph. Phenylalanine
v.
Valine Histidine Alanine Tg. Tyrosine Ac. Arsinins
Hi. AI.
method over the techniques described by other workers are speed of separation, clarity and compactness of amino acid zones, and the ready wailability, a t low cost, of the solvents required. Table 111 shows thst 8ome of the amino acids can be separated and estimated by more than one solvent mixture. This has aerved as a check both on the values obtsjned for these amino acids and also on the accuracy of the method. The separation of lysine from the other hasic amino acids has engaged the attention of many workers in the past. Consden, Gordon, and Martin (5) and Dent (4) succeeded in separating the above amino acids from a mixture, by two-dimensional chramatography using phenol and collidine as the developing solvents. McFarren (16) recornmended the use of lutidine saturated with pH 6.2 buffer for the same purpose by the undimensional technique. I n the present method the separation of lysine from other amino acids has been achieved by a cheap solvent system consisting of phenol-l-butsnol-acetic acid-water. This solvent mixture is easy to handle, unlike collidine and lutidine which possess an unpleasant odor. Very few solvents have so.far been suggested in the literature for the separation of leucine and isoleucine from each other and from other amino acids. Consden, Gordon, and Martin ( S ) used benzyl aleohol-water or l-butanol-water or a mixture of the two in equal proportions in unidimensional chromatograms, for this purpose. Edman (5)and Wretlind ($3) suggested the use of pyridine-amyl alcohol-water mixture. Work (B)reported that tert-amyl alcohol saturated with water was satisfactory for the separation of leucine, isoleucine, and phenylalanine, when the paper strip was irrigated for 3 days. McFarren ( 1 5 ) recommended the use of benzyl aleohol-l-butanol saturated with p H 8.4 buffer in conjunction with a filter paper buffered a t the same pH. None of the above solvent systems proved quite satisfactory in the authors’ hands for this purpose. The separation of leucine, isoleucine, and phenylalanine was finally achieved by
(1) Balston, J. N., and Talbot, B.E., “A Guide to Filter Paper and Cellulose Powder Chromatography.” H. Reeve Angel & CO.. London, 1952. (2) Block, R. J.. and Bolling, D., “Amino Acid Composition of Proteins and Foods,” 2nd ed.. C. C Thomas. Springfield. Ill., 1950. (3) Consden. R., Gordon, A. H., and Martin. A. J. P..Biochm. J . , 38, 224 (1944). (4) Dent. C. E., B i o c h a . J.. 43, 169 (1948). ( 5 ) Edman, P., A ~ k i vK a i Minmd. Geol., 22, 3, I (1945). (6) Giri, K. V., Current Sci. (India), 20, 296. (1950). (7) Giri. X. V.. and Rao, N. A. N., J . IndianImt. Sci., 34.95 (1952). (4)Ibid.. 35, 343 (1953). (9) Giri, K. V., Krishnamurthy, K., and Venkitasuhramanyan. T. A., current S C ~ . (rndia)., 2 1 , i i ( 1 ~ 5 2 ) . (IO) Zbid.. p. 44. (11) Giri. K. V., Krishnamurthy, K.. and Yenkitmubramanyan. T. A.. J. Indian Inst. Sci., 34,209 (1952). (12) Krishnamurthy. K., and Swaminethan,M., Cuwent Sci. (India), 23, 223 (1954). (13) Krishnamurthy, K.. and Swaminathan, M., J . Sci. I d . Rcseavch (India). 13B, 374 (1954). (14) Krishnamurthy. K.. and Swarninathan,M., Science and Cultwe (rndiaj. 20, 51 (1954). (15) McFarren. E. F.. ANAL.CHEM.,23, 168 (1951). (161 MoFarren. E. F.. and Mills, J. A,, Ibid.. 24, 650 (1952). (17) Partridge. S. M., Biochom. J . , 42, 238 (1948). (18) Proom. H., and W’oiwood. A. J.. J . Gen. Mirrobiol.. 5, 681 (1951). (19) Rutker, L., Notwe. 161, 435 (1948). (20) Ssifer, A.. and Oreskes. I.. ANAL.CHEM.,25, 1539 (1953). (21) Thompson, J. F.. Zaeharius, R. M., and Steward. F. C., Plant Phzlsiol., 26, 375 (1951). (22) Work. E., Biochim. et Bzophw. Acta. 3, 400 (1949). (23) Wretlind. K. A. J.. Acta Phwiol. Scond., 13, 45 (1947).
.
R E ~ E ~ Vfor E Drwiew October 6,1954. Accepted April 28. 1955
rreuisiuii
iii
A-nay ciiiissiuii apecrrugrapny
I n the article on “Precision in X-Ray Emission Spectrography” [Liebhafsky, H. A., Pfeiffer, H. G., and Zemany, P. D., ANAL. CHEM.,27,1257 (1955)], in the third paragrapli from the end the last sentence should read: Further, when sl 8 ~ an increase in the number of the counts will not necessarily reduce SA.
>>
Determination of Nordlhydroguaiaretic Acid in Creosote Bush I n the article on “Determination of Nordihydroguaiaretic Acid in Creosote Bush” [Page, J. 0.. ANAL. CHEM.,27, 1266 (1955)1, the first line of data. on unrecrystdlized nordihydroguaiaretic acid tetraacetate should read: NDG.4, twice reermt. m.p. 186-70 c.
0.P702
1.5214
1.5237
-1.5
66.52
6.65
.
