Application of high-performance size-exclusion chromatography to study the autoxidation of unsaturated triacylglycerols

A combination of solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) was used to study the autoxidation of triacylglycerol (TAG) mixtures separated from low-erucic acid rapeseed oil and butter oil. The samples were autoxidized in the dark at 40°C for four weeks. The polar compounds of the autoxidated samples were separated by SPE (NH₂ stationary phase), and the polar fraction was further characterized by HPSEC with a series of three size-exclusion columns and an evaporative light-scattering detector. The polar fraction contained TAG polymers, polar TAG monomers (PTAG) and diacylglycerols. Peroxide values and anisidine values of the samples were also measured. By using three different types of TAG mixtures, it could be demonstrated that the PTAG content of the TAGs increases during autoxidation. A slight increase was also detected in polymer content. The correlation between PTAG content and the comparative measurements was considered significant. The results indicate that the measurement of PTAG and polymeric material content by HPSEC analysis can be used when studying the autoxidation level of edible oils and in characterizing the autoxidation products of different molecular sizes.     

Analysis of lipid classes by solid-phase extraction and high-performance size-exclusion chromatography

An improved method to analyze lipid classes of edible oils and fats by solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) is presented. A mixture of lipid standards was fractionated by the solid-phase extraction procedure (NH₂ phase) into polar and nonpolar fractions; these were then submitted to analysis by HPSEC. The size-exclusion chromatographic columns were three styrene/divinylbenzene columns with pore sizes of 100 Å and 50 Å. Light-scattering was used for the detection system, and the parameters of the detector were optimized to minimize the difference between the responses of the compounds studied. With this procedure it was possible to separate the following lipid classes: triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids, sterols, sterol esters, tocopherols and carotenoids. Quantitative analysis was studied for a light-scattering detector with several lipid standards of different molecular weights and unsaturation levels.     

Ethanolysis of castor and cottonseed oil: A systematic study using classical catalysts

Several classical catalytic systems for the transesterification reaction have been used to produce FA ethyl esters (FAEE) from castor and cottonseed oils The effects of the amount and nature of the catalyst, and of the reaction temperature, on the yields of FAEE were determined. The most efficient transesterification of castor oil was achieved in the presence of methoxide and acid catalysts, whereas for cottonseed oil, which has a composition that is much more similar to most vegetable oils than is castor oil, the highest yields of FAEE were obtained following base-catalysed ethanolysis.     

Determination of mono-, di-, and triglycerides by molecular distillation and thin-layer chromatography

Analysis of mixtures of mono-, di-, and triglycerides by molecular distillation and thin-layer chromatography is described. Mono- and diglycerides undergo appreciable acyl migration through the effect of heat during molecular distillation. Nevertheless this technique may be used for the quantitative analysis of mixtures of mono-, di-, and triglycerides, provided there are no substances present which catalyze, disproportionation. Thin-layer chromatographic (TLC) analysis of mono-, di-, and triglycerides is fast and simple and can be carried out on a micro-scale with a high degree of accuracy and precision. It also is extremely sensitive, permitting the quantitative estimation of as little as 0.1% of a single component in a mixture. This research was supported by the Hormel Foundation and General Foods Corporation, Tarrytown, N.Y. Presented at fall meeting, American Oil Chemists’, Society, New York, October 17–19, 1960.     

Industrial high-performance liquid chromatography purification of docosahexaenoic acid ethyl ester and docosapentaenoic acid ethyl ester from single-cell oil

The use of polyunsaturated fatty acids (PUFA) as medicine or in functional diets requires high purity. An industrial purification method for PUFA from Schizochytrium sp. SR21 oil was investigated. This oil contains fewer unwanted components than fish oils. Docosahexaenoic acid and docosapentaenoic acid ethyl esters (DHA-E and DPA-E) were prepared by treatment of this oil with ethanol and 1 N potassium hydroxide in hexane. DHA-E and DPA-E were purified by an industrial high-performance liquid chromatography (HPLC) plant. The separation plant consists of two columns (400 mm i.d., 1,000 mml) with temperature-controlled water jackets and double-plunger (four heads) injection and eluent pumps. This plant was computer-controlled and equipped with an explosion-prevention system. The packed material was octadecylsilica (reverse-phase ODS), and the eluent was methyl alcohol/water (98:2). DHA-E and DPA-E from single-cell oil were highly purified by this industrial HPLC method in a one-step process. The DHA-E and DPA-E obtained were better than 99% purity.     

Sunflower oil used for frying: Combination of column,gas and high-performance size-exclusion chromatography for its evaluation

The alterations of a sunflower oil were evaluated by column, gas and high-performance size-exclusion chromatography after being used for deep-fat frying fifteen repeated and discontinuous times. Polar compounds increased significantly (6.2 ± 0.3% to 18.7 ± 0.8% in oil). Linoleic acid decreased (53.8 ± 0.2 to 48.1 ± 0.8 mg/100 mg oil) while oleic acid remained unaltered after 15 fryings. Saturated fatty acids such as palmitic and stearic, also remained unaltered. Triglyceride polymers (0.1 ± 0.0 to 2.4 ± 0.2 mg/100 mg oil), triglyceride dimers (1.0 ± 0.2 to 6.7 ± 0.3 mg/100 mg oil) and oxidized triglycerides (3.4 ± 0.2 to 7.6 ± 0.3 mg/100 mg oil) increased significantly in the oil used 15 times to fry potatoes. These thermoxidative compounds correlated well with the number of fryings (r=0.9864, r=0.9535 and r=0.9758, respectively). Diglyceride compounds remained unaltered, while free fatty acids increased from 0.4 ± 0.0 to 0.6 ± 0.0 mg/100 mg oil. Both of these, which are characteristic of hydrolytic alteration, did not correlate significantly (r=0.5985 and r=0.4261, respectively) with the number of fryings. These data suggest that a thermoxidative process, rather than a hydrolytic one, took place in this study.     

体积排阻色谱法测定介孔材料的孔结构

分别合成了球形的苯基桥键和乙烷桥键两种新型杂化介孔材料.选用已知平均相对分子质量且具有窄相对分子质量分布的葡聚糖为探针溶质,采用动态光散射法测定了粒径,与经验公式计算结果接近.通过体积排阻法对两种材料的孔隙率和孔径分布进行测定并与氮气吸附法结果进行比较,实现介孔材料孔结构快速测定.结果表明,体积排阻色谱法测定苯基桥键和...     

逆体积排阻层析法测定层析介质的孔径分布

针对层析介质的孔径分析,基于刚性球状分子进入柱状孔的假设,分别采用高斯正态分布和对数正态分布描述孔径分布,利用简化的单孔分配因子模型,建立了孔径分布函数和分配因子Kd的关联,通过体积排阻层析实验测定系列标准物的Kd,从而拟合得到孔径分布信息,建立了逆体积排阻层析法。以葡聚糖作为分子大小的标准物,测定了5种典型层析介质(SP Sepharose FAST FLOW、Q Sepharose FAST FLOW、ToyopearlDEAE-650M、Streamline DEAE和Sephadex G-150)的Kd,计算和比较了不同介质的孔径分布,分析了介质的可吸附孔表面积等结构参数,证实了逆体积排阻层析法分析层析介质孔径分布的可行性和实用性。     

Heated fat-based oil substitutes, oleic and linoleic acid-esterified propoxylated glycerol

Four oils [triolein, trilinolein, oleic acid-esterified propoxylated glycerol (EPG-08 oleate), and linoleic acid-esterified propoxylated glycerol (EPG-08 linoleate)], each without added antioxidants, were heated for 12 h/d at approximately 190°C in a small deep-fat fryer until the polymer concentration exceeded 20%, as determined by high-performance size-exclusion chromatography. Increases in the free fatty acid content, total acid value, food oil sensor value, and p-anisidine value during heating indicated that significant thermal oxidation had occurred in each oil. Capillary supercritical fluid chromatography (SFC) was used to determine the substrate concentration of each oil after each heating interval. The average, apparent first-order reaction rate constant (as determined by SFC) for trilinolein was 0.0348±0.0034 h⁻¹, while the rate for EPG-08 linoleate was 0.0253±0.0032 h⁻¹. The average apparent reaction rate constant for triolein was 0.0256±0.0011 h⁻¹, while the rate for EPG-08 oleate was 0.0252±0.0008 h⁻¹. Triolein contained >20% polymer after 60 h of heating, EPG-08 oleate contained >20% polymer after 36 h of heating, and both trilinolein and EPG-08 linoleate contained >20% polymer after 24 h of heating.     

Separation of wax esters from olive oils by high-performance liquid chromatography

To detect adulteration of olive oil with solvent-extracted oils, the determination of the wax ester content has become more important during recent years. Hence, a greater number of wax ester analyses need to be performed by quality control laboratories. The most common method in use requires a liquid chromatographic (LC) separation of the less polar fraction, which contains the wax esters, from the glyceride matter on a hand-filled silica gel column. The aim of this project was to verify the possibility of replacing LC with high-performance liquid chromatography by taking advantage of the greater reliability and repeatability of this technique, as well as of the possibility of making the separation automatic. The paper describes how to perform the analysis and the statistical test that was carried out; furthermore, a comparison has been made with the usual method and results are in good agreement.     

Lipase-catalyzed transesterification of rapeseed oil and 2-ethyl-1-hexanol

Lipase-catalyzed transesterification (alcoholysis) of lowerucic acid rapeseed oil and 2-ethyl-1-hexanol without an additional organic solvent was studied in stirred batch reactors. Of a number of commercially available enzymes investigated, the best results were obtained with aCandida rugosa lipase. The optimal transesterification conditions were an oil/alcohol molar ratio of 1∶2.8, a minimum of 1.0% (w/w) added water, and with a temperature of 37–55°C. Under the optimal conditions, a nearly complete conversion was obtained in one hour with 14.6% (w/w) lipase, whereas 0.3% (w/w) lipase required 10 h for similar results. The enzyme was inactivated at 60°C.     

Separation and quantitation of mono-, di-, and triglycerides and free oleic acid using thin-layer chromatography with flame-ionization detection

α-Monoolein was prepared from glycidol and oleic acid by the regioselective opening of glycidol in the presence of an anionic resin. During the reaction, the lipochemical synthesis medium becomes enriched in monoolein, the effective emulsifying agent. This mixture can be analyzed by thinlayer chromatography coupled with flame-ionization detection (FID). The products and reagents do not need to be derivatized. Diglyceride and triglyceride by-products affecting the selectivity of the reaction also could be detected using this technique. Cholesterol was used as an internal standard. The factors influencing the separation, including the hydrogen flow rate, scan speed, and the composition of the developing solvent, were investigated. The degree of separation is highly sensitive to the hexane/diethyl ether ratio of the developing solvent. Good separation of triglyceride, oleic acid, the two diglycerides, cholesterol, α-monoolein, and glycidol was obtained with the mixture hexane/diethyl ether/formic acid (65:35:0.04, by vol). Detector response, detection limits, and rod-to-rod variations also were examined. A range of rod loads giving a straightforward relationship between FID response and amount of compound loaded (relative to oleic acid and α-monoolein) was defined. The accuracy of the quantification was illustrated by analysis of a mixture of oleic acid and α-monoolein standards of known composition. Presented at the AOCS conference, New Trends in Lipid and Lippprotein Analysis, La Grande Motte, France, September 1993.     

Identification and quantification of feruloylated mono-, di-, and triacylglycerols from vegetable oils

The use of HPLC-MS to separate and identify the feruloylated acylglycerols formed during the transesterification of ethyl ferulate with TAG was examined. Novozym 435 (Candida antarctica lipase B)-catalyzed transesterifications of ethyl ferulate and soybean oil resulted in a mixture of feruloylated MAG, DAG, and TAG and diferuloylated DAG and TAG. These feruloylated acylglycerols have recently garnered much interest as cosmeceutical ingredients. The ratio of the various feruloylated acylglycerol species in the resultant oils is presumed to affect the oil's cosmetic efficacy as well as its physical (formulation) properties. Thus, it was desirable to develop an analytical method to separate, identify, and quantify the individual feruloylated acylglycerols to determine their relative ratios. The feruloylated acylglycerols were successfully separated and identified by HPLC-MS using a phenyl-hexyl reversed-phase column developed with a water/methanol/1-butanol gradient. The chromatograms of the feruloylated acylglycerols from soybean oil were convoluted by myriad fatty acids; therefore, feruloylated acylglycerols from triolein were studied as a model reaction. Hydrolysis of the feruloylated acylglycerols from triolein catalyzed by Lipase PS-C “Amano” I (Burkholderia cepacia), which showed no hydrolysis reactivity toward ethyl ferulate, allowed for the chromatographic assignment of the feruloyl acylglycerol positional isomers.     

Soybean oil triacylglycerol analysis by reversed-phase high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

Soybean oil triacylglycerols from genetically modified soybean lines were conclusively identified by reversed-phase high-performance liquid chromatography coupled with mass spectrometry with atmospheric pressure chemical ionization. Atmospheric pressure chemical ionization is a soft ionization technique which gives simple spectra for triacylglycerols. Spectral identification of the triacylglycerols was based on the molecular [M+1]⁺ ion and the 1(2)-, 2(3)- and 1(3)-diacylglycerol fragments. Triacylglycerols identified in high-stearic and high-palmitic soybean varieties were quantitated by reversed-phase high-performance liquid chromatography with flame-ionization detection. There was excellent agreement between the fatty acid composition calculated from the triacylglycerol composition and the fatty acid composition obtained by gas chromatography of the transmethylated oils. The oils of the modified soybean varieties, compared to typical soybean oil, contained increased content of triacylglycerols known to be more oxidatively stable, such as linoleoyloleoylstearoyl, linoleoylpalmitoylstearoyl, and linoleoyldipalmitoyl glycerols, and less triacylglycerols like trilinoleoylglycerol, known to decrease oxidative stability. This study showed that the atmospheric pressure chemical ionization technique is suitable for mass spectral identification of neutral molecules, such as triacylglycerols, which do not contain a chargeable functional group.     

Quantitative determination of glycerol,mono- and diglycerides by gas liquid chromatography

Standard chemical procedures for the determination of glycerol, mono- and diglycerides in food products are lengthy, particularly when more than one component has to be determined in the same sample. Gaschromatographic techniques are applicable to these compounds when they are converted into their trimethylsilyl derivatives. A simple, rapid and quantitative gas chromatographic procedure has now been developed which is based on the addition of an internal standard (cholesteryl acetate) to samples which are subsequently silylated. This procedure has been applied to the direct determination of glycerol, mono- and diglycerides in emulsifiers, shortenings and other samples. Presented at the AOCS Spring Meeting, San Francisco, California, April 1969.     

The triacylglycerol structure of olive oil determined by silver ion high-performance liquid chromatography in combination with stereospecific analysis

The compositions of positionssn-1,sn-2 andsn-3 of triacylglycerols from “extra-virgin” olive oil (Olea europaea) were determined. The procedure involved preparation of diacyl-rac-glycerols by partial hydrolysis with ethyl magnesium bromide; 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated by highperformance liquid chromatography (HPLC) on silica, and their fatty acid compositions were determined. The same procedure was also carried out on the five main triacylglycerol fractions of olive oil after separation according to the degree of unsaturation by HPLC in the silver ion mode. Although stereospecific analysis of the intact triacyl-sn-glycerols indicated that the compositions of positionssn-1 andsn-3 were similar, the analyses of the molecular species demonstrated marked asymmetry. The data indicate that the “1-random, 2-random, 3-random” distribution theory is not always applicable to vegetable oils.     

Some esters of mono-, di-, and tricarboxystearic acid as lubricants: Preparation and evaluation

Conditions were determined for selective alkaline transesterification of methyl 9(10)-carbomethoxystearate. With 2,2-dimethylpentanol and 2-ethylhexanol, the corresponding alkyl 9(10)-carbomethoxystearates were obtained in good yield. These two mixed alkyl diesters, along with other alkyl esters of mono-9(10)-, di- and tricarboxystearic acids prepared by conventional methods, were evaluated as lubricants. Several esters compared favorably with dioctyl sebacate used as the control. The esters had high viscosity indices and low American Society for Testing and Materials slopes. The pour point of purified dimethylpentyl 9(10)-carbomethoxystearate is below −70 C. Several esters have antiwear properties when added to the control oil.     

Some esters of mono-, di-, and tricarboxystearic acid as plasticizers: Preparation and evaluation

Methyl mono-, di-, and tricarboxystearates were prepared by either a two-step hydroformylation-oxidation reaction or direct hydrocarboxylation of unsaturated vegetable oil methyl esters. Procedures were developed for preparing alkyl dicarbomethoxy-, dicarboethoxy-, and dicarbobutoxystearates. These triesters, along with some di- and tetraesters from mono- and tricarboxystearic acids, were evaluated as primary plasticizers for polyvinyl chloride (PVC). Except for butyl carbobutoxystearate, the esters were compatible at the 32% level and had properties equal or superior to those of dioctyl phthalate. Methyl and butyl diesters of carboxystearic acid had undesirable migration and volatility properties. The migration and volatility properties of some tri- and tetraalkyl esters were equal to or better than the controls. Of the various esters tested, methyl dicarbomethoxystearate containing 13–49% methyl tricarbomethoxystearate was an efficient plasticizer for PVC at the 32% level.     

Comparison of analyses of surfactants in cosmetics using high-performance liquid chromatography and high-performance capillary electrophoresis

Separation of anionic, cationic, and amphoteric surfactants containing n-dodecyl groups and hydrophilic moieties was done by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE), and an ultraviolet-visible detector. Quantitation of surfactants in commercial cosmetic and toiletry products was also done using similar methods. Conditions used to separate mixtures of surfactants by HPLC are as follows: stationary phase: ODS 2; mobile phase: MeOH/H₂O (80∶20, vol/vol) containing 1.0M NH₄Cl, 0.003 M tetrabutylammonium hydrogen sulfate, and 0.005 M diammonium phosphate buffer solution at pH=6.0. Conditions for HPCE are as follows: an uncoated capillary (100 μm i.d., 110 cm in length, effective length of 75 cm) with 25 kV of applied voltage, and an aqueous buffer containing 80 mM sodium borate/20 mM NaOH at pH=9.2. Surfactants were eluted within 15 min. The accuracy of both techniques was evaluated by analyzing the recovery ratio of surfactants in the mixture.