HPLC analysis of phospholipids by evaporative laser light-scattering detection

High-performance liquid chromatography (HPLC) for analysis of phospholipids has traditionally employed ultraviolet detection of the eluted compounds. The evaporative laser light-scattering detector (ELSD) offers new opportunities for quantitative analysis of lipids. Phospholipids were isolated from crude and degummed oils prepared from soybeans subjected to storage at high moisture content. Analytical and preparative separations of phospholipids by normal-phase HPLC were accomplished. Major class fractions were analyzed by transmethylation and capillary collumn chromatography for fatty acid composition, and by reverse-phase C-18 HPLC (RP-HPLC) for molecular species composition. The RP-HPLC-ELSD system was limited to the analysis of phosphatidylcholine and phosphatidylethanolamine.     

Separation and quantitation of hydroxy and epoxy fatty acid by high-performance liquid chromatography with an evaporative light-scattering detector

A new high-performance liquid chromatography technique with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of hydroxy and epoxy fatty acids. This method employs a gradual binary gradient (hexane/isopropanol) and ELSD detection. The minimum limit of detection is about 1 μg and ratio of mass to signal is essentially linear in the range of 10 to 200 μg. This high-performance liquid chromatography (HPLC) technique is able to separate various positional isomers of mono-hydroxy and dihydroxy fatty acids and can also discriminate between monohydroxy, epoxy, epoxyhydroxy, dihydroxy and trihydroxy fatty acids.     

Argentation supercritical fluid chromatography for quantitative analysis of triacylglycerols

A method for quantitative analysis of neutral lipids has been developed. Four different techniques have been combined for this purpose—supercritical fluid chromatography (SFC), silver ion chromatography, packed microcolumns and miniaturized evaporative light-scattering detection (ELSD). The development and optimization of the method are discussed. The separation of a series of vegetable, fish and hydrogenated oils was demonstrated. Application of eluent composition programming resulted in excellent separation of complex samples. Packed microcolumn argentation SFC provides at least as high a separation power as corresponding high-performance liquid chromatography methods. The combination of packed microcolumn SFC and miniaturized ELSD constitutes a powerful analytical system for the quantitative analysis of triacylglycerols.     

Quantitative determination of monoglycerides and diglycerides by high-performance liquid chromatography and evaporative light-scattering detection

Neutral lipid classes were separated with normal-phase high-performance liquid chromatography, and mono- and diglycerides were determined with an evaporative light-scattering detector (ELSD). The 1,3-diacylglycerols were resolved from the 1,2-diacylglycerol positional isomers, although some 1,3-diacylglycerols of low molecular weight interfered with the 1,2-diacylglycerols of high molecular weight. For monoglycerides, the separations between 1-(and 3-)acyl and 2-acylglycerols were optimized only between those pairs with identical fatty acyl groups. Samples were dissolved in a solvent mixture and analyzed without derivatization. The results (monoglyceride) obtained from this method agreed well with those derived from gas chromatographic and supercritical fluid chromatographic methods. The universal nature of the ELSD makes this method applicable to oils and emulsifiers containing both saturated and unsaturated fatty acyl moieties.     

Analysis of nonionic surfactants in bench-scale biotreater samples

The effluents and activated sludges used in benchscale biotreater units have been analyzed for nonionic alcohol ethoxylates and their residues. Separate bench-scale units were fed linear alcohol ethoxylates (AE), highly branched and branched nonylphenol ethoxylates. Effluents and sludges were first pretreated by a foam sublation technique to provide a gross separation of surfactants from the environmental matrix. This step was followed by normal-phase high-performance liquid chromatography (HPLC) with either fluorescence detection (FD) or evaporative light-scattering detection (ESLD). The AEs were derivatized with phenylisocyanate and analyzed by normal-phase HPLC coupled with FD. At extremely low surfactant levels, pretreatment of large sample volumes resulted in interferences on derivatization. Hence, a normal-phase HPLC method with ELSD was developed. Although some interferences do appear using ELSD, this method appears to be a more viable alternative to derivatization/FD for very low levels of AE. HPLC with FD and ELSD detection methods are more quantitative and provide information on the polyoxyethylene chain than is possible with traditional methods like cobalt-thiocyanate active substance. Presented at the 82nd AOCS Annual Meeting & Expo, May 1991, Chicago, Illinois.     

Analysis of tocopherols in vegetable oils by high-performance liquid chromatography: Comparison of fluorescence and evaporative light-scattering detection

A comparison of the responses of an evaporative light-scattering detector (ELSD) and a fluorescence detector for tocopherols in vegetable oils by high-performance liquid chromatography is presented. The tocopherols were separated from acylglycerols by gel-permeation chromatography (GPC). The tocopherol fraction was collected off a set of four GPC columns with a mobile phase of methylene chloride before separation on a normal-phase silica column with a mobile phase of hexane/isopropanol, 99.7∶0.3 (vol/vol). An internal standard of 5,7 dimethyltocol, which was detected by both the ELSD and fluorescence detector, was used to obtain quantitative data. The fluorescence detector was ten times more sensitive than the ELSD. γ-Tocopherol was the major tocopherol detected in the vegetable oils studied and ranged from 24.1–93.3 mg/100 g. The amounts of tocopherols found in the vegetable oils agreed favorably with the literature values.     

The Identification and Quantification of Steryl Glucosides in Precipitates from Commercial Biodiesel

There have been several discoveries of unexpected precipitates in manufacturing facilities, transport vessels, and storage tanks containing commercial biodiesel. In some cases these have been formed during storage at temperatures above the cloud point of the fuel. High performance liquid chromatography (HPLC) and mass spectrometry (MS) methods were applied to 24 field receipt samples of solids from such biodiesels. The analyses revealed the presence of steryl glucosides (SG), common phytosterols (plant sterol) found in crude soybean oil and many other plant materials, in these biodiesel precipitates. Quantitative analysis of the solids revealed SG concentrations as high as 68 wt% of the provided material (which had not been previously washed with solvent to remove entrained biodiesel). In some samples no SG were present. In others they constituted all of the non-biodiesel material present. Monacylglycerols and diacylglycerols, the products of incomplete transesterification of triacylglycerols, were also present in some samples. The normal phase and reverse phase methods described in this report could be used to analyze SG quantitatively from biodiesel precipitates with an HPLC instrument equipped with either an evaporative light-scattering detector (ELSD) or a more common UV detector operating at 205 nm. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Chromatographic Separation of Synthesized Phenolic Lipids from Krill Oil and Dihydroxyphenyl Acetic Acid

The separation and characterization of novel biomolecules, phenolic lipids, obtained by the enzymatic transesterification in organic solvent-free media of krill oil with 3,4-dihydroxyphenylacetic acid were investigated. The experimental findings showed that by increasing the polarity of the gradient eluent and by decreasing the solvent strength of the mobile phase, from methanol to acetonitrile, a higher resolution was obtained. The use of a shorter column and smaller particle packing size resulted in an enhancement of the efficiency, with decreases in both separation time and solvent consumption. Overall, the evaporative light-scattering detector (ELSD) showed better repeatability of the resolution (R), theoretical plate number (n), plates per meter (N) and the retention time values as compared to that of the UV detection at 210 and 280 nm. In terms of detection and repeatability, ELSD was shown to be a more appropriate tool for the quantitative analysis of the components of krill oil and its esterified phenolic lipids than UV detection. Fourier transform infrared spectroscopy analysis tentatively confirmed the nature of the separated compounds. In addition, the structural analyses of novel biomolecules by HPLC–MS–APCI/ESI suggested the formation of two phenolic monoacylglycerols.     

The analysis of lipids via HPLC with a charged aerosol detector

Because most lipid extracts are a mixture of saturated and unsaturated molecules, the most successful strategies for the quantitative analysis of lipids have involved the use of so-called “mass” or universal detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC “mass” detector, a charge aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents charging the aerosol particles, and measuring the current from the charged aerosol flux. In the present study, the CAD was evaluated with several normal phase and reverse phase HPLC methods commonly used for the quantitative analysis of lipid classes and lipid molecular species. The CAD detected common lipids such as triacylglycerols, diacylglycerols, glycolipids, phospholipids, and sterols. Lower molecular weight lipids such as free FA had smaller peak areas (50–80% lower). FAME were not detected by the CAD, probably because they were completely evaporated and did not form aerosol particles. The minimum limits of detection of the CAD with lipids varied with different mobile phase solvents. Using solvent systems that were predominantly hexane, the minimum limits of detection of triacylglycerols, cholesterol esters, and free sterols were about 1 ng per injection and the mass-to-peak area ratio was nearly linear from the range of about 1 ng to about 20 mg per injection. Three other solvents commonly used for HPLC lipid analysis (methanol, isopropanol, and acetonitrile) caused higher levels of background noise and higher minimum limits of detection. These experiments indicate that the CAD has the potential to become a valuable tool for the quantitative HPLC analysis of lipids. Long-term studies are needed to evaluate full instrument performance.     

Reversed-Phase high-performance liquid chromatographic analysis of triacylglycerol autoxidation products with ultraviolet and evaporative light-scattering detectors

The oxidation products of triacylgycerol standards (trilinolenin, trilinolein and triolein) and a natural mixture of rapeseed oil triacylglycerols were analyzed with a reversed-phase high-performance liquid chromatographic method. The oxidation products were detected with ultraviolet (UV) and evaporative light-scattering detectors (ELSD). The chromatographic profiles obtained with these two detectors were similar for all samples except triolein. ELSD is a mass detector and can detect the oxidation products of triolein and other compounds that do not have conjugated dienes in their structure. The two detectors can be used in series. The sensitivity of ELSD approached that of the UV detector used. ELSD seems to be a good universal detector type for monitoring autoxidation products of edible oils.     

Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection

Glycolipids from edible plant sources were accurately quantified by silica-based, normal-phase high-performance liquid chromatography using an evaporative light-scattering detector. Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, ceramide monohexoside, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol) were separated and determined with a binary gradient system consisting of chloroform and methanol/water (95∶5, vol/vol) without any interference from other lipid classes and pigments. The described method was applied to 48 edible plants available in Japan including cereals, legumes, vegetables, and fruits. Examined plant species contained glycolipids in wide concentration ranges, such as 5–645 mg/100 g tissue.     

Fractionation of soybean phospholipids by high-performance liquid chromatography with an evaporative light-scattering detector

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from 23 soybean lines with a wide range of fatty acid compositions were resolved into seven fractions by high-performance liquid chromatography (HPLC). Fraction identities were assigned from fatty acid compositions determined by gas chromatography (GC). A mass detector, i.e., an evaporative light-scattering detector, was used for HPLC quantification. The detector response was a power function of PC and PE concentrations. Various correction methods were applied to the detector response to obtain the best agreement between phospholipid (PL) fatty acid compositions determined by GC and that calculated from the corrected HPLC fraction percentages. The corrected HPLC fraction composition also was compared with that calculated from stereospecific distribution data using a 1-random-2-random hypothesis. Correlation between PL-fatty acid and HPLC-fraction percentages showed that genetic modification of soybean oil composition caused changes in PL species, which alter physical properties and may alter the physiological functions of PL in biomembranes.     

Rapid Detection and Quantification of Triacylglycerol by HPLC–ELSD in Chlamydomonas reinhardtii and Chlorella Strains

Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thin-layer chromatography (TLC), and then analyzing the fatty acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG accumulation in two Chlamydomonas reinhardtii (21 gr and CC503) and three Chlorella strains (UTEX 1230, CS01 and UTEX 2229) grown under conditions of nitrogen depletion was measured. The TAG levels were found to be 3–6 % DW (Chlamydomonas strains) and 7–12 % DW (Chlorella strains) respectively by both HPLC–ELSD and TLC/GC methods. HPLC–ELSD resolved the major lipid classes such as carotenoids, TAG, diacylglycerol (DAG), free fatty acids, phospholipids, and galactolipids in a 15-min run. Quantitation of TAG content was based on comparison to calibration curves of trihexadecanoin (16:0 TAG) and trioctadecadienoin (18:2 TAG) and showed linearity from 0.2 to 10 μg. Algal TAG levels >0.5 μg/g DW were detectable by this method. Furthermore TAG content in Chlorella kessleri UTEX 2229 could be detected. TAG as well as DAG and TAG content were estimated at 1.6 % DW by HPLC–ELSD, while it was undetectable by TLC/GC method.     

In-line and off-line trapping of lipids extracted from chicken liver by supercritical carbon dioxide

This supercritical fluid extraction study determined the retentive properties of neutral alumina sorbent as an in-line trap for lipids in the dynamic state over a pressure range of 490–680 bar and temperatures of 40 and 80°C. Lipids were extracted from a chicken liver matrix using supercritical carbon dioxide over a 40-min period at a flow rate of 3 L/min (expanded gas), then were quantified by high-performance liquid chromatography using an evaporative light-scattering detector. Approximately 30 and 18%, respectively, of the total extracted lipids were trapped on the in-line alumina sorbent bed at 40°C as the operating pressure increased from 490 to 680 bar, while the remaining lipids were trapped off-line after CO₂ decompression. The major lipid classes trapped in-line were fatty acids and cholesterol, whereas only minor amounts of the less polar lipid classes such as sterol esters and triacylglycerols were retained. At 80°C and 680 bar, less than 1.5% of the extracted total lipids was trapped in-line, indicating the lack of adsorptive selectivity for lipids by alumina under these conditions.     

Effect of absorbent in solid‐phase extraction on quantification of phospholipids in palm‐pressed fiber

Palm‐pressed fiber (PPF) is a by‐product of palm oil milling and it has been found to contain a high percentage of phospholipids (PL). The aim of this work was to evaluate the best solid‐phase extraction (SPE) method to purify PL from PPF. The purified PL were analyzed using high‐performance liquid chromatography (HPLC) with an evaporative light‐scattering detector (ELSD). The oil was extracted from PPF using the Folch method and classes of PL were purified using SPE. Different solvent phases and normal‐phase SPE cartridges [silica (Si), aminopropyl‐bonded silica (NH₂) and diol‐bonded silica (2OH)] at the same ratio of oil to sorbent mass were used to study the separation of PL from the extracted oil. The recovery of each standard PL was performed in a model oil system with the same amount of standard PL being added, and the repeatability of the SPE method was investigated. The isolation of PL by SPE diol cartridge and subsequent analysis by HPLC/ELSD have shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and lysophosphatidylcholine in PPF. This method provided a high yield and rapid separation of PL in PPF with less contamination from other lipid groups.     

Analysis of tocopherols and phytosterols in vegetable oils by HPLC with evaporative light-scattering detection

Methods were developed for the separation, detection, and quantification of tocopherols and phytosterols by high-performance liquid chromatography with an evaporative light-scattering detector. Four tocopherols— α, β, γ and δ—and four phytosterols—campesterol, β-sitosterol, brassicasterol, and stigmasterol—were analyzed in soybean, sunflower, low-erucic acid rapeseed (LEAR) and corn oils. The use of an evaporative light-scattering detector, in conjunction with modification of methods from the literature to prepare and analyze tocopherols and phytosterols by HPLC, showed consistent results between trials and levels of these minor constituents. Presented at the Annual American Oil Chemists' Society Meeting, May 3–7, 1989, Cincinnati, OH.     

Rapid analysis of oxidized cholesterol derivatives by high-performance liquid chromatography combined with diode-array ultraviolet and evaporative laser light-scattering detection

Extensive evidence of the deleterious biological effects of oxidized 5-cholesten-3β-ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light-scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5-cholestan-5α,6α-epoxy-3β-ol (5α-epoxycholesterol), 5-cholestan-5β,6β-epoxy-3β-ol (5β-epoxycholesterol), and 5-cholestan-3β,5α,6β-triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5-cholesten-3β,7α-diol (7α-hydroxycholesterol), 5-cholesten-3β,7β-diol (7β-hydroxycholesterol), 5-cholesten-3β-ol-7-one (7-ketocholesterol), 5α- and 5β-epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α- and 7β-hydroxycholesterols and 3,5-cholestadien-7-one. On the other hand, 7α- and 7β-hydroxycholesterols, 7-ketocholesterol, 5α- and 5β-epoxycholesterols and 3,5-cholestadien-7-one were observed in copper-oxidized low-density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.     

Extraction of Algal Lipids and Their Analysis by HPLC and Mass Spectrometry

Algae are a promising source of biofuel but claims about their lipid content can be ambiguous because extraction methods vary and lipid quantitation often does not distinguish between particular lipid classes. Here we compared methods for the extraction of algal lipids and showed that 2-ethoxyethanol (2-EE) provides superior lipid recovery (>150–200 %) compared to other common extraction solvents such as chloroform:methanol or hexane. Extractions of wet and dry algal biomass showed that 2-EE was more effective at extracting lipids from wet rather than dried algal pellets. To analyze lipid content we used normal-phase HPLC with parallel quantitation by an evaporative light scattering detector and a mass spectrometer. Analysis of crude lipid extracts showed that all major lipid classes could be identified and quantified and revealed a surprisingly large amount of saturated hydrocarbons (HC). This HC fraction was isolated from extracts of bioreactor-grown algae and further analyzed by HPLC/MS, NMR, and GC/MS. The results showed that the sample consisted of a mixture of saturated, straight- and branched-chain HC of different chain lengths. These algal HC could represent an alternative biofuel to triacylglycerols (TAG) that could feed directly into the current petroleum infrastructure.     

脂肪酶促乌桕脂组分甘油解及其反应机理研究

Glycerolysis of Chinese vegetable tallow （CVT） fraction was investigated using a 1,3-specific lipase from Rhizopus arrhizus as catalyst. Based upon a binary gradient HPLC with an evaporative light-scattering detector （ELSD）, the contents of free fatty acids （FFA）, monoglycerides （MG）, diglycerides（DG） and triglycerides （TG） with their positional isomers during the glycerolysis were determined. The effects of water content and the ratio of glycerol to oil on the product distribution of glycerolysis were studied. Under the optimum reactant conditions： 250 units lipase per gram oil at 37℃ with 1：2 molar ratio of oil to glycerol in a solvent-free system, after 24 h reaction, the product consisted of 7.2% TG, 25.6% MG, 56.1% DG and 4.9% FFA （all by mass）. Furthermore, the mechanism of glycerolysis was discussed in detail.     

油菜秸秆高温水解液中单糖组分的高效液相色谱测定

建立了高温水中CO₂催化油菜秸秆水解体系中单糖的高效液相色谱（HPLC）分析的方法。其分析采用Prevail^TM ES糖柱,蒸发光散射检测器（ELSD）,流动相为乙腈/水（体积比80∶20）,等度洗脱,流速0.8 mL/min,柱温35 ℃,漂移管温度95 ℃,干燥空气作为载气,流速为3.0 L/min。20 min内阿拉伯糖、木糖、果糖、甘露糖、半乳糖和葡萄糖6种单糖得到了较好的分离,各物质线性回归方程的相关系数R²为0.999 1~0.999 7,其平均回收率为95.3 %~108.0 %,相对标准偏差（RSD）均小于3 %。