Myung Ho Hyun

Liquid chromatographic resolution of racemic amino acids and their derivatives on a new chiral stationary phase based on crown ether

Abstract A new high-performance liquid chromatography chiral stationary phase (CSP) was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel. The new CSP was employed in separating the two enantionmers of various natural and unnatural racemic α-amino acids and their derivatives. All natural and unnatural racemic α-amino acids tested were resolved with reasonable separation factors on the new CSP, except for proline, which does not contain a primary amino group. Racemic α-amino acid derivatives were also tested for their separability on the new CSP. In general, N-monoalkyl amides of α-amino acids were resolved better than the corresponding free α-amino acids. However, N,N-dialkyl amides and esters of α-amino acids were not resolved as well as the corresponding free α-amino acids, except for phenylglycine derivatives. In the case of phenylglycine, both N-monoalkyl and N,N-dialkyl amides of phenylglycine were resolved better than phenylglycine itself and its ester derivative.

Liquid chromatographic resolution of racemic amines and amino alcohols on a chiral stationary phase derived from crown ether

Abstract A chiral stationary phase (CSP) prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel was successfully employed in resolving various racemic amines and amino alcohols including therapeutically active compounds such as amphetamine, phenylethanolamine, octopamine and norepinephrine. Related compounds such as α-amino carbonyl compounds were also resolved on the CSP. In order to see the effect of organic and acidic modifiers in the mobile phase and the temperature of the column on the enantioselectivity exerted by the CSP, four racemic compounds which resolved well on the CSP were selected and their resolutions were investigated with the variation of organic and acidic modifiers in the mobile phase and the temperature of the column. From the results, the chromatographic factors such as capacity factors ( k ′), separation factors ( α ) and resolution factors ( R s ) were expected to be controlled to some extent by varying organic and acidic modifiers in the mobile phase and the temperature of the column.

Optimization of Phosphatase- and Redox Cycling-Based Immunosensors and Its Application to Ultrasensitive Detection of Troponin I

The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium-tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl(2), and the optimum conditions for antigen-antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen-antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (L-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (L-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.

Separation of the stereoisomers of racemic fluoroquinolone antibacterial agents on a crown-ether-based chiral HPLC stationary phase

SummaryA chiral stationary phase derived from (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid has been successfully used for the direct separation of the enantiomers of recemic fluoroquinolines containing a primary amino group being investigated as antibacterial agents. The chromatographic resolution behavior was found to depend on the content and the type of acidic and organic modifiers in the mobile phase and on the column temperature.

Liquid chromatographic direct resolution of β-amino acids on a chiral crown ether stationary phase

A chiral stationary phase (CSP) prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel was used for the direct resolution of β-amino acids. To determine the optimum mobile phase composition, two β-amino acids were selected and their resolutions on the CSP were performed with variation of the types and contents of organic and acidic modifiers in the aqueous mobile phase. The chromatographic resolution behaviors were found to be dependent on the types and contents of organic and acidic modifiers in the aqueous mobile phase. From the results of these experiments, a possible optimum mobile phase composition was proposed to be a mixed solvent of 50% methanol in water containing acetic acid (10 mM). The chromatographic resolution behaviors were also found to be dependent on the column temperature, the retention (k1) and the separation factors (α) being greater at lower temperatures. At the optimum mobile phase composition, all β-amino acids tested in this study were resolved quite well on the CSP with baseline separation.

New ligand exchange chiral stationary phase for the liquid chromatographic resolution of α- and β-amino acids

Abstract A new ligand exchange chiral stationary phase (CSP) has been developed by covalently bonding ( R )- N , N -carboxymethyl undecyl phenylglycinol mono-sodium salt onto silica gel and applied in the resolution of α- and β-amino acids. In the resolution of α-amino acids, the new CSP was better in some cases than the old one, which was previously developed by covalently bonding ( S )- N , N -carboxymethyl undecyl leucinol mono-sodium salt onto silica gel, but worse in some other cases than the old one in terms of the separation factors ( α ). However, the new CSP was always much better than the old one in terms of the resolution factors ( R s ). In the resolution of β-amino acids, the new CSP was always much better than the old one in terms of both the separation and resolution factors. In an effort to characterize the new CSP, the chromatographic behaviors for the resolution of selected α- and β-amino acids were investigated with the variation of the content of organic modifier and Cu(II) concentration in aqueous mobile phase and the column temperature.

Liquid Chromatographic Chiral Separations by Crown Ether‐Based Chiral Stationary Phases

Abstract Liquid chromatographic chiral stationary phases (CSPs) based on chiral crown ethers have been successfully utilized in the separation of the enantiomers of various racemic compounds containing a primary amino group. Especially, CSPs based on chiral crown ethers incorporating chiral binaphthyl unit or tartaric acid unit have been most successful. In this review, the development of CSPs based on chiral crown ethers incorporating a chiral binaphthyl unit or a tartaric acid unit, their applications to the resolution of various primary and non‐primary amino compounds with the variation of the type and the content of organic, acidic, and inorganic modifiers in an aqueous mobile phase or with a non‐aqueous mobile phase and the efforts to improve the chiral recognition efficiency or the stability of the CSPs have been discussed.

Preparation and application of a new ligand exchange chiral stationary phase for the liquid chromatographic resolution of α-amino acid enantiomers

A new liquid chromatographic ligand exchange CSP has been prepared by covalently bonding (S)-N,N-carboxymethyl undecyl leucinol monosodium salt onto silica gel and employed in resolving various alpha-amino acids. The new CSP was quite good in resolving various a-amino acids and the resolution results were dependent on the type and content of organic modifier in the mobile phase. From these results, a chiral recognition model using a lipophilic interaction between the tethering alkyl group of the CSP and the substituent at the chiral center of alpha-amino acids was proposed. The liquid chromatographic resolution of alpha-amino acids on the new CSP was also found to be dependent on the Cu(II) concentration in the mobile phase and the column temperature.

Preparation and evaluation of a chiral stationary phase bearing both π-acidic and -basic sites

Abstract A new chiral stationary phase has been prepared evaluated. It contains both π-acidic -basic sites. When used to chromatograph racemic analytes which also contain π-acidic -basic sites, two simultaneous π-π interactions occur. The impact of such interactions on the chromatographic behavior of the enantiomers of several homologous series of analytes was determined is explained in terms of competing chiral recognition mechanisms.

Preparation and application of a new modified liquid chromatographic chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

As an effort to improve the chiral recognition efficiency of a previously reported chiral stationary phase (CSP) based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid, a new CSP was prepared by simply replacing the amide N-H hydrogens of the tethering groups of the old CSP with methyl groups. The new CSP was superior to the old one in the resolution of racemic primary amines. However, in the resolution of alpha-amino acids and amino alcohols, the new and the old CSPs were complementary with each other. The elution orders on the new CSP were sometimes opposite to those on the old one. Consequently, the chiral recognition mechanism on the new CSP was presumed to be different from that on the old one. The chiral recognition behavior of the new CSP were investigated with four selected analytes and found to be dependent to some extent on the content of organic and acidic modifiers in aqueous mobile phase and the column temperature.