Analysis of Cellular Fatty Acids of Bacteria bv Gas-Liquid Chromatography 



should be made to insure satisfactory separation of tiiese two compounds. If tliese two compo- 

 nents are resolved on a non-polar phase material, other esters which are present in a bacterial 

 fatty acid sample will, in general, also be well resolved. 



C. Procedure 



For analysis, 1-3 /il of the bacterial fatty acid methyl ester sample is injected into the 

 column, and a tracing (chromatogram) of the separated components of the mixture is recorded 

 on a strip chart. The retention time (time from injection to the center of the eluted peak) of each 

 peak is recorded and compared to retention times of those in a reference standard. The presence 

 of specific fatty acids and their relative concentration compared to those of other acids in the 

 sample are the bases for ditTerentiation {2). 



IDENTIFICATION 



A significant amount of information on the identity of the fatty acids in tlie bacterial 

 sample can be obtained by comparing GLC retention data to highly purified reference standards 

 on both non-polar (i.e.. OV-101 ) and polar [i.e.. ethylene-glycol-adipate (EGA)] stationary-phase 

 columns. On non-polar phases, fatty acid methyl esters are separated by boiling points. Thus, 

 unsaturated acids elute before their saturated homologs, saturated branched-chain acids before 

 their saturated straight-chain homologs, cyclopropare acids before their straight-chain homologs, 

 and 2-hydroxy acids before their 3-hydroxy homologs. On polar columns such as EGA, the 

 elution sequence is reversed for saturated acids and their unsaturated homologs. Iso- and anteiso- 

 isomers of saturated branched-chain acids also separate to some degree on polar columns (S). 

 Thus identical retention time matches on both polar and non-polar phase materials give strong 

 preliminary identification. In addition, the presence of unsaturated acids can be confirmed by 

 bromination or hydrogenation. Treatment of the methyl ester sample with bromine results in the 

 addition of this compound across the double bonds to produce a much less volatile species 

 (bromo-methyl ester), which does not appear in the chromatogram under the initial conditions of 

 GLC upon subsequent GLC analysis. Similarly, hydrogenation converts unsaturated esters to 

 their corresponding saturated esters, which causes the GLC peaks representing esters of unsatu- 

 rated acids to disappear and causes the peaks representing saturated acid esters to increase in size. 

 The presence of a hydroxy acid can be confinned by treating the methyl ester sample with an 

 anhydride (e.g., acetic, trifluoroacetic) to form the diester derivative. Upon subsequent analysis 

 by GLC, the peak representing the methyl ester derivative will disappear from the chromatogram. 

 and a new peak representing the diester derivative will appear. The diester will elute from the 

 column more quickly (shorter retention time) as a result of its increased volatility. 



A. Hydrogenation of Methyl Esters 



1. After analysis by GLC, reduce the methyl ester sample almost to dryness under nitro- 

 gen. 



2. Add approximately 0.5 ml of a 3:1 mixture of chloroform;methanol down the side of 

 the tube and mix well. 



3. Transfer the sample to a 10-ml vacuum hydrolysis tube (Kontes Glass Company). 



4. Add a small amount (5 mg) of catalyst (5% platinum on charcoal) and a small, Tetlon- 

 coated stirring bar. 



5. Connect the tube to a ring stand and suspend the tube over a magnetic stirrer. Close the 

 upper part of the tube and mix the contents by stirring. 



120 



