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.     

A high performance size-exclusion chromatographic method for evaluating heated oils

High performance size-exclusion chromatography (HPSEC) was used to measure compounds with high-molecular weight (MW) formed during the heating of oil. Formation of the high-MW compounds is believed to be a reliable indicator of heat abuse in oils. The HPSEC method employs two μ-spherogel size-exclusion columns (500 and 1000 ?) in a series to separate the high-MW compounds which are detected at a wave-length of 234 nm by using a variable wavelength detector. The method was examined in the following study. Two sources of soybean oil were heated under laboratory conditions at 182 ± 2 C for eight 7-hr days. Samples were taken periodically and tested by using HPSEC. Oil samples from two commercial deep-fat frying operations were similarly tested. In all cases, size, number and apparent MW of the compounds formed increased with increasing frying time. The HPSEC procedure was compared with a method involving separation of polar and nonpolar components in a used frying fat by means of column chromatography on silica gel. This is Journal Paper No. J-11947 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project No. 2568.     

Size-exclusion chromatography of high molecular weight water-soluble polymers

A preparative-scale, aqueous size-exclusion chromatography system was constructed for fractionation of large molecular weight polymers. Calibration of molecular weight to elution volume was accomplished without polymer standards by using an eluent viscosity detector in series with a refractometer. The system was found to have a hydrodynamic size-separation resolution that ranged from 500 to 3500 Å.     

High performance size exclusion chromatography of fatty acids,mono-, di- and triglyceride mixtures

A high performance size exclusion chromatographic (HPSEC) method is described for the separation and quantitation of fatty acids, mono-, di- and triglyceride mixtures. The various lipid components were separated on two columns packed with 5 μm styrene/divinylbenzene copolymer and connected in series. Toluene was employed as eluant, and components were monitored by refractometry. A formula derived for calculation of total weighted correction factors (WCF) for the various lipid classes based on known values of correction factors of simple lipid components and the fatty acid composition of the sample allowed quantitation of lipid mixtures containing a variety of different molecules. The precision of the experiments is such that the relative standard deviation for each lipid component was 1–5%, and a component could be detected at 0.05% level.     

Determination of nonvolatile components of heated soybean oils separated with high-efficiency mixed-bed polystyrene/divinylbenzene columns

Whole heated soybean oils and their polar fractions were analyzed for nonvolatile components by high-performance size-exclusion chromatography (HPSEC) with evaporative light scattering detection (ELSD). High molecular-weight (MW) polymer compounds with MW ≥ trimer were efficiently separated with new 3-μm mixed-bed styrene/divinylbenzene copolymer columns. Peaks of high MW polymer components in the new column system appeared to be sharper and more symmetrical than those obtained with other columns. In the model systems studied, continuous addition of water to partially simulate frying conditions resulted in a significant increase (up to 30%) in the polar lipid content of the heated oils evaluated. Due to relatively high concentrations of monomeric triglycerides (84.6–93.5%) present in the whole unfractionated oils, small but erratic variations in the compositional distribution of components were observed in oils containing different amounts of added water. On the other hand, HPSEC-ELSD analyses of the polar fractions (monomeric triglycerides, 25.4–62.6%) showed significant changes in the content and composition of nonvolatile components with the amount of water added. In general, prolonged heating with increasing amounts of water accelerated hydrolysis and polymerization of heated soybean oils. Discrepancies in total polymerization of heated soybean oils. Discrepancies in total polymeric materials obtained from HPSEC composition data for whole oils and polar fractions are discussed in terms of nonuniformity in sample matrices, detection limitations for minor components, and a nonlinear ELSD response rationale.     

Molecular weight distribution of cellulose as its tricarbanilate by high performance size exclusion chromatography

Cellulose tricarbanilates (CTCs) were prepared from a range of cellulose samples (cotton linters, wood pulps, Avicel, amorphous cellulose, and cellulose II) for molecular weight distribution (MWD) studies by high performance size exclusion chromatography (HPSEC). The HPSEC columns were calibrated using CTC standards with the aid of a microcomputer. CTCs were prepared by reaction of cellulose samples with phenylisocyanate in pyridine at 80°C. For some samples, e.g., cellulose II, activation with liquid ammonia and pyridine was necessary prior to reaction in pyridine. All samples tested were also derivatised in dimethylsulfoxide at 70°C, although for high molecular weight (MW) cellulose samples some MW reduction occurred in this solvent. Conditions were determined for optimum precipitation of CTCs in aqueous methanol without coprecipitation of low MW impurities.     

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.     

Ethanolysis of rapeseed oil: Quantitation of ethyl esters,mono-, di-, and triglycerides and glycerol by high-performance size-exclusion chromatography

High-performance size-exclusion chromatography (HPSEC) was used to evaluate the influence of different variables affecting the transesterification of rapeseed oil (RSO) with anhydrous ethanol and sodium ethoxide as catalyst. The effect of temperature, ethanol/RSO molar ratio, catalyst concentration, and time can be interpreted by observing the variations of the reaction medium composition. HPSEC has made the quantitation of ethyl esters, mono-, di-, and triglycerides and glycerol possible. The best results for laboratory-scale reactions were obtained at 80°C with a 6:1 molar ratio of EtOH/RSO and 1% of NaOEt by weight of RSO.     

Exclusion chromatography using porous glass. II. Application to hydrophilic polymers

A chromatograph employing five columns packed with porous glass of pore size 1250 Å to 75 Å provided peak retention volumes (V_R) that were reproducible and essentially independent of sample size and flow rate when aqueous eluents were used. Calibration was carried out with a series of dextran fractions and polystyrene sulfonate samples, both of moderately narrow molecular weight distribution. The universal calibration method, based on hydrodynamic volume, was tested for four different polymer types. All four types produced a common curve within experimental error, which indicates that absolute molecular weight distributions may be derived from aqueous exclusion chromatography data for at least these polymer types. Additional study using a higher salt concentration produced hydrodynamic-volume plots that superposed with those above. The use of the same set of porous glass columns with polystyrene standards in three different organic solvents produced calibration curves that agreed well with the aqueous curves after corrections were made for differences in available pore volumes.     

Physicochemical characterization of galactosyldiglycerides and their quantitation in wheat flour lipids by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method was developed for analyzing digalactosyldiglycerides (DGDG) and monogalactosyldiglyceride (MGDG) in polar lipids fractionated from lipid extracts of wheat or flour. Wheat lipid samples were prepared by solvent extraction, then fractionated on a silica gel packed open column. A Spherisorb ODS (octadecyl silane) column with methanol/water elution system was used for separation of glycolipids in the polar lipid fractions. The detection limit of the refractive index detector with interferometric optics was 0.25μg for both DGDG and MGDG. Separating on nonpolar bonded phase columns permitted us to differentiate, based on fatty acid composition and position, among components within the specific glycolipid classes. Semipreparative HPLC on analytical columns was used to subfractionate the polar lipids. The glycolipids were collected for functional group characterization. Approximately 35% of each DGDG subfraction was accounted for as carbohydrate. The absence of phosphorus precluded phospholipids. Fatty acid analysis by gas chromatography showed the first DGDG to be linoleic acid, whereas the second DGDG peak was composed of linoleic, oleic and palmitic acids. Mass spectrometric analysis of the first DGDG peak showed linoleic acid in both the SN-1 and 2 positions. Mass spectrometric analysis revealed that palmitic or oleic acid in the second peak was preferentially located on the SN-1 position; linoleic acid was on the SN-2 position. Contribution no. 80-207J, Department of Grain Science and Industry, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS. Part of a dissertation submitted by T.N. Tweeten in partial fulfillment of the PhD degree. Honored Student Award Presentation at AOCS annual meeting, San Francisco, April 1979.     

Sample pretreatment for aggregate-free PVC molecular weight measurement by size-exclusion chromatography

The procedure of aggregate-free poly(vinyl chloride) (PVC) solutions for size-exclusion chromatography (SEC) at room temperature using tetrahydrofuran (THF) as the mobile phase is proposed. PVC was dissolved in 1,2,4-trichlorobenzene (TCB) at 130 or 140°C for 6 h, precipitated in methanol, filtrated, and dried. The pretreated PVC was again dissolved in THF and SEC measurements were performed at room temperature. High molecular weight (MW) PVC required a higher pretreatment temperature of 140°C. The existence of aggregates sometimes could not be observed by a refractive index detector, but a light-scattering detector attached to the SEC columns could detect them clearly. The slope of the relationship between the MW and retention volume at the high MW region was steep compared with that at the other part of the peak and it was also a good indicator of the existence of aggregates. The pretreatment of PVC resulted in the decrease in MW averages, which was attributed to the disappearance of aggregates and not to the degradation of PVC. The pretreatment at 150°C resulted in the degradation of PVC. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68: 1801–1809, 1998     

Physicochemical characterization of carrageenans—A critical reinvestigation

Kappa‐, iota‐, and lambda‐carrageenan (food grade) were analyzed by static light scattering (MALS in batch mode) in 0.1M NaNO₃ at 25 and 60°C, earlier heated up to 90°C or not. At 25°C, there was a strong tendency for a concentration‐dependent aggregation in the order lambda < kappa < iota. At 60°C, all samples were molecularly dispersed. The strongly temperature‐dependent refractive index increments (equilibrium dialysis) differ. Data interpretation in terms of the wormlike chain model using the Skolnik‐Odijk‐Fixman approach led to an intrinsic persistence length around 3 to 4 nm and expansion factors as high as 1.5 and above in a thermodynamically good solvent for all three types. Triple‐detector HPSEC (DRI, MALS, viscometry) on the three commercial samples plus a degraded (by acidic hydrolysis) kappa‐carrageenan in the same solvent/eluant at 60°C yielded a uniform and slightly curved [η]‐M relationship for 5 × 10³ ≤ M/(g mol) ≤ 3 × 10⁶ and a nearly identical molar mass dependence of the radius of gyration. HPSEC at 25°C on kappa‐carrageenan confirmed formation of soluble aggregates. Special emphasis was put on analytical and methodological aspects. The reliability of the experimental data was demonstrated by analogous measurements on dextran calibration standards. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Extraction of Astaxanthin from Shrimp Waste using Response Surface Methodology and a New Hybrid Organic-Inorganic Monolith

A 17-run Box-Behnken design (BBD) with three factors was used to improve the conditions for extracting astaxanthin from shrimp waste. The astaxanthin level was determined by high performance liquid chromatography (HPLC) using a C₁₈ column and a UV detector at 476 nm. The mean extraction yield of astaxanthin at the improved conditions (extraction time 1.4 h, extraction temperature 72.4°C, and liquid-solid ratio 27.3) was 98.7 ± 2.6 µg g^?1. A solid-phase extraction (SPE)-HPLC method was developed with a new hybrid organic-inorganic hybrid monolith for further purification of astaxanthin from shrimp waste. The SPE recoveries were ranging from 81.3 ± 2.4% to 86.5 ± 3.3%, and the intra-day and inter-day relative standard deviations (RSDs) of the proposed method were less than 4.3 ± 0.5% and 4.8 ± 0.2%, respectively. BBD increased the extraction efficiency and shortened extraction times significantly. SPE-HPLC with hybrid monolith showed good selectivity and purified astaxanthin from shrimp waste.     

Improved Methods for the Fatty Acid Analysis of Blood Lipid Classes

Two improved methods have been developed for preparation of fatty acid methyl esters (FAME) from major O-ester lipid classes in blood, i.e., cholesterol ester, triacylglycerol, and glycerophospholipids. The methods involve simple operations, and use neither harmful solvents such as chloroform or benzene nor highly reactive volatile reagents such as acetyl chloride. The FAME synthesis reaction proceeds under mild temperature conditions. The methods include (1) extraction of lipids from 0.2 ml of blood with 0.2 ml of tert-butyl methyl ether and 0.1 ml of methanol, (2) separation of the total lipids into lipid classes using a solid-phase extraction column or thin-layer chromatography, and (3) methanolysis of each lipid class at room temperature or at 45 °C. In all the operations, solvent concentration is performed only once prior to gas–liquid chromatography (GC). No noticeable differences in composition determined by GC have been found between FAME prepared by the present methods and those prepared by a conventional method involving lipid extraction with chloroform/methanol. The mild reaction and simplified procedures of the present methods enabled safe and reproducible analysis of the fatty acid compositions of the major ester-lipid classes in blood.     

A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction

A modified procedure for extraction of total lipids from whey protein concentrates was developed such that stable emulsion with extracting solvents was avoided and the solvent system remained monophasic. Nonlipid contaminants from the extract were removed using gel filtration instead of traditional aqueous washing to prevent any loss of polar lipids. The extraction of total lipids by the modified procedure was complete and comparable with a reference procedure. Traditional thin-layer chromatography is tedious and more qualitative than quantitative for lipid class separation. Total lipids were further separated into free fatty acids, phospholipids, cholesterol ester, triacylglycerol, cholesterol, diacylglycerol, and monoacylglycerol, using modified solid phase extraction procedure. Columns with 2 g amino propyl packing allowed separation of up to 80 mg of total lipids into lipid classes gravimetrically. The values for anhydrous milk fat for all lipid classes agreed with those in the literature. Separation of total lipids into lipid classes with solid phase extraction is easy, quantitative, and can also be performed on a preparative scale.     

Factorial Design Optimization of Solid-Phase Microextraction for High-Performance Liquid Chromatography−Ultraviolet Spectrometry Analysis of Polycyclic Aromatic Hydrocarbons in Cigarette Filter Tar

A simple and rapid procedure for the determination of polycyclic aromatic hydrocarbons (PAHs) in cigarette filter tar using solid-phase microextraction (SPME) was developed. The analysis was carried out using high-performance liquid chromatography equipped with an ultraviolet detector. The effects of the SPME experimental parameters on the extraction recovery were studied simultaneously using a central composite design (CCD) after a 2^6?2 fractional factorial experimental design. The SPME variables of interest were the extraction temperature, the extraction time, and the stirring speed, as well as the pH and the concentrations of NaCl (%, w/v) and acetonitrile (ACN). The optimal SPME conditions were as follows: an extraction temperature of 65°C, an extraction time of 50 min, a stirring speed of 800 rpm, 0% NaCl (w/v), 10% ACN in the sample, and a source pH of 8.0. The extraction calibration plots were linear over the range of 0.25?20 ng mL^?1 (r² > 0.9912) and the limits of detection (LODs) for the 6 PAHs studied were from 0.17–5.02 ng cigarette^?1. The relative standard deviation (RSD) ranged from 7.1–13.5% for intra-day variation and from 8.5–18.4% for inter-day variation. The performance of the proposed method was evaluated for extraction and determination PAHs in real samples (various brands of cigarettes). The total amounts of all of the studied PAHs found in the filter tar of the three brands of cigarettes were 320.2, 17.9, and 66.7 ng cigarette^?1, respectively.     

High-resolution gas chromatographic determination of alkanols in oils extracted from olives

Polar columns with stationary phases characterized by high thermal stability have attracted a great deal of interest in the study of lipid compounds that have, until recently, been studied with nonpolar columns. Such polar columns have also proved useful in the study of the entire unsaponifiable matter of a lipid and in determining the alkanols of extra-virgin olive oil, husk oil and their relative mixtures. The proposed procedure permits the study of alkanols without having to isolate them from the other classes of compounds present in the unsaponifiable matter by means of thin-layer chromatography.     

Analysis of Polycyclic Aromatic Hydrocarbons in Egyptian Petroleum Condensate Oils

The polycyclic aromatic hydrocarbons (PAH) in Egyptian condensates are analyzed for the first. A solid phase extraction (SPE) followed by gas chromatography-mass selective detection was used for their analysis. The method was calibrated for optimal extraction conditions. Excellent recoveries were found (78–114%) for the PAHs that were identified using a variety of standards and GC-MS spectra. The solid-phase extracted PAH fraction was further separated by HPLC on a Ag(I) mercaptopropanosilica gel to reduce the complexity of the sample by separating the PAHs based on the number of aromatic rings. The analytes were quantified using GC with a flame ionization detector. For this kind of sample SPE is a more convenient separation technique than an open column. PAHs containing two to four rings in the concentration range 0.6–11 μg/L were measured. Some preliminary geochemical hypotheses based on the analyzed PAHs and the previously analyzed S-containing aromatic compounds were formed as to the depositional environment and source rock type.     

Separation of Oxygen and Nitrogen by Porous Cyanometallates

The adsorption-based separation in porous solids takes place through steric, kinetic, or equilibrium effect selectivity. In this contribution the oxygen-nitrogen separation by four porous frameworks representative of cyanometallates was studied by inverse gas chromatography. The following materials were considered: Cd₃[Co(CN)₆]₂ (cubic), Zn₃[Co(CN)₆]₂ (rhombohedral), Zn₃K₂[Fe(CN)₆]₂ (rhombohedral) and Co[Fe(CN)₅NO] (cubic). Chromatographic separation profiles from gases mixtures using columns of these materials were recorded. For columns prepared from rhombohedral zinc hexacyanocolbaltate(III) excellent separation of O₂ and N₂ was observed. Such behavior was attributed to a kinetic-based selectivity related to the size and shape for the pore windows of this material. The porous framework of this zinc phase is formed by ellipsoidal cavities (12.5 × 9 × 8 Å) communicated by elliptical windows of ～ 5 Å. For Cd₃[Co(CN)₆]₂ and Co[Fe(CN)₅NO] also kinetic-based selectivity was observed while for Zn₃K₂[Fe(CN)₆]₂ the K⁺ ion located close to the cavity windows hinders the porous windows accessibility for nitrogen and oxygen molecules. All the samples to be studied were characterized from X-ray diffraction, infrared spectroscopy, termogravimetric and adsorption data.     

Differences in Oxidation Kinetics Between Conjugated and Non-Conjugated Methyl Linoleate

The oxidation kinetics of conjugated methyl linoleate was compared with that of non-conjugated methyl linoleate under mild oxidation conditions (30 °C in the dark). Samples of methyl 9-cis,11-trans-linoleate, methyl 10-trans,12-cis linoleate and methyl 9-cis,12-cis linoleate were assayed separately and in mixtures. For comparative purposes, methyl α-linolenate and methyl oleate were also used. Two complementary analytical approaches were selected to monitor the progress of oxidation, (1) the traditional follow-up of residual substrate by gas liquid chromatography, and (2) an analytical procedure by high-performance size-exclusion chromatography (HPSEC) for direct measurement of the oxidation compounds formed. The HPSEC method enabled us to quantitate oxidized monomers, dimers and polymers concomitantly in a rapid and direct analysis. Results showed that conjugated methyl linoleate samples oxidized later than their non-conjugated counterparts, and showed a very different oxidation pattern. Thus, formation of oxidized monomers was negligible and the first and major compounds formed were polymerization products. Also, under the conditions used, non-conjugated and conjugated methyl linoleate samples in 1:1 mixtures led to decreased oxidation rate of non-conjugated methyl linoleate and increased oxidation rate of conjugated methyl linoleate. This study supports the view that oxidation kinetics of conjugated dienes differ substantially from that of methylene-interrupted dienes.