Short communication: effect of number of extractions on percentage of long-chain fatty acids from fresh alfalfa.

Accurate determination of fatty acids in fresh forage is very important when studying biohydrogenation. Fatty acids from fresh alfalfa were extracted by hexane:isopropanol (H:IP, 3:2 vol/vol) in 3 sequential extractions. The percentage and profile of fatty acids from each of the 3 extractions were evaluated by a randomized complete block design with repeated measures in space. Samples of fresh alfalfa were randomly harvested and immediately submerged in liquid nitrogen. For the first extraction, approximately 5 g of the frozen alfalfa was mixed with 18 mL of H:IP per gram of material. Samples were then centrifuged and the supernatant was collected. The second and third extractions were done by adding H:IP to the pellet (3 mL/g of the original sample weight), mixing for 2 min, and then centrifuging. Samples were submerged in H:IP and stored in the dark at 8 degrees C at all times. The solvent from each extraction was partially evaporated and the fatty acids methylated by methanolic HCl. Repeated extractions increased the percentage of total fatty acids recovered from the samples. The concentration of fatty acids in the alfalfa after 3 extractions was 4.0%. The first, second, and third extractions resulted in 92.7, 4.8, and 2.6% of the total fatty acids extracted, respectively. There was no effect of extraction on the proportion of 16:0, 18:0, 18:1, and 18:2 fatty acids. However, the proportion of 18:3 in the extract decreased from the first to the second extraction and the ratio of saturated to unsaturated fatty acid increased from the first to the second extraction. The results of this experiment revealed that the profile of fatty acids can vary with the number of extractions performed. The higher amount of 18:3 in the first extraction may reflect the higher proportion of linolenic acid in the more easily extracted plant fractions.