Regulation 8(2)
1. The method described below enables the presence of residues of biphenyl, 2-hydroxybiphenyl (orthophenylphenol) or sodium biphenyl-2-yl oxide (sodium orthophenylphenate) in the peel of citrus fruit to be detected. The sensitivity limit of this method, in absolute terms, is approximately 5 μg. for biphenyl and 1 μg. for 2-hydroxybiphenyl or sodium biphenyl-2-yl oxide, which is the equivalent of 5 mg. of biphenyl and 1 mg. of 2-hydroxybiphenyl respectively in the peel of 1 kg. of citrus fruit.
2. An extract is prepared from the peel using dichloromethane in an acid medium. The extract is concentrated and separated by thin layer chromatography using silica gel. The presence of biphenyl, 2-hydroxybiphenyl or sodium biphenyl-2-yl oxide is shown by fluorescence and colour tests.
3. The following reagents shall be used–
(a)cyclohexane (analytical reagent grade);
(b)dicholoromethane (analytical reagent grade);
(c)hydrochloric acid 25 per centum (weight/volume);
(d)silica gel GF 254 (Merck or equivalent);
(e)0.5 per centum (weight/volume) solution of 2,4,7-trinitrofluorenone (TNF) (Fluka, BDH or equivalent) in acetone;
(f)0.1 per centum (weight/volume) solution of 2,6-dibromo—p-benzoquinone-chlorimine in ethanol (stable for up to one week if kept in the refrigerator);
(g)concentrated solution of ammonia, specific gravity: 0.9;
(h)standard 1 per centum (weight/volume) solution of pure biphenyl in cyclohexane;
(j)standard 1 per centum (weight/volume) solution of pure 2-hydroxybiphenyl in cyclohexane.
4. The following apparatus shall be used–
(a)a mixer;
(b)a 250 ml. flask with ground glass joint and with a reflux condenser;
(c)a reduced pressure evaporator;
(d)micropipettes;
(e)a thin layer chromatographic apparatus with plates measuring 20×20 cm.;
(f)an ultra-violet lamp (254 nm.), the intensity of which should be such that a spot of 5 μg. of biphenyl is visible;
(g)equipment for pulversing reagents;
(h)an oven.
5. The analysis shall be carried out as follows–
(a)Preparation and extraction. All the fruit in the sample for analysis is cut in half. Half of each piece of fruit is kept for quantitative determination of the residue of any biphenyl or 2-hydroxybiphenyl present. Pieces of peel are taken from the other halves to give a sample of about 80 g. These pieces are chopped, crushed in the mixer and placed in the 250 ml. flask; to this is added 1 ml. of 25 per centum hydrochloric acid and 100 ml. dichloromethane. The mixture is heated under reflux for 10 minutes. After cooling and rinsing of the condenser with about 5 ml. of dichloromethane, the mixture is filtered through a fluted filter. The solution is transferred to the evaporator and some anti-bumping granules are added. The solution is concentrated at reduced pressure at a temperature of 60°C. to a final volume of about 10 ml. If a rotary evaporator is used, the flask should be kept in a fixed position to avoid loss of biphenyl through the formation of a film of the product on the upper wall of the flask.
(b)Chromatography: 30 g. of silica gel and 60 ml. of water are placed in a mixer and mixed for one minute. The mixture is then spread on to 5 chromatographic plates to form a layer approximately 0.25 mm. thick. The plates covered with this layer are subjected to a stream of hot air for 15 minutes and then placed in an oven where they are kept for 30 minutes at a temperature of 110°C.
After cooling, the surface layer of each plate is divided into lanes, 2 cm. wide, by parallel lines penetrating the silica gel down to the surface of the glass plate. 50 μl. of the extract to be analysed are applied to each lane as a narrow band of contiguous spots approximately 1.5 cm. from the lower edge of the plate. At least one lane is kept for the controls consisting of a spot of 1 μl. (that is, 10 μg.) of the standard solutions of biphenyl and 2-hydroxybiphenyl, one standard per lane. The chromatographic plates are developed in a mixture of cyclohexane and dichloromethane (25 : 95) in tanks previously lined with filter paper.
(c)Detection and identification: The presence of biphenyl and 2-hydroxybiphenyl is shown by the appearance of spots in ultra-violet light (254 nm.). The sodium biphenyl-2-yl oxide will have been converted to 2-hydroxybiphenyl during the extraction in an acid medium, and its presence cannot therefore be distinguished from that of 2-hydroxybiphenyl. The products are identified in the following manner–
(i)biphenyl gives a yellow spot in daylight when sprayed with the TNF solution;
(ii)2-hydroxybiphenyl gives a blue spot when sprayed with the solution of 2,6-dibromo—p-benzoquinonechlorimine, followed by rapid passage through a stream of hot air and exposure to an ammonia-saturated atmosphere.
1. The method described below gives a quantitative analysis of the residues of biphenyl in whole citrus fruit. The accuracy of the method is ±10 per centum for a biphenyl content greater than 10 mg. per kg. of fruit.
2. After distillation in an acid medium and extraction by cyclohexane, the extract is subjected to thin layer chromatography on silica gel. The chromatogram is developed and the biphenyl is eluted and determined spectrophotometrically at 248 nm.
3. The following reagents shall be used–
(a)concentrated sulphuric acid solution;
(b)silicone-based anti-foaming emulsion;
(c)cyclohexane (analytical reagent grade);
(d)hexane (analytical reagent grade);
(e)ethanol (analytical reagent grade);
(f)anhydrous sodium sulphate;
(g)silica gel GF 254 (Merck or equivalent);
(h)standard 1 per centum (weight/volume) solution of pure biphenyl in cyclohexane: dilute with cyclohexane to obtain the following three solutions–
(i)0.6 μg/μl;
(ii)1 μg/μl;
(iii)1.4 μg/μl.
4. The following apparatus shall be used–
(a)a 1 litre mixer;
(b)a 2 litre distillation flask with a modified Clevenger-type separator as shown in the diagram in Schedule 6 and a cooled reflux condenser;
(c)a 10 ml. graduated flask;
(d)50 μl. micropipettes;
(e)a thin layer chromatographic apparatus with 20×20 cm. plates;
(f)an oven;
(g)a centrifuge with 15 ml. conical tubes;
(h)an ultra-violet spectrophotometer.
5. The analysis shall be carried out as follows–
(a)Preparation and extraction: All the fruit in the sample for analysis is cut in half. Half of each piece of fruit is kept for qualitative analysis for residues of biphenyl, 2-hydroxybiphenyl or sodium biphenyl-2-yl oxide. The other halves are put all together and shredded in a mill or crushed until a homogeneous mixture is obtained. From this at least two sub-samples of 200 g. are taken for analysis in the following manner. Each sub-sample is placed in a mixer with 100 ml. of water and mixed at slow speed for several seconds. Water is added until the volume of the mixture reaches ¾ of the capacity of the mixer, and the mixture is then mixed for 5 minutes at full speed. The resulting purée is transferred to the 2 litre distillation flask. The mixer is rinsed with water and the rinsings added to the contents of the flask. (The total quantity of water to be used in mixing and rinsing is 1 litre.) To the mixture are added 2 ml. sulphuric acid, 1 ml. anti-foaming emulsion and several anti-bumping granules. The separator and reflux condenser are fitted on to the flask. Distilled water is poured into the separator until the water level is well past the lower arm of the lateral return tube, followed by 7 ml. cyclohexane. Distillation is carried out for about 2 hours. The lower aqueous layer in the separator is discarded and the upper layer is collected in the 10 ml. graduated flask. The separator is rinsed with about 1.5 ml. of cyclohexane and the rinsings added to the contents of the flask, which are then brought up to volume with cyclohexane. Finally a little anhydrous sodium sulphate is added and the mixture is shaken.
(b)Chromatography: 30 g. of silica gel and 60 ml. of water are placed in a mixer and mixed for one minute. The mixture is then spread on to 5 chromatographic plates to form a layer approximately 0.25 mm. thick. The plates covered with this layer are subjected to a stream of hot air for 15 minutes and then placed in an oven where they are kept for 30 minutes at a temperature of 110°C. After cooling, the surface layer of each plate is divided into 4 lanes, 4.5 cm. wide, by parallel lines penetrating the silica gel down to the surface of the glass plate. 50 μl. of the extract to be analysed are applied to one lane of each plate as a narrow band of contiguous spots approximately 1.5 cm. from the lower edge of the plate. 50 μl. of the standard solutions (i) (ii) and (iii), corresponding respectively to 30, 50 and 70 μg. levels of biphenyl are applied in the same way to the three remaining lanes, one solution to each lane.
If a large number of samples are being analysed at one time, standard solutions need not be applied to every plate. Reference may be made to a standard curve provided that this curve has been prepared from the average values obtained from 5 different plates to which the same standard solutions have been applied.
(c)Development of chromatograms and elution: The chromatograms are developed with hexane to a height of 17 cm. in tanks previously lined with filter paper. The plates are air dried. By illuminating the plates with ultra-violet light (254 nm.), the areas of silica gel containing biphenyl are located and marked off in rectangles of equal area.
The entire layer of silica gel within the areas thus marked off is immediately scraped from the plate with a spatula. The biphenyl is extracted by mixing the silica gel with 10 ml. of ethanol and shaking several times over a period of 10 minutes. The mixture is transferred to the centrifuge tubes and centrifuged for 5 minutes at 2,500 revolutions per minute.
A control sample of silica gel is taken by the same method using an area of the same size. If a series of analyses are made, this control area is taken from an unused lane of a plate and below the solvent front; if a single analysis is made the control sample is taken from an area below one of the positions at which the standard biphenyl is located.
(d)Spectrophotometric determination: The supernatant liquid is decanted into the spectrophotometer cells and the absorption determined at 248 nm. against a control extract from a chromatographic area free from biphenyl.
6. A standard curve is drawn, plotting the biphenyl values of 30, 50 and 70 μg. against the corresponding absorptions, as determined on the spectrophotometer. This gives a straight line which passes through the origin. This graph allows the biphenyl content of the samples to be read directly in mg. per kg. from the absorption value of their extracts.
1. The method described below enables a quantitative analysis of the residues of 2-hydroxybiphenyl and sodium biphenyl-2-yl oxide in whole citrus fruit to be made. The method gives results which for a 2-hydroxybiphenyl or sodium biphenyl-2-yl oxide content of the order of 12 mg. per kg. are low by an average value of between 10 per centum and 20 per centum.
2. After distillation in an acid medium and extraction by di-isopentyl ether, the extract is purified and treated with a solution of 4-aminophenazone. A red colour develops, the intensity of which is measured spectrophotometrically at 510 nm.
3. The following reagents shall be used–
(a)70 per centum (weight/weight) orthophosphoric acid;
(b)silicone-based anti-foaming emulsion;
(c)di-isopentyl ether (analytical reagent grade);
(d)purified cyclohexane: shake 3 times with a 4 per cent (weight/volume) solution of sodium hydroxide, wash 3 times with distilled water;
(e)4 per centum (weight/volume) sodium hydroxide solution;
(f)buffer solution at pH 10.4: into a 2 litre graduated flask put 6.64 g. of boric acid, 8.00 g. of potassium chloride and 93.1 ml. of N sodium hydroxide solution; mix and bring up to calibration mark with distilled water;
(g)reagent I: dissolve 1.0 g. of 4-aminophenazone (4-amino-2, 3-dimethyl-1-phenyl-5-pyrazolone; 4-aminoantipyrin) in 100 ml. of distilled water;
(h)reagent II: dissolve 2.0 g. of potassium ferricyanide in 100 ml. of distilled water. Reagents I and II must be kept in brown glass flasks and are only stable for approximately 14 days;
(j)silica gel;
(k)standard solution: dissolve 10 mg. of pure 2-hydroxybiphenyl in 1 ml. of 0.1 N NaOH; dilute to 100 ml. with a 0.2 M sodium borate solution (1 ml.=100 μg. 2-hydroxybiphenyl). For the standard curve, dilute 1 ml. to 10 ml. with the buffer solution.
4. The following apparatus shall be used–
(a)a shredding or crushing mill;
(b)a mixer;
(c)a 1 litre distillation flask with a modified Clevenger-type separator as shown in the diagram in Schedule 6 and a reflux condenser;
(d)an electrically controlled heating mantle;
(e)a 200 ml. separating funnel;
(f)graduated cylinders of 25 and 100 ml.;
(g)graduated flasks of 25 and 100 ml.;
(h)1 to 10 ml. pipettes;
(j)0.5 ml. graduated pipettes;
(k)a spectrophotometer with 4 or 5 cm. cells.
5. All the fruit in the sample for analysis is cut in half. Half of each piece of fruit is kept for qualitative analysis for residues of biphenyl, 2-hydroxybiphenyl or sodium biphenyl-2-yl oxide. The other halves are put all together and shredded in a mill or crushed until a homogeneous mixture is obtained. From this at least two sub-samples of 250 g. are taken for analysis in the following manner–
Each sub-sample is placed in a mixer with 500 ml. of water and mixed until a very fine homogeneous mixture is obtained in which the oily cells are no longer perceptible. A sample of 150 to 300 g. of the purée is taken, depending on the presumed 2-hydroxybiphenyl content and placed in the 1 litre distillation flask with a quantity of water sufficient to dilute the mixture to 500 g. in the flask. After the addition of 10 ml. of 70 per centum orthophosphoric acid, several anti-bumping granules and 0.5 ml. of anti-foaming emulsion, the separator and the reflux condenser are fitted on to the flask. 10 ml. of di-isopentyl ether are placed in the separator and the flask is heated gently in the electrically controlled heating mantle until the mixture boils. Emulsion formation is minimised if the mixture is boiled gently for the first 10 to 20 minutes. The rate of heating is then gradually increased until the mixture boils steadily and one drop of water reaches the trapping solvent every 3 to 5 seconds. After distilling for 6 hours, the contents of the separator are poured into the 200 ml. separating funnel, and the separator and the condenser are rinsed with 60 ml. of cyclohexane and then with 60 ml. of water. The rinsings are added to the contents of the separating funnel. The mixture is shaken vigorously and when the phases have separated the aqueous phase is discarded.
To extract the 2-hydroxybiphenyl, the organic phase is shaken vigorously 5 times, each time for 3 minutes, with 10 ml. of 4 per centum sodium hydroxide. The alkaline solutions are combined, adjusted to pH 9–10 with orthophosphoric acid in the presence of phenolophthalein paper, and diluted to 100 ml. with distilled water. A pinch of silica gel is added in order to clarify the solution which will have a slightly cloudy appearance. The solution is then shaken and filtered through a dry, fine-grain filter. Since colouring is developed with the maximum of accuracy and precision using quantities of 2-hydroxybiphenyl of between 10 and 70 μg. an aliquot sample of between 0.5 and 10 ml. of solution is taken with a pipette, taking into account the quantities of 2-hydroxybiphenyl which might be expected to be found. The sample is placed in a 25 ml. graduated flask; to this are added 0.5 ml. of reagent I, 10 ml. of the buffer solution and then 0.5 ml. of reagent II. The mixture is made up to the calibration mark with the buffer solution and shaken vigorously.
After 5 minutes the absorption of the red colouring at 510 nm. is measured spectrophotometrically against a control containing no extract. The colour does not lose intensity within 30 minutes. Evaluation is made by reference to a standard curve drawn from determinations using the standard 2-hydroxybiphenyl solution under the same conditions.
6. For each analysis it is recommended that the spectrophotometric determination be made with two different volumes of the neutralised alkaline extract.
Untreated citrus fruit give by this method a “blank” reading of up to 0.5 mg. per Kg. for oranges and 0.8 mg. per Kg. for lemons.