Optimization of the column loadability for the preparative HPLC Separation of soybean phospholipids

A simple and rapid preparative-scale separation method was investigated in order to obtain pure soybean phospholipids. Because of technical and economical reasons, two coarse, irregular silica gels were selected. Comparing both stationary phases, a ternary mixture of hexane, 2-propanol and water yielded a different elution order of the phospholipids at analytical sample loads, in spite of the chemical similarity of these packing materials. During scaleup, it became obvious that the retention characteritics were largely influenced by the sample load, thus making the 15–35 μm RSiL inappropriate for preparative-scale separations of phospholipids. Moreover, the column loadability could be increased by controlling the flow rate. Hence, a solvent program was elaborated which enabled a column loadability of up to 2% by weight of the stationary phase. Using analytical high performance liquid chromatography, it was shown that the method proposed yielded over 90% pure phospholipids at a recovery of nearly 80%. Research assistant of the Belgian National Fund for Scientific Research (N.F.W.O.).     

Analysis of soybean lecithins and beef phospholipids by HPLC with an evaporative light scattering detector

Linear (r > 0.99) calibration curves were obtained for 10–150 μg of phosphatidylethanolamine (PE), 10–75 μg of phosphaditylinositol (PI), phosphaditylserine (PS) and lysophosphatidylethanolamine, 10–100 μg of phosphatidic acid (PA) and 10–250 μg of phosphatidylcholine (PC) by high-performance liquid chromatography analyses with an evaporative light scattering detector, a Zorbax 7-μm silica column and gradient elution with two solvents. One solvent (A) contained 415 mL isooctane (IOCT), 5 mL tetrahydrofuran (THF), 446 mL isopropanol (IPA), 104 mL CHCl₃ and 30 mL H₂O; and the other solvent (B) contained 216 mL IOCT, 4 mL THF, 546 mL IPA, 154 mL CHCl₃ and 80 mL H₂O. The gradient in which 100% A linearly changed to 100% B in 20 min followed by 12 min of 100% B and then a linear change to 100% A during 5 min separated PE, PS and PC in soybean lecithins and beef lipids, but failed to resolve PI and PA. In these same samples, less polar lipids were separated from phospholipids (PL) by elution from Bond-Elut silica columns with diethyl ether/hexane (20:80, vol/vol), and PL were recovered by elution with methanol. This procedure is useful for concentration of minor lipid components. Levels of PE, PI-PA, PS and PC were higher in granular than in liquid lecithin, and PC was the most abundant PL in soybean lecithins and beef lipids.     

High-performance liquid chromatographic separations of soy phospholipids

High-performance liquid chromatography methods to quantitate soy phospholipids vary as to which phospholipids are analyzed, degree of method ruggedness, precision, time, and standards. Fluid and deoiled soy lecithins were analyzed by three different high-performance liquid chromatography methods, including the American Oil Chemists’ Society (AOCS) Method Ja 7b-91. The other methods include an isocratic mixed phase (normal-phase column and reverse-phase solvent) method with ultraviolet detection, and a binary gradient normal-phase (proposed International Lecithin and Phospholipid Society) method with light-scattering detection. A set of 20 analyses were repeated on three different days for fluid and deoiled product by the three methods. The statistical comparison involved the selected methods and the phosphatidylcholine and phosphatidylethanolamine measured data, which were the common analytes among the methods. The precision for the mixed phase method and the Proposed International Lecithin and Phospholipid Society method was better than that for the AOCS method. Selection of reference standards was an important issue in defining the results. Column conditioning varied by 2–3 h for the Proposed International Lecithin and Phospholipid Society method, 16 h for the mixed phase method, and 2–3 d for the AOCS method. The ruggedness for the methods showed the following descending order: proposed International Lecithin and Phospholipid Society, mixed phase, AOCS.     

Antioxidant effect of soy lecithins on vegetable oil stability and their synergism with tocopherols

The antioxidant effect of lecithins was tested on several oils and fats varying in FA composition and tocopherol content. Standard lecithins, when added at a level of 1% w/w, exhibited a good protective effect against oxidation. This effect was observed to depend on the phospholipid content of the tested lecithins and the FA composition of the tested oils. Better results were obtained with lecithin samples containing high proportions of PC and PE. Indeed, the main antioxidant mechanism of lecithins was due to a synergistic effect between amino-alcohol phospholipids and γ- and δ-tocopherols. No synergism was observed with α-tocopherols, especially when the tested oil was rich in linoleic acid. Therefore, the antioxidant protection of lecithins was not effective for sunflower oil. Finally, the use of fractionated or enriched lecithins was not clearly advantageous compared to standard oil lecithins.     

Effects of expander process on the phospholipids in soybean oil

Crude oils were extracted from soybean flakes and collets by conventional and expander processes, respectively. The phospholipids were removed by degumming, and the lecithins were produced by using commercial procedures. The effects of the expander process on the degumming efficiencies were evaluated. The differences in the phosphatide compositions of the oils and the lecithins produced from expander and conventional processes were compared by high-performance liquid chromatography. The phosphorus content indicated that expander-processed oil contained more phosphorus (985 ppm) than the conventional oil (840 ppm). However, the phospholipids in the expander-processed oil were more hydratable than those in the conventional oil. After degumming, the phosphorus content in the expander-processed and conventional oil were reduced by 93.2 and 78.6%, respectively. The expander-processed lecithin contained 74.3% acetone-insoluble matter (AI), and the conventional lecithin contained 65.8%. More phosphatidylcholine was found in the expander-processed lecithin (39.78%, based on AI) than in the conventionally processed lecithin (34.19%). The phosphatidylinositol contents of the expander-processed lecithin and the conventional lecithin are almost the same (19.95 and 19.97%). The phosphatidylethanolamine in the expander-processed lecithin (12.36%) was lower than that in the conventional lecithin (18.07%).     

Enrichment of phospholipids from neutral lipids in peanut oil by high-performance liquid chromatography

Phospholipids from crude peanut oil were enriched on a 2-cm silica column and subsequently separated from neutral lipids within the chromatographic system without prior concentration. Hexane effectively removed the bulk neutral lipids, leaving the adsorbed phospholipids on the silica precolumn. Individual phospholipids were separated from the remaining neutral lipids and from each other by using two mixed solvents and a gradient program. This method separates the phospholipids in approximately 27 min after the desired enrichment level has been reached. The research reported in this paper was a cooperative effort by the Agricultural Research Service of the United States Department of Agriculture and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7625.     

Preparative separation of phospholipids from soybean by NP-HPLC

Soybean phospholipids-phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC)-were separated by normal-phase high performance liquid chromatography. The system was operated in a gradient mode. The experimental variables were gradient time and mobile phase composition. The experimental results showed that PE, PI and PC were resolved by three step-change gradient modes which employed ternary systems of hexane/1-propanol/water (58/40/2 and 56/40/4 by vol.%) and methanol/1-propanol/water (80/18/2, by vol.%) for the gradient times of 10, 30 and 76 min, respectively.     

Characterization of phospholipids from glyphosate-tolerant soybeans

The phospholipids from three control and two glyphosate-tolerant soybean cultivars were isolated by extraction of soy flakes with hexane and characterized after separation by high-pressure liquid chromatography. In addition, several lots of commercial fluid lecithin were analyzed and the results were compared to values published in the literature. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid were identified as major components in these cultivars and in the commercial lecithin samples. The results showed that glyphosate-tolerant soybeans yield lecithin comparable and equivalent to conventional soybean cultivars.     

高效液相色谱法测定卵磷脂中磷脂酰胆碱的含量

研究建立了高效液相色谱法测定3种卵磷脂样品中磷脂酰胆碱含量的分析方法。采用硅胶色谱柱(5μm,150mm×4．6mm),检测波长为207nm,流动相:乙腈-甲醇-85％磷酸(30:70:0．6,体积比),以磷脂酰胆碱峰面积定量。大豆、葵花和胡麻卵磷脂中磷脂酰胆碱的含量分别为64．3％、53．3％、59．8％。平均回收率为99．1％,RSD=1．48％。该方法简便快速、结果可靠。     

A simple and cost-effective gram-scale chromatographic method for the purification of soybean phospholipids

A method was developed for the preparative fractionation of soybean lecithin to enable the study of the functional properties of pure soybean phospholipids. Hereby, a coarse and irregularly shaped silica gel was used as the stationary phase, whereas the mobile phase consisted of three mixtures of hexane, 2-propanol, and water with increasing polarity. These solvents were included in a step gradient, which was formed by an isocratic pump connected to a solvent switcher. With this system, two grams of soybean lecithin were fractionated. The purity was evaluated by analytical high-performance liquid chromatography, and the recovery was estimated from concentration determinations by flow injection analysis. From these results, it was concluded that 60 to 75% of the three major soybean phospholipids could be recovered with a purity of at least 93%. Only 1.5 L of solvents were needed for both the column equilibration and the elution of all soybean phospholipids. Senior research associate of the Belgian National Fund for Scientific Research (N.F.W.O.).     

High-performance liquid chromatography analysis of peanut phospholipids. I. Injection system for simultaneous concentration and separation of phospholipids

This paper discusses the details of a high-performance liquid chromatography method for the simultaneous concentration and separation of phospholipids or other trace compounds by direct oil injection using two different solvent systems. The system equilibrates and concentrates phospholipids on a silica column using hexane. At the same time, an analytical column is equilibrating and separating phospholipids using two binary solvent mixtures. This system eliminates a preconcentration step previously accomplished by solid-phase extraction, open-column chromatography, and other previously used methodology. Other advantages include: a 40% reduction in analysis time, elimination of a second transfer of labile compounds, decreased solvent use, and a simpler array of solvents to separate phospholipids. The method described has broader applications, such as trace organic compounds in water supplies, and trace metals with appropriate modifications for the particular analysis.     

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.     

农药中间体一氯频吶酮液相色谱分析方法的研究

采用Agilent5nmCl8液相色谱分析方法对农药中间体一氯频吶酮进行定量分析,结果表明:一氯频吶酮线性相关系数为R2=0.9999;平均回收率99.0%;标准偏差为;变异系数为0.18,结果表明此方法具有分离效果好,操作简单、快速、灵敏度高,重现性好等特点.     

A preferred solvent system for high-performance liquid chromatographic analysis of soybean phospholipids with evaporative light-scattering detection

Normal-phase high-performance liquid chromatography separations of phospholipids (PL) depend heavily on the variation in mobile phases. Incorporation of pure tetrahydrofuran to a mobile phase of chloroform, methanol, and ammonium hydroxide improved the separation of polar phospholipids. However, impurities were often found to be present in tetrahydrofuran that caused severe peak interferences with PL analytes. The use of a common chromatographic solventtert.-butyl methyl ether, in lieu of tetrahydrofuran in mobile phases, eliminated problems associated with solvent contamination and yielded reproducible results. The modified method has been applied for the quantitative analysis of phospholipids in crude soybean oils.     

从大豆油脚中提取精制磷脂的研究

本文介绍了从大豆油脚中提取精制磷脂的方法,并讨论了丙酮用量、萃取温度、脱油次数以及活性白土用量、脱色温度等因素对产品质量的影响。得出丁最佳工艺条件,产品收率可达97％左右。     

从大豆粉末中提取卵磷脂的超声辅助萃取新工艺

采用超声波为辅助手段,以卵磷脂得率为评价指标,用乙醇提取大豆粉末中卵磷脂。结果表明,最佳提取工艺条件为:乙醇浓度为80%,提取温度为40℃,超声时间为50 min,固液比为1∶3.0(g/mL),萃取次数为2次时,可以有效地提取大豆粉末中卵磷脂达12.85%。     

Separation and analysis of phospholipids in different foods with a light-scattering detector

A method for the separation of phospholipids (PL) from total lipids by solid-phase extraction (SPE) with reversephase C₈ cartridges is described. The method was validated with a standard mixture of PL and applied to natural food matrixes, such as egg, chicken meat, salami, and ripened cheese. The recovery of PL ranged between 93 and 99.7% and was evaluated by an organic phosphorus spectrophotometric determination. The egg powder PL fraction obtained by SPE contained about 20% (w/w) nonpolar PL material when 100–150 mg of lipids were loaded onto the cartridge. Higher percentages of nonphospholipid components (30–43%) were obtained when the amount of lipids loaded was below or above the 100–150 mg range. The purified PL fractions were analyzed by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. Good HPLC performance was observed even with low-purity SPE fractions (43% nonphospholipid material).     

Removal of nonhydratable phospholipids from soybean oil

On the basis of the literature concerning the chemical and physicochemical properties of phosphatidic acid and phosphatidates, an experimental investigation of the conditions under which nonhydratable phospholipids (Mg/Ca-phosphatidates) are removable from water degummed soybean oil was carried out. The experiments were carried out by mixing water-degummed oil at different fixed pH values with buffer solutions with and without admixture of Ca⁺⁺, Mg/Ca-binding reagents and surfactants. The results indicate that the nonhydratable phospholipids can be removed in a chemically nonconverted state as a component of micelles or of mixed emulsifiers. Furthermore, the nonhydratable phospholipids are removable by conversion into dissociated form, i.e., by removal of Mg and Ca from the phosphatidates, which can be accomplished by acidulation or by treatment with Mg/Ca-complexing or Mg/Ca-precipitating reagents. Alkali-refining experiments have shown that removal or chemical conversion of the nonhydratable phospholipids result in reduced emulsion formation and in improved separation of the deacidified oil from the emulsion layer and the soapstock. Presented at the ISF-AOCS Congress, Chicago, September 1970.     

HPLC analysis of phospholipids in crude oil for evaluation of soybean deterioration

Damage to soybeans due to pre-harvest stress, storage, and export shipment has been related to an increase in the nonhydratable phospholipid content of crude oil. Phospholipids in crude soybean oil extracted from such distressed soybeans have been analyzed by gradient high-performance liquid chromatography. Crude oil was fractionated by solid phase extraction using sequential elution for recovery of phosphatides. High-performance liquid chromatography of the concentrated phospholipids was accomplished on a Lichrosorb Si-60 10 μ column, 250×4.6 mm with ultraviolet detection at 206 nm. A 20-min solvent gradient of 2-propanol/hexane/water (42∶56∶2, 51∶38∶11) gave retention profiles of phospholipid distribution (major subclasses) that changed with impact of stress applied to plant or seed. Soybeans stored at high moisture levels (16% and 20% moisture) for up to 28 days yielded oils having phosphorus contents which decreased in direct relationship to days of storage. Retention profiles were unusable for fractions isolated from oils with phosphorus content below 100 ppm. Data show that during progressive damage, the content of phosphatidylcholine and phosphatidylinositol decreased while the phosphatidic acid content increased. Presented at the Annual American Oil Chemists' Society meeting, May 8–12, 1988, Phoenix, AZ.     

Evaluation of self degumming properties of phospholipids in soybean oil using HPLC

Freshly extracted crude soybean oil was obtained from a commercial soybean oil refinery, divided into aliquots, stored at 24 C and 5 C for varying lengths of time up to two weeks, and analyzed for total phosphorus. The concentrations of phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylcholine (PC) were determined by phosphorus analysis following separation by HPLC. Portions of the aliquots were degummed, and total phosphorus and individual phospholipids were determined as described above. Total phosphorus decreased 41% in crude oil stored at 24 C and 34% in crude oil stored at 5 C over two weeks. PE and PC precipitated more readily than PI and PA. Total phosphorus remaining in degummed oil was least after 12 hr storage of the crude oil and increased as the storage period became longer. The PE, PI and PA concentrations showed similar trends in degummed oil; however the PC concentration was least at 0 hr and increased thereafter. These results are discussed in terms of phospholipid-phospholipid interactions and possible interactions between phospholipids and other compounds present in the oil. Presented at the AOCS meeting in Philadelphia in May 1985.