Internal standard solution– Dissolve an accurately weighed quantity of nonadecane in hexanes to obtain a solution having a known concentration of about 12 mg per mL.
Standard solution– Dissolve accurately weighed quantities of Methyl Laurate RS, Methyl Oleate RS, Methyl Myristate RS, Methyl Palmitate RS, Methyl Linoleate RS, Methyl Caproate RS, Methyl Caprylate RS, Methyl Caprate RS, Methyl Palmitoleate RS, Methyl Stearate RS, and Methyl Linolenate RS in hexanes to obtain a solution having a known concentration of each methyl ester as given below. Transfer 1.0 mL of Internal standard solution to 5.0 mL of this solution, and mix.
Chromatographic system– The gas chromatograph is equipped with a flame-ionization detector maintained at 300º and a 0.25-mm×30-m fused silica capillary column coated with a 0. 25-μm film of phase G16. The injection port is maintained at 250º.The column temperature is initially held ai 120º for 3 minutes, then programmed to rise to 220º at the rate of 50º per minute, where it is maintained for an additional 12 minutes. The carrier gas is helium flowing at a rate of about 1 mL per minute. Chromatograph the Standard solution, and record the responses as directed under Procedure: the relative retention times are about 0.39 for methyl caproate, 0.56 for methyl caprylate, 0.76 for methyl caprate, 0.94 for methyl laurate, 1.0 for nonadecane (Internal standard),1.1 for methyl myristate, 1.3 for methyl palmitate, 1.35 for methyl palmitoleate, 1.65 for methyl stearate, 1.7 for methyl oleate, 1.8 for methyl linoleate, and 2.0 for methyl linolenate; the resolution, R, between the methyl stearate and methyl oleate peaks is not less than 1.5; the tailing factor for each of the methyl ester peaks in the Standard solution is not greater than 2.0, and the relative standard deviation for replicate injections for each of the methyl ester peaks is not more than 5.0%.
Test solution– Transfer about 100 mg of Extact, accurately weighed, to a pressure-proof, screw-capped vial, and add 3.0 mL of a solution of sulfuric acid in methanol (5 in 100). Heat at 100º in an oil bath for 2 hours, shaking from time to time. Allow to cool, and add 1.0 mL of Internal standard solution, 10.0 mL of water, 1g of sodium chloride, and 5 mL of hexanes. Shake well, allow the layers to separate completely, and use the hexanes layer.[NOTE- Store in a refrigerator until ready to use.]
Procedure– Calculate the percentage of each fatty acid in the portion of Extract taken by the formula:
in which W is the weight, in mg, of Extract taken to prepare the Test solution; and the other terms are as denfined there in.