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A colorimetric method for determination of pyrocatechol and related substances
ANALYTICAL
7, 315-321
BIOCHEMISTRY
A Calorimetric of Pyrocatechol I’. MADHUSUDANAN From
the Department
(1964)
Method and
Received
Determination
Related
NAIR’
of Biochemistry,
for
Substances
C. S. VAIDYANATHAN
AND
Indian Institute June
of Science, Bangalore,
India
5, 1963
The formation of colored products by the addition of ammonium molybdate or tungstate to solutions containing pyrocatechol or other catecholic compounds was first reported by Rae (1) and has been successfully employed by Evans (2) for the detection of such substances. When this procedure was used in an attempt to estimate pyrocatechol in enzyme reaction mixtures, it was observed t.hat the presence of trichloroacetic acid caused a considerable increase in the intensity of the color. By making use of this finding and aft#er a detailed study of the factors affecting the color production, a rapid and sensitive method was developed for the estimation of pyrocatechol. A method for the separation of pyrocatechol from interfering sub&nnces by circular paper chromatography is also presented. METHODS
Apparatus
Absorbance measurements were made either with a Beckman model DK-2 spectrophotometer or a Klett-Summerson photoelectric colorimetex with Klett filter No. 54 (526-580). The latter was used for routine determinations of pyrocat,echol and for the investigation of factors affecting color formation. All the chemicals were reagent grade: 1. Standard pyrocatechol: 60 mg of pyrocatechol was dissolved in 100 ml of distilled water. This solution can be stored in refrigerator without decomposition for at least a week. Suitable dilutions of this stock solution were made to give working standards of varying concentrations. 2. Sodium tunystate: a 10% solution in water. 1 Present address: Atlantic Canada, Dalhousie University,
Regional Halifax,
Laboratory, Nova Scotia. 315
National
Research
Council
of
3. Sodium nitrite:
a 0.5% solution in water (prepared fresh daily). the developing solvent, first recommended by Reio (3) for paper chromatography for phenolic substances was a mixture of 80 parts by volume of methyl ethyl ketone, 4 parts of acetone, 12 parts of water, and 2 parts of formic acid (100%). 4. Solvent
for chromatography:
Procedure
To 1 ml of pyrocatechol solution containing 50 mg of trichloroacetic acid were added in succession 1 ml of sodium tungstate solution, 0.5 ml of 0.5 N hydrochloric acid, and 1 ml of sodium nitrite solution. A yellow coloration was produced and reached maximum intensity in about 3 min. After 5 minutes, 2 ml of 0.5 N sodium hydroxide was added giving a cherry red color. After 15 min, the optical density of the solution was measured. The time lapse for the measurement of the color is not very critical because it is stable for about 12 hr. Paper Chromatography
The test solution was spotted on the circumference of a circle 4 cm in diameter drawn around the center of a Whatman No. 1 filter paper circle (24-cm diameter), and dried in a current of air. The chromatogram was developed according to the method of Giri and Rao (4, 5), then air-dried and examined under ult.raviolet light of 366 mp. Adrenaline, noradrenaline, and dihydroxyphenylalanine gave fluorescent bands. Pyrocatechol appeared as an absorbance band. The Rf values of pyrocatechol, dihydroxyphenylalanine, adrenaline, and noradrenaline were 0.98, 0.60, 0.77, and 0.86, respectively. Their position could also be located by means of a guide strip sprayed with diazotized sulfanilic acid. Each band was then excised and eluted with 3 ml of 5% trichloroacetic acid. A l-ml aliquot was used to estimate the content of catecholic compounds by the method outlined above. RESULT8
Eflect of Trichloroacetic Acid
The effect of different concentration of trichloroacetic acid on the color intensity is shown in TabIe 1. It has been observed t.hat addiCon of 300 mg of trichloroacetic acid in a final volume of 6 ml produced the maximum coloration. The absorption spectrum of the colored complex formed by the reaction between pyrocatechol and tungstate in the presence of nitrite and trichloroacetic acid is illustrated in Fig. 1. It shows a maximum absorption at 510 mp.
PYROCATECHOL
TABLE EFFECT Concentrations trichloroacetio acid added 6 ml, %
OF TRICHLOROACETIC
of
317
DETERMINATION
1 ACID
CONCENTRATION
Amount of trichloroacetic acid in 1 ml, mg
in
m&t readings
-
0
5
8.33
10 15
16.66
6
8
8 20 86 90 70
25.00 33.32 50.00 66.64
20 30 40
10 rg of catechol was present in the l-ml aliquot taken for developing the color.
7. P x g 4
60
-
50
-
40
-
30-
20
IO I I 0
350
400
450 Wavelengih
I
,
500
550
(rnfi)
FIG. 1. Absorption spectrum of colored complex formed by reaction of catechol with tungstate reagent. Color was developed as mentioned under Methods. Catechol concentration used in this case was 30 pg.
Specificity of the Color Reaction Of the compounds tested only those with an aromatic ring containing two vicinal hydroxyl groups gave a red color with tungstate in the presence of nitrite (Table 2). Effect of Interfering
Substances
Aromatic amines might be expected to interfere because of their interaction with nitrite in the reagent. The results given in Table 3 show that
318
NAIR
AND
VAIDYANATHAN
TABLE SPECIFICITY
OF THE
2 COLOR
REACTION
Substance tested (IO ,a mas taken in each case)
Pyrocntechol o-Cresol Resorcinol Hydroquinone Phloroglucinol o-Aminophenol Salicylic acid Adrenaline Noradrenaline Dopa 3-Hydrosytyramine Pyrocatachuic Protocatachuic PyrogallolQ Gallic acida a These
two
compounds
gave
Iilett
-
a light
OF AROMATIC Substances
90 0 0 0 0 0 0 81 90 82 0 65 65 -
acid acid
yellow TABLE
EFFECT
readings
AMINES added
color
on addition
of NaOH.
3 ON
THE
COLOR Iilett
Control Anthranilic acid Aniline hydrochloride a-?Saphthylamine Sulfanilic acid o-Aminophenol
RENTION readings
90 IG 10 50 54 90
A l-ml solution containing 60 pg of pyrocatechol was dispensed into different tubes; 350 pg of each of the above-mentioned substances in 1 ml water was added to 1 ml of &echo1 solution followed by 1 ml of 307c trichloroacetic acid solution. The mixture was made up to 6 ml with distilled water and 1 ml of aliquot was taken for color development. In the control, 1 ml of amine solution was replaced by 1 ml of water.
all those tested did, in fact, reduce the intensity of the color formed. This interference could be removed by adding excess formaldehyde (Table 4). Reducing substances like ferrous sulfate, glutathione, and sodium bisulfite at a final concentration of 10M4M were without effect on the color reaction. Standard
Graph for Pyrocatechol
Different amounts of pyrocatechol (60, 120, 180, 240, 300 pg) from the stock solution were dispensed into separat,e test tubes and the volumes
PTROCATECHOL
T,4BLE EFFECT
319
DETERXINATIO?G
OF DIFFERENT CONCENTRATIONS OF INTERFERENCE CMJSED
4 OF FORMALDEHYDE BY ANTHRhNILIC
Klctt
ON REVERSAL &ID
readings
15 30 40 80 90 90
Control 0.2 0.5 0.8 1.0 3.0
A l-ml solution containing 60 pg of pyrocatechol was dispensed into different tubes; 360 pg of anthranilic acid in 1 ml water was also added to these tubes. Different volumes of formaldehyde (37-4ly,), as mentioned above, were added to these tubes. In the control tube formaldehyde was omitted. The volume was made up to 6 ml with distilled water and a l-ml aliquot was taken for color development.
made to 5 ml with distilled water; 1 ml of 30% trichloroacetic acid was added to each and a l-ml aliquot from the mixtures was taken for estimation. The color intensity was measured with a Klett-Summerson photoelectric calorimeter using green filter (540 mp). The results obtained after submitting the stock solutions to paper chromatography show that there was quantitative recovery of catcchol after elution from the paper and the calorimetric readings closely corresponded with those represented in the standard graph (Fig. 2). DISCUSSION
The intensification by trichloroacetic acid of the color given by pyrocatechol in the presence of tungstate and sodium nitrite considerably enhances the sensitivity of the method and facilitates the rapid and accurate determination of microgram amounts of pyrocatechol. The concentration of trichloroacetic acid in the system is, however, a critical factor and needsto be carefully controlled by suitable dilution (Table 1). The chromogenic reaction is group specific and is given by substances which are structurally relat’ed to pyrocatechol, e.g., adrenaline, noradrenaline, and dihydroxyphenylalanine (Table 2). It is, therefore, necessary to separate mixtures of catccholic compounds by paper chromatography before estimation. Another difficulty is the interference by aromatic amines (Table 3). Thus in a study of the enzymic conversion of anthranilic acid to catechol it was found that the presence of excess anthranilic acid completely suppressedthe color formation. However, this interference can be overcome by the addition of excess formaldehyde, which has no effect on the intensity of the color (Table 4). Substances present in the biological extracts caused no interference.
NAIR
AND
VAIDYANATHBN
‘4
420 i 360 t
micrograms
of Catechol
Fro. 2. Comparison of color intensities obtained for different amounts of catechol with tungstate reagent: (*@I values for untreated catechol; (A-A) colorimetric estimation of catechol after submitting to paper chromatography.
Varying amounts of pyrocatechol added to boiled extracts of Tecoma stuns leaves could be accurately estimated with complete recovery of the added amounts. Since pyrocatecho) and related substances can be readily separated by circular paper chromatography, the effects of interfering substances which might be present in biological extracts may be avoided. Moreover the successful chromatographic separation of pyrocatechol from other related substances would permit the simultaneous determination of a number of catecholic compounds, in metabolic studies. This method has been successfully employed by the authors in the study on the formation of catechol from anthranilic acid in .Yecoma stans leaves, and in the metabolism of various catecholic compounds in plants. Also this method can be used for the estimation of catecholic compounds like adrenaline and noradrenaline in animals as well.
PI-ROCATECHOL
DETERMINATIOX
321
SUMMARY
A coiorimetric assay for the quantitative determination of catecholic compounds was developed. The method was based on the observation that a red color was formed when nitrite was added to a solution containing pyrocatechol and sodium tungstate. Aromatic amines interfere with the reaction but this could be overcome by the addition of formaldehyde. When interfering substances are present along with pyrocatechol, it can be readily separated by paper chromatography and estimated after elution from the filter paper. REFERENCES 1. 2. 3. 4.
RAE, J., Pharm. J. 125, 451 (1930) ; cited from Chem. Abstr. 25, 980 (1931). EVANS, W. C., Biochem. J. 41, 373 (1947). REIO, L., J. Chromntog. 1, 338 (1958). GIRI, K. V., AND RAO, N. A. N., J. Indian Inst. Sci. 34A, 95 (1952). 5. GIRI, K. V., AND RAO, N. A. N., Nature 169, 923 (1952).
7, 315-321
BIOCHEMISTRY
A Calorimetric of Pyrocatechol I’. MADHUSUDANAN From
the Department
(1964)
Method and
Received
Determination
Related
NAIR’
of Biochemistry,
for
Substances
C. S. VAIDYANATHAN
AND
Indian Institute June
of Science, Bangalore,
India
5, 1963
The formation of colored products by the addition of ammonium molybdate or tungstate to solutions containing pyrocatechol or other catecholic compounds was first reported by Rae (1) and has been successfully employed by Evans (2) for the detection of such substances. When this procedure was used in an attempt to estimate pyrocatechol in enzyme reaction mixtures, it was observed t.hat the presence of trichloroacetic acid caused a considerable increase in the intensity of the color. By making use of this finding and aft#er a detailed study of the factors affecting the color production, a rapid and sensitive method was developed for the estimation of pyrocatechol. A method for the separation of pyrocatechol from interfering sub&nnces by circular paper chromatography is also presented. METHODS
Apparatus
Absorbance measurements were made either with a Beckman model DK-2 spectrophotometer or a Klett-Summerson photoelectric colorimetex with Klett filter No. 54 (526-580). The latter was used for routine determinations of pyrocat,echol and for the investigation of factors affecting color formation. All the chemicals were reagent grade: 1. Standard pyrocatechol: 60 mg of pyrocatechol was dissolved in 100 ml of distilled water. This solution can be stored in refrigerator without decomposition for at least a week. Suitable dilutions of this stock solution were made to give working standards of varying concentrations. 2. Sodium tunystate: a 10% solution in water. 1 Present address: Atlantic Canada, Dalhousie University,
Regional Halifax,
Laboratory, Nova Scotia. 315
National
Research
Council
of
3. Sodium nitrite:
a 0.5% solution in water (prepared fresh daily). the developing solvent, first recommended by Reio (3) for paper chromatography for phenolic substances was a mixture of 80 parts by volume of methyl ethyl ketone, 4 parts of acetone, 12 parts of water, and 2 parts of formic acid (100%). 4. Solvent
for chromatography:
Procedure
To 1 ml of pyrocatechol solution containing 50 mg of trichloroacetic acid were added in succession 1 ml of sodium tungstate solution, 0.5 ml of 0.5 N hydrochloric acid, and 1 ml of sodium nitrite solution. A yellow coloration was produced and reached maximum intensity in about 3 min. After 5 minutes, 2 ml of 0.5 N sodium hydroxide was added giving a cherry red color. After 15 min, the optical density of the solution was measured. The time lapse for the measurement of the color is not very critical because it is stable for about 12 hr. Paper Chromatography
The test solution was spotted on the circumference of a circle 4 cm in diameter drawn around the center of a Whatman No. 1 filter paper circle (24-cm diameter), and dried in a current of air. The chromatogram was developed according to the method of Giri and Rao (4, 5), then air-dried and examined under ult.raviolet light of 366 mp. Adrenaline, noradrenaline, and dihydroxyphenylalanine gave fluorescent bands. Pyrocatechol appeared as an absorbance band. The Rf values of pyrocatechol, dihydroxyphenylalanine, adrenaline, and noradrenaline were 0.98, 0.60, 0.77, and 0.86, respectively. Their position could also be located by means of a guide strip sprayed with diazotized sulfanilic acid. Each band was then excised and eluted with 3 ml of 5% trichloroacetic acid. A l-ml aliquot was used to estimate the content of catecholic compounds by the method outlined above. RESULT8
Eflect of Trichloroacetic Acid
The effect of different concentration of trichloroacetic acid on the color intensity is shown in TabIe 1. It has been observed t.hat addiCon of 300 mg of trichloroacetic acid in a final volume of 6 ml produced the maximum coloration. The absorption spectrum of the colored complex formed by the reaction between pyrocatechol and tungstate in the presence of nitrite and trichloroacetic acid is illustrated in Fig. 1. It shows a maximum absorption at 510 mp.
PYROCATECHOL
TABLE EFFECT Concentrations trichloroacetio acid added 6 ml, %
OF TRICHLOROACETIC
of
317
DETERMINATION
1 ACID
CONCENTRATION
Amount of trichloroacetic acid in 1 ml, mg
in
m&t readings
-
0
5
8.33
10 15
16.66
6
8
8 20 86 90 70
25.00 33.32 50.00 66.64
20 30 40
10 rg of catechol was present in the l-ml aliquot taken for developing the color.
7. P x g 4
60
-
50
-
40
-
30-
20
IO I I 0
350
400
450 Wavelengih
I
,
500
550
(rnfi)
FIG. 1. Absorption spectrum of colored complex formed by reaction of catechol with tungstate reagent. Color was developed as mentioned under Methods. Catechol concentration used in this case was 30 pg.
Specificity of the Color Reaction Of the compounds tested only those with an aromatic ring containing two vicinal hydroxyl groups gave a red color with tungstate in the presence of nitrite (Table 2). Effect of Interfering
Substances
Aromatic amines might be expected to interfere because of their interaction with nitrite in the reagent. The results given in Table 3 show that
318
NAIR
AND
VAIDYANATHAN
TABLE SPECIFICITY
OF THE
2 COLOR
REACTION
Substance tested (IO ,a mas taken in each case)
Pyrocntechol o-Cresol Resorcinol Hydroquinone Phloroglucinol o-Aminophenol Salicylic acid Adrenaline Noradrenaline Dopa 3-Hydrosytyramine Pyrocatachuic Protocatachuic PyrogallolQ Gallic acida a These
two
compounds
gave
Iilett
-
a light
OF AROMATIC Substances
90 0 0 0 0 0 0 81 90 82 0 65 65 -
acid acid
yellow TABLE
EFFECT
readings
AMINES added
color
on addition
of NaOH.
3 ON
THE
COLOR Iilett
Control Anthranilic acid Aniline hydrochloride a-?Saphthylamine Sulfanilic acid o-Aminophenol
RENTION readings
90 IG 10 50 54 90
A l-ml solution containing 60 pg of pyrocatechol was dispensed into different tubes; 350 pg of each of the above-mentioned substances in 1 ml water was added to 1 ml of &echo1 solution followed by 1 ml of 307c trichloroacetic acid solution. The mixture was made up to 6 ml with distilled water and 1 ml of aliquot was taken for color development. In the control, 1 ml of amine solution was replaced by 1 ml of water.
all those tested did, in fact, reduce the intensity of the color formed. This interference could be removed by adding excess formaldehyde (Table 4). Reducing substances like ferrous sulfate, glutathione, and sodium bisulfite at a final concentration of 10M4M were without effect on the color reaction. Standard
Graph for Pyrocatechol
Different amounts of pyrocatechol (60, 120, 180, 240, 300 pg) from the stock solution were dispensed into separat,e test tubes and the volumes
PTROCATECHOL
T,4BLE EFFECT
319
DETERXINATIO?G
OF DIFFERENT CONCENTRATIONS OF INTERFERENCE CMJSED
4 OF FORMALDEHYDE BY ANTHRhNILIC
Klctt
ON REVERSAL &ID
readings
15 30 40 80 90 90
Control 0.2 0.5 0.8 1.0 3.0
A l-ml solution containing 60 pg of pyrocatechol was dispensed into different tubes; 360 pg of anthranilic acid in 1 ml water was also added to these tubes. Different volumes of formaldehyde (37-4ly,), as mentioned above, were added to these tubes. In the control tube formaldehyde was omitted. The volume was made up to 6 ml with distilled water and a l-ml aliquot was taken for color development.
made to 5 ml with distilled water; 1 ml of 30% trichloroacetic acid was added to each and a l-ml aliquot from the mixtures was taken for estimation. The color intensity was measured with a Klett-Summerson photoelectric calorimeter using green filter (540 mp). The results obtained after submitting the stock solutions to paper chromatography show that there was quantitative recovery of catcchol after elution from the paper and the calorimetric readings closely corresponded with those represented in the standard graph (Fig. 2). DISCUSSION
The intensification by trichloroacetic acid of the color given by pyrocatechol in the presence of tungstate and sodium nitrite considerably enhances the sensitivity of the method and facilitates the rapid and accurate determination of microgram amounts of pyrocatechol. The concentration of trichloroacetic acid in the system is, however, a critical factor and needsto be carefully controlled by suitable dilution (Table 1). The chromogenic reaction is group specific and is given by substances which are structurally relat’ed to pyrocatechol, e.g., adrenaline, noradrenaline, and dihydroxyphenylalanine (Table 2). It is, therefore, necessary to separate mixtures of catccholic compounds by paper chromatography before estimation. Another difficulty is the interference by aromatic amines (Table 3). Thus in a study of the enzymic conversion of anthranilic acid to catechol it was found that the presence of excess anthranilic acid completely suppressedthe color formation. However, this interference can be overcome by the addition of excess formaldehyde, which has no effect on the intensity of the color (Table 4). Substances present in the biological extracts caused no interference.
NAIR
AND
VAIDYANATHBN
‘4
420 i 360 t
micrograms
of Catechol
Fro. 2. Comparison of color intensities obtained for different amounts of catechol with tungstate reagent: (*@I values for untreated catechol; (A-A) colorimetric estimation of catechol after submitting to paper chromatography.
Varying amounts of pyrocatechol added to boiled extracts of Tecoma stuns leaves could be accurately estimated with complete recovery of the added amounts. Since pyrocatecho) and related substances can be readily separated by circular paper chromatography, the effects of interfering substances which might be present in biological extracts may be avoided. Moreover the successful chromatographic separation of pyrocatechol from other related substances would permit the simultaneous determination of a number of catecholic compounds, in metabolic studies. This method has been successfully employed by the authors in the study on the formation of catechol from anthranilic acid in .Yecoma stans leaves, and in the metabolism of various catecholic compounds in plants. Also this method can be used for the estimation of catecholic compounds like adrenaline and noradrenaline in animals as well.
PI-ROCATECHOL
DETERMINATIOX
321
SUMMARY
A coiorimetric assay for the quantitative determination of catecholic compounds was developed. The method was based on the observation that a red color was formed when nitrite was added to a solution containing pyrocatechol and sodium tungstate. Aromatic amines interfere with the reaction but this could be overcome by the addition of formaldehyde. When interfering substances are present along with pyrocatechol, it can be readily separated by paper chromatography and estimated after elution from the filter paper. REFERENCES 1. 2. 3. 4.
RAE, J., Pharm. J. 125, 451 (1930) ; cited from Chem. Abstr. 25, 980 (1931). EVANS, W. C., Biochem. J. 41, 373 (1947). REIO, L., J. Chromntog. 1, 338 (1958). GIRI, K. V., AND RAO, N. A. N., J. Indian Inst. Sci. 34A, 95 (1952). 5. GIRI, K. V., AND RAO, N. A. N., Nature 169, 923 (1952).