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Indirect oxidimetric determination of certain α-amino acids with vanadium(V)
136
ANALYTICA CHIMICA ACTA
INDIRECT OXIDIMETRIC DETERMINATION OF CERTAIN a-AMINO ACIDS WITH VANADIUM(V) S. P. RAO, H. S. R A T H I AND J, N. GAUR
Department o/Chemistry, Jaswant College, Jodhpur (India) (Received November 2Ist, 196o)
a-Amino acids which are smoothly oxidised quantitatively b y certain 1,2-glycol splitting reagents such as periodic acid 1 and lead tetraacetate 2 are resistant to certain other oxidants such as cerium(IV) 3 and vanadium(V). The de-aminated products of the amino acids with nitrous acid, i.e. the a-hydroxy acids, can however be oxidised easily by these oxidants. TAKAHASHI et al. 3 have developed an indirect method for the determination of a-amino acids via oxidation of the de-amination products but the results are not strictly stoichiometric and empirical factors calculated from pure compounds are required. Studies on the oxidation of organic compounds by vanadate solution in these laboratories have shown that vanadate is a milder and more specific oxidant for organic compounds than cerium(IV). The present communication describes a study of the oxidation of the de-amination products of the a-amino acids with a view to developing a method for their determination; the reactions are more stoichiometric than with cerium(IV). It has also been shown that pure vanadium pentoxide can be used as a primary standard. ~XP~RIMEHTAL
Preparation and standardisation o/vanadate solution/tom vanadium pentoxide Earlier workers*, 5 have recommended either ammonium vanadate or sodium metavanadate for the preparation of vanadate solutions. BISHOP AND CRAWFORD 5 have shown that pure ammonium vanadate can be used as a primary standard but it is generally preferred to standardise the solution before use. RAO et al. 4 converted ammonium vanadate to the sodium salt by boiling with slight excess of sodium carbonate till the smell of ammonia ceased and decomposed excess carbonate with acid but this method is laborious. Therefore it was thought better to use vanadium pentoxide for preparing sodium vanadate solution. The vanadium pentoxide used in the present investigation was an A.R. sample supplied by Baker which was stated to have I o o % assay. Analytically pure vanadium pentoxide can also be prepared by decomposing twice crystallized ammonium metavanadate in a platinum crucible in presence of air at a temperature of 4oo°-5oo ° and storing the material in a desiccator. Vanadium pentoxide which consists of cinnabar red rhombic crystals, is only slightly soluble in water giving an acidic reaction, but dissolves freely in alkaline solution to form colourless vanadate solution. On acidification the solution turns yellow but does not reprecipitate at low concenAnal. Chim. Acta, 25 (1961) 136-138
INDIRECT OXIDIMETRY OF 0~-AMINO ACIDS WITH VANADIUM(V)
137
trations (c[. As~O:~). Solutions of exact strength can be prepared by this method and are free from ammonium ions. According to JANDER AND JAHR6, polyvanadates exist as an equilibrium mixture in the solution depending on its pH. But this does not affect the usual oxidising properties of the vanadate solution. The vanadate solution was prepared by shaking a weighed quantity of vanadium pentoxide in approximately I.O N sodium hydroxide until dissolution was complete. The solution was then diluted to the mark in a volumetric flask. The strengths of the solutions were checked by titrating against a standard ferrous ammonium sulphate solution, the strength of which was determined by potassium dichromate solution, N-phenylanthranilic acid being used as internal indicator. The results of the standardisation given in Table I indicate that the vanadium pentoxide can be used satisfactorily as a primary standard. TABLE I Va~)adium pentoxi~taken (g)
Vanadium penloxide~und (g)
9.o856 8.8792 lO.1224
9.o91o 8.86oo lO.134o
Amino acid solutions Fresh 0.02 M solutions of pure amino acids (DL-alanine, DL-norleucine, and 3-glutamic acid) were prepared by dissolving the requisite amount of pure amino acids in distilled water.
General procedure [or de-amination and oxidation o~ amino acids To an aliquot of the amino acid solution in a 25o-ml conical flask fitted with a reflux condenser, approximately I ml of 3o% sodium nitrite solution and IO ml of 3 N sulphuric acid together with 2o ml of distilled water were added and the solution was boiled till a drop of the solution gave no colour with starch-iodide paper. A known excess of o.I N vanadate solution was added and the overall acid concentration was adjusted to approximately 12 N. The solution was refluxed on a water bath for 3o min and then cooled. The excess of vanadate in the solution was determined by back-titration with standard ferrous ammonium sulphate solution potentiometrically or with N-phenylanthranilic acid as indicator. From the amount of vanadate consumed the equivalents of oxygen consumed per mole of the amino acid was determined. The results are given in Table II. T A B L E II
A mino acid
oL-Alanine DL-Norleucine D-Glutamic acid
A mount of o.oz M amino acid (too
A m o u n t of o.x N vanadate solution (too
5-o 4.o 3.5
5 5 5
Equivalents o] oxygen ¢onsumed per mole of amino acid found calculated 2.15 2.0 3 4-3 2
2 2 4
A n a l . C h i m . A c t a , 25 (1961) 136-I38
138
s . P . RAO, H. S. RATHI, J. N. GAUR
From Table II it is evident that the equivalents of oxygen consumed correspond to the oxidation to formic acid and higher fatty acids according to the general equations: R C H (NH,) C O O H
HNO~ -+ R C H (OH) C O O H + N2 + H , O O
R C H (OH) C O O H
> RCOOH + HCOOH
and that the reactions are more or less stoichiometric. DISCUSSION
It has been shown that the oxidation of the de-amination products of the a-amino acids with vanadate provides a more satisfactory method for their determination than oxidation by cerium(IV) and the use of empirical corrections can be eliminated. The success of the method depends to a very great extent on complete elimination of nitrous acid, on preventing the loss of the decomposition products of a-hydroxy acids, and on preventing further oxidation of formic acid. That formic acid is not oxidJsed to a measurable extent under these conditions is well established and the loss of volatile decomposition products, i.e. the aldehydes and the formic acid, can be minimised by circulating cold water through the reflux condenser. The method can also be applied for the determination of a mixture of two or more amino acids. ACKNOWLEDGEMENT
The authors express their thanks to Prof. K. P. H A L D A R , Head of the Department of Chemistry, for his keen interest in the work. SUMMARY A m e t h o d for p r e p a r i n g s t a n d a r d v a n a d a t e solution u s i n g p u r e v a n a d i u m p e n t o x i d e a s p r i m a r y s t a n d a r d is described. T h e d e t e r m i n a t i o n of c e r t a i n a - a m i n o acids b y o x i d a t i o n of their d e - a m i n a tion p r o d u c t s w i t h s t a n d a r d v a n a d a t e solution is studied. Rt~SUM]~ U n e m 6 t h o d e est propos6e p o u r le dosage de c e r t a i n s acides o~-amin6s; on t r a i t e la solution k a n a l y s e t p a r u n e s o l u t i o n de v a n a d i u m ( V ) , d o n t o n t i t r e l'exc~s p a r le fer(II). ZUSAMMENFASSUNG B e s c h r e i b u n g einer M e t h o d e zur B e s t i m m u n g gewisser o~-Aminosguren d u r c h B e h a n d l u n g m i t einer Vanadium-(V)-16sung, deren U e b e r s c h u s s m i t einer Eisen-(II)-16sung zurficktitriert wird. REFERENCES 1 2 3 4
L. R. T. V. E. 6 H.
MALAPRADE, Bull. soc. chim. France, 43 (1928) 683. CRIEGEE, Ann., 481 (193 o) 236. TAKAHASHI, K. IKIMOTO AND S . SAKURAI, Repts. Inst. Ind. Science, Univ. Tokyo, 5 (1955) 121. P. RAO, B. V. S. R. MURTY AND G. G. RAO, Z. anal. Chem., 147 (1955) 161. BIsHoP AND A. B. CRAWFORD, Analyst, 75 (195o) 273. REMY, Treatise on Inorganic Chemistry, Vol. II, Elsevier, A m s t e r d a m , x956, p. ioo. Anal. Chim. Acta, 25 (196I) I 3 6 - 1 3 8
ANALYTICA CHIMICA ACTA
INDIRECT OXIDIMETRIC DETERMINATION OF CERTAIN a-AMINO ACIDS WITH VANADIUM(V) S. P. RAO, H. S. R A T H I AND J, N. GAUR
Department o/Chemistry, Jaswant College, Jodhpur (India) (Received November 2Ist, 196o)
a-Amino acids which are smoothly oxidised quantitatively b y certain 1,2-glycol splitting reagents such as periodic acid 1 and lead tetraacetate 2 are resistant to certain other oxidants such as cerium(IV) 3 and vanadium(V). The de-aminated products of the amino acids with nitrous acid, i.e. the a-hydroxy acids, can however be oxidised easily by these oxidants. TAKAHASHI et al. 3 have developed an indirect method for the determination of a-amino acids via oxidation of the de-amination products but the results are not strictly stoichiometric and empirical factors calculated from pure compounds are required. Studies on the oxidation of organic compounds by vanadate solution in these laboratories have shown that vanadate is a milder and more specific oxidant for organic compounds than cerium(IV). The present communication describes a study of the oxidation of the de-amination products of the a-amino acids with a view to developing a method for their determination; the reactions are more stoichiometric than with cerium(IV). It has also been shown that pure vanadium pentoxide can be used as a primary standard. ~XP~RIMEHTAL
Preparation and standardisation o/vanadate solution/tom vanadium pentoxide Earlier workers*, 5 have recommended either ammonium vanadate or sodium metavanadate for the preparation of vanadate solutions. BISHOP AND CRAWFORD 5 have shown that pure ammonium vanadate can be used as a primary standard but it is generally preferred to standardise the solution before use. RAO et al. 4 converted ammonium vanadate to the sodium salt by boiling with slight excess of sodium carbonate till the smell of ammonia ceased and decomposed excess carbonate with acid but this method is laborious. Therefore it was thought better to use vanadium pentoxide for preparing sodium vanadate solution. The vanadium pentoxide used in the present investigation was an A.R. sample supplied by Baker which was stated to have I o o % assay. Analytically pure vanadium pentoxide can also be prepared by decomposing twice crystallized ammonium metavanadate in a platinum crucible in presence of air at a temperature of 4oo°-5oo ° and storing the material in a desiccator. Vanadium pentoxide which consists of cinnabar red rhombic crystals, is only slightly soluble in water giving an acidic reaction, but dissolves freely in alkaline solution to form colourless vanadate solution. On acidification the solution turns yellow but does not reprecipitate at low concenAnal. Chim. Acta, 25 (1961) 136-138
INDIRECT OXIDIMETRY OF 0~-AMINO ACIDS WITH VANADIUM(V)
137
trations (c[. As~O:~). Solutions of exact strength can be prepared by this method and are free from ammonium ions. According to JANDER AND JAHR6, polyvanadates exist as an equilibrium mixture in the solution depending on its pH. But this does not affect the usual oxidising properties of the vanadate solution. The vanadate solution was prepared by shaking a weighed quantity of vanadium pentoxide in approximately I.O N sodium hydroxide until dissolution was complete. The solution was then diluted to the mark in a volumetric flask. The strengths of the solutions were checked by titrating against a standard ferrous ammonium sulphate solution, the strength of which was determined by potassium dichromate solution, N-phenylanthranilic acid being used as internal indicator. The results of the standardisation given in Table I indicate that the vanadium pentoxide can be used satisfactorily as a primary standard. TABLE I Va~)adium pentoxi~taken (g)
Vanadium penloxide~und (g)
9.o856 8.8792 lO.1224
9.o91o 8.86oo lO.134o
Amino acid solutions Fresh 0.02 M solutions of pure amino acids (DL-alanine, DL-norleucine, and 3-glutamic acid) were prepared by dissolving the requisite amount of pure amino acids in distilled water.
General procedure [or de-amination and oxidation o~ amino acids To an aliquot of the amino acid solution in a 25o-ml conical flask fitted with a reflux condenser, approximately I ml of 3o% sodium nitrite solution and IO ml of 3 N sulphuric acid together with 2o ml of distilled water were added and the solution was boiled till a drop of the solution gave no colour with starch-iodide paper. A known excess of o.I N vanadate solution was added and the overall acid concentration was adjusted to approximately 12 N. The solution was refluxed on a water bath for 3o min and then cooled. The excess of vanadate in the solution was determined by back-titration with standard ferrous ammonium sulphate solution potentiometrically or with N-phenylanthranilic acid as indicator. From the amount of vanadate consumed the equivalents of oxygen consumed per mole of the amino acid was determined. The results are given in Table II. T A B L E II
A mino acid
oL-Alanine DL-Norleucine D-Glutamic acid
A mount of o.oz M amino acid (too
A m o u n t of o.x N vanadate solution (too
5-o 4.o 3.5
5 5 5
Equivalents o] oxygen ¢onsumed per mole of amino acid found calculated 2.15 2.0 3 4-3 2
2 2 4
A n a l . C h i m . A c t a , 25 (1961) 136-I38
138
s . P . RAO, H. S. RATHI, J. N. GAUR
From Table II it is evident that the equivalents of oxygen consumed correspond to the oxidation to formic acid and higher fatty acids according to the general equations: R C H (NH,) C O O H
HNO~ -+ R C H (OH) C O O H + N2 + H , O O
R C H (OH) C O O H
> RCOOH + HCOOH
and that the reactions are more or less stoichiometric. DISCUSSION
It has been shown that the oxidation of the de-amination products of the a-amino acids with vanadate provides a more satisfactory method for their determination than oxidation by cerium(IV) and the use of empirical corrections can be eliminated. The success of the method depends to a very great extent on complete elimination of nitrous acid, on preventing the loss of the decomposition products of a-hydroxy acids, and on preventing further oxidation of formic acid. That formic acid is not oxidJsed to a measurable extent under these conditions is well established and the loss of volatile decomposition products, i.e. the aldehydes and the formic acid, can be minimised by circulating cold water through the reflux condenser. The method can also be applied for the determination of a mixture of two or more amino acids. ACKNOWLEDGEMENT
The authors express their thanks to Prof. K. P. H A L D A R , Head of the Department of Chemistry, for his keen interest in the work. SUMMARY A m e t h o d for p r e p a r i n g s t a n d a r d v a n a d a t e solution u s i n g p u r e v a n a d i u m p e n t o x i d e a s p r i m a r y s t a n d a r d is described. T h e d e t e r m i n a t i o n of c e r t a i n a - a m i n o acids b y o x i d a t i o n of their d e - a m i n a tion p r o d u c t s w i t h s t a n d a r d v a n a d a t e solution is studied. Rt~SUM]~ U n e m 6 t h o d e est propos6e p o u r le dosage de c e r t a i n s acides o~-amin6s; on t r a i t e la solution k a n a l y s e t p a r u n e s o l u t i o n de v a n a d i u m ( V ) , d o n t o n t i t r e l'exc~s p a r le fer(II). ZUSAMMENFASSUNG B e s c h r e i b u n g einer M e t h o d e zur B e s t i m m u n g gewisser o~-Aminosguren d u r c h B e h a n d l u n g m i t einer Vanadium-(V)-16sung, deren U e b e r s c h u s s m i t einer Eisen-(II)-16sung zurficktitriert wird. REFERENCES 1 2 3 4
L. R. T. V. E. 6 H.
MALAPRADE, Bull. soc. chim. France, 43 (1928) 683. CRIEGEE, Ann., 481 (193 o) 236. TAKAHASHI, K. IKIMOTO AND S . SAKURAI, Repts. Inst. Ind. Science, Univ. Tokyo, 5 (1955) 121. P. RAO, B. V. S. R. MURTY AND G. G. RAO, Z. anal. Chem., 147 (1955) 161. BIsHoP AND A. B. CRAWFORD, Analyst, 75 (195o) 273. REMY, Treatise on Inorganic Chemistry, Vol. II, Elsevier, A m s t e r d a m , x956, p. ioo. Anal. Chim. Acta, 25 (196I) I 3 6 - 1 3 8