Commission Regulation (EEC) No 000/90 (repealed)Dangos y teitl llawn

22. SORBIC ACID U.K.

1. PRINCIPLE OF METHODS U.K.

1.1. Determination by ultraviolet absorption spectrophotometry U.K.

Sorbic acid (trans, trans, 2,4-hexadienoic acid) extracted by steam distillation is determined in the wine distillate by ultraviolet absorption spectrophotometry. Substances that interfere in the ultraviolet are removed by evaporation to dryness using a lightly alkali, calcium hydroxide. Thin layer chromatography is used for confirmation of levels (1 mg/l) less than 20 mg/l.

1.2. Determination by gas chromatography U.K.

Sorbic acid extracted in ethyl ether is determined by gas chromatography with an internal standard.

1.3. Identification of traces by thin-layer chromatography U.K.

Sorbic acid extracted in ethyl ether is separated by thin layer chromatography and its concentration is evaluated semi-quantitatively.

2. DETERMINATION BY ULTRAVIOLET ABSORPTION SPECTROPHOTOMETRY U.K.

2.1. Reagents U.K.

2.1.1. Crystalline tartaric acid, C₄H₆O₆. U.K.

2.1.2. Calcium hydroxide, Ca(OH)₂, solution, approximately 0,02 M. U.K.

2.1.3. Reference sorbic acid solution, 20 mg/l: U.K.

Dissolve 20 mg of sorbic acid, C₆H₈O₂, in approximately 2 ml of 0,1 M sodium hydroxide solution. Pour into a 1 000 ml volumetric flask, and make up to the mark with water. It is also possible to dissolve 26,8 mg of potassium sorbate, C₆H₇KO₂, in water and make up to 1 000 ml with water.

2.2. Apparatus U.K.

2.2.1. Steam distillation apparatus (see chapter ‘Volatile acidity’). U.K.

2.2.2. Water bath at 100 °C. U.K.

2.2.3. Spectrophotometer enabling absorbence measurements to be made at a wavelength of 256 nm and having a quartz cell with a 1 cm optical path. U.K.

2.3. Procedure U.K.

2.3.1. Distillation U.K.

Place in the flask of the steam distillation apparatus 10 ml of wine and add 1 to 2 g tartaric acid (2.1.1). Collect 250 ml of the distillate.

2.3.2. Preparation of the calibration curve U.K.

Prepare, by dilution of the reference solution (2.1.3), four dilute reference solutions with 0,5, 1,0, 2,5 and 5 mg of sorbic acid per litre. Measure their absorbences with the spectrophotometer at 256 nm using that of distilled water as a blank. Plot a curve showing the variation of absorbence as a function of concentration. The variation is linear.

2.3.3. Determination U.K.

Place 5 ml of the distillate in an evaporating dish of 55 mm diameter, add 1 ml of calcium hydroxide solution (2.1.2). Evaporate to dryness on a water bath.

Dissolve the residue in several ml of distilled water, transfer completely to a 20 ml volumetric flask and make up to the mark with rinsing water. Measure the absorbence at 256 nm using the spectrophotometer against a blank consisting of a solution obtained by diluting 1 ml of calcium hydroxide solution (2.1.2) to 20 ml with water.

Plot the value of the measured absorbence on the calibration curve and from this find the concentration C of sorbic acid in the solution.

Note: In this method the loss due to evaporation can be neglected and the absorbence measured on the treated distillate diluted ¼ with distilled water. U.K.

2.4. Expression of results U.K.

2.4.1. Calculation U.K.

The sorbic acid concentration in the wine expressed in mg per litre is given by 100 × C

where

C

=

concentration of sorbic acid in the solution analysed by spectrophotometry expressed in mg per litre.

3. DETERMINATION BY GAS CHROMATOGRAPHY U.K.

3.1. Reagents U.K.

3.1.1. Ethyl ether, (C₂H₅)₂O, distilled just before use. U.K.

3.1.2. Internal reference solution: solution of undecanoic acid, C₁₁H₂₂O₂, in 95 % vol ethanol at a strength of 1 g/l. U.K.

3.1.3. Aqueous solution of sulphuric acid, H₂SO₄ (ρ₂₀ = 1,84 g/ml) diluted 1:3 (v/v). U.K.

3.2. Apparatus U.K.

3.2.1. Gas chromatograph fitted with a flame ionization detector and a stainless steel column (4 m × ⅛ inch) previously treated with dimethyldichlorosilane and packed with a stationary phase consisting of a mixture of diethyleneglycol succinate (5 %) and phosphoric acid (1 %) (DEGS — H₃PO₄) or of a mixture of diethyleneglycol adipate (7 %) and phosphoric acid (1 %) (DEGA — H₃PO₄) bonded on Gaschrom Q 80 — 100 mesh. U.K.

Treatment of column with DMDCS — pass through the column a solution containing 2 to 3 g of DMDCS in toluene. Immediately wash with methanol, followed by nitrogen and then wash with hexane followed by more nitrogen. It is now ready to be packed.

Operating conditions:

Oven temperature: 175 °C.

Temperature of the injector and detector: 230 °C.

Carrier gas: nitrogen (flow rate = 200 ml/min).

3.2.2. Microsyringe, 10 µl capacity graduated in 0,1 µl. U.K.

Note: Other types of columns that give a good separation can be used, particularly capillary columns (e.g. FFAP). The working method described is given as an example. U.K.

3.3. Procedure U.K.

3.3.1. Preparation of sample to be analysed U.K.

Into a glass test tube of approximately 40 ml capacity and fitted with a ground glass stopper, introduce 20 ml of wine, add 2 ml of the internal reference solution (3.1.2) and 1 ml of dilute sulphuric acid (3.1.3).

After mixing the solution by repeatedly turning the tube over, add to its contents 10 ml of ethyl ether (3.1.1). Extract the sorbic acid in the organic phase by shaking the tube for five minutes. Leave to settle.

3.3.2. Preparation of the reference solution U.K.

Select a wine for which the chromatogram of the ether extract shows no peak corresponding to the elution of sorbic acid. Overload this wine with sorbic acid at a concentration of 100 mg per litre. Treat 20 ml of the sample prepared in this way according to the procedure described in 3.3.1.

3.3.3. Chromatography U.K.

Using a microsyringe, inject into the chromatograph in turn 2 µl of the ether-extract phase obtained in 3.3.2 and 2 µl of the ether-extracted phase obtained in 3.3.1.

Record the respective chromatograms: check the identity of the respective retention times of the sorbic acid and the internal standard. Measure the height (or area) of each of the recorded peaks.

3.4. Expression of results U.K.

3.4.1. Calculation U.K.

The concentration of sorbic acid in the analysed wine, expressed in mg per litre, is given by:

where

H

=

height of the sorbic acid peak in the reference solution

h

=

height of the sorbic acid peak in the sample for analysis

I

=

height of the internal standard peak in the reference solution

i

=

height of the internal standard peak in the sample for analysis

^Note: The sorbic acid concentration may be determined in the same way from measurements of the areas under the respective peaks. U.K.

4. IDENTIFICATION OF TRACES OF SORBIC ACID BY THIN LAYER CHROMATOGRAPHY U.K.

4.1. Reagents U.K.

4.1.1. Ethyl ether, (C₂H₅)₂O. U.K.

4.1.2. Aqueous sulphuric acid solution, H₂SO₄ (ρ₂₀ = 1,84 g/ml), diluted 1:3 (v/v). U.K.

4.1.3. Reference solution of sorbic acid in an approximately 10 % vol ethanol/water mixture containing 20 mg per litre. U.K.

4.1.4. Mobile phase: hexane-pentane-acetic acid (20:20:3) (C₆H₁₄/C₅H₁₂/CH₃COOH, ρ₂₀ = 1,05 g/ml). U.K.

4.2. Apparatus U.K.

4.2.1. Precoated 20 × 20 cm plates for thin layer chromatography coated with polyamide gel (0,15 mm thick) with the addition of a fluorescent indicator. U.K.

4.2.2. Cell for thin layer chromatography. U.K.

4.2.3. Micropipette or microsyringe for delivering volumes of 5 µl to within ± 0,1 µl. U.K.

4.2.4. Ultraviolet lamp (254 nm). U.K.

4.3. Procedure U.K.

4.3.1. Preparation of sample to be analysed U.K.

Into a glass test tube of approximately 25 ml capacity and fitted with a ground glass stopper, place 10 ml of wine, add 1 ml of dilute sulphuric acid (4.1.2) and 5 ml of ethyl ether (4.1.2). Mix by repeatedly turning the tube over. Leave to settle.

4.3.2. Preparation of dilute reference solutions U.K.

Prepare five dilute reference solutions from the solution in 4.1.3 containing 2, 4, 6, 8 and 10 mg sorbic acid per litre.

4.3.3. Chromatography U.K.

Using a microsyringe or micropipette, deposit 5 µl of the ether-extracted phase obtained in 4.3.1 and 5 µl of each of the dilute reference solutions (4.3.2) at points 2 cm from the lower edge of the plate and 2 cm apart from each other.

Place the mobile phase (4.1.4) in the chromatograph tank to a height of about 0,5 cm and allow the atmosphere in the tank to become saturated with solvent vapours. Place the plate in the tank. Allow the chromatogram to develop over 12 to 15 cm (development time approximately 30 minutes). Dry the plate in a current of cool air. Examine the chromatogram under a 254 nm ultraviolet lamp.

The spots indicating the presence of sorbic acid will appear to be dark violet against the yellow fluorescent background of the plate.

4.4. Expression of results U.K.

A comparison of the intensities of the spots produced by the sample to be analysed and by the reference solutions will enable a semi-quantitative assessment to be made of the sorbic acid concentration between 2 and 10 mg per litre. A concentration of 1 mg per litre could be determined with the deposition of 10 µl of the sample solution to be analysed.

Concentrations above 10 mg per litre could be determined with the deposition of less than 5 µl of the solution to be analysed (measured out using a microsyringe).