Hajime Seino

Enzymatic synthesis of carbohydrate esters of fatty acid (I) esterification of sucrose, glucose, fructose and sorbitol

The authors attempted to synthesize carbohydrate esters of fatty acids enzymatically in order to overcome the problems associated with the chemical processes for the synthesis of commercial sucrose esters. The enzymes used were lipases from microorganisms belonging toRhyzopus, Enterbacterium, Aspergillus, Pseudomonas, Chromobacterium, Candida, Mucor andPenicillium. Fatty acids (stearic, oleic and linoleic) and carbohydrates (sucrose, glucose, fructose and sorbitol) used for the reaction were obtained from commercial sources. The enzyme reaction was performed by mixing the enzyme and the substrates in the buffer solution and incubating at 40 C; after freeze-drying the mixture, the products were extracted and subjected to thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). It was observed by TLC and HPLC that carbohydrate esters of fatty acid were produced by the enzyme reaction, and their structures were confirmed by infra red (IR) and nuclear magnetic resonance (NMR) spectrometries. The lipase fromCandida cylindracea was the most enzyme active on the synthesis of carbohydrate esters. The optimum conditions for its activity were as follows: molar ratio of carbohydrate to fatty acid: 0.05mol/l : 0.2mol/l; amount of lipase: 4g/l; pH of the reaction mixture: 5.4 in phosphate buffer; reaction period: 72 hr.

Influences of operating conditions on determination of fatty acid methyl esters by gas chromatography.

The compositions of standard mixtures of methyl laurate, myristate, plamitate and stearate were determined by gas liquid chromatography, varying the operating conditions such as sample size, column temperature and flow rate of the carrier gas. The influences of these operating conditions on analytical values were examined. The sample size had a significant influence on the analytical values. The degree of influence was dependent on the column temperature and the composition of the sample. It was also observed that the flow rate of the carrier gas affected the analytical values.

Determination of fatty acid composition by gas chromatography: I. Analysis with use of thermal conductivity detector

For the purpose of establishing a standard method for the gas chromatographic determination of fatty acid composition, a collaborative study team has carried out replicate analyses of specified samples using gas chromatographs equipped with thermal conductivity detectors and has examined the entire set of experimental data by a statistical method. From the results of the four collaborative works it was found that deviation of analytical values from exact composition and interlaboratory scattering of data may be considerably decreased by the following means: (a) enlarging the size of narrow peaks (less than 5 mm at a half height) or peaks with low height by adjusting the attenuator range or chart speed; (b) correcting the analytical values by using correction factors determined from analysis of known mixtures having composition similar to that of an unknown sample.

Determination of fatty acid composition by gas chromatography: II. Analysis with use of flame ionization detector

In this study known mixtures of four or five fatty acid methyl esters were analyzed collaboratively by gas chromatography with flame ionization detectors. The experimental data was treated statistically to examine inter- and intralaboratory scattering. More-over the effect of the use of correction factors was investigated. Even if only the specific analytical values that scattered a little were chosen, the averages of such values did not always approach the actual values. In some laboratories a sort of regularity was observed in the deviation of analytical values from real values throughout the analyses of four samples. The application of correction factors to the analytical values obtained by these laboratories resulted in a considerable decrease of interlaboratory scattering and deviation from the real values. When a constant amount of sample was injected, intralaboratory scattering was decreased, whereas interlaboratory scattering was not. Injection of large sample sizes caused deviation. From this collaborative study it was recommended that 0.5–1.0 μl of 20% solution be injected.