Identification and quantification of fatty acids

< To understand the quality and composition of fat, identification and quantification of fatty acids present are important. Though fatty acid esters could be identified by TLC, quantification is best done with gas-liquid chromatography (GLC)



Principle
Fatty acids are made volatile by converting them into methyl esters. The conversion of fatty acids in methyl esters is carried out directly by trans esterification i.e., from glycol ester to methyl ester. Esterification can also be  done after saponification. The esters are identified and quantified by injecting into GLC and comparing with a set of standard esters.

Materials

» 10%BCl₃ or 14% BF₃ in methanol
» Saturated NaCl Solution
» Na₂SO₄ (anhydrous)
» Hexane
» Nitrogen Gas
» GLC
Column                             -                   Pretested 10% silar 10^oC on gas chrom Q 100 to 200 mesh (6’ x ¼” – internal diameter 4mm)
Detector                            -                   FID
Injector temp                    -                   280^oC
Detector temp                  -                   250^oC
Carrier gas                       -                   Nitrogen at 50mL/min
Column/oven temp         -                   165^oC    

Procedure

1.	To 150 to 300mg of oil, in a culture tube add 3mL of 10% BCl3 or 14% BF3.
2.	Add boiling chips, cover the tube with aluminum foil and heat at 83oC for 6min.
3.	Transfer the contents to a 30mL separating funnel and also collect the washing of the tube into the separating funnel. Washing is done four times with 1mL portions of hexane.
4.	Shake and allow to separate.
5.	Add 4mL saturated NaCl solution.
6.	Shake and collect hexane layer over anhydrous Na2SO4.
7.	Rinse funnel with 2mL hexane, collect the hexane layer and combine the hexane extracts.
8.	Filter the hexane extract through filter paper (Whatman No. 4)
9.	Reduce the volume of the filtrate to 2-3mL by drying with a slow stream of nitrogen.
10.	Inject an aliquot to pre-conditioned GLC.
11.	Inject standard methyl esters separately and calculate the retention time for individual esters.

Calculation

Identify peaks by relative position on the chart. Esters appear in order of increasing number of carbon atoms and of increasing unsaturation for the same number of carbon atoms. Calculate the peak area of each acid in the sample and compare with the standards. From the quantity injected and the attenuation, the amount of fatty acid content can be calculated. The latest computerized GLC models give concentration directly.



Notes

1.	If free fatty acids alone are to be determined, incorporate methyl urea (520-540mg) in the tube to prevent transesterification of fatty acids of glycerides. This will ensure reduction of trans-esterification to 0.02-0.05%.
2.	An alternative method for trans-esterification of fat is given below: Take 5-50mg of lipid sample in 15 x 45mm culture tube with a cap (Evaporate the solvent if the sample is in the solvent). Add 0.25/0.5mL of 0.4N sodium methoxide in anhydrous methanol and cover with a cap. Immerse the capped tube to a depth of 12mm for 30sec in a water bath at 65°C. Esterification is considered complete when the mixture of lipid and reagent becomes homogeneous. Continue heating for another 90sec. Remove the tube and cool to room temperature. Quickly add to this, 1g mixture of silica gel G and anhydrous CaCl2 (1:1 w/w). Mix the contents thoroughly with a glass rod. Add 5mL of hexane/carbon disulphide and shake for 1-2min. centrifuge for a short time. Withdraw the clear solvent layer. Wash with the solvent to have a good recovery. Evaporate the solvent at low heat to 0.5mL final volume. This material now contains the methyl esters of fatty acids that can be injected into a GLC for identification and quantification.
3.	Instead of trans-esterification, saponification followed by esterification can also be done as follows. Saponify 1g fat with 25mL of 4% methanolic KOH for 30min (Reflux with an air condenser over a boiling water bath). After saponification, remove methanol in a flash evaporator below 50°C. Dissolve the residue in sufficient water. Transfer to a separating funnel. Add petroleum ether (60-80°C), shake, and remove the petroleum ether layer to get rid of unsaponifiable matter. Hydrolyze the saponified material with HCl and extract the free fatty acid with petroleum ether. Repeat the extraction with petroleum ether four times. Pool the ether extracts and dry over anhydrous Na2SO4. Evaporate to dryness under vacuum at 50°C. Add 20-fold methanol –HCL and reflux for 2h over a water bath. Remove excess methanol by rotary evaporation. Add petroleum ether and transfer to a separating funnel. Shake with 0.5N sodium carbonate and remove the aqueous layer for washing. The resultant petroleum ether extract contains the methyl esters of fatty acids which are then injected into a GLC for quantification.
4.	Any unesterified liquid component will spoil the column in most cases and hence the esterification must be checked before injecting into GLC. The check is done by carrying out the separation by TLC (5 x 20cm glass plates) using silica gel G in a solvent system of petroleum ether – diethyl ether – acetic acid (80:20:1 v/v). the prominent spot for esters and the absence of spots for triglycerides confirm the completion of esterification.

References
1. Metcalfe, L D, Schmitz, A A and Pelke, J R (1966) Anal Chem 38 514.
2. Chapman Jr., G W (1979) JAOCS 56 77.
3. Munshi, S K (1987) In: Manual in Modern Analytical Technique in Agricultural Biochemistry (Ed Sudarshan Singh) Punjab Agricultural University Ludhiana p 266.