Modified aluminas as chromatographic supports for high-performance liquid chromatography

This review begins by describing the relevant properties of alumina as a support material for chemically bonded stationary phases in HPLC. The most common chemical modification processes are summarized as well as the advantages and disadvantages of each method. In order to more fully understand the chemically modified alumina surface, some spectroscopic approaches are outlined for characterization of the bonded phases. Finally, a number of successful applications are described for a variety of chemically modified aluminas in order to illustrate their potential usefulness and to compare their chromatographic behavior to the more conventional silica-based materials.     

Sample preparation for high-performance liquid chromatography in the clinical laboratory

Techniques for the preparation of clinical specimens for high-performance liquid chromatography are described. Liquid samples containing high enough concentrations of analytes may be injected directly or after centrifugation. Proteins may be removed by precipitation with organic, anionic or cationic precipitants or by ultrafiltration. Compounds having large partition coefficients may be extracted with an organic solvent. Extraction may also be performed following derivatization, ion-suppression, ion-pairing, salt addition or complex formation. Solid phase extraction may be off-line using disposable cartridges or on-line with an Advanced Automated Sample Processor. The advantages and disadvantages of each method and the trends in sample preparation techniques are discussed.     

Purification of synthetic oligodeoxyribonucleotides by ion-exchange high-performance liquid chromatography

Synthetic oligodeoxyribonucleotides ranging from 11 to 37 nucleotides in length and with varying base compositions, prepared by both the phosphotriester and phosphite procedures, have been purified by ion-exchange high-performance liquid chromatography on Whatman Partisil 10/SAX columns using phosphate buffer gradients. The effects of different buffer systems on elution times and resolution have been evaluated. Oligomer composition and length had a marked effect on the resolution achieved. In general the use of formamide buffers gave the best results, particularly in the case of 2'-deoxyguanosine-rich sequences. These methods have also been successfully applied to the purification of mixtures of synthetic oligodeoxynucleotides.     

Polymeric supports for affinity chromatography and high-performance affinity chromatography

Affinity chromatography is the most selective chromatographic method for the purification of biologically active materials. It is based on the biospecific interaction of the substrates with a ligand, which is chemically immobilized onto a suitable matrix (support). Different matrices provided by natural and synthetic polymers are used for the preparation of affinity supports. In this communication we describe and compare the properties of various supports based on polysaccharides, polyacrylamides and inorganic materials. In particular, we discuss the utility of different silica derivatives (especially primary hydroxyl silica) for the immobilization of ligands and high-performance affinity chromatography.     

Monitoring the quality of the preparation allapinin by high-performance liquid chromatography

Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 129–130, January–February, 1990.     

Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides

Biologically active peptides synthesized by the solid phase methodology of Merrifield were purified by reversed-phase high-performance liquid chromatography using newly developed preparative radially compressed cartridges fitting Waters Assoc . Prep LC 500 liquid chromatograph. Cartridges were handpacked with Vydac C18, C4 or diphenyl derivatized silicas (pore size 300 A) of different particle sizes (10-20 micron). Large scale purification of gram amounts of gonadotropin releasing hormone analogs (agonist and antagonist) as well as amidated human pancreatic tumor growth hormone releasing factor (a 40-peptide) illustrate the resolutive power of this technique applied to the isolation of more than 300 synthetic peptides in our laboratory over the last two years. Difficult separations were achieved by changing supports (C18, C4, diphenyl) as well as mobile phase composition: (triethylammonium phosphate pH 2.25 or 6.5, 0.1% trifluoroacetic acid, ammonium acetate pH 6.5 and acetonitrile). Protected amino acids and peptides amenable to normal-phase chromatography on Vydac spherical underivatized silica were purified economically by the reversed-phase mode. It is understood that this general, convenient and versatile strategy may be applicable to the preparative scale isolation of any other class of compounds usually separated on reversed-phase high-performance liquid chromatography.     

Synthesis and characterization of novel internal surface reversed-phase silica supports for high-performance liquid chromatography

An original synthetic method was developed for the preparation of a family of six novel deactivated restricted-access materials (RAMs), belonging to the group of the internal surface reversed-phase (ISRP) supports. The supports (ISRP-RAM phases A-F) have an alkyl-chain (14 methylenes) with two embedded ureido groups bound only to the internal surfaces of the porous silica, and polyvinyl alcoholic groups (PVA, 100,000-->22,000 molecular weight) chemically bound to the external surfaces. The average pore diameters of the prepared ISRP-RAM supports, calculated by inverse size-exclusion chromatography, ranged between 49 A and 88 A, and were able to exclude macromolecules heavier than about 24000 Da (such as serum proteins) from the pores. The novel supports were designed for the determination of a semi-synthetic anticancer drug of the camptothecin family in human plasma, but they represent universal ISRP-RAM supports not limited to such class of compounds.     

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.     

Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography

This review deals with HPLC method to be used for the determination of synthetic phenolic antioxidants added to various foods. Sample preparation, isolation techniques, separation systems as well as detection methods used in applied food analysis procedures are discussed.     

Automated normal-phase preparative high-performance liquid chromatography as a substitute for flash chromatography in the synthetic research laboratory

An automated normal-phase preparative HPLC system was developed in order to omit time-consuming flash column chromatography in the synthetic research laboratory. The system is equipped with steel columns packed with spherical 12 microm silica and is able to separate samples in a range of 0.1-10 g depending on the column diameter and chromatographic problem. It was designed to be used as an open access instrument in the research department. The general users select from binary gradient programs after running an analytical TLC with the raw product. The HPLC instrument was fully controlled by the Chromeleon software from Dionex. A Gilson 215 robot served as injector/collector.     

New application of high-performance liquid chromatography for analysis of urinary C-peptide

A successful application of high-performance liquid chromatography for analysis of urinary C-peptide is described. Samples (1.0 ml of human urine) were first subjected to gel chromatography to remove interfering substances, and then applied to a reversed-phase column (LiChrosorb RP-18, 7 micron). The detection of C-peptide was performed using a highly specific radioimmunoassay. With the newly developed techniques, at least four forms of immunoreactive C-peptide were detected in human urine. One of these peptides was indistinguishable from authentic C-peptide. The present study has clearly demonstrated the heterogeneity of urinary C-peptide.     

Thermotropic laterally attached liquid crystalline polymers: I. New stationary phases for high-performance liquid chromatography

Up to now thermotropic liquid crystalline side chain polymers have been seldom used as stationary phases in high-performance liquid chromatography (HPLC). The preparation of a new class of surface modified silica gels is reported. They are obtained by coating on the silica support liquid crystalline polysiloxanes with mesogenic side groups laterally attached to the polymer backbone through a flexible spacer. Their chromatographic behavior in reversed-phase HPLC is described for the separation of polycyclic aromatic hydrocarbons. The results show excellent planarity and rod shape recognition capabilities. Comparisons with low-molecular-mass liquid crystalline-bonded silica and longitudinally attached liquid crystalline polymer-coated stationary phase are also reported. Finally, comparisons to commercially available C₁₈ phases are described for the separation of complex mixtures.     

Nucleotide preparation from cells and determination of nucleotides by ion-pair high-performance liquid chromatography.

Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.     

Quantitative analysis of the Maksikold multicomponent anticatarrhal preparation by pH-gradient high-performance liquid chromatography

A procedure was developed for the rapid analysis of a new multicomponent anticatarrhal medication Maksikold by high-performance liquid chromatography (HPLC). The possibility of the simultaneous determination of all active substances in the preparation, including ascorbic acid, is an advantage of the proposed procedure. For the efficient resolution of the peaks of analytes and interfering additives, a mobile phase with the pH varied in the course of an experimental run was used. The procedure was used to analyze pilot samples of the preparation. The results obtained exhibit a high precision.     

Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulphamonomethoxine (SMMX), sulphadimethoxine (SDMX), sulphisozole (SIZ), nalidixic acid (NA), oxolinic acid (OXA), piromidic acid (PMA), furazolidone (FZ) and sodium nifurstyrenate (NFSA) in cultured fish was developed. The drugs were extracted with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on an Inertsil ODS column (150 x 4.6 mm I.D.) using 5 mM aqueous oxalic acid-acetonitrile (55:45) as the mobile phase with detection at 265 nm (0.04 a.u.f.s.). The calibration graphs were rectilinear from 1 to 20 ng for OXA, from 2 to 50 ng for SMMX, SDMX, SIZ, NA, PMA and FZ and from 5 to 100 ng for NFSA. The recoveries of each drug added to fish were 65.0-89.5%. The detection limits were 0.02 micrograms/g for OXA, 0.05 micrograms/g for SMMX, SDMX, SIZ, NA, PMA and FZ and 0.1 micrograms/g for NFSA.     

The preparation and evaluation of superior bonded phases for reversed-phase,high-performance liquid chromatography

Summary The preparation of efficient chemically bonded phases based upon interaction of silica gel with trichlorosilanes is reported. Chlorinated solvents are found to yield superior products whilst reaction at elevated temperatures is shown to be unnecessary. A new capping procedure, involving methanolysis and subsequent reaction with trimethylchlorosilane, is shown to be more effective than existing procedures.