Computer treatment of gas-liquid chromatographic data, with special reference to fatty acid methyl esters

A computer program is described which analyzes output punched directly onto paper tape from a gas-liquid chromatograph. Although this program was written specifically for samples of fatty acid methyl esters derived from adipose tissue triglycerides which are eluted within 1 hr, modification of the dimension statements in the program would enable it to deal with samples which require a longer time to come off the column. The salient features of the rationale of the program are discussed in detail, particularly the procedures for base line correction and for estimating the contributions from components which are not perfectly separated in the column. Examples are given of the program in practice, of comparing the results it gives with those obtained by manual triangulation of the areas on a recorder chart, and of indicating the range of column load over which we have found that it operates satisfactorily. A sample computer print-out from the program is presented and interpreted.     

Quantitative gas-liquid chromatographic analysis of rodent milk triglycerides

A comparison has been made of the milk and adipose tissue triglycerides of rabbits and guinea pigs provided with one diet and of rats and mice provided with another. Both intact triglycerides and component fatty acids were analyzed by gas-liquid chromatography. Good correlation of the data obtained by the two techniques was obtained by calculating the average chain length of the fatty acid moieties. Little difference was found in the triglyceride composition of the adipose tissue of the different species. However, wide variation in the triglyceride composition of the milk was found between the species: the average fatty acid chain length in milk was 11.7 for rabbits, 14.2 for rats, 15.3 for mice, and 17.2 for guinea pigs. The corresponding values for adipose tissue were in the range 16.9-17.4 in all animals. The significance of enzymes that synthesize short-chain fatty acids in mammary gland is discussed.     

Preparation of fatty acid methyl esters for gas-liquid chromatography

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

One-step rapid extractive methylation of plasma nonesterified fatty acids for gas chromatographic analysis

The present report describes a one-step method for the derivatization and extraction of nonesterified fatty acids in plasma with subsequent analysis by conventional capillary gas-liquid chromatography or gas-liquid chromatography-mass spectrometry. The procedure requires 200 microliters of citrated plasma, dilution with 200 microliters of methanol containing a suitable internal standard, and rapid methylation (10 min) with ethereal diazomethane. An aliquot (60%) of the ether layer is subsequently removed, taken to dryness with nitrogen gas, and the residue is dissolved in a small volume of hexane (usually 50 microliters) for chromatographic analysis (taking 1 microliter for on-column injection). Samples are ready for analysis within 15 min after initial preparation of the plasma. The method has been found to be simple and rapid, providing clean fatty acid profiles. Although the method has been tested with 200 microliters of rat and human plasma, it can easily be adapted to a 40 microliters plasma sample if the esterified plasma extract is suspended in a smaller volume of hexane and/or a larger aliquot of the extract were to be injected into the gas chromatograph through use of a splitless injector.     

Gas chromatographic analysis of free fatty acids. Part 2: Determination of free fatty acids during anaerobic putrefaction

By using tailor-made capillary columns the changes of the free fatty acid spectrum during anaerobic putrefaction of piggery waste and sludge at pH 5.0–5.5 were examined. Free fatty acids with 18 to 22 carbon atoms were the main components before putrefaction. With progressive fermentation time the share of short chain acids formed from higher acids was increased.     

A gas-liquid chromatographic method for steric analysis of epoxy acids

A gas-liquid chromatographic method for determination of the absolute configuration of the two chiral carbon atoms of epoxy fatty acids was developed. The method involved (1) conversion of the saturated epoxy ester into a pair of regioisomeric allylic alcohols by consecutive treatments with selenophenoxide anion and hydrogen peroxide, (2) oxidative ozonolysis performed on the (-)-menthoxycarbonyl derivatives of the allylic alcohols, and (3) steric analysis of the resulting two 2-hydroxy acids (methyl esters, (-)-menthoxycarbonyl derivatives) by gas-liquid chromatography using appropriate reference compounds. Application of the method for steric analysis of several synthetic epoxyoctadecanoates as well as (+)-vernolic acid derived from Vernonia galamensis is described.     

Essential fatty acids in tissue phospholipids and triglycerides of the zinc-deficient rat

This study addressed the possibility that zinc deficiency has different effects on the fatty acid composition of triglyceride compared to total phospholipid. Male weanling Sprague-Dawley rats were maintained for 6 weeks on a semisynthetic diet deficient in zinc (3 mg/kg zinc). Control rats (40 mg/kg zinc) were pair-fed. Lipid fractionation and fatty acid analysis were by thin-layer and gas chromatography, respectively. In zinc-deficient rats, the percentage of linoleic acid was increased or that of arachidonic acid was decreased in total phospholipids of plasma, liver, and testis, and in skin total lipids. Saturated and monounsaturated fatty acids were increased in the triglyceride of liver but decreased in the triglyceride of epididymal fat of zinc deficient rats. Essential fatty acids, as a proportion of total fatty acids, were decreased in triglyceride of liver but increased in triglyceride of epididymal fat of zinc-deficient rats. Our fatty acid data from tissue total phospholipids therefore support the concept that linoleic acid desaturation is impaired in zinc deficiency.     

Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters

The transient rise in plasma triacylglycerol fatty acids after single-dose ingestion of fish oil as triacylglycerols, free acids, or ethyl esters with linseed oil as an absorption standard was used to determine the relative absorption of fish oil fatty acids in eight men. As free acids, the fish oil fatty acids were well absorbed (greater than or equal to 95%). As triacylglycerols, eicosapentaenoic acid (1.00 g) and docosahexaenoic acid (0.67 g) were absorbed only 68% and 57% as well as the free acids. The ethyl esters were absorbed only 20% and 21% as well as the free acids. The incomplete absorption of eicosapentaenoic and docosahexaenoic acids from fish oil triacylglycerols correlates well with known in vitro pancreatic lipase activity.     

High-performance liquid chromatographic separation of fatty acids as pentafluorobenzyl esters

Using ultraviolet detection (254 nm), pentafluorobenzyl esters have been shown to be suitable derivatives for the semi-preparative separation of fatty acids by number of double bonds on silica columns and by chain length on reversed-phase columns. The two chromatographic systems are entirely complementary in that a critical pair in one of the two systems can be completely separated in the other system, thus allowing the isolation of any given fatty acid from a complex mixture following two sequential injections. The complete separation of pentafluorobenzyl cis-9,10-methylene-hexadecanoate and petafluorobenzyl heptadec-10-enoate in both systems has also been achieved.     

Effect of hypoglycemia on the brain free fatty acid level and the uptake of fatty acids by phospholipids

The effect of hypoglycemia on the uptake of [1-¹⁴C]arachidonate and [1-¹⁴C]oleate into a synaptosomal and microsomal glycerophospholipids was investigated. In the presence of ATP, Mg²⁺ and CoA, rat brain synaptosomes and micorsomes catalyze the transfer of arachidonate and oleatc into glycerophospholipids. Arachidonate was mainly incorporated into phosphatidylinositol (PI) and phosphatidylcholine (PC), whereas oleate was incorporated into phosphatidylcholine and phosphatidylethanolamine (PE).Hypoglycemia was produced by intraperitoneal injection of 10 or 100 units of crystalline insulin per kg body weight. Two hours after injection the blood glucose level decreased to 10–20 mg%. The content of brain phospholipids was slightly decreased but the change was not statistically significant. The level of free fatty acids (FFA) was increased. More pronounced and reproducible changes were found when hypoglycemia was produced by injection of 100 units of insulin per/kg body weight. Changes in brain cortex were similar to those observed in microsomes and synaptosomes. Hypoglycemia affected the incorporation of arachidonic acid into glycerophospholipids of brain membranes. Uptake of [1-¹⁴C]arachidonate was decreased selectively by 50% (into phosphatidic acid /PA/) when hypogiycemia was produced by injection of 10 units of insulin per kg body weight. The Higher dose of insulin 100 units per kg body weight produced a 20% inhibition of arachidonate incorporation into synaptosomal PI and a 13% decrease of incorporation into microsomal phosphatidylcholine. Incorporation of [1-¹⁴C]oleate into membrane phospholipids was not changed by hypoglycemic insult. It is proposed that the disturbances in fatty acid level, particularly arachidonate, and decreased uptake of arachidonic acid by synaptosomal glycerophospholipids may be responsible for alteration of membrane function and changes of synaptic processes.     

Separation of triglycerides and free fatty acids on Sephadex LH-20

Free fatty acids and triglycerides have been separated by elution with chloroform or chloroform + 0.2% ($\text{v}\text{v}$) acetic acid from columns of Sephadex LH-20. Loads of up to 10 mg/gm dry weight Sephadex were used, and recovery of artificial and natural mixtures was generally better than 95%.     

Total cholesterol, fatty acids, and plasmalogens can be reliably quantitated by analysis on chromarods after the methylation step required for fatty acid analysis by gas-liquid chromatography

A new method of simultaneous quantitation of total fatty acids, plasmalogens, and cholesterol using thin-layer chromatography with flame-ionization detection (TLC-FID) is presented. This method only requires methanolysis of the crude lipid extracts, an operation usually performed for fatty acid analysis by gas-liquid chromatography (GLC), and both analyses can thus be carried out in one step. Because of its resolution power and sensitivity, the TLC-FID technique can be used to analyze microsamples routinely assayed in capillary GLC (less than 15 micrograms of total fatty acids). The data obtained by capillary GLC and those provided by TLC-FID after careful individual calibration of the rods are almost identical. Reproducibility of the TLC-FID measurements is lower for fatty acid methyl esters and dimethylacetals, but equivalent for cholesterol quantitation, if the samples are analyzed twice on chromarods.     

Stable gas-liquid chromatography column for fatty acid analysis.

A mixed gas-liquid chromatography column of FFAP and Apolar-7CP has excelelnt thermal stability and shows excellent characteristics for the separation of the methylesters of fatty acids from Salmonella enteritidis call wall lipids.