Commission Implementing Regulation (EU) No 299/2013Show full title

“ANNEX IX 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 of olive oil (as described in the Appendix) in the ultraviolet.

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. The specific absorbance at 232 nm and 268 nm in iso-octane or 232 nm and 270 nm in cyclohexane for a concentration of 1 g per 100 ml in a 10 mm cell is calculated.

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. Before use it is recommended that the wavelength and absorbance scales of the spectrometer be checked as follows. U.K.

3.1.1. Wavelength scale: This may be checked using a reference material consisting of an optical glass filter containing holmium oxide which has distinct absorption bands. The reference material is designed for the verification and calibration of the wavelength scales of visible and ultraviolet spectrophotometers having nominal spectral bandwidths of 5 nm or less. The holmium glass filter is measured in the absorbance mode against an air blank, over the wavelength range of 640 to 240 nm. For each spectral bandwidth (0,10 – 0,25 – 0,50 – 1,00 – 1,50 – 2,00 and 3,00), a baseline correction is performed with an empty cell holder. The wavelengths of the spectral bandwidth are listed in the certificate of the reference material in ISO 3656. U.K.

3.1.2. Absorbance scale: This may be checked using a reference material consisting of 4 solutions of potassium dichromate in perchloric acid sealed in four UV quartz cells to measure the linearity and photometric accuracy reference in the UV. The potassium dichromate filled cells (40 mg/ml, 60 mg/ml, 80 mg/ml and 100 mg/ml) are measured against a perchloric acid blank. The net absorbance values are listed in the certificate of the reference material in ISO 3656. U.K.

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

3.3. 25 ml graduated flasks. U.K.

3.4. Analytical balance, capable of being read to the nearest 0,0001 g. U.K.

4. REAGENTS U.K.

Use only reagents of recognized analytical grade, unless otherwise stated.

Solvent: Iso-octane (2,2,4-trimethylpentane) for the measurement at 232 nm and 268 nm or cyclohexane for the measurement at 232 nm and 270 nm, having an absorbance less than 0,12 at 232 nm and less than 0,05 at 250 nm against distilled water, measured in a 10 mm cell.

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 (to the nearest 1 mg) 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 quartz cell 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.

NOTE: It may not be necessary to measure the absorbance over the full wavelength range. U.K.

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:

Κλ

=

specific extinction at wavelength λ,

Ελ

=

extinction measured at wavelength λ;

c

=

concentration of the solution in g/100 ml;

s

=

thickness of the quartz cells in cm.

The results are to be expressed to two decimal places.

6.2. Variation of the specific extinction (ΔΚ) U.K.

Spectrophotometric analysis of olive oil in accordance with the official method in the Union legislation involves also the determination of the variation of the absolute value of the specific extinction (ΔΚ), which is given by:

where Km is the specific extinction at wavelength m, the wavelength for maximum absorption depends on the solvent used: 270 for cyclohexane and 268 for iso-octane.

Appendix

OLIVE OIL CHARACTERISTICS

a Total isomers which could (or could not) be separated by capillary column.
b Or where the median defect is less than or equal to 3,5 and the fruity median is equal to 0.
c Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be lampante olive oil if the total aliphatic alcohol content is less than or equal to 350 mg/kg or if the erythrodiol and uvaol content is less than or equal to 3,5 %.
d Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be crude olive-pomace oil if the total aliphatic alcohol content is above 350 mg/kg and if the erythrodiol and uvaol content is greater than 3,5 %.
e K 270 if solvant is cyclohexane, K 268 if solvant is iso-octane.”
Category		Fatty acid methyl esters (FAMES) and fatty acid ethyl esters (FAEEs)	Acidity (%) (*)	Peroxide index mEq 02/kg (*)	Waxes mg/kg (**)	2 glyceril monopalmitate (%)	Stigmastadiene mg/kga	Difference: ECN42 (HPLC) and ECN42 (theoretical calculation)	K232 (*)	K270 (*) 'K 270 or K 268e'	Delta-K (*)e	Organoleptic evaluation Median defect (Md) (*)	Organoleptic evaluation Fruity median (Mf) (*)
1.	Extra virgin olive oil	Σ FAME + FAEE ≤75 mg/kg or 75 mg/kg <Σ FAME + FAEE ≤150 mg/kg and (FAEE/FAME) ≤1,5	≤ 0,8	≤ 20	≤ 250	≤ 0,9 if total palmitic acid % ≤ 14 %	≤ 0,10	≤ 0,2	≤ 2,50	≤ 0,22	≤ 0,01	Md = 0	Mf > 0
						≤ 1,0 if total palmitic acid % > 14 %
2.	Virgin olive oil	—	≤ 2,0	≤ 20	≤ 250	≤ 0,9 if total palmitic acid % ≤ 14 %	≤ 0,10	≤ 0,2	≤ 2,60	≤ 0,25	≤ 0,01	Md ≤ 3,5	Mf > 0
						≤ 1,0 if total palmitic acid % > 14 %
3.	Lampante olive oil	—	> 2,0	—	≤ 300c	≤ 0,9 if total palmitic acid % ≤ 14 %	≤ 0,50	≤ 0,3	—	—	—	Md > 3,5b	—
						≤ 1,1 if total palmitic acid % > 14 %
4.	Refined olive oil	—	≤ 0,3	≤ 5	≤ 350	≤ 0,9 if total palmitic acid % ≤ 14 %	—	≤ 0,3	—	≤ 1,10	≤ 0,16	—	—
						≤ 1,1 if total palmitic acid % > 14 %
5.	Olive oil composed of refined and virgin olive oils	—	≤ 1,0	≤ 15	≤ 350	≤ 0,9 if total palmitic acid % ≤ 14 %	—	≤ 0,3	—	≤ 0,90	≤ 0,15	—	—
						≤ 1,0 if total palmitic acid % > 14 %
6.	Crude olive-pomace oil	—	—	—	> 350d	≤ 1,4	—	≤ 0,6	—	—	—	—	—
7.	Refined olive-pomace oil	—	≤ 0,3	≤ 5	> 350	≤ 1,4	—	≤ 0,5	—	≤ 2,00	≤ 0,20	—	—
8.	Olive-pomace oil		≤ 1,0	≤ 15	> 350	≤ 1,2	—	≤ 0,5	—	≤ 1,70	≤ 0,18	—	—