SOP: Determination of Ascorbic Acid by the Xylene Extraction Method
GIFSQ — Global Institute for Food Safety & Quality
Principle
The method follows the direct colorimetric method, except that the excess of dye is taken up in xylene and the colour is measured in the xylene layer. It is particularly suitable for fresh materials and stored products containing considerable interfering substances, and enables the determination of true ascorbic acid. Formaldehyde and peroxide modifications correct for non-ascorbic acid reducing substances, sulphite and iron.
Apparatus required
- Blender
- Volumetric flasks
- Stoppered conical flasks
- Colorimeter or spectrophotometer (reading at 520 nm)
Reagents
a. Acetate buffer, pH 4: mix 1 litre of 50% sodium acetate (CH₃COONa·3H₂O) with 1 litre of glacial acetic acid.
b. Dye: dissolve 125 mg of 2,6-dichlorophenol-indophenol in warm distilled water, cool, make up to 100 ml and filter. Dilute 18 ml to 100 ml with water (1 ml of dye = 0.1 mg of ascorbic acid). The stock solution of dye may be stored in a refrigerator for about a week.
c. Metaphosphoric acid (HPO₃): dissolve 30 g of sticks or pellets of HPO₃ in distilled water and dilute to 1000 ml.
d. Xylene: use redistilled xylene. Xylene used in the method may be recovered by shaking with a 20% solution of NaOH to neutralize acetic acid, followed by distillation.
e. Standard ascorbic acid solution: weigh exactly 100 mg of ascorbic acid and make up to 100 ml with 3% HPO₃. Dilute 10 ml to 100 ml (1 ml = 0.1 mg of ascorbic acid).
f. 3% hydrogen peroxide: dilute 30% hydrogen peroxide 10 times.
g. Formaldehyde.
h. Anhydrous sodium sulphate (for drying the xylene layer).
Sample preparation (extraction)
Macerate 100 g of sample with 100 g of 3% HPO₃ in a blender
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Weigh a portion of the macerate containing 10 to 15 mg of ascorbic acid (10 to 20 g of macerate), transfer to a 100-ml volumetric flask, make up to the mark with 3% HPO₃, and filter
Procedure
Standard curve
Pipette 0.0, 0.5, 0.75, 1, 1.5 and 2 ml of standard ascorbic acid solution into 50-ml stoppered conical flasks and make up to 2 ml with 3% HPO₃
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Add 2 ml of acetate buffer, 3 ml of dye and 15 ml of xylene in rapid succession
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Stopper the flasks and shake vigorously for 10 sec to extract the excess dye into the xylene; allow to separate
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Pipette out the water layer below the xylene layer and discard it
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To the xylene layer, add a few crystals of anhydrous Na₂SO₄ to remove traces of moisture
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Measure the colour at 520 nm, setting the instrument to 100% transmittance using xylene as blank
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Plot absorbance against concentration to get the standard curve
Basic method
Take 2 ml of filtrate in a stoppered conical flask; add 2 ml of buffer, 3 ml of dye and 15 ml of xylene in rapid succession (ascorbic acid is unstable after addition of the pH 4 buffer, and other reducing substances may react with the dye, so the additions must be rapid)
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Stopper and shake for 10 to 15 sec
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Pipette out the water layer, add anhydrous Na₂SO₄ to the xylene, and measure the colour as in the standard curve
Formaldehyde modification (for heat-treated or long-stored materials)
Flask 1 (total ascorbic acid, A): 2.0 ml sample, 2.0 ml buffer, 1.0 ml water — then 3.0 ml dye and 15.0 ml xylene; add in rapid succession, stopper and shake
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Flask 2 (non-ascorbic acid reducing substances, B): 2.0 ml sample, 2.0 ml buffer, 1.0 ml formaldehyde (40%) — allow to stand for 10 min, then add 3 ml of dye and 15 ml of xylene; stopper and shake
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Measure the colour in the xylene layer as in the basic method
Peroxide modification (for sulphite-treated products or products stored long in metal containers)
Flask 1 (total ascorbic acid, A): 2.0 ml sample, 2.0 ml buffer, 2.0 ml water — then 3.0 ml dye and 15.0 ml xylene
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Flask 2 (non-ascorbic acid reducing substances, C): 2.0 ml sample, 2.0 ml buffer, 2.0 ml 3% hydrogen peroxide — then 3.0 ml dye and 15.0 ml xylene
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Add in rapid succession, stopper and shake; measure the colour in the xylene layer as in the basic method
Calculation
Find the ascorbic acid concentration from the standard curve and calculate:
Total ascorbic acid, mg per 100 g or ml = (Ascorbic acid in sample from curve × Volume made up × 100) / (ml of solution taken × Wt or volume of sample taken)
Calculate the non-ascorbic acid reducing substances in the formaldehyde or hydrogen peroxide modification as in the case of total ascorbic acid.
True ascorbic acid = Total ascorbic acid (A) − Non-ascorbic acid reducing substances (B or C)
Where,
- A = total ascorbic acid from the basic method (flask 1)
- B = non-ascorbic acid reducing substances from the formaldehyde modification (flask 2)
- C = non-ascorbic acid reducing substances from the peroxide modification (flask 2)
Precautions
- Add buffer, dye and xylene in rapid succession: ascorbic acid is unstable after addition of the pH 4 buffer, and other reducing substances may react with the dye.
- Discard the water layer below the xylene layer before measuring the colour.
- Remove traces of moisture from the xylene layer with anhydrous sodium sulphate.
- Set the instrument to 100% transmittance using xylene as blank.
References
- Robinson, W.B. and E. Stotz, J. Biol. Chem., 160, 217 (1945).
Source flags: the source print reads “(OF 275 peroxide” for reagent 6 — read as “3% hydrogen peroxide” (dilute 30% ten times), consistent with the 3% strength used in the peroxide modification. The total-ascorbic-acid calculation expression is reconstructed from garbled print; only the final relationship (True ascorbic acid = A − B, or A − C) is legible in the source.