SOP: Identification of Ether-Extractable Preservatives by Thin Layer Chromatography

GIFSQ — Global Institute for Food Safety & Quality

Principle

The sample is acidified and the ether-extractable organic preservatives (benzoic acid, p-hydroxybenzoic acid, p-chlorobenzoic acid, the esters of p-hydroxybenzoic acid, sorbic acid, dehydroacetic acid, salicylic acid, cinnamic acid) are extracted with ether, purified, separated by thin layer chromatography, and identified from their Rf values and sublimation temperatures (Table 12-6).

Apparatus required

• TLC apparatus: glass plates, spreader, chromatographic chamber, stand for plates
• Rotary evaporator
• Heating block
• Desiccator

Reagents

a. Silica Gel G.

b. Dilute HCl (1+4).

c. Ethyl ether : petroleum ether mixture (50:50).

d. 2 N sodium carbonate solution (weak alkali).

e. 2 N HCl.

f. n-Butanol saturated with 2 N ammonia (developing solvent).

Procedure

Mix 50 g of Silica Gel G with 90 ml of water and prepare thin layers (0.25 mm) on 20 × 20 cm glass plates using the spreader

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Allow the TLC plates to air dry, activate by heating at 100 °C for 30 min, and cool in a desiccator

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Acidify squashes, cordials and syrups with dilute HCl (1+4); turn solid and semi-solid products (chutneys, jams) or viscous products (sauce, ketchup) into solution by blending with water, and acidify with dilute HCl (1+4)

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Extract the solution with an equal amount of the ethyl ether : petroleum ether mixture (50:50)

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Wash the ether extract with weak alkali (2 N sodium carbonate) in a separating funnel

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Acidify the alkali extract with 2 N HCl and re-extract with an equal volume of the ether mixture

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Evaporate the ether solution to a small volume using a rotary evaporator and use the concentrate for spotting

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Apply 20 µl of each of the extracts about 1 to 2 cm from the edge of the plate

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Place in the chromatographic chamber containing n-butanol saturated with 2 N ammonia and develop for a distance of 12 cm; allow the plates to air dry

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Place the plates on a heating block and raise the temperature slowly — preservatives sublimate at different temperatures; record the Rf values and sublimation temperatures and identify the preservatives from Table 12-6

References

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

Cavello, M. & O. Schettino — as cited in the book for the TLC identification of preservatives.