ISO 6867:2000 pdf download

ISO 6867:2000 pdf download.Animal feeding stuffs – Determination ofvitamin E content- Method using high-performance liquid chromatography.
When the layers have separated, remove the stopper, wash the sides of the stopper with a few millilitres of light petroleum (4.5) and insert the adjustable tube (see 5.4), positioning the lower open end so that it is just above the level of the interface.
By application of a slight pressure of inert gas (4.10) to the side arm tube, transfer the upper, light petroleum layer to a 1 litre separating funnel (see 5.4).
Add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 mm.
Allow the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube (see 5.4) as before.
Again, add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 mm.
Again, allow the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube as before.
Wash the combined light petroleum extracts with four 100 ml portions of water using at first only gentle inversion then only gentle shaking in order to keep emulsion formation to a minimum.
Transfer the washed extract through a medium/fast filter paper containing 30 g of anhydrous sodium sulfate (4.8) into a 1 litre flask suitable for vacuum evaporation (5.3).
Rinse the separating funnel with two 20 ml portions of light petroleum (4.5) and add the rinsings through the filter to the evaporation flask.
Wash the filter further with two 25 ml portions of light petroleum (4.5) and collect the washings in the evaporation flask.
Evaporate the light petroleum extract to dryness under vacuum at a temperature not exceeding 40 DC.
Care should be taken to ensure that the flask is removed from the rotary evaporator immediately after reaching the point of dryness; prolonged drying may lead to loss of vitamin E from the extract residue.
If the vitamin E concentration of the light petroleum extract is sufficiently high, the extract may be made up to a fixed volume with light petroleum and an aliquot part taken for the rotary evaporation stage.
Restore atmospheric pressure by admitting inert gas (4.10).
8.4 Determination
8.4.1 Dissolve the residue from 8.3 in a minimum volume of hexane (4.4) and transfer quantitatively to a 25 ml volumetric flask.
Rinse the evaporation flask with three small portions of hexane (4.4), transferring the rinsings to the volumetric flask. Dilute to volume with hexane and mix.
If necessary, filter the sample extract through a membrane filter (5.5) or centrifuge.
8.4.2 Inject 20 p1 of the sample extract onto the column of the liquid chromatograph (5.1) and measure the area of the DL-a-tocopherol peak. The following HPLC conditions are offered for guidance; other conditions may be used provided that they give equivalent results:
liquid chromatographic column (5.1.3): 250 mm x 4,6 mm, silica 5 pm or 10 pm packing, or equivalent;
— mobile phase (4.11): mixture of hexane (4.4) and 1 ,4-dioxan (4.7), 970:30 (by volume); flow rate: 1,5m1/min;
detector (5.1.4): fluorescence detector (excitation 295 nm, emission 330 nm).
Reverse-phase chromatography may also be used provided that the efficiency of the column is sufficient to allow the separation of DL-a-tocopherol from other tocopherols and sample co-extractives. If reverse-phase chromatography is used, sample and standard solutions should be made up in an appropriate solvent, for example methanol (4.13).
8.4.3 Calculate the mean peak area from replicate injections of the sample extract and determine the DL-a-tocopherol concentration in micrograms per millilitre of the extract, either
a) by reference to the mean peak areas obtained from replicate injections of DL-ct-tocopherol standard solution of concentration within 5 % of the concentration in the sample extract, or
b) by reference to a calibration curve prepared as in 8.5.
8.5 Calibration
8.5.1 Preparation of DL-ct-tocopherol standard solutions
8.5.1.1 DL-a-tocopherol stock standard solution
Dissolve approximately 100 mg, weighed to the nearest 0,1 mg, of DL-a-tocopherol (4.6) in 100 ml of hexane (4.4).
The stock standard solution is stable for 1 week when stored at 4 °C in an airtight amber glass flask.
8.5.1.2 DL-a-tocopherol working standard solution: single-point calibration
Prepare a working standard by diluting the stock standard (8.5.1.1) with hexane (4.4) to give a concentration approximately equal to that expected in the sample extract. Alternatively, proceed as in 8.5.1.3.
8.5.1.3 DL-a-tocopherol working standard solution: multi-point calibration
Prepare a range of calibration working standards containing 2 pg/mI, 4 pg/mI, 6 pg/mI, 8 pg/mI and 10 pg/mI of
DL-a-tocopherol by diluting the stock standard solution (8.5.1 .1) with hexane (4.4).
Prepare working standards daily.
8.5.2 UV check of DL-cx-tocopherol standard substance
Weigh, to the nearest 0,1 mg, 100 mg of DL-cx-tocopherol (4.6) in a 100 ml volumetric flask. Dissolve in ethanol (4.12). Dilute to the mark with the same solvent and mix.
Dilute 2,0 ml of this solution to 25,0 ml with ethanol (4.12 ) and measure the UV spectrum of the resulting solution against ethanol (4.12) in the spectrometer (5.7) at wavelengths of between 250 nm and 320 nm. The absorption maximum should be at 292 nm.