Silica High-performance Liquid Chromatography Research Articles

Use of principal component analysis for studying the separation of pesticides on polyethylene-coated silica columns

The retention time of 27 pesticides was determined on an polyethylene-coated silica high-performance liquid chromatography column (PEEsil) using water–methanol mixtures as eluents. Linear correlations were calculated between the logarithm of the capacity factor and the methanol concentration in the eluent (C) and the relationship between the slope and intercept values of the above correlation, the hydrophobicity, specific hydrophobic surface area of the pesticides and their retention parameters on porous graphitized carbon column was elucidated by using principal component analysis followed by two-dimensional nonlinear mapping. The most polar pesticides showed irregular retention behaviour of the PEEsil column, their retention decreased with increasing concentration of methanol in the eluent. This irregular retention was tentatively explained by the silanophile effect: at higher methanol concentrations the polar substructures of pesticides have a higher probability of binding to the polar adsorption centers on the silica surface not covered by the hydrophobic ligand increasing in this manner the retention capacity of the support. Principal component analysis showed that PEEsil support has retention characteristics different from those of traditional reversed-phase supports.

Synthesis and characterization of a strong cation exchanger based on polymer-coated silica for high-performance liquid chromatography

A strong cation exchanger based on silicone polymer-coated silica (PC-SCX) was synthesized by the following three-step procedure: (1) coating porous silica with a silicone polymer; (2) introduction of epoxy groups with allyl glycidyl ether; (3) introduction of sulfonic groups with sulfanilic acid. The surface structure of PC-SCX was characterized by solid-state 13C-nuclear magnetic resonance spectrometry (NMR) and elemental analysis. In comparison with conventional silica-based cation exchanger, PC-SCX showed an improved durability and a better reproducibility in different synthetic batches. PC-SCX was applied to separation of several different classes of standard cationic compounds, such as biogenic amines, water-soluble vitamins and an antibacterial agent.

Triacylglycerol composition and structure in genetically modified sunflower and soybean oils

AbstractChanges in composition were examined in oils extracted from genetically modified sunflower and soybean seeds. Improvements were made to the analytical methods to accomplish these analyses successfully. Triacylglycerols (TAG) were separated on two 300 mm × 3.9 mm 4µ Novapak C18 high‐performance liquid chromatography (HPLC) columns and detected with a Varex MKIII evaporative light‐scattering detector. Peaks were identified by coelution with known standards or by determining fatty acid composition of eluted TAG by capillary gas chromatography (GC). Stereospecific analysis (fatty acid position) was accomplished by partially hydrolyzing TAG with ethyl magnesium bromide and immediately derivatizing the resulting diacylglycerols (DAG) with (S)‐(+)‐1‐(1‐naphthyl)ethyl isocyanate. The derivatized sn‐1,2‐DAG were completely resolved from the sn‐2,3‐DAG on two 25 mm × 4.6 mm 3 µ silica HPLC columns. The columns were chilled to −20°C to obtain baseline resolution of collected peaks. The distribution of fatty acids on each position of the glycerol backbone was derived from the fatty acid compositions of the two DAG groups and the unhydrolyzed oil. Results for the sn‐2 position were verified by hydrolyzing oils with porcine pancreatic lipase, isolating the resulting sn‐2 monoacylglycerols by TLC, and determining the fatty acid compositions by GC. Results demonstrated that alterations in the total fatty acid composition of these seed oils are determined by the concentration of TAG species that contain at least one of the modified acyl groups. As expected, no differences were found in TAG with fatty acid quantities unaffected by the specific mutation. In lieu of direct metabolic or enzymatic assay evidence, the authors’ positional data are nevertheless consistent with TAG biosynthesis in these lines being driven by the mass action of available acyl groups and not by altered specificity of the acyltransferases, the compounds responsible for incorporating fatty acids into TAG.

Optimized high-performance liquid chromatographic method for determination of lamotrigine in serum with concomitant determination of phenytoin, carbamazepine, and carbamazepine epoxide.

Lamotrigine (LG), phenytoin (PY), carbamazepine (CM), and carbamazepine epoxide (CE) are measured with an optimized procedure that uses thin sorbent extraction disks and a highly selective, sterically protected bonded silica high-performance liquid chromatography (HPLC) column. Routinely, serum (200 microliters at pH 6.8 with cyheptamide as internal standard) is applied to an Empore octyl (C8) solid-phase extraction disk to isolate the drugs. a water wash removes interferences, and the retained drugs are eluted with a small volume of solvent. The eluate is directly injected onto a Zorbax Stable Bond cyanopropyl HPLC column with quantification at 214 nm. Evaporation-concentration steps are unnecessary. Overall, for all drugs, between-run precision coefficients of variation (n = 16 each) ranged from 2.1% to 4.9% at concentrations from 0.75 to 20.5 mg/l; extraction recoveries fell within a range of 96% to 110% at concentrations of 2, 10, and 30 mg/l tested for each drug; the lowest limit of detection was 0.15 to 0.35 mg/l. The analytical response was linear for each drug > 80 mg/l (LG) and > 50 mg/l (PY, CM, and CE). Optimization graphs are presented to illustrate the rationale for selection of test parameters for a robust method. In addition, a comparison study between two commercial laboratories demonstrates accuracy problems associated with LG testing.

Synthesis of a new phosphoglycolipid with biological activity towards platelets

Various synthetic as well as naturally occurring compounds have been found to exhibit platelet-activating factor (PAF)-like activity or to act as specific PAF inhibitors. In this work we have synthesized a new phosphoglycolipid, methyl 2,3,4-tri- O-acetyl-6-(1′- O-stearoyl-2′- O-acetyl- dl-glycero-3′-phosphoryl)-α- d-glucopyranoside ammonium salt, using a combination of known synthetic steps. This phosphoglycolipid was first purified on TLC (Rf 0.7, using chloroform/methanol/water, 65:25:4, v/v/v as solvent system). It was further purified onto a high performance liquid chromatography silica column with an elution system that contained acetonitrile and methanol (retention time 13.5 min). Its identification was based on chemical determinations and electrospray mass spectrometry analysis. The above compound induced washed platelet aggregation with an EC 50 value at 2 × 10 −4 M. The aggregation curve was biphasic, the first wave of which was through the PAF way while the second one was through the ADP way. Treatment with acetylhydrolase resulted in a rapid decrease of the first wave of aggregation and in a slow decrease of the second wave. In lower concentrations, the phosphoglycolipid inhibited PAF- and thrombin-induced aggregation with IC 50 values of the order of 10 −7 M. In conclusion, this phosphoglycolipid has a diverse biological activity. The PAF-like activity of this new lipid enforces the conception that PAF is a member of a large family consisting of lipid mediators.

Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites.

Bulk and emulsion copolymerizations ofn-butyl acrylate and poly(methyl methacrylate) macromonomer

Bulk and emulsion copolymerizations of an -unsaturated poly(methyl methacrylate) (PMMA) macromonomer with n-butyl acrylate (n-BA) were investigated. The reactivity of PMMA macromonomer in bulk copolymerization with n-BA was found to be lower than that of methyl methacrylate monomer with n-BA. The incorporation of PMMA macromonomer into poly(butyl acrylate) (PBA) latex particles by miniemulsion copolymerization was proved by high performance liquid chromatography-silica adsorption spectroscopy. Dynamic mechanical studies showed that PMMA macromonomer was grafted to the PBA backbone, and the degree of grafting increased as the ratio of PMMA macromonomer to n-BA increased. Microphase separation of the PMMA macromonomer grafts was observed at higher ratio of macromonomer (higher or equal to 10% weight of macromonomer based on total polymer phase). The n-BA/PMMA macromonomer copolymer behaved completely differently from the physical blend of PBA and PMMA macromonomer particles of the same composition.

Blood cardiolipin in haemodialysis patients. Its implication in the biological action of platelet-activating factor

Bovine heart cardiolipin specifically inhibits rabbit platelet aggregation induced by PAF in vitro. In the past we have reported that patients with primary glomerulonephritis have increased PAF levels in plasma (Iatrou et al., 1995b). In this work we investigate the existence of cardiolipin in the blood of end-stage renal patients due to primary glomerulonephritis and we study its implication in the biological study of PAF. Lipids from blood samples of end-stage renal patients were extracted, fractionated onto silicic acid column and onto High Performance Liquid Chromatography (HPLC) cation exchange column. PAF fraction was removed and phospholipids were separated from the rest lipid fraction with current counter distribution and furthermore fractionated onto HPLC silica column. The results show: 1. cardiolipin is present in the blood of end-stage renal patients. 2. Blood cardiolipin specifically inhibits PAF-induced aggregation in washed rabbit platelets. 3. Scatchard plot analysis of PAF binding, in the presence of unlabelled PAF and in the presence of cardiolipin, shows that rabbit platelets possess two different types of binding sites. One of which is saturable and of high affinity, kD = 0.103 ± 0.03 nM (SEM, n = 3) with 337 ± 94 binding sites per platelet for PAF and kD = 0.087 ± 0.02 nM with 371 ± 92.7 binding sites per platelet for cardiolipin while the other one has almost infinite binding capacity. 4. Blood cardiolipin competes [ 3H]PAF binding in rabbit platelets. This work shows that cardiolipin exists in the blood of end-stage renal patients and specifically inhibits PAF-induced aggregation as well as PAF binding in rabbit platelets. The possible implication of the biological actions of cardiolipin in the anticardiolipin-antiphospholipid syndrome is also discussed.

Determination by Chromatographic Methods of the Adsorption Rate Constant of HSA on Immobilized Polyclonal and Monoclonal Antibodies

High performance liquid chromatography (HPLC) was used to determine the association rate constants of antigen-antibody reactions at the solid-liquid interface. The adsorption of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a HPLC silica support was studied by frontal analysis. The theoretical model assumes irreversible adsorption during the time scale of the experiment. The shape of the breakthrough curves reveals the presence of several kinetic adsorption steps: the model fits well the first part of the elution profile. Frontal analysis gives the apparent adsorption rate constant, which includes the mass transfer in the stagnant fluid phase. This effect can be accounted for by carrying out studies as a function of flow rate on columns packed with adsorbents having various densities of immobilized antibody. Within experimental error, the effective rate constant for the irreversible adsorption process is in good agreement with previous experiments performed by saturating the column with repeated pulse injections.

High-performance liquid chromatography separation and light-scattering detection of phospholipids from cooked beef

A sensitive high-performance liquid chromatography (HPLC) method for the separation and quantitative analysis of major phospholipids (PLs) in biological systems is described. PLs were purified by solid-phase extraction with an amino (NH 2) phase. Separation of PLs was carried out on an HPLC silica gel column, with a mobile phase consisting of chloroform, methanol and ammonium hydroxide, and detection was performed with a light-scattering evaporative detector. HPLC analysis of PLs extracted from ground beef cooked under different conditions and capillary gas chromatography of the fatty acid methyl esters showed that cooking treatments did not have a significant effect on the PL composition and fatty acid contents of the single PLs in ground beef.

Comparison of four methods for the determination of polycyclic aromatic hydrocarbons in airborne particulates

High-performance liquid chromatography (HPLC) with ultraviolet and fluorescence detection and capillary gas chromatography (GC) with flame ionization and mass spectrometric (MS) detection were used to determine nineteen polycyclic aromatic hydrocarbons (PAHs) in airborne particulates. Sixteen of them are included in the prioritary pollutants list of the US Environmental Protection Agency. Five C 18-bonded silica HPLC columns and five GC capillary columns were checked to select the best conditions for the PAH mixtures. Samples were extracted by adding an organic solvent and sonication. The recoveries obtained were > 75%. These results are compared with those obtained using Soxhlet extraction. The method was applied to the determination of PAHs at five sampling sites in the city of Valencia during 1 week. The results confirm that the best detection limits are obtained by HPLC-fluorescence, which is also the simplest, shortest and most economical method. In spite of its high maintenance cost, GC-MS in the single-ion monitoring mode is also suitable for the determination of PAHs in real samples using a low-volume system.

Isolation and characterization of sucrose polyesters

AbstractVarious chromatographic techniques for isolation and separation of highly esterified sucrose polyesters (SPE) of olive oil are described. High‐performance size‐exclusion chromatography was used to check the purity of the samples, particularly to show that SPE were free from unreacted fatty acid methyl esters. Thin‐layer chromatography (TLC), Iatroscan TLC flame‐ionization detection (FID) and highperformance liquid chromatography (HPLC) on reversed‐phase were applied for separation of octa‐, hepta‐ and hexaesters of sucrose. Pure fractions from the total mixture, obtained by column chromatography, were identified by infrared and nuclear magnetic resonance spectroscopy. When necessary, specific reactions were applied; particularly, silylation and lead acetate‐dichlorofluorescein (in toluene) spray were used to ascertain the degee of esterification of sucrose. Finally, octa‐, hepta‐ and hexaesters of sucrose were quantitated by silica column chromatography, TLC/FID and HPLC on reversed‐phase.

Antimutagenic and bactericidal substances in the fruit body of a Basidiomycete Agaricus blazei, Jun-17

The fruit body of a Basidiomycete Agaricus blazei, Jun-17 (Himematsutake) was extracted with hexane and chloroform-methanol (2:1, v/v), and the antimutagenic effect of the extracts was examined using an Ames/Salmonella/microsome assay. Both extracts of Agaricus inhibited the mutagenicity of benzo[a]pyrene(B[a]P). The hexane extract was purified by silica gel column chromatography and high performance liquid chromatography (HPLC), and linoleic acid was isolated as a main substance having antimutagenic activity. Fr. IIa, IIb, IIc and IIb, which reduced the number of His+ revertant colonies induced by B[a]P, were separated from the chloroform-methanol extract by silica gel column chromatography and HPLC. An antimutagenic substance in Fr. IIa was linoleic acid. From Fr. IIb, a bactericidal, not antimutagenic, substance was isolated and identified as 13-hydroxy cis-9, trans-11-octadecadienoic acid (13ZE-LOH). Antimutagenic substances in Fr. IIc and IId were not purified. The possible source and mechanism of formation of 13ZE-LOH are discussed.

Structure of ten free N-glycans in ripening tomato fruit. Arabinose is a constituent of a plant N-glycan.

The concentration-dependent stimulatory and inhibitory effect of N-glycans on tomato (Lycopersicon esculentum Mill.) fruit ripening was recently reported (B. Priem and K.C. Gross [1992] Plant Physiol 98: 399-401). We report here the structure of 10 free N-glycans in mature green tomatoes. N-Glycans were purified from fruit pericarp by ethanolic extraction, desalting, concanavalin A-Sepharose chromatography, and amine-bonded silica high performance liquid chromatography. N-Glycan structures were determined using 500 MHz 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and glycosyl linkage methylation analysis by gas chromatography-mass spectrometry. A novel arabinosyl-containing N-glycan, Man alpha 1-->6(Ara alpha 1-->2)Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc, was purified from a retarded concanavalin A fraction. The location of the arabinosyl residue was the same as the xylosyl residue in complex N-glycans. GlcNAc[5']Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAc[5']Man2GlcNAc(Fuc)GlcNAc were also purified from the weakly retained fraction. The oligomannosyl N-glycans Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, and Man8GlcNAc were purified from a strongly retained concanavalin A fraction. The finding of free Man5GlcNAc in situ was important physiologically because previously we had described it as a promoter of tomato ripening when added exogenously. Mature green pericarp tissue contained more than 1 microgram of total free N-glycan/g fresh weight. Changes in N-glycan composition were determined during ripening by comparing glycosyl and glycosyl-linkage composition of oligosaccharidic extracts from fruit at different developmental stages. N-Glycans were present in pericarp tissue at all stages of development. However, the amount increased during ripening, as did the relative amount of xylosyl-containing N-glycans.

Comparison of adsorption properties of Florisil and silica in high-performance liquid chromatography: II. Retention behaviour of bi- and trifunctional model solutes

The retention of several bi- and trifunctional model solutes using different adsorbents, Florisil and silica, and different binary eluent systems containing isopropanol, dioxane or tetrahydrofuran as modifiers in n-heptane was investigated by high-performance liquid chromatography. The retention parameters are presented as log k′-log k′ relationships obtained for solutes in different chromatographic systems. The selectivity of the systems investigated and the influence of modifiers on the adsorptive properties of the adsorbents are discussed.

Comparison of adsorption properties of Florisil and silica in high-performance liquid chromatography: I. Retention behaviour of monofunctional model solutes

Earlier investigations demonstrated some special properties of Florisil and a greater selectivity than silica for the adsorption of some pairs of organic compounds. Differences in selectivity of the two adsorbents may be characterized by graphical correlations of retention parameters for these adsorbents. Consideration of relationship between log k′ Florisil and log k′ silica allows the interpretation of some factors that influence the adsorptive behaviour of organic compounds. The retention behaviour of several monofunctional aromatic compounds (quinoline bases, nitroarenes, primary and secondary aromatic amines and phenols) was therefore investigated by high-performance liquid chromatography in chromatographic systems using Florisil and silica and binary mobile phases consisting of heptane and a polar modifier (isopropanol, 1,4-dioxane or tetrahydrofuran).

A Study of Phospholipids and Galactolipids in Pollen of Two Lines of Brassica napus L. (Rapeseed) with Different Ratios of Linoleic to Linolenic Acid

The phospholipids and galactolipids of the pollen-coat and internal domains of two lines of Brassica napus, Wesroona and IXLIN, with different linoleic/linolenic acid ratios (18:2/18:3) have been characterized by normal phase silica high performance liquid chromatography and gas liquid chromatography. The polar lipids of the pollen-coat are similar to leaf lipids in the high proportion of galactolipids (almost 50%) and the fatty acids; 18:3, palmitic (16:0) and hexadecatrienoic (16:3). In contrast, the pollen internal domain, although rich in 18:3, 18:2 and 16:0, is composed primarily of phosphatidyl-choline, -ethanolamine, and -inositol whose 18:2/18:3 ratio is correlated with that of the seed generation. The difference between the two divergent 18:2/18:3 ratio lines is most evident in the internal domain phospholipids. The 18:2/18:3 ratio of the galactolipids of both pollen domains is not significantly effected by the line genotype. The results are interpreted in terms of the previously described ;prokaryotic' and ;eukaryotic' plant desaturation pathways (PG Roughan, CR Slack [1982] Annu Rev Plant Physiol 33: 97-132). We propose that the eukaryotic pathway is the major desaturation pathway providing polyunsaturated fatty acids to the haploid-specified internal domain in which the IXLIN genotype modifies the activity of the sn-2 linoleoyl phosphatidylcholine desaturase/s of the endoplasmic reticulum. In the diploid-specified pollen-coat, our evidence suggests that a combination of the prokaryotic and eukaryotic pathways contribute polyunsaturated fatty acids.

Identification and determination of 1 alpha,25-dihydroxyvitamin D3 in rat skin by high-performance liquid chromatography and radioreceptor assay.

Endogenous 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in normal rat skin was identified by using thermal isomerization to convert the metabolite into its pre-isomer, high-performance liquid chromatography (HPLC) and displacement potency with a chick intestinal cytosol receptor. When the metabolite in normal rat skin was determined by a radioreceptor assay after purification by Sep-Pak silica cartridge column chromatography and HPLC, the concentration was 71.0 +/- 6.6 pg/g of wet tissue (mean +/- S.D.). It is also shown that [3H]-1,25-(OH)2D3 and [3H]-25-hydroxyvitamin D3 administered intravenously in the mouse are located in the skin. These results suggest that the metabolite may play an important role in the skin.

O-258 - A serial determination of arachidonic acid metabolites in biological samples: Separation and fluorometric detection using SEP-PAK and octadecylsilyl silica-high performance liquid chromatography

O-258 - A serial determination of arachidonic acid metabolites in biological samples: Separation and fluorometric detection using SEP-PAK and octadecylsilyl silica-high performance liquid chromatography

High-speed size characterization of chromatographic silica by flow/hyperlayer field-flow fractionation

Flow/hyperlayer field-flow fractionation (FFF) has been successfully applied to the rapid size-based fractionation and characterization of various 3-μm and 5-μm commercial high-performance liquid chromatography silica supports. Because highj-resolution information on both average particle diameter and size distribution was obtained in less than 3 min by using a relatively simple apparatus, the method is attractive for the routine characterization of chromatographic packing material. Flow/hyperlayer FFF is not only faster than most sedimentation methods, it is ideally suited for the characterization of porous particles since separation is based solely on diameter and is independent of density.