Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

Separtation of metal tetrakis(p-tolyl)porphine complexes by reversed-phase high-performance liquid chromatography

Summary The feasibility of reversed-phase high-performance liquid chromatography for the separation of several metal complexes ofmeso-tetrakis(p-tolyl)porphine (TTP) is described. A combination of an octadecyl-bonded stationary phase with a non-aqueous polar mobile phase, such as an acetone-acetonitrile mixture, has proved effective for the separation. Thus, the TTP complexes of Mg, VO, Ni, Cu, Zn, and Pd and also TTP free acid were successfully separated in about 10min on a Li-Chrosorb RP-18 column (7m, 250×4mm i.d.) with a 7030 (vol/vol) mixture of acetone and acetonitrile at a flow-rate of 1 mlmin^–1.     

Separation of polypeptide antibiotics by reversed-phase high-performance liquid chromatography

Summary The influence of different reversed-phase packings and the addition of acidic modifiers to the mobile phase was observed on the separation of basic and neutral polypeptide antibiotics by gradient elution. A dependence of pore size, coverage, reaction type and endcapping of the packings was not observed. Nevertheless, not all reversed-phase packings were suitable for the separation of polypeptides, especially of basic molecules. The addition of phosphoric or perchloric acid to the mobile phase prevented adsorption of the basic polypeptide antibiotics on the stationary phase.     

Determination of pheophytinatonickel(II) by reversed-phase highperformance liquid chromatography

Summary HPLC determination of pheophytinatonicke(II) (Pheo-Ni) prepared by the replacement of magnesium(II) in chlorophyll with nicke(II) is described. The good separation of PheoNi was obtained by using chemically bonded C₁₈ as the stationary phase and acetone-methanol (50∶50, vol/vol) as the mobile phase. Conventional spectrophotometric method was also used for the determination of PheoNi. For the synthetic samples prepared by mixing (pheophytinato a) nicke(II) [(Pheo-a) Ni] and (pheophytinato b) nicke(II) [(Pheo-b) Ni], analytical values obtained by the spectrophotometric method were very high compared to those obtained by HPLC. In the proposed HPLC method, (Pheo-a) Ni and (Pheo-b). Ni could be determined in the concentration range of 0.028–30μg/ml and 0.038–30μg/ml with relative standard deviations (n=10) of 3.1% and 0.8%, respectively.     

Monitoring the preparation of NAD analogs by reversed-phase high-performance liquid chromatography

Summary Pig brain NAD glycohydrolase immobilized on Affi-Gel 10 or nylon 6 was used for the conversion of NAD into 3-acetylpyridine adenine dinucleotide (APAD) or 3-aminopyridine adenine dinucleotide (AAD). A reversed-phase chromatographic system consisting of a C₁₈ Resolve column and phosphate buffer (pH 6.2)-methanol as the mobile phase was used to monitor the production of APAD and AAD.     

Nucleotide pools and energy charges of smooth molluscan muscles analysed by reversed-phase,ion-pair,high-performance liquid chromatography

Summary A method is described for the analysis of the 5 mono-, di- and triphosphates of adenosine, guanosine, uridine and cytidine, as well as uridinediphosphate-glucose and cyclic AMP. Separation is achieved by reversed-phase ion-pair chromatography with linear gradient elution. Application of this method to the analysis of nucleotides in smooth molluscan muscles is described, including the determination of cAMP-levels and the calculation of energy charges for all of the four nucleotide systems.     

Temperature effect in separation of fullerene by high-performance liquid chromatography

Summary The effect of column temperature, especially at low temperatures, on the separation of fullerenes on monomeric and polymeric octadecyl silica (ODS) bonded phases has been studied. Decreasing the column temperature induces an increase in selectivity. The best temperature for the separation of fullerenes was determined for both types of ODS phase with n-hexane eluent. The selectivity for higher fullerenes on monomeric phases becomes similar to that on polymeric phases to low temperature. It has been found that as the carbon content of monomeric phases is increased, the selectivity also becomes similar to polymeric phases.     

Determination of the five major opium alkaloids by reversed-phase high-performance liquid chromatography on a base-deactivated stationary phase

Summary A reversed phase HPLC method for the separation of the five major alkaloids fromPapaver somniferum L., morphine, codeine, thebaine, papaverine and noscapine, has been developed and validated. By use of a basedeactivated silica-based stationary phase excellent peak shape was achieved for each substance. The five alkaloids were quantified by internal standardization within 20 min and with good precision. The method is applicable to opium and to poppy straw.     

On-column endcapping and derivatization in reversed-phase high-performance liquid chromatography

Summary On-column endcapping and derivatization or regeneration of C₈ and C₁₈ reversed-phase HPLC columns with newly introduced reagents were studied. These treatments can increase column life expectancy by restoring retention times and original chromatographic characteristics of the columns. This is illustrated by examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Determination of the curcuminoid pigments in turmeric (Curcuma domestica Val) by reversed-phase high-performance liquid chromatography

Summary A rapid, simple and reproducible reversed-phase high-performance liquid chromatographic method was developed for the quantitative determination of curcumin, demethoxycurcumin and bis-demethoxycurcumin in ethanolic extracts of turmeric. The pigments were separated on a styrene-divinylbenzene copolymer column (Hamilton PRP-1), using an acetonitrile-water (5545, v/v) mobile phase. The pigments were monitored with a diode-array detector at 425 nm. The limit of detection was 10.2 ng curcumin, 11.1 ng demethoxycurcumin and 6.2 ng bis-demethoxycurcumin. Comparison of HPLC and spectrophotometric results for the determination of the total curcuminoid content for a number of turmeric samples, reveal that the spectrophotometric method invariably yielded higher results, indicating an overestimation of curcuminoids.     

Determination of thiopental in human serum and plasma by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic method for the analysis of thiopental in 100l of human serum or plasma is described. The procedure involves protein precipitation with acetonitrile. The supernatant is directly injected into a chromatograph containing a reversed-phase CLC-ODS (Shimadzu) column. A 5050 (v/v) mixture of water-acetonitrile, at a flow-rate of 1.0ml/min is used as the mobile phase. Detection is carried out ata wavelength of 280nm. Total analysis time per sample is 10min. The assay was found to be linear in the range of 0.1 to 120g/ml. Reproducibility was good, with intra-assay coefficients of variation from 1.780 to 3.208% and inter-assay coefficients of variation from 3.241 to 4.860%. The absolute recoveries were 97.4 to 101,4%. Other drugs were tested for potential interference with the assay, but none was found.     

Optimized separation of pesticide enantiomers by chiral high-performance liquid chromatography

Summary A computer-assisted method is described for optimization of multi-component, mobile phase selection for separating enantiomers of four pesticides in normal-phase HPLC. The method is based on the triangle, solvent-selection concept using a statistical scanning method. The optimization of the separation over the experimental region is based on a special polynomial estimation from seven experimental runs, and resolution (R_s) is used as the selection criterion. Excellent agreement was obtained between predicted and experimental data.     

Size-exclusion and high-performance liquid chromatography separation of peptides from peptic haemoglobin hydrolysate obtained by ultrafiltration

Summary Gel filtration (size-exclusion) and high-performance liquid chromatography have been used to separate peptic peptides from haemoglobin hydrolysate. Elution profiles on Sephadex G-25 displayed nine fractions with molecular weights lower than 6500 daltons. Each fraction was analysed for total amino acid content and showed less than 1% free amino acids. Reversed phase HPLC, using ammonium acetate buffer and acetonitrile as solvent, was applied to each fraction in order to obtain pure peptide peaks. The importance of acquiring a better knowledge of such an hydrolysate is discussed. Various potential applications of this type of hydrolysate, some of them already being undertaken, are envisaged.     

Determination of ephedra alkaloids by high-performance liquid chromatography

Summary Two simple methods were developed for the simultaneous determination of six alkaloids (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine and methyl-pseudoephedrine) inEphedrae Herba by high-performance liquid chromatography. The first method was carried out by using a Cosmosil 5C₁₈-MS column with a gradient solvent system consisting of a phosphate buffer and acetonitrile, and detection at 210 nm. The contents of alkaloids in non-pretreated ephedra herb extracts could be determined easily in 50 min. Alternatively, the alkaloids could be determined within 35 minutes by using a Cosmosil 5C₁₈-MS column with an isocratic solvent system of a sodium dodecyl sulfate-acetonitrile solution. The two methods are compared and discussed.     

Study of silica gel and copper silicate gel solubilities in high performance liquid chromatography

Summary After summarizing published results on silica gel and alkyl-bonded silica gel solubilities, the experimental solubility of unmodified silica and copper silicate gel (used in ligand exchange chromatography) in watermethanol-ammonia and water-acetonitrile-ammonia mixtures are given. These results demonstrate that silica gel solubility, measured by a static method, varies greatly with the water volume fraction of a ternary mixture. Curiously, no influence from the organic nature of the solvent was demonstrated. This observation is inconsistent with the fact that the copper silicate gel used in ligand-exchange chromatography with water-methanol-ammonia as mobile phase is attacked more rapidly than with water-acetonitrile-ammonia.An explanation based upon the variations of acid-base properties of silicic acid versus mobile phase dielectric constant values in conjunction with the methanol protophilic properties is proposed. Finally, it is shown that chromatographic columns filled with copper silicate gel have lifetimes of several months, if they are used with a procolumn (of copper silicate gel) and with acetonitrile as organic solvent with a volume fraction greater than 0.5 in the mobile phase.     

Evaluation of octadecyl-bonded alumina for separations of proteins and peptides by reversed-phase high-performance liquid chromatography

Summary A recently-developed octadecyl-bonded alumina (ODA) stationary phase was evaluated for the separation of peptides and proteins by reversed phase high performance liquid chromatography. Using standard water-acetonitrile mobile phase gradients containing 0.1 % trifluoroacetic acid, the average peak capacity obtained for the separation of a mixture of ribonuclease a, cytochrome c, lysozyme and carbonic anhydrase on an ODA column are similar to that obtained on a widely used octadecylsilane (ODS) column. However, overall chromatographic resolution of the components of this mixture on ODA is inferior to that obtained on ODS. Cytochrome c peak areas were found to be 50% smaller on the ODA column than on ODS. On the other hand, both peak capacities and resolutions of octapeptide mixtures were found to be generally superior on the ODA column, and peak areas for a representative octapeptide were found to be virtually identical for both ODA and ODS columns. The differences in the results obtained on the ODA and ODS columns for these separations are attributed to the smaller pore size and unique fused-microplatelet shape of the ODA particles. Comparisons of the separations of the tryptic digest of cytochrome c on the ODS and ODA columns demonstrate that the ODA phase is potentially as useful as ODS for peptide mapping applications.     

High-performance liquid chromatography of sugars on copper (II) modified silica gel

Summary A sensitive method of sugar analysis by HPLC is described in which a copper (II) modified silica gel as stationary phase is used. Detection is based on UV absorption of a complex formed between the sugar, copper, and ammonia.     

Purification of CIS-trimedlure isomers by high-performance liquid chromatography

Summary High-performance liquid chromatographic procedures were developed to make it possible to obtain the fourcis-trimedlure isomers (V, W, X, and Y) in pure form. Trimedlure-V and-Y were each readilt separated from the four-componentcis-trimedlure mixture through high-performance liquid chromatographic analysis on 5-μm and 3-μm silica, but trimedlure-W and-X were not adequately resolved. Chromatography of 5-μm silica of the mixtures obtained by epimerization of thetrans-trimedlure isomers, C and B₂, yielded the respective epimers, trimedlure-W and-X, in pure form.     

Retention behavior of dipeptide isomers in reversed-phase high-performance liquid chromatography

Summary The effects of eluent pH and organic modifier concentration on the capacity factor (k) and selectivity of dipeptide isomers were investigated. It has been observed that the variation in the logarithm of the capacity factor of the dipeptide isomers is linearly dependent on the organic modifier concentration (C_b), however, the selectivity is almost independent of it. Both capacity factor and selectivity were seriously affected by the pH of the eluent. Both the capacity factor and the intercept of the ln k vs. C_b plot increased with increasing van der Waals volume of the non-polar amino acid subunit of the dipeptides.     

Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography

Summary A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to analyze various fatty acids in recombinant mouse L cells. These fatty acids were the metabolites of oleic acid. A process was developed to extract fatty acids from the cell samples before RP-HPLC analysis. The samples were first saponified with 0.5 M NaOH in 96% ethanol then extracted with acidified ethyl acetate. After extraction, the sample was dried and dissolved in HPLC-grade methanol. After centrifugation to remove insoluble impurities, the sample was applied to a C₁₈RP-HPLC column using a gradient of acetonitrile (ACN)-H₂O. The eluted fatty acids were monitored by ultraviolet (UV) absorption at 195 nm and identified by retention time and adsorption spectrum comparison. This method successfully resolved various fatty acids and provided a tool for the elucidation of the fatty acid metabolic pathway in the cells.