Takenori Tanimura

Direct enantiomeric separation of β-amino acids and β-amino alcohols by ligand-exchange chromatography

Three underivatized β-amino acids and various aromatic β-amino alcohols were separated into enantiomers by high-performance liquid chromatography using octadecylsilanized silica coated with N-n-dodecyl-l-hydroxyproline as the stationary phase and acetate buffer containing copper(II) as the mobile phase.

Resolution of DL-Valine by Countercurrent Solvent Extraction with Continuous Sample Feeding

Abstract DL-Valine was resolved completely by a solvent extraction system with a continuous sample feeding process called continuous countercurrent fractional extraction (CFE). The two-phase system contained n-butanol and water with a copper(II) complex of N-n-dodecyl-L-hydroxproline as a resolving reagent. The upper (alcohol-rich) phase and the lower (water-rich) phase proceeded in the segmented extraction columns countercurrently; the D-isomer was recovered in the upper phase, while the L-isomer was recovered in the lower phase. The optical purity of the enantiomers obtained was consistently 99.5% or higher. The D-isomer extracted in the upper phase was recovered completely by using a backextraction column in series after the extraction column, allowing the upper phase to be used repeatedly for the resolution. The efficiency of the CFE system was estimated at more than 24 theoretical stages.

A highly efficient carboxylic acid analyzer and its application

A new highly efficient carboxylic acid analyzer, employed with liquid chromatography and a specific detection method, has been designed for the quantitative analysis of carboxylic acids in foods and biological samples. This apparatus is particularly advanced in terms of its sensitivity and resolution as a result of the extensive modifications to our previous system. The applications of this analyzer to the analyses of juices, beer, human serum and urine are presented.

Quantification of the isomerization of Asp residue in recombinant human αA-crystallin by reversed-phase HPLC

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.

Spectrophotometric Determination of Carboxylic acids with 2-Mitrophenylhydrazine in Aqueous Solution

Abstract Carboxylic acids were determined spectrophotometrically by converting them into 2-nitrophenylhydrazides. The coupling reaction of carboxylic acids with 2-nitrophenylhydrazine was effected by a water-soluble carbociimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and proceeded efficiently in aqueous solution buffered at pH 4.5 with pyridine and hydrochloric acid. The yields of hydrazides from acetic and benzoic acids were nearly quantitative. The reaction mixture was then made strongly alkaline, and after heating to reduce the blank absorbance, the absorbance of the hydrazide was measured at around 540 nm. Calibration curves of various carboxylic acids were linear between 0.02 and 0.5 μmol through the origin.

Retention behaviour of a porous styrene-divinylbenzene copolymer in the separation of fatty acids and related compounds

Abstract Using a porous styrene-divinylbenzene copolymer for the chromatographic separation of straight-chain fatty acids, we found the following properties: (1) a linear relationship between the logarithm of the capacity factor and the number of methylene groups in the alkyl chain, (2) almost identical slopes of the lines for three groups of fatty acid analogues and benzoic acid esters and (3) a linear relationship between the slope and the concentration of methanol in the mobile phase.

Spectrophotometry Determination of Carboxylic Acids by Ferric Hydroxamate Formation with Water-Soluble Carbodiimide

Abstract Carboxylic acids in aqueous solution were determined spectrophotometrically as ferric hydroxamates. A water-soluble carbodiimide, l-cyclohexyl-3-(2-morpho-linyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate, was used as a cuopling agent to form hydroxamic acids. The coupling reaction was carried out at 37°C for 30 min in a solution buffered at pH 5.3 with pyridine and hydrochloric acid. Calibration curves of benzoic, acetic, citric, and a-ketoglutaric acids were linear between 0.25 and 8 μmol.

A RAPID HPLC DETERMINATION OF THE ISOMERIZATION LEVEL OF ASP-RESIDUES WITHIN SYNTHETICα-A-CRYSTALLIN FRAGMENTS

ABSTRACT We describe a rapid HPLC method for measuring the isomerization levels of Asp-residues within peptides. This method can detect the isomerization of Asp-residue at a level of less than 1% without performance of acid hydrolysis. Using this method, we further studied the isomerization of Asp-residues in the αA-crystallin f(55–65) fragment of sequence TVLDSGISEVR (T6). Four isomers of T6 peptides, which contain L-Asp, L-isoAsp, D-Asp, and D-isoAsp, respectively, were synthesized. Following incubation of any one of these peptides, four possible isomers of T6 peptides were found in an aqueous buffer. The isoAsp/ Asp ratios of peptides in these solutions were found to reach 3.6–3.8 after incubation at 90°C for 72 hr.

Flow injection determination of drugs by specific detection of carboxylic acids

The spectrophotometric determination of various drugs having a carboxyl function is described. The method involves flow injection and specific detection of carboxylic acids based on the formation of 2-nitrophenyl-hydrazide derivatives mediated by a water-soluble carbodiimide. Using this system, the linear calibration range spanned three orders of magnitude from 125 pmol to 200 nmol and both hydrophilic and hydrophobic drugs could be determined easily at a sampling rate of 100 per hour. A number of drug preparations were also analysed with a coefficient of variation of less than 1%(n= 10).

Chiral Separation of Carboxymethyl Derivatives of Amines by Liquid Chromatography on Reversed-phase Silica Gel Coated with N-n-Dodecyl-L-hydroxyproline

Carboxymethyl derivatives of amines containing a chiral α-carbon were separated into enantiomers by high-performance liquid chromatography using octadecylsilanized silica gel coated with N-n-dodecyl-L-hydroxyproline as the stationary phase and an aqueous solution containing copper(II) as the mobile phase. Detection was by post-column reaction involving derivatization to tertiary amine and chemiluminescence reaction of ruthenium bipyridine complex.