Commission Regulation (EEC) No 2568/91Show full title

ANNEX IX U.K. SPECTROPHOTOMETRIC INVESTIGATION IN THE ULTRAVIOLET

FOREWORD U.K.

Spectrophotometric examination in the ultraviolet can provide information on the quality of a fat, its state of preservation and changes brought about in it by technological processes.

The absorption at the wavelengths specified in the method is due to the presence of conjugated diene and triene systems. These absorptions are expressed as specific extinctions E^1 % _1 cm (the extinction of 1 % solution of the fat in the specified solvent, in a thickness of 1 cm) conventionally indicated by K (also referred to as ‘extinction coefficient’).

1. SCOPE U.K.

The method describes the procedure for performing a spectrophotometric examination [X1 of olive oil in] the ultraviolet.

Editorial Information

X1 Substituted by Corrigendum to Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis (Official Journal of the European Communities No L 248 of 5 September 1991).

2. PRINCIPLE OF THE METHOD U.K.

The fat in question is dissolved in the required solvent and the extinction of the solution is then determined at the specified wavelengths with reference to pure solvent. Specific extinctions are calculated from the spectrophotometer readings.

3. EQUIPMENT U.K.

3.1. A spectrophotometer for measuring extinction in the ultraviolet between 220 and 360 nm, with the possibility of reading individual nanometric units. U.K.

3.2. Rectangular quartz cuvettes, with covers, having an optical length of 1 cm. When filled with water or other suitable solvent the cuvettes should not show differences between them of more than 0,01 extinction units. U.K.

3.3. 25 ml graduated flasks. U.K.

[F1 3.4. Chromatography column having an upper part 270 mm in length and a diameter of 35 mm and a lower part 270 mm in length and a diameter of approximately 10mm.] U.K.

Textual Amendments

F1 Substituted by Commission Regulation (EEC) No 183/93 of 29 January 1993 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.

4. REAGENTS U.K.

4.1. Spectrophotometrically pure iso-octane (2,2,4-trimethylpentane). With reference to distilled water this should have a transmittance of not less than 60 % at 220 nm and not less than 95 % at 250 nm, or U.K.

• spectrophotometrically pure cyclohexane: with reference to distilled water this should have a transmittance of not less than 40 % at 220 nm and not less than 95 % at 250 nm[F1 .]

• [F2 . . . . .]

Textual Amendments

F2 Deleted by Commission Regulation (EEC) No 183/93 of 29 January 1993 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.

4.2. Basic alumina for column chromatography prepared and checked as described in Appendix I. U.K.

4.3. n-hexane, for chromatography. U.K.

5. PROCEDURE U.K.

5.1. The sample in question must be perfectly homogeneous and without suspected impurities. Oils which are liquid at ambient temperature are to be filtered through paper at a temperature of approximately 30 °C, hard fats are to be homogenized and filtered at a temperature of not more than 10 °C above the melting point. U.K.

5.2. Weigh accurately approximately 0,25 g of the sample so prepared into a 25 ml graduated flask, make up to the mark with the solvent specified and homogenize. The resulting solution must be perfectly clear. If opalescence or turbidity is present filter quickly through paper. U.K.

5.3. Fill a cuvette with the solution obtained and measure the extinctions at an appropriate wavelength between 232 and 276 nm, using the solvent used as a reference. U.K.

The extinction values recorded must lie within the range 0,1 to 0,8. If not the measurements must be repeated using more concentrated or more dilute solutions as appropriate.

5.4. When a determination of specific extinction is required after passage over alumina, proceed as follows. Place 30 g of basic alumina in suspension in hexane in the chromatography column. After the adsorbent has settled remove the excess hexane down to approximately 1 cm above the top of the alumina. U.K.

Dissolve 10 g of the fat, homogenized and filtered as described in 5.1, in 100 ml of hexane and pour the solution into the column. Collect the eluate and evaporate off all the solvent under vacuum at a temperature below 25 °C.

Proceed immediately as specified in 5.2 using the fat so obtained.

6. EXPRESSION OF THE RESULTS U.K.

6.1. Record the specific extinctions (extinction coefficients) at the various wavelengths calculated as follows: U.K.

where:

K_λ

=

specific extinction at wavelength _λ;

E_λ

=

extinction measured at wavelength λ;

c

=

concentration of the solution in g/100 ml;

s

=

thickness of the cuvette in cm.

The results are to be expressed to two decimal places.

6.2. Spectrophotometric analysis of olive oil in accordance with the official method in the EEC regulations specifies determination of the specific extinction in iso-octane solution at wavelengths of 232 and 270 nm and the determination K, which is given by: U.K.

where K_m is the specific extinction at wavelength m, the wavelength for maximum absorption around 270 nm.

APPENDIX I Preparation of the alumina and testing its activity

A.1.1. Preperation of the alumina U.K.

Place alumina which has been previously desiccated in a furnace at 380 to 400 °C for three hours into a hermetically sealed container, add distilled water in the ratio of 5 ml per 100 g of alumina, immediately close the container, shake repeatedly, and then allow to rest for at least 12 hours before use.

A.1.2. Checking the activity of the alumina U.K.

Prepare a chromatographic column with 30 g of alumina. Working as described in paragraph 5.4 pass a mixture consisting of:

• 95 % virgin olive oil having a specific extinction of less than 0,18 at 268 nm,

• 5 % ground-nut oil treated with earth in the refining process, having a specific extinction of not less than 4 at 268 nm

through the column.

If after passage through the column the mixture has a specific extinction of more than 0,11 at 268 nm the alumina is acceptable, if not the level of dehydration must be increased.

APPENDIX II Calibration of the spectrophotometer

A.2. The equipment must be checked at intervals (at least every six months) for both wavelength response and the accuracy of the response. U.K.

A.2.1. The wavelength may be checked using a mercury vapour lamp or by means of suitable filters. U.K.

A.2.2. In order to check the response of the photocell and the photomultiplier proceed as follows: weigh 0,2000 g of pure potassium chromate for spectrophotometry and dissolve in 0,05 N potassium hydroxide solution in a 1 000 ml graduated flask and make up to the mark. Take precisely 25 ml of the solution obtained, transfer to a 500 ml graduated flask and dilute up to the mark using the same potassium hydroxide solution. U.K.

Measure the extinction of the solution so obtained at 275 nm, using the potassium hydroxide solution as a reference. The extinction measured using a 1 cm cuvette should be 0,200 ± 0,005.