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Direct titrimetric microdetermination of l -asparagine and dl -valine
MICROCHEMICAL
JOURNAL
Direct
(1969)
14,385-390
Titrimetric L-Asparagine
I. Determination
-Asparagine
of of
Microdetermination and D L-Saline
Each
Other
and without
DL-Saline
of
in
Presence
Allahabad-2,
India
Separating
0. C. SAXENA Chemical
Laboratories,
Um’versity Received
of Allahabad,
January
27, 1969
Literature concerning the determination of L-asparagine and DL-valine is not very plentiful; and no direct and simple method for their quantitative determination either separately or in presence of each other was found. However, L-asparagine is generally determined chromatographitally (I, 2 ) titrimetrically (3, 5) ; manometrically (7) ; microbiologically and enzymically (9). DL-Valine is, generally, determined titri(8); metrically by oxidizion with potassium permanganate (12) ; by a modified potentiometric ninhydrin method (13) ; by oxidizion with ninhydrin to aldehydes (14) ; by circular chromatography (10) ; by direct photometry (I I ) ; by partition chromatography on silica gel (6) ; and by gas chromatography (4). Most of the methods are difficult, lengthy, and time consuming with the exception of only one direct photometric method (II) described for DL-valine. But there is no successful method which determined them either after complete separation from a mixture of these two or in one solution without separation. The present method deals with the direct determination of L-asparagine and DL-valine in micro quantities separately, as well as in presence of each other in the same solution without separation. Separately, L-asparagine and DL-valine were determined by direct titration with indium sulfate and potassium tellurite using congo red and xylenol orange as indicators, respectively. From potentiometric titrations and results of analysis it was confirmed that complexes are formed between indium and L-asparagine, and between tellurium and DL-VZiliIle in the ratios 1: 3 and 1:4, respectively. Probably the following reactions take place : 385
386
SAXENA COOH H-i-N coon
I\ H
H,C
3 H-L-NH, I&q),
+
7”“” /I N--F-H :” H CH, N-H
AH,
t
H-&-COOH
-
3 H,SO,
&ONH,
and
K,TeO,
t
4
W \
H3c \ Hm:: C-C-N / IA W
7"""
F-F-NH2 H,C H
-
W,HOO,
+ 2 KOH + HO y/
\~-j~~,““’
H J-i-” s Micro
722
’ A3
‘CH
9
(fe)
EXPERIMENTAL
METHODS
Reagents used
L-Asparagine, DL-valine, and potassium tellurite (E. Merck grade); indium sulfate, congo red, and xylenol orange (B.D.H. grade). Apparatus
Micro Micro
pipette (graduated of l-ml capacity with LC = 0.01. burette (graduated) of 2-ml capacity with LC = 0.01. PROCEDURE
Direct Determination of L-Asparagine and DL-vdine Separately L-Asparugine. A known volume of a standard solution of L-asparagine
was taken in a beaker and diluted to about 30 ml. A few drops (l-2) of congo red solution were added in the beaker and the whole solution assumed a red color. This solution was titrated against a standard solution of indium sulfate from a microburette. The endpoint was marked by a sharp change in color from red to violet. DL-Vuline. A known volume of a standard solution of DL-valine was taken in a beaker and diluted to about 30 ml. A few drops of a solution of xylenol orange weer added and the whole solution was either rose colored or pink red depending on the concentration of DL-valine and xylenol orange. The pink red solution was titrated against a standard solution of potassium tellurite run from a microburette, where at the endpoint a sharp change in color took place from pink red to purple.
ASPARAGINE
Determination Other without
AND
of L-Asparagine Separation
387
VALINE
and m-Valine
in Presence
of Each
Known volumes of standard solutions of L-asparagine and DL-valine were taken in a beaker in the ascending and descending order from the top, respectively. A few drops of xylenol solution were added after dilution with distilled water to about 30 ml, the whole solution was pink. Then a standard solution of indium sulfate was run from a microburette, when the first change was marked by a light rose color. To this same solution and with those few drops of the indicator xylenol orange a standard solution of potassium tellurite was allowed to run in to titrate DL-valine. At the endpoint a purple red color appeared sharply. Hence, in the same solution first L-asparagine was titrated an dthen DL-valine using the same indicator. RESULTS
AND
DISCUSSIONS
The results are shown in Tables l-3. Ranges for L-asparagine and DL-valine varied from 6.0537 X 1O-4 to 18.1608 X 1W mg/liter; and from 5.8478 X lo+ to 29.2390 X lo-* mg/liter, respectively. TABLE MICRODETERMINATION
1 OF
L-Asparagine L-Asparagine 0.02 M (ml) 0.2 0.3 0.4 0.5 0.6
Inn60 4) 3 0.0084 M (ml) 0.16 0.24 0.32 0.40 0.48
(X lo-” -____
Found
6.0056 9.0084 12.0112 15.0140 18.0168
6.0537 9.0804 12.1074 15.1340 18.1608
0.5 1.0 1.5 2.0 2.5
0.12 0.24 0.36 0.48 0.60
0.8
OF DL-VALINE
DL-Valine ____--__--__-_
(ml)
Error (%)
2
MICRODETERMINATION
KzTe03 0.0104 M
mg/liter)
Taken
TABLE
DL-Valine 0.01 M (ml)
L-ASPARAGINE
(X 10e4 mg/liter)
Taken
Found
Error (%I _____
5.8575 11.7150 17.5725 23.4300 29.2875
5.8478 11.6956 17.5434 23.3912 29.2390
0.16
-
(ml)
0.16 0.24 0.32 0.48
0.2 0.3 0.4 0.6
M
IndS04) 3
0.0084
(ml)
0.02 M
L-Asparagine
6.0056 9.0084 12.0112 18.0168
Taken
L-Asparagine
MICRODETERMINATION
3
6.0537 9.0804 12.1074 18.1608
Found
(X 10-4 mg/liter)
2.5 2.0 1.5 0.5
(ml)
DbValine 0.01 M
OF L-ASPARAGINE AND DL-VALINE EACH OTHER WITHOUT SEPARATION
TABLE
0.6 0.48 0.36 0.12
(ml)
K2Te03 0.0104 M
29.2875 23.4300 17.5725 5.8575
Found
29.2390 23.3912 17.5434 5.8478
Taken
DL-Valine (X 1O-* mg/liter)
IN PRESENCE OF
-
ASPARAGINE
AND VALINE
389
Since the complex between indium sulfate and L-asparagine is formed in the ratio of 1:3, the calculations were done accordingly by multiplying the values obtained by 3. But in the case of tellurium and DL-valine the complex is formed in the ratio of 1:4 hence the values obtained are multiplied by 4. In separate determinations of -valine two different indicators congo red and L-asparagine and D. xylenol orange were used, which give a sharp change from red to violet and from pink to purple, respectively. Peculiarity of this method lies in determining L-asparagine and DL-valine together without separating and titrating in one solution using only xylenol orange as indicator. Further, it is peculiar that pink color developed by adding xylenol orange to a mixture of L-asparagine and DL-valine, under these concentration ratios, L-asparagine dominates. Hence, the reason why, L-asparagine is titrated first against a standard solution of indium sulfate, a light rose color marks the endpoint. Then, in the same solution without adding any further indicator DL-valine is titrated against standard solution of potassium tellurite, when purple color is developed at the endpoint. An important precaution is taken while titrating L-asparagine: the total volume in each titration should be the same, and, also, every time the same number of drops of the indicator should be added. Results show the maximum error in the case of L-asparagine and DL-valine by 0.8 and 0.16% respectively. Present method has advantages over other methods that these amino acids have been determined separately and in presence of these two without separating quantitatively in micro amounts, are simple in itself, accurate, less time consuming. REFERENCES 1. Auclair, .I. L. and Dubreuil, R., Simple ultramicromethod for the quantitative estimation of amino acids by paper partition chromatography. Can. J. Zool. 30, 109 (1952). 2. Barbiroli, G., Paper chromatographic separation and &termination of 22 amino acids. Mikrochem. Ichnoanal. Acta 4, 652 (1965). 3. Das, M. N. and Palit, S. R., Some applications of glycolic, titration. 1. Estimation of organic base. J. Indian Chem. Sot. 31, 34 (1954). 4. Darbre, A. and Blau, K., Quantitative estimation of some amino acids by gas chromatography. Biochem. J. 88, 81 (1963). 5. Ekebald, P., Perchloric acid titration of amino acids in acetic acid. Svensk Farm. Tidskr. 57, 185 (1953). 6. Kandatsu, M. and Naito, H., Determination of neutral amino acids by partition chromatography on silica gel. Nippon Nogeikagaku Kaishi. 33, 170 (1959). 7. Krebs, H. A., Manometric determination of L-aspartic acid and L-asparagine. Biochem.
J. 47, 605 (1950).
390
SAXENA
8. Mondolfo,
V., Distribution of amino acids biological meth28, 333 (1949). Mardashev, S. R. and Manaeve, V. V., New method for the quantitative determination of amino decraboxylic acids and their amides. Biokhimiya 15,465 (1950). Rao, N. A. N. and Wadhwaui, T. K. Quantitative estimation of amino acids by circular paper chromatography. 1. Indian Inst. Sci. 37a, 130 (1955). Roberts, H. R. and Kolor, M. G., Accuracy of quantitative paper chromatography in amino acid determination by means of direct photometry. Anal. Chem. 29, 1800 (1957). Sjollema, N. and Dienske, J. W., Formation of nitrate, ammonia and fatty acids from L-amino acids and the formation carbonate by oxidation with potassium permanganate. Rec. Trav. Chim. 52, 229 (1933). Troll, W. and Cannon, R. K., A modified photometric ninhydrin method for the analysis of amino acid imino acids. .I. Biol. Chem. 290, 803 (1953). Turba, F. and Schrader, E. V., Determination of homologous monoarninomonocarboxylic acids. Beielstein Naturwissenschaften 34, 57 (1947). ods. Boll.
9.
10. Il.
12.
13. 14.
V., and Comboni, Zst. Sieroterap.
Milan.
JOURNAL
Direct
(1969)
14,385-390
Titrimetric L-Asparagine
I. Determination
-Asparagine
of of
Microdetermination and D L-Saline
Each
Other
and without
DL-Saline
of
in
Presence
Allahabad-2,
India
Separating
0. C. SAXENA Chemical
Laboratories,
Um’versity Received
of Allahabad,
January
27, 1969
Literature concerning the determination of L-asparagine and DL-valine is not very plentiful; and no direct and simple method for their quantitative determination either separately or in presence of each other was found. However, L-asparagine is generally determined chromatographitally (I, 2 ) titrimetrically (3, 5) ; manometrically (7) ; microbiologically and enzymically (9). DL-Valine is, generally, determined titri(8); metrically by oxidizion with potassium permanganate (12) ; by a modified potentiometric ninhydrin method (13) ; by oxidizion with ninhydrin to aldehydes (14) ; by circular chromatography (10) ; by direct photometry (I I ) ; by partition chromatography on silica gel (6) ; and by gas chromatography (4). Most of the methods are difficult, lengthy, and time consuming with the exception of only one direct photometric method (II) described for DL-valine. But there is no successful method which determined them either after complete separation from a mixture of these two or in one solution without separation. The present method deals with the direct determination of L-asparagine and DL-valine in micro quantities separately, as well as in presence of each other in the same solution without separation. Separately, L-asparagine and DL-valine were determined by direct titration with indium sulfate and potassium tellurite using congo red and xylenol orange as indicators, respectively. From potentiometric titrations and results of analysis it was confirmed that complexes are formed between indium and L-asparagine, and between tellurium and DL-VZiliIle in the ratios 1: 3 and 1:4, respectively. Probably the following reactions take place : 385
386
SAXENA COOH H-i-N coon
I\ H
H,C
3 H-L-NH, I&q),
+
7”“” /I N--F-H :” H CH, N-H
AH,
t
H-&-COOH
-
3 H,SO,
&ONH,
and
K,TeO,
t
4
W \
H3c \ Hm:: C-C-N / IA W
7"""
F-F-NH2 H,C H
-
W,HOO,
+ 2 KOH + HO y/
\~-j~~,““’
H J-i-” s Micro
722
’ A3
‘CH
9
(fe)
EXPERIMENTAL
METHODS
Reagents used
L-Asparagine, DL-valine, and potassium tellurite (E. Merck grade); indium sulfate, congo red, and xylenol orange (B.D.H. grade). Apparatus
Micro Micro
pipette (graduated of l-ml capacity with LC = 0.01. burette (graduated) of 2-ml capacity with LC = 0.01. PROCEDURE
Direct Determination of L-Asparagine and DL-vdine Separately L-Asparugine. A known volume of a standard solution of L-asparagine
was taken in a beaker and diluted to about 30 ml. A few drops (l-2) of congo red solution were added in the beaker and the whole solution assumed a red color. This solution was titrated against a standard solution of indium sulfate from a microburette. The endpoint was marked by a sharp change in color from red to violet. DL-Vuline. A known volume of a standard solution of DL-valine was taken in a beaker and diluted to about 30 ml. A few drops of a solution of xylenol orange weer added and the whole solution was either rose colored or pink red depending on the concentration of DL-valine and xylenol orange. The pink red solution was titrated against a standard solution of potassium tellurite run from a microburette, where at the endpoint a sharp change in color took place from pink red to purple.
ASPARAGINE
Determination Other without
AND
of L-Asparagine Separation
387
VALINE
and m-Valine
in Presence
of Each
Known volumes of standard solutions of L-asparagine and DL-valine were taken in a beaker in the ascending and descending order from the top, respectively. A few drops of xylenol solution were added after dilution with distilled water to about 30 ml, the whole solution was pink. Then a standard solution of indium sulfate was run from a microburette, when the first change was marked by a light rose color. To this same solution and with those few drops of the indicator xylenol orange a standard solution of potassium tellurite was allowed to run in to titrate DL-valine. At the endpoint a purple red color appeared sharply. Hence, in the same solution first L-asparagine was titrated an dthen DL-valine using the same indicator. RESULTS
AND
DISCUSSIONS
The results are shown in Tables l-3. Ranges for L-asparagine and DL-valine varied from 6.0537 X 1O-4 to 18.1608 X 1W mg/liter; and from 5.8478 X lo+ to 29.2390 X lo-* mg/liter, respectively. TABLE MICRODETERMINATION
1 OF
L-Asparagine L-Asparagine 0.02 M (ml) 0.2 0.3 0.4 0.5 0.6
Inn60 4) 3 0.0084 M (ml) 0.16 0.24 0.32 0.40 0.48
(X lo-” -____
Found
6.0056 9.0084 12.0112 15.0140 18.0168
6.0537 9.0804 12.1074 15.1340 18.1608
0.5 1.0 1.5 2.0 2.5
0.12 0.24 0.36 0.48 0.60
0.8
OF DL-VALINE
DL-Valine ____--__--__-_
(ml)
Error (%)
2
MICRODETERMINATION
KzTe03 0.0104 M
mg/liter)
Taken
TABLE
DL-Valine 0.01 M (ml)
L-ASPARAGINE
(X 10e4 mg/liter)
Taken
Found
Error (%I _____
5.8575 11.7150 17.5725 23.4300 29.2875
5.8478 11.6956 17.5434 23.3912 29.2390
0.16
-
(ml)
0.16 0.24 0.32 0.48
0.2 0.3 0.4 0.6
M
IndS04) 3
0.0084
(ml)
0.02 M
L-Asparagine
6.0056 9.0084 12.0112 18.0168
Taken
L-Asparagine
MICRODETERMINATION
3
6.0537 9.0804 12.1074 18.1608
Found
(X 10-4 mg/liter)
2.5 2.0 1.5 0.5
(ml)
DbValine 0.01 M
OF L-ASPARAGINE AND DL-VALINE EACH OTHER WITHOUT SEPARATION
TABLE
0.6 0.48 0.36 0.12
(ml)
K2Te03 0.0104 M
29.2875 23.4300 17.5725 5.8575
Found
29.2390 23.3912 17.5434 5.8478
Taken
DL-Valine (X 1O-* mg/liter)
IN PRESENCE OF
-
ASPARAGINE
AND VALINE
389
Since the complex between indium sulfate and L-asparagine is formed in the ratio of 1:3, the calculations were done accordingly by multiplying the values obtained by 3. But in the case of tellurium and DL-valine the complex is formed in the ratio of 1:4 hence the values obtained are multiplied by 4. In separate determinations of -valine two different indicators congo red and L-asparagine and D. xylenol orange were used, which give a sharp change from red to violet and from pink to purple, respectively. Peculiarity of this method lies in determining L-asparagine and DL-valine together without separating and titrating in one solution using only xylenol orange as indicator. Further, it is peculiar that pink color developed by adding xylenol orange to a mixture of L-asparagine and DL-valine, under these concentration ratios, L-asparagine dominates. Hence, the reason why, L-asparagine is titrated first against a standard solution of indium sulfate, a light rose color marks the endpoint. Then, in the same solution without adding any further indicator DL-valine is titrated against standard solution of potassium tellurite, when purple color is developed at the endpoint. An important precaution is taken while titrating L-asparagine: the total volume in each titration should be the same, and, also, every time the same number of drops of the indicator should be added. Results show the maximum error in the case of L-asparagine and DL-valine by 0.8 and 0.16% respectively. Present method has advantages over other methods that these amino acids have been determined separately and in presence of these two without separating quantitatively in micro amounts, are simple in itself, accurate, less time consuming. REFERENCES 1. Auclair, .I. L. and Dubreuil, R., Simple ultramicromethod for the quantitative estimation of amino acids by paper partition chromatography. Can. J. Zool. 30, 109 (1952). 2. Barbiroli, G., Paper chromatographic separation and &termination of 22 amino acids. Mikrochem. Ichnoanal. Acta 4, 652 (1965). 3. Das, M. N. and Palit, S. R., Some applications of glycolic, titration. 1. Estimation of organic base. J. Indian Chem. Sot. 31, 34 (1954). 4. Darbre, A. and Blau, K., Quantitative estimation of some amino acids by gas chromatography. Biochem. J. 88, 81 (1963). 5. Ekebald, P., Perchloric acid titration of amino acids in acetic acid. Svensk Farm. Tidskr. 57, 185 (1953). 6. Kandatsu, M. and Naito, H., Determination of neutral amino acids by partition chromatography on silica gel. Nippon Nogeikagaku Kaishi. 33, 170 (1959). 7. Krebs, H. A., Manometric determination of L-aspartic acid and L-asparagine. Biochem.
J. 47, 605 (1950).
390
SAXENA
8. Mondolfo,
V., Distribution of amino acids biological meth28, 333 (1949). Mardashev, S. R. and Manaeve, V. V., New method for the quantitative determination of amino decraboxylic acids and their amides. Biokhimiya 15,465 (1950). Rao, N. A. N. and Wadhwaui, T. K. Quantitative estimation of amino acids by circular paper chromatography. 1. Indian Inst. Sci. 37a, 130 (1955). Roberts, H. R. and Kolor, M. G., Accuracy of quantitative paper chromatography in amino acid determination by means of direct photometry. Anal. Chem. 29, 1800 (1957). Sjollema, N. and Dienske, J. W., Formation of nitrate, ammonia and fatty acids from L-amino acids and the formation carbonate by oxidation with potassium permanganate. Rec. Trav. Chim. 52, 229 (1933). Troll, W. and Cannon, R. K., A modified photometric ninhydrin method for the analysis of amino acid imino acids. .I. Biol. Chem. 290, 803 (1953). Turba, F. and Schrader, E. V., Determination of homologous monoarninomonocarboxylic acids. Beielstein Naturwissenschaften 34, 57 (1947). ods. Boll.
9.
10. Il.
12.
13. 14.
V., and Comboni, Zst. Sieroterap.
Milan.