SOP: Determination of Sorbic Acid by Column Chromatography and Spectrophotometry

GIFSQ — Global Institute for Food Safety & Quality

Principle

Sorbic acid is distilled from the sample, extracted into chloroform, separated from interfering substances on a silicic acid column, and measured by its absorbance at 254 nm against a standard.

Apparatus required

• Two-neck 500-ml distilling flask with ground-glass joints, with a 125-ml separating funnel fixed to one neck and a Friedrich’s condenser with ground-glass joint to the other
• 125-ml separating funnel
• Chromatographic tube of 1.5 cm diameter and 25 cm long, with a narrow tube at one end
• 1000-ml separating funnel
• Spectrophotometer
• Steam bath

Reagents

a. Silicic acid, chromatographic grade.

b. Bromocresol green indicator: 0.25% in methanol.

c. Solvents: (i) 70% methanol; (ii) chloroform–iso-octane (1+4) saturated with 70% methanol — mix 100 ml of chloroform and 400 ml of iso-octane, transfer to a 1000-ml separating funnel, add 20 ml of 70% methanol, shake vigorously, let the layers separate, drain off the methanol layer, and filter the chloroform–iso-octane layer through fluted filter paper into a suitable container.

d. Sorbic acid standard solution: dissolve 50 mg of sorbic acid in iso-propanol and make to 500 ml with the same solvent (1 ml = 0.1 mg); dilute 5 ml of this solution to 250 ml with iso-propanol to get the working standard (1 ml = 0.2 mg, as printed).

e. Magnesium sulphate (50 g portions).

f. Sodium chloride.

g. 1 N ammonium hydroxide.

Note: the working standard dilution as printed (5 ml of a 0.1 mg/ml solution made to 250 ml) arithmetically gives 0.002 mg/ml, not the printed 0.2 mg/ml — carried as printed, flagged; consult the printed book.

Procedure

Weigh an aliquot of liquid sample containing 1.5 to 2.0 mg of sorbic acid, transfer to the distillation flask, and add enough water to make to 50 ml

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For solid samples, weigh a portion containing 15–20 mg of sorbic acid, blend in a blender with water, transfer to a 500-ml volumetric flask, dilute to volume with water, filter, and pipette 50 ml to the distillation flask

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Add 50 g of magnesium sulphate and distil until 40–45 ml of distillate collects in a measuring cylinder; transfer the distillate to a 250-ml separating funnel

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Add another 50 ml of water to the distillation flask through the separating funnel; collect a second 40–45 ml of distillate and transfer to the separating funnel; wash the condenser with hot water, transfer the washings to the funnel, and let cool

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Saturate the distillate with NaCl and extract the sorbic acid with five 40-ml portions of chloroform; after each extraction, transfer the chloroform layer to a second separating funnel containing 10 ml of distilled water

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Filter the chloroform layer through a wad of cotton and combine the chloroform extracts; evaporate on a steam bath under a current of air to about 25 ml

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Transfer to a 50-ml beaker and evaporate as before to about 10 ml; remove the last 10 ml of chloroform in a current of air at a temperature not exceeding 37.5 °C

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Dissolve the residue immediately in 2 ml of the chloroform–iso-octane mixture

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Chromatographic separation: to 5 g of silicic acid in a mortar, add 1 ml of bromocresol green indicator, one drop of 1 N ammonium hydroxide, and the maximum quantity of 70% methanol that the silicic acid will hold without becoming sticky when slurried with the chloroform–iso-octane mixture; mix thoroughly, add enough chloroform–iso-octane mixture to first form a paste and then a slurry

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Place a thin wad of cotton at the constricted end of the tube and pour the slurry over it; remove the excess solvent by air pressure (5–7 lb) or mild suction; do not let the column dry below the surface of the gel

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Transfer 2 ml of the sorbic acid extract to the column; wash the beaker thrice with 2-ml portions of the chloroform–iso-octane solvent, adding each washing to the column after the extract and each washing has passed into the column

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Add the chloroform–iso-octane solvent and allow it to percolate through the column under air pressure or mild suction; discard the acid that comes initially and collect the sorbic acid band in a 50-ml volumetric flask; make to volume with the chloroform–iso-octane solvent

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Pipette an aliquot containing 0.15–0.20 mg of sorbic acid to a 100-ml volumetric flask and dilute to 100 ml with iso-propanol

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Measure the absorbance of the sample and of the standard at 254 nm with a spectrophotometer

Calculation

mg of sorbic acid per 100 g (liquid samples) = [0.2 × OD of sample × 100 × 50 × 100] ÷ [OD of standard × 5 × ml taken for final dilution × Wt of sample taken]

Where,

• OD of sample = the absorbance of the sample solution at 254 nm
• OD of standard = the absorbance of the standard solution at 254 nm
• ml taken for final dilution = the volume of the sorbic acid band solution taken for the final dilution to 100 ml (ml)
• Wt of sample taken = the weight of the sample taken (g)

For solid samples, the weight taken for blending, the volume made up, and the aliquot taken for estimation are included in the same expression.

Note: the factor 5 in the denominator sits in an ambiguous position in the printed layout (it falls between “OD of standard” and “ml taken for final dilution” in the garbled text) — carried as shown; consult the printed book. The book reports a recovery of 93% by this method in apple cider (printed as “93°”).

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Ramsey, L.L. & W.I. Patterson, J. Assoc. Offic. Agri. Chem., 31, 139 (1948); Johnson, D.W., J. Assoc. Offic. Agri. Chem., 41, 586 (1958) — as cited in the book.