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Extra info for Methods of Biochemical Analysis, Volume 8
3) with Apiezon L at 270°C. (Fig. 8. 2- c24 '26 f 0 10 20 30 I 40 I 50 I 1 I 1 60 70 80 90 Time in minutes 100 110 120 Fig. 17. Separation of long chain fatty acid esters. ; pressure, 42 cm. Hg above atmospheric; gas flow, 29 ml. ; filling, 4 g. ; amount introduced, approx. 2 mg. Detection by COZ after combustion. The lower components, which eluted before CMmethyl ester, originate from the impure triacontanoic acid. The best conditions found to date for separations of the various ranges of fatty acids are summarized in Table I1 (page 7).
Portions of ether; the ether solution is then dried over anhydrous sodium sulfate. The ether solution is evaporated to small volume in a conical centrifuge tube, and excess freshly distilled diazomethane (Ardnt (2)) in ether is added. After standing for 30 minutes excess diazomethane and ether are evaporated on the water bath (in a fume hood), and the residue is dissolved in a few pl, of methyl tri- or pentadecanoate, taken up in a micropipet, and applied to the gas chromatogram. In Figure 21 is s h a m a separation of methyl esters of mono- and dicarboxylic acids using a column of Apiezon M (or L) grease a t 197" with a low flow rate of gas.
There can be little doubt that with the use of integrators the accuracy of determination of any single acid could be improved from the present 3-4% error to less than 1%. B. CALCULATION OF PERCENTAGE COMPOSITION When it is desired to know the absolute amounts of acids in the isolated mixture, the load placed on the column must be known ac- FATTY ACIDS BY GAS-LIQUID CHROMATOGRAPHY 41 curately so that the total peak area obtained can be related to the area expected for a given mass of material. , Ce-C22(so that the actual load is unimportant), and to calculate the percentage of any component as 100 X corrected peak area of component + total corrected area The correction factors will vary from detector to detector and should be determined to an accuracy at least equal to that of rea measurement.