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Interaction of substances during the process of paper chromatography
VOL. 20 (z956)
SHORT COMMUNICATIONS
393
Interaction of substances during the process of paper chromatography The f u n d a m e n t a l principle of paper c h r o m a t o g r a p h y is t h a t the c o m p o u n d s move independently of each other on the p a p e r strip, and their quality is thus determined by their position. Consequently, the s u b s t a n c e s a p p e a r as discrete spots. In contrast, there frequently a p p e a r more stretched b a n d s - - h o m o g e n e o u s or s t r u c t u r e d - - o r the spots are of the so-called " c o m e t " shape, instead of being discrete spots 1-6. These p h e n o m e n a m a y be explained partly by dissociation of the compounds, p a r t l y by interaction of the substances (eventually t h a t of the substances and the solvent). I t m a y be noted t h a t the same p h e n o m e n o n is encountered in the paper chromatog r a p h y of both inorganic ~-9 and organic substances. HACKMAN AND LAZARUS in referring to one of our papers 4 - - where the p h e n o m e n a occurring during the simultaneous c h r o m a t o g r a p h i n g of different amino acids were explained as being due to their i n t e r a c t i o n - - w r o t e as followsl°: " i f this explanation is correct then the interpretation of p a p e r c h r o m a t o g r a p h y would become extremely complicated, results of earlier workers might have to be reassessed and the usefulness of paper c h r o m a t o g r a p h y for the q u a n t i t a t i v e and qualitative analysis of amino acids wouhl be considerably reduced". First of' all, it m a y be noted t h a t the basis of this conclusion is not the explanation b u t the p h e n o m e n o n itself. The impressive results and vast use/ulness o/ paper chromatography as a microanalytical method--especially i~: a~:alysis o[ amino a c i d s - - i s beyond doubt. However, the results of the paper c h r o m a t o g r a p h y m a y be considered adequate only if the f u n d a m e n t a l condition mentioned above, i.e. the independent m o v e m e n t of the substances, is fulfilled. HACKMAN AND LAZARUS suggest interaction between the glutamic acid and the phenol and effect of change of p H as a possible explanation for the phenomenon. We can also confirm the multiple spots p h e n o m e n o n w i t h glutamic acid alone, using phenol as solvent. In our opinion, however, the interactiou of the a m i n o acids is proved by the following experiments. On simultaneous c h r o m a t o g r a p h i n g of the glycine and glutamic acid (solvent: butanol-acetic acid) three spots appeared, at the mol ratios: 3:1, 4 : I , 5:1, b u t when the glycine and glutamic acid were separately c h r o m a t o g r a p h e d only a single spot appeared in every case. The arginine and glutamic acid being simultaneously c h r o m a t o g r a p h e d with phenol, the change of R F values is most pronounced at I: 4 tool ratio, according to FRANKEL'S statement, i.e. these amino acids form a so-called molecule c o m p o u n d at this mol ratio H. The change of R F values and stretching of spots m a y be observed if the amino acids are soluble either in buffer solution or water, and is m o s t pronounced at p H 3.-' and 7. (The p H of the saturated solution of the molecule c o m p o u n d is 3.2.) Naturally, the p h e n o m e n o n cannot be observed at e x t r e m e low and high p H values owing to the decomposition of the molecule compound. However, the multiple spots p h e n o m e n o n a p p e a r s in the presence of larger a m o u n t of amino acids only. In these and the above-mentioned experiments the a m o u n t of a m i n o acids were a b o u t lO-2O 7 and in no case more t h a n 5o ~,. Our explanation of the role of amino and carboxyl groups in the interaction is consistent with the following experiment also: if these groups are blocked by metal complex formation*the interaction (change of R F values) cannot be observed TM. I n our opinion the c h r o m a t o g r a p h y of amino acid-metal complexes instead of free amino acids is one of the most promising m e t h o d s for the more exact p a p e r c h r o m a t o g r a p h y of the a m i n o acids.
Institute for Inorganic and Analytical Chemistry, Pediatric Department, University of Szeged, Szeged (Hungary)
M. T. BECK P. ~BREV
1 j. W. H. LUGG AND B. T. OVERELL, Nature, 16o (1947) 87. S. ARONOFF, Science, 11o (1949) 590. 3 G. ZlMMERMANN, Z. anal. Chem., I38 (I953) 32I. 4 i . T. BECK AND P. ~BREY, 2Xiagyar K~m. Foly6irat, 59 (1953) 350; ,4cta Chim..4cad. Sci. H)o~g., 4 (1954) 231. 5 p. ]~-BREY AND M. T. BECK, Kis~rletes Orvostudomd~zy, 7. ([955) 14.5. e G. B. WEST AND J. F. RILEY, Nature, 174 (I954) 882. 7 E. C. MARTIN, Anal. Chim. Acta, [3 (1955) 353. s M. T. BECK, Acta Chim. Acad. Sci. Hung., 3 (19.53) 187. 9 B. ERDEM AND H. ERLENMEYER, Helv. Chim..4cta, 37 (1954) 222o. 10 R. H. HACKMAN AND M. LAZARUS, Biochim. Biophys..4cta, 17 (1955) 147. ii M. FRANKEL, Biochem. Z., 242 (1931) 67. lz M. T. BECK AND J. CS/~.SZ/~R, .4cta Chim. ,4cad. Sci. Hung., 7 (1955) 465 • Received J a n u a r y 12th, 1956
SHORT COMMUNICATIONS
393
Interaction of substances during the process of paper chromatography The f u n d a m e n t a l principle of paper c h r o m a t o g r a p h y is t h a t the c o m p o u n d s move independently of each other on the p a p e r strip, and their quality is thus determined by their position. Consequently, the s u b s t a n c e s a p p e a r as discrete spots. In contrast, there frequently a p p e a r more stretched b a n d s - - h o m o g e n e o u s or s t r u c t u r e d - - o r the spots are of the so-called " c o m e t " shape, instead of being discrete spots 1-6. These p h e n o m e n a m a y be explained partly by dissociation of the compounds, p a r t l y by interaction of the substances (eventually t h a t of the substances and the solvent). I t m a y be noted t h a t the same p h e n o m e n o n is encountered in the paper chromatog r a p h y of both inorganic ~-9 and organic substances. HACKMAN AND LAZARUS in referring to one of our papers 4 - - where the p h e n o m e n a occurring during the simultaneous c h r o m a t o g r a p h i n g of different amino acids were explained as being due to their i n t e r a c t i o n - - w r o t e as followsl°: " i f this explanation is correct then the interpretation of p a p e r c h r o m a t o g r a p h y would become extremely complicated, results of earlier workers might have to be reassessed and the usefulness of paper c h r o m a t o g r a p h y for the q u a n t i t a t i v e and qualitative analysis of amino acids wouhl be considerably reduced". First of' all, it m a y be noted t h a t the basis of this conclusion is not the explanation b u t the p h e n o m e n o n itself. The impressive results and vast use/ulness o/ paper chromatography as a microanalytical method--especially i~: a~:alysis o[ amino a c i d s - - i s beyond doubt. However, the results of the paper c h r o m a t o g r a p h y m a y be considered adequate only if the f u n d a m e n t a l condition mentioned above, i.e. the independent m o v e m e n t of the substances, is fulfilled. HACKMAN AND LAZARUS suggest interaction between the glutamic acid and the phenol and effect of change of p H as a possible explanation for the phenomenon. We can also confirm the multiple spots p h e n o m e n o n w i t h glutamic acid alone, using phenol as solvent. In our opinion, however, the interactiou of the a m i n o acids is proved by the following experiments. On simultaneous c h r o m a t o g r a p h i n g of the glycine and glutamic acid (solvent: butanol-acetic acid) three spots appeared, at the mol ratios: 3:1, 4 : I , 5:1, b u t when the glycine and glutamic acid were separately c h r o m a t o g r a p h e d only a single spot appeared in every case. The arginine and glutamic acid being simultaneously c h r o m a t o g r a p h e d with phenol, the change of R F values is most pronounced at I: 4 tool ratio, according to FRANKEL'S statement, i.e. these amino acids form a so-called molecule c o m p o u n d at this mol ratio H. The change of R F values and stretching of spots m a y be observed if the amino acids are soluble either in buffer solution or water, and is m o s t pronounced at p H 3.-' and 7. (The p H of the saturated solution of the molecule c o m p o u n d is 3.2.) Naturally, the p h e n o m e n o n cannot be observed at e x t r e m e low and high p H values owing to the decomposition of the molecule compound. However, the multiple spots p h e n o m e n o n a p p e a r s in the presence of larger a m o u n t of amino acids only. In these and the above-mentioned experiments the a m o u n t of a m i n o acids were a b o u t lO-2O 7 and in no case more t h a n 5o ~,. Our explanation of the role of amino and carboxyl groups in the interaction is consistent with the following experiment also: if these groups are blocked by metal complex formation*the interaction (change of R F values) cannot be observed TM. I n our opinion the c h r o m a t o g r a p h y of amino acid-metal complexes instead of free amino acids is one of the most promising m e t h o d s for the more exact p a p e r c h r o m a t o g r a p h y of the a m i n o acids.
Institute for Inorganic and Analytical Chemistry, Pediatric Department, University of Szeged, Szeged (Hungary)
M. T. BECK P. ~BREV
1 j. W. H. LUGG AND B. T. OVERELL, Nature, 16o (1947) 87. S. ARONOFF, Science, 11o (1949) 590. 3 G. ZlMMERMANN, Z. anal. Chem., I38 (I953) 32I. 4 i . T. BECK AND P. ~BREY, 2Xiagyar K~m. Foly6irat, 59 (1953) 350; ,4cta Chim..4cad. Sci. H)o~g., 4 (1954) 231. 5 p. ]~-BREY AND M. T. BECK, Kis~rletes Orvostudomd~zy, 7. ([955) 14.5. e G. B. WEST AND J. F. RILEY, Nature, 174 (I954) 882. 7 E. C. MARTIN, Anal. Chim. Acta, [3 (1955) 353. s M. T. BECK, Acta Chim. Acad. Sci. Hung., 3 (19.53) 187. 9 B. ERDEM AND H. ERLENMEYER, Helv. Chim..4cta, 37 (1954) 222o. 10 R. H. HACKMAN AND M. LAZARUS, Biochim. Biophys..4cta, 17 (1955) 147. ii M. FRANKEL, Biochem. Z., 242 (1931) 67. lz M. T. BECK AND J. CS/~.SZ/~R, .4cta Chim. ,4cad. Sci. Hung., 7 (1955) 465 • Received J a n u a r y 12th, 1956