Biological systems contain a wide range of fatty acids in cell membranes and stored fat. In particular, the cell membrane of microbial organisms consists of a bilayer of glycerophospholipids in which the fatty acids are esterified to glycerol (Carlsson 1973, p.288).
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Microbial fatty acids are unique from one species to another. Analysis and characterisation of these fatty acids from various microbes has been used to create a reference library. This makes it easy to compare experimental results with the data in the library. The fatty acid composition depends on the growth conditions of the microbe. Low temperatures result in greater amounts of unsaturated fatty acids while higher temperatures result in branched chain fatty acids (Ottenstein & Bartley 1971, p.681).
Other factors such as pH and salinity affect the amounts of other fatty acids, which ultimately provide a good basis for identification of the bacterium. Various techniques can be used quantitatively to separate and analyse fatty acids for microbe identification such gas chromatography, DNA sequencing, HPLC etc (Larsson, Sonesson & Imenez n.d,p.730).
Principle of gas chromatography
Gas chromatography equipment can be used to separate and analyse fatty acids that do not decompose on vaporisation. It consist of a column packed with a stationary phase and a mobile phase (a carrier gas) on which the analyte (fatty acids) are carried. Each component in the mixture has a unique retention time within the column. According to Truant (2002), “the polar stationary phase in the column separates the components of a mixture causing them to elute at different times i.e. retention time” (p.78). The components are detected electronically as they leave the column
The fatty acids are converted to methyl ester derivatives for GC separation. This is achieved by reacting dried bacterial samples with methanolic HCL in methanol at about 800C. The contents are then mixed with water followed by low speed centrifugation. The upper phase is then used for gas chromatography analysis (Rogosa & Love 1968, p.290).
Factors that contribute to sensitivity
The sensitivity of this method depends on both the nature of the compound and the detector used. Good results are obtained when the two are correctly matched. A flame ionisation detector is usually used since it is stable and has a wide dynamic range. However, flame ionisation detector has low sensitivity for very small amounts of the fatty acids found in bacteria. To overcome this problem, electron capture detector is used. The fatty acid is converted to a halogen-containing derivative which is readily detected by ECD even at very low concentrations (Gehrke & Lamkin 1961, p.86).
Factors that contribute to specificity
The fatty acids derivatives have specific retention time within the column. This allows separation of the mixture and consequently good resolution of the peaks on the chromatogram. The type of material used to coat the lining will directly determine what sort of compounds will be adsorbed on the static phase. Therefore, different columns are designed for specific compounds.
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Advantages of gas chromatography
Gas chromatography allows for rapid separation of volatile fatty acids regardless of their origin. The sample size required is usually small and the results are obtained within a short time interval. Gas chromatography is highly sensitive which allows separation and detection of samples in the range of microgram and sub-microgram. According to Gehrke & Lamkin (1961),”high sensitivity is achieved by the use of electron capture detector instead of flame ionization detector or thermal conductivity detector” (p.86).
Disadvantages of gas chromatography
Gas chromatography is not suitable for analysis of high molecular fatty acids and underivatized fatty acids. These polar compounds form hydrogen bonds/are adsorbed on the capillary walls of the stationary phase resulting to poorly resolved peaks. The adsorption may be irreversible thereby destroying the capillary permanently. The presence of other volatile compounds other than the fatty acids interferes with the peaks making quantitative analysis impossible.
List of References
Carlsson, J., 1973. Simplified gas chromatographic procedure for identification of bacterial metabolic products. Journal of Applied Microbiology, 25, pp.287-289.
Gehrke, C., & Lamkin, W., 1961. Quantitative determination of steam-volatile fatty acids by gas-liquid chromatography. Journal of Agriculture and Food Chemistry, 9(2), pp.85-88.
Larsson, L., Sonesson, A., & Imenez, J., n.d. Ultrasensitive analysis of microbial fatty acids using gas chromatography with electron capture detection. European Journal of Clinical Microbiology & Infectious Diseases, 6(6), pp.729-731.
Ottenstein, D., & Bartley, D., 1971. Separation of free acids C2-C5 in dilute aqueous solution column technology. Journal of Chromatogram Science, 9(1), pp. 674-681.
Rogosa, M., & Love, L., 1968. Direct quantitative gas chromatographic separation of C2-C6 fatty acids, methanol, and ethyl alcohol in aqueous microbial fermentation media. Journal of Applied Microbiology, 16(2), pp.285-290.
Truant, A., 2002. Manual of commercial methods in clinical microbiology. New York: Prentice Hall.