Analysis of Cellular I'atty Acids of Bacteria by Gas-Liquid Chromatography 



5. Remove tubes from the water bath and cool to room temperature prior to extraction. 

 For additional information regarding other esterification procedures see reference 4. 



Extraction of Methyl Esters 



1. Add 10 ml of a 1:1 mixture of ethyl ether:hexane to the tube and tighten cap. Mix well, 

 i.e., invert the tube 10-20 times and shake firmly for about 10 sec. 



2. Allow the phases or layers to separate. Transfer the ether:hexane phase (top layer) to a 

 50- or 100-ml beaker. CAUTION: Avoid transferring aqueous phase along with the ether: 

 hexane phase. 



3. Add another 10 ml of ether:hexane to the aqueous phase (bottom layer) and repeat the 

 extraction procedure of Step C-1. 



4. Allow the phases to separate, and combine the ether:hexane layer with the first fraction 

 already in the beaker. 



5. Place the beaker under a gentle stream of tlowing nitrogen gas in a chemical hood, and 

 evaporate to reduce the combined 20 ml of solvent to approximately 0.5 ml. CAUTION: 

 Never allow the sample to go to complete dryness during evaporation of solvent. 



6. Add a small amount (80-100 mg) of Na2S04 to the beaker to remove residual moisture, 

 and then transfer the sample to a 13- x 100-mm screw-capped tube. Rinse the beaker with a 

 small volume of hexane, and then add the rin.se to the sample tube. 



7. Reduce the final volume (sample plus solvent) in the test tube to approximately 0.1 ml 

 under a gentle tlow of nitrogen gas in the hood. The methyl ester sample is then analyzed by 

 GLC or is stored at- 20° C. 



GAS-LIQUID CHROMATOGRAPHY 



A. Equipment 



The gas chromatographs used in this laboratory for cellular fatty acid analysis are equipped 

 with tlame-ionization detectors (FID) and temperature programs. Glass rather than metal col- 

 umns is used to avoid possible destruction of hydroxy acids (2). A non-polar stationary-phase 

 material such as the methyl silicones (SE-30, OV-1, OV-101) is used as the principal analytical 

 column. These materials have excellent separating efficiency, temperature stability, and low 

 column bleed, and are readily available from several commercial sources. A 3% concentration of 

 one of these phases coated on an inert support (i.e., 1 00- 1 20 mesh Gas-Chrom 0) is packed into a 

 0. 1 6 in (4.06 mm I.D. ) x 12 ft (3.66 m) glass column. The column is placed into the instrument 

 without connecting it to the detector and "conditioned" overniglit at 290"C with a low carrier 

 gas flow (20 cc/min) passing through the column. After conditioning, the column is connected to 

 the detector, checked for leaks, and temperature programmed from IOO°-270°C several times 

 prior to sample analysis. The carrier gas is adjusted to about 60 cc/min, and the instrument is set 

 as follows: initial temperature, I60°C; final temperature, 270°C; temperature program rate, 

 5°C/min. Under these conditions, excellent separation of a fatty acid methyl ester mixture 

 containing up to 23 components can be accomplished within 30 minutes. 



B. Column Control 



A measure of the efficiency of column separation on non-polar phases is base line or near 

 base line resolution of the methyl esters of palmitoleic ( 16: 1) and palmitic (16:0) acids. Appro- 

 priate adjustments of carrier gas tlow, initial column temperature, and temperature program rates 



