Yuzo Yanagihara

Development of a c18-bonded vinyl alcohol copolymer gel for reversed-phase high-performance liquid chromatography

Abstract The fundamental characteristics of a polymer-based C18 gel developed for reversed-phase high-performance liquid chromatography were investigated. The octadecyl polymer (ODP) gel obtained by introduction of octadecyl groups in place of the hydroxy groups of vinyl alcohol copolymers was packed into a 150 mm x 6 mm I.D. column and the performance was compared with that of commercial polystyrene and ODS gels. The ODP gel displayed excellent tolerance towards acidic, alkaline and buffered eluents, with little of the shrinkage and swelling shown by conventional polystyrene and modified polystyrene gels with varying eluent polarity, thus making it appropriate for a wide range of eluents, including water, ethanol and acetonitrile. The ODP column exhibited a theoretical plate number with alkyl alcohol standards that was higher than those of most ODS columns, and showed none of the adsorption and peak tailing that tend to occur with basic substances on ODS columns, owing to the complete absence of residual silanol groups in the ODP gel.

Determination of free catecholamines in human urine by direct injection of urine into a liquid chromatographic column-switching system with fluorimetric detection

An ion-exchange chromatographic method combined with ion exclusion was developed for the determination of free catecholamines in human urine. Catecholamines were separated by ion exclusion from most acidic and neutral impurities by filtration through an anion-exchange column with a hydrophilic matrix (Asahipak ES-502N) and the excluded catecholamines were separated by ion-exchange chromatography on a column of weakly acidic ion exchanger with a hydrophilic matrix (Asahipak ES-502C), connected in series to the Asahipak ES-502N column with a four-way automatic valve. A sodium succinate-borate buffer of pH 6.7 (0.035 mol of succinic acid, 0.0075 mol of borate and 0.5 mmol of ethylenediaminetetraacetate were dissolved in 1 kg of water and the pH of the solution was adjusted to 6.7 with sodium hydroxide) was used as the mobile phase, and the temperature of both columns was kept at 30 degrees C. The catecholamines in the eluate were determined fluorimetrically by post-column derivatization with glycylglycine. A diluted urine sample was injected directly onto the first column. The first column was back-flushed with the mobile phase for 52.5 min after the elution of the catecholamines from the first to the second column. Then the columns were washed with the mobile phase for 10 min in the normal direction before the next sample was injected into the first column. Samples could be analysed every 70 min and 5 pmol/ml of epinephrine, 5 pmol/ml of norepinephrine and 25 pmol/ml of dopamine in human urine could be determined.

Chromatographic properties of a vinyl alcohol copolymer gel column for the analysis of non-ionic surfactants

Abstract A high-performance size-exclusion chromatographic column packed with Asahipak GS-310 vinyl alcohol copolymer gel was investigated for its applicability to the analysis of non-ionic surfactants with mobile phases containing water and aceto-nitrile or other organic solvents in various ratios. The surfactant series consisted of R(OCH2CH2)nOH, where R is an alkylaryl group and n = 1–100. In the water-rich region, with acetonitrile concentrations of 0–30%, surfactants either were not eluted or their elution was extremely delayed, indicating a hydrophobic interaction between the gel and the alkylaryl group in the surfactant. In the acetonitrile-rich region, with acetonitrile concentrations of 60–100%, the surfactants were eluted relatively rapidly in order of decreasing molecular weight, without a fine separation between compounds with different n values. These results indicate that the organic solvent effetively inhibited the hydrophobic interaction and that size exclusion was the predominant separation mechanism. In the intermediate region, with 30–60% acetonitrile concentrations, the surfactants were eluted in the same order as for the acetonitrile-rich region, but less rapidly and with a fine separation between n values. The results indicate that the separation mechanism is mainly hydrophobic interaction between the alkylaryl groups and the gel, the hydrophobicity of the gel being too weak for effective interaction with the oxyethylene groups. The results indicate that the Asahipak GS-310 hydrophilic polymer gel column can be effectively employed for the practical, efficient high-performance liquid chromatographic analysis of non-ionic surfactants.

Estimation of catecholamines by ion-exchange chromatography on Asahipak ES-502C, using glycylglycine as the post-derivatizing agent.

The estimation of catecholamines in human urine was carried out by ion-exchange chromatography on a column of a weakly acidic ion exchanger with an hydrophilic matrix. The catecholamines were first adsorbed onto Amberlite CG-50 (buffered at pH 6.5 with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid solution. They were then separated from impurities that responded to fluorometric detection by isocratic elution from a column of Asahipak ES-502C, a cross-linked vinyl alcohol copolymer with carboxymethyl groups, at 60 degrees C. The mobile phase was 0.05 M sodium succinate buffer pH 5.25 containing 0.015 M borate and 0.5 mM ethylenediaminetetraacetate. Isoproterenol was used as the internal standard; epinephrine, norepinephrine, isoproterenol and dopamine were eluted in this order. One sample could be analyzed every 35 min. The detection limits were 0.2 ng for epinephrine and norepinephrine, 0.6 ng for dopamine. The elution pattern was quite reproducible; the elution volumes of the catecholamines had not changed after 500 determinations.

High-performance liquid chromatographic analysis of peptides on an asahipak gs-320 column packed with hydrophilic polymer gel

Abstract Investigation of the chromatographic characteristics of various amino acids and peptides on an Asahipak GS-320 column, developed for high-performance gel filtration chromatography, revealed that these substances, which possess hydrophobic sites, are retained by adsorption and eluted in order of increasing hydrophobicity. The use of organic solvents as eluents was studied and practical separations of amino acids and peptides by isocratic elution were obtained.

New, stable polyamine-bonded polymer gel column

Abstract The Asahipak NH2P-50 columm, packed with polyamine-bonded polymer gel, was investigated in terms of mono- and oligosaccharide separation efficiency, reproducibility and column durabibility. Elution consistently occurred in the order mono-, di- and trisaccharide, and elution volume increased with increasing acetonitrile concentration. Chromatograms obtained over a period of one week of some 170 saccharide analyses on a single NH2P-50 column were virtually indistinguishable, while under the same conditions the efficiency of an amino-bonded silica gel column steadily decreased. The NH2P-50 column was amenable to both acidic and alkaline eluents, with no decrease in column efficiency after extended alternating passage of acidic and alkaline solutions. It also exhibited high separation efficiency with eluent containing tetrapropylammonium hydroxide and acetic acid solution.

Characteristics of C4- and C8-bonded vinyl alcohol copolymer gels for reversed-phase high-performance liquid chromatography

Abstract The fundamental characteristics of C 4 - and C 8 -bonded polymer (C4P and C8P) gels developed for reversed-phase high-performance liquid chromatography and obtained by introduction of butyryl and octanoyl groups, respectively, at the hydroxyl groups of vinyl alcohol copolymers were investigated and compared with the characteristics of a previously developed C 18 -bonded polymer (ODP) gel obtained in a similarf manner and with those of commercial C 4 -, C 8 - and C 18 -bonded silica gels. For both alkyl alcohols and standard proteins, the retention strength on the polymer gels clearly increased with increasing number of carbons in the bonded alkyl group and thus in the order C4P, C8P, ODP. The C4P and C8P gels, like the ODP gel and in clear contrast to the silica gels, displayed excellent tolerance toward acidic, alkaline and buffered eluents. They also exhibited minimal shrinking and swelling effects with variations in eluent polarity, as measured by their solvent regain.

Amphipathic character of hydrophilic polymer gel columns

Abstract An investigation of the Asahipak GS series of aqueous high-performance liquid chromatographic columns, particularly of the characteristics of the gels in organic solvents, showed that two of the columns, GS-310 and GS-510, are amenable to chromatographic analysis with both hydrophilic and non-polar organic solvents, such as methanol and chloroform, as the gels swell more in organic solvents than in distilled water. Experiments confirmed that it is possible to perform gel permeation chromatographic analysis of polystyrene in the organic mobile phase on these two columns, as well as of water-soluble polymers in the aqueous mobile phase.

Separation of ascorbic acid, dehydroascorbic acid, diketogulonic acid and glucose by isocratic elution from a column of a hydrophilic gel

High-performance liquid chromatography on an Asahipak GS-320 hydrophilic gel column with tartrate buffer (0.015 M, pH 3.0) containing 2 mM ethylenediaminetetraacetate and 0.05% beta-thiodiglycol as the eluent allowed the separation of glucose, diketogulonic acid, dehydroascorbic acid and ascorbic acid within 30 min. Fluorimetric monitoring of these compounds in the eluate with benzamidine at alkaline pH and at 90 degrees C in the presence of potassium sulphite allowed the determination of nanogram amounts of ascorbic acid, dehydroascorbic acid and diketogulonic acid. This method was applied to the determination of ascorbic acid in fruit juice.

Peptide behaviour and analysis on a chemically stable C18-bonded vinyl alcohol copolymer column with alkaline and acidic eluents

C18-bonded vinyl alcohol copolymer (ODP) gel showed no weight loss or decrease in column efficiency for alkyl alcohols after being immersed in aqueous solutions of pH 2 and 10 at 50 degrees C for 48 h, and only a 1% weight loss and a slight decrease in alkyl alcohol retention volumes after similar immersion at pH 13. The chemical stability of the ODP gel was further demonstrated in analyses of acidic, neutral and basic peptides on an ODP column with eluents of pH 3-10, which showed that the peptides differ considerably in the sensitivity of their retention behaviour to eluent pH, even though hydrophobic interaction invariably appeared to be the main retention mechanism. The ODP column was therefore applied to the analysis and alignment of lysilendopeptidase (LEP) peptides derived from reduced and S-carboxymethylated carboxyl proteinase (Rcm-P-CP) of Pseudomonas sp. 101. One acid-soluble and seven alkaline-soluble LEP peaks were found in analyses on the ODP column using acidic and alkaline eluents, respectively. The chymotryptic peptides of Rcm-P-CP were first separated on an ODS column with an acidic eluent, and the eight eluates which contained lysine residue, as determined by amino acid analysis, were then analysed on the ODP column with an alkaline eluent, resulting in a further separation of each into several peaks and thus in the recovery of fractions of pure peptides. The LEP peptide alignment was then determined by overlapping the sequences of the chymotryptic peptides with the C- and N-terminal regions of the LEP peptides.