ISO 5509:2000 pdf download

ISO 5509:2000 pdf download.Animal and vegetable fats and oils —Preparation of methyl esters of fatty acids.
Boil under reflux until the droplets of fat disappear, swirling the flask gently every 30 s to 1 mm to prevent a solid ring of sodium hydroxide forming around the walls of the flask. This usually takes 5 mm to 10 mm, but in certain exceptional cases it may take longer. See A.3 and A.4. Add the appropriate amount (see Table 1) of the methanolic boron trifluoride solution (3.3.3) through the top of the condenser.
Proceed in accordance with either 3.6.3 or 3.6.4.
3.6.2.3 Introduce the test portion into the appropriate flask (see Table 1). Add the appropriate amount (see Table 1) of the methanolic boron trifluoride solution (3.3.3) into the flask. Fit the condenser (3.4.2) to the flask.
Proceed in accordance with either 3.6.3 or 3.6.4.
3.6.3 Preparation of the methyl esters in isooctane solution (mainly for gas liquid chromatography purposes)
3.6.3.1 Continue boiling for 3 mm. In the case of oils with long-chain fatty acids, such as fish oils, continue boiling for 30 mm.
3.6.3.2 Add the appropriate amount (see Table 1) of isooctane (3.3.4) to the boiling mixture through the top of the condenser.
3.6.3.3 Remove the flask from the heat source and remove the reflux condenser. IMMEDIATELY, without allowing the flask to cool, add 20 ml of sodium chloride solution (3.3.5). Stopper the flask and shake it vigorously for at least 15 s.
3.6.3.4 Add more of the saturated sodium chloride solution (3.3.5) to bring the liquid level of the mixture into the neck of the flask. Allow the two phases to separate.
3.6.3.5 Transfer 1 ml to 2 ml of the upper isooctane layer into a 4 ml vial (3.4.5) and add a small amount of anhydrous sodium sulfate (3.3.6) to remove any traces of water.
The isooctane solution thus obtained may be injected as follows:
a) directly onto a packed column for gas-liquid chromatography (see A.5);
b) after appropriate dilution with isooctane for capillary column systems prior to the injection (see A.6); c) after dilution with a lower boiling solvent such as heptane for the special case of capillary on-column injection.
3.6.4 Preparation of dry methyl esters (for TLC or IR spectroscopy)
3.6.4.1 Continue boiling for 3 mm.
3.6.4.2 Add the appropriate amount (see Table 1) of hexane (3.3.8) to the boiling mixture through the top of the condenser.
3.6.4.3 Remove the flask from the heat source and remove the reflux condenser. IMMEDIATELY, without allowing the flask to cool, add 20 ml sodium chloride solution (3.3.5). Stopper the flask and shake it vigorously for at least 15 s.
3.6.4.4 Transfer the saline solution and hexane layer to a 250 ml separation funnel (3.4.6). Add about 30 ml of the saturated sodium chloride solution. Allow the two phases to separate. Retain the hexane solution. Extract the saline solution twice with 50 ml portions of hexane (3.3.8).
3.6.4.5 Combine the hexane solution and the two extracts and wash them with 20 ml portions of water (3.3.1) until no free acid is obtained, using the methyl red solution (3.3.9) as indicator.
Dry over anhydrous sodium sulfate (3.3.6). Filter the solution and evaporate the solvent cautiously on a water bath under a stream of nitrogen (3.3.7) or use a rotary evaporator (3.4.7).
If the remaining portion contains a considerable amount of short-chain methyl esters (C6 to Cl 0), a substantial loss of these can hardly be avoided. For test portions less than 500 mg, it is preferable to reduce proportionally the volumes of sodium chloride solution, solvent and water. See A.6.
4 Trimethylsulfonium hydroxide (TMSH) method
WARNING — The method described involves the use of potentially hazardous reagents. Normal precautions shall be taken for eye protection and for protection from the dangers of corrosive chemical burns. Trimethylsulfonium hydroxide may be poisonous.
4.1 Principle
The test sample is dissolved in t-butyl methyl ether and the methyl esters are prepared by trans-esterification with trimethylsulfonium hydroxide (TMSH). Immediate injection in the gas chromatograph at an injector temperature of above 250 °C. In the presence of short-chain fatty acids (4 to 8 carbon atoms), the use of valeric acid methyl ester is recommended as an internal standard (see 4.2).
4.2 Applicability
This rapid method is only for the preparation of methyl esters for GLC. It is applicable to all fats and oils including milk fat and blends containing milk fat. Isomerization of unsaturated fatty acids has not been observed.
The method can be applied to compounds containing the chemical configurations listed in 3.2, but it is not known whether an entire conversion into methyl esters will take place. Also free fatty acids are only esterified by about 70 % to 80 %.
Lipids containing hydroxy groups are partially converted to the corresponding 0-methyl ether derivatives which may interfere with fatty acid methyl esters (FAME) in GLC separation. Therefore the TMSH derivatization method is not recommended without limitation for lipids containing hydroxy groups. On the other hand it may be of some diagnostic value for the analysis of such lipids by GLC/mass spectrometry.
The TMSH procedure cannot be applied when cold-on-column injection is used in GLC analysis. Moreover the use of polar stationary phases (cyanopropyl siloxanes) is not recommended.
For the determination of short-chain fatty acids (C4 to C8), the use of valeric acid methyl ester (methyl pentanoate) as an internal standard is recommended, provided there is no valeric acid in the sample.
4.3 Reagents
Use only reagents of recognized analytical grade.
4.3.1 t-Butyl methyl ether.
4.3.2 Trimethylsulfonium hydroxide (TMSH) 2), methanolic solution, 0,2 mol/l.
The solution remains stable for at least 2 months if stored at 4 °C in small quantities in a closed tube.
NOTE Reference [3] gives a method of preparation.
4.3.3 Internal standard stock solution, for butyric and/or caproic acid determination only.
Weigh, to the nearest 0,1 mg, about 250 mg of valeric acid methyl ester into a 50 ml volumetric flask. Use isooctane to dissolve the sample and to dilute to the mark.