SOP: Estimation of Ascorbic Acid, Dehydroascorbic Acid and Diketogulonic Acid (2,4-Dinitrophenylhydrazine Method)
GIFSQ — Global Institute for Food Safety & Quality
Principle
2,4-Dinitrophenylhydrazine forms an osazone with dehydroascorbic acid and diketogulonic acid, which dissolves in concentrated H₂SO₄ forming an intense red solution measurable colorimetrically at 520 nm. Ascorbic acid is estimated after oxidation with bromine to dehydroascorbic acid, followed by osazone formation. Three prepared solutions separate the fractions: solution 1 gives ascorbic acid + dehydroascorbic acid + diketogulonic acid, solution 2 gives dehydroascorbic acid + diketogulonic acid, and solution 3 gives diketogulonic acid.
Apparatus required
- Blender
- Volumetric flasks
- Test tubes
- Gas washing bottle
- Constant-temperature water bath (37° C)
- Ice bath
- Colorimeter or spectrophotometer (reading at 520 nm)
- Filter or centrifuge
Reagents
a. 9 N H₂SO₄: dilute 250 ml of H₂SO₄ to 1 litre with water.
b. 2% 2,4-dinitrophenylhydrazine: dissolve 2 g of 2,4-dinitrophenylhydrazine in 100 ml of 9 N H₂SO₄. Store in an amber-coloured bottle in a refrigerator. Filter before use. The solution is stable for 2 weeks.
c. 20% metaphosphoric acid (HPO₃): dissolve 100 g of HPO₃ in 500 ml of distilled water. Store in a refrigerator; stable for 2 weeks; preferably prepare fresh. Prepare 5% or 10% HPO₃ solutions from the stock by dilution with water.
d. 2% thiourea in 5% metaphosphoric acid: dissolve 10 g of thiourea in 500 ml of 5% HPO₃.
e. 85% H₂SO₄: dilute 900 ml of H₂SO₄ to 1000 ml with water.
f. Bromine.
g. Ascorbic acid standard: dissolve 100 mg of ascorbic acid in 100 ml of 5% HPO₃ (1 ml = 1 mg of ascorbic acid).
h. Hydrogen sulphide (Kipp’s).
i. Carbon dioxide (Kipp’s).
j. Stannous chloride.
Sample preparation
Total ascorbic acid (solution 1): blend 100 g of sample and 100 g of 10% HPO₃ in a blender to a uniform slurry. Weigh a portion of the slurry estimated to contain 1 to 2 mg of ascorbic acid. Transfer to a 100-ml volumetric flask and dilute to volume with 5% HPO₃. Mix and filter or centrifuge to get a clear liquid. To the filtrate, add 1 or 2 drops of bromine and shake gently until the solution becomes yellow; if the solution does not become yellow, add another drop and shake. Remove the excess bromine by passing air or nitrogen (saturated with water) through the solution until it is colourless. Measure the volume and add thiourea to give a concentration of 1% (0.4 g for 40 ml of extract).
Dehydroascorbic acid and diketogulonic acid (solution 2): weigh a portion of the sample containing 5 to 10 μg of ascorbic acid per ml. Assuming the sample contains 50 mg/100 g ascorbic acid and the final volume to be made up to is 250 ml, weigh 2.5 to 5.0 g of the sample, add 1.25 g of stannous chloride and 12.5 ml of HPO₃, grind the sample and make up to 250 ml with 5% HPO₃. Filter and use the filtrate for osazone formation.
Diketogulonic acid (solution 3): pipette 100 ml of the filtrate from step 2 into a gas washing bottle. Pass H₂S for 15 min; to minimize odour, pass the exhaust H₂S through NaOH. To 40 ml of the H₂S-saturated solution, add 0.4 g of thiourea to give a concentration of 1%. Shake until dissolved and filter. Bubble CO₂ into the filtrate for 5 min. (The H₂S reduces dehydroascorbic acid to ascorbic acid, which does not react with 2,4-dinitrophenylhydrazine.) In heated and/or stored samples, the resulting solution is coloured and it is not possible to measure the diketogulonic acid.
Procedure
Formation of osazone
For each of solutions 1, 2 and 3, take 3 test tubes and mark them 1, 2 and 3 (blank, sample, sample):
Tube 1 (blank): 4.0 ml sample; add no DNPH reagent yet
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Tubes 2 and 3 (samples): 4.0 ml sample + 1.0 ml of 2% 2,4-dinitrophenylhydrazine reagent
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Incubate at 37° C ± 0.5° C for exactly 6 hr in a water bath
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Remove from the water bath and keep in an ice bath
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Add 5.0 ml of 85% H₂SO₄ to all tubes, drop by drop with thorough mixing, and allow to cool
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Add 1.0 ml of 2% 2,4-dinitrophenylhydrazine reagent to tube 1 (the blank)
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Remove the tubes from the ice bath and allow to stand at room temperature for 30 min
Colour measurement
Measure the colour at 520 nm, setting the instrument to 100% transmittance with the blank in place in each set
Standard curve
To 50 ml of standard ascorbic acid solution (1 ml = 1 mg) in a conical flask, add 2 to 3 drops of bromine until the colour becomes yellow, and transfer to a gas washing bottle
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Pass water-saturated air through the solution to remove unreacted bromine
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Pipette 10 ml of this solution into a 500-ml volumetric flask, add 5.0 g of thiourea and make up to volume with 5% HPO₃ (1 ml = 20 μg)
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Pipette 5, 10, 20, 25, 30, 40, 50 and 60 ml of this solution into separate 100-ml volumetric flasks and make up to the mark with 5% HPO₃ containing 1% thiourea, giving solutions of 1, 2, 4, 5, 6, 8, 10 and 12 μg per ml respectively
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Form osazone for each of the seven standards as in the sample, and measure the colour at 520 nm
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Plot % transmittance as ordinate against concentration of ascorbic acid as abscissa on semilogarithmic paper, or absorbance against concentration on ordinary graph paper
Calculation
Read the concentration of ascorbic acid in each of solutions 1, 2 and 3 from the standard curve, and calculate using the expression:
mg/100 g = (μg as read from the graph × Volume made up × 100) / (ml of sample taken × Wt of sample × 1000)
Determine ascorbic acid, dehydroascorbic acid and diketogulonic acid using the following relationship:
- Diketogulonic acid = solution 3
- Dehydroascorbic acid = solution 2 − solution 3
- Ascorbic acid = solution 1 − solution 2
Where,
- Solution 1 gives: ascorbic acid + dehydroascorbic acid + diketogulonic acid
- Solution 2 gives: dehydroascorbic acid + diketogulonic acid
- Solution 3 gives: diketogulonic acid
Precautions
- Incubate for exactly 6 hr at 37° C ± 0.5° C.
- Add the 85% H₂SO₄ drop by drop with thorough mixing and allow to cool.
- Add the DNPH reagent to the blank only after the ice bath step.
- In heated and/or stored samples, the solution after H₂S treatment is coloured and diketogulonic acid cannot be measured.
References
- Roe, J.H., M.B. Mills, M.J. Osterling and C.M. Dameron, J. Biol. Chem., 174, 201 (1949).
- Roe, J.H. and M.J. Osterling, J. Biol. Chem., 152, 511 (1944).
- Roe, J.H., J. Biol. Chem., 116, 609 (1936).
- Roe, J.H. and C.A. Kuether, J. Biol. Chem., 147, 399 (1943).
- Methods of Vitamin Assay, The Association of Vitamin Chemists, Interscience Publishers, New York, 3rd edn., p. 328 (1966).
Source flags: the printed relationship lines (“Diketogulonic acid … / Dehydroascorbic acid = 2−3 / Ascorbic acid = 1−2”) are garbled in the extraction; the relationships shown follow the book’s own scheme printed for solutions 1, 2 and 3. “5 yg” read as 5 μg; “50 mg/1C0 g” read as 50 mg/100 g. The source lists eight standard volumes (5, 10, 20, 25, 30, 40, 50 and 60 ml) but then says “form osazone for each of the seven standards” — mismatch carried as printed, flagged for verification. The source prints “Koe, J.H. & C.A. Kuether” — read as Roe, J.H. The 12.5 ml of HPO₃ in solution 2 has no stated strength in the source.