Gas Chromatography - Applications > Free Fatty Acids from Milk > Page 65
The separation of the gasoline components is shown in figure 41. The stationary phase used was Petrocol (the trade name for a special poly(dimethysiloxane)) that is actually intra-column polymerized and thus bonded to the surface and, as a result, is very thermally stable. The alkane chains in the polymer contribute strong dispersive properties to the stationary phase. The necessary high efficiency was obtained by using a column, 100 m long, 250 mm I.D. carrying a film of stationary phase 0.5 mm thick. The column was held at 35oC after in injection for 15 min. and then programmed to 200oC at 2oC/min and finally held at 200oC for 5 min. To ensure that there was no condensation in the detector, the FID was held at 250 oC (50oC) above the maximum column temperature . The sample size was 0.1 ml which was split 100-1 onto the column and so the total charge on the column was about 1 mg. Helium was used as the carrier gas at a linear velocity of 20 cm/sec. The value of the open tubular column is clearly demonstrated.
Free Fatty Acids from Milk
An example of the use of the packed column in natural product analysis is the separation and determination of the free fatty acids in whole milk. An example of such an analysis is shown in figure 42. This analysis requires a rather lengthy procedure for sample preparation but, at the same time, avoids a derivatization procedure that can easily give incorrect, low values for the fatty acid content. Due to their relatively high volatility, the lower fatty acids can be lost as vapor during the procedure. Losses can also occur as a result of their incomplete derivatization. The sample preparation developed by Supelco involved mixing 10 ml of the milk with 10 ml ethanol, 3 ml of 28% ammonium hydroxide, 25 ml of petroleum ether and 25 ml of diethyl ether. The mixture is then well shaken and allowed to stand for about 20 minutes. The ether phase is separated and carefully evaporated to dryness under a stream of nitrogen. The residue is treated with 3 ml of 0.5n NaOH in methanol and heated on a steam bath for 15 minutes. 5 ml of water is added and then 2N HCl until a pH of about 2.0 is reached. The fatty acids are then extracted with a mixture of 5 ml of petroleum ether and 5 ml of diethyl ether. If a quantitative estimation is required, then an internal standard would be added and the solution diluted to a known volume and an aliquot placed on the column.