ISO 5509 pdf download – Animal and vegetable fats and oils — Preparation of methyl esters of fatty acids

ISO 5509 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 tO 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 trifluonde 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 I). Add the appropnate amount (see Table 1) 01 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 fatly acIds, such as fish Oils, continue boiling for 30 mm.
3.6.3.2 Add the appropriate amount (see Table I) 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 allovnnq 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 13.3.5) to bring the l.iid level of the mixture Into the neck of the flask. Allow the two phases to separate.
3.6.35 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) dwectly 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)
3.6.4i Continue boiling for 3 mm.
3.642 Add the appropriate amount see Table 1) of hexarie (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 15s.
3.6.4.4 Transter the saline solution and hexane layer to a 250 ml sepafahon 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 oh water (3.3.1) until no free acid Is obtained, using the methyl red solutIon (3.3.9) as indicator.
Dry over anhlydrous sooium 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 (06 to ClO). 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.
4.2Applicability
This rapid method is only for the preparation of methyl esters for GLC. it is applicable to all fats and oils includingmilk 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 knownwhether an entire conversion into methyl esters will take place.Also free fatty acids are only esterified by about70% to 80 %.
Lipids containing hydroxy groups are partially converted to the corresponding O-methyl ether derivatives which mayinterfere with fatty acid methyl esters (FAME) in GLC separation. Therefore the TMSH derivatization method is notrecommended without limitation for lipids containing hydroxy groups. On the other hand it may be of somediagnostic value for the analysis of such lipids by GLCmass spectrometry.