Synthesis and function of helical polyphenylacetylenes are briefly reviewed. Optically active helical polyphenylacetylenes were synthesized by polymerization of the corresponding chiral phenylacetylene monomers and their chiral recognition abilities were evaluated as a chiral stationary phase for HPLC. The polyphenylacetylenes showed high chiral recognition and could resolve some racemic compounds. Conceptually new helical polyphenylacetylenes were found to be induced on optically inactive polyphenylacetylenes bearing functional groups upon complexation with optically active small molecules capable of interacting with the polymers. The complexes exhibited a characteristic induced circular dichroism in the UV-visible region in organic solvents as well as in water, which could be used to determine the absolute configuration of chiral molecules. A unique helical polyacetylene bearing a β-cyclodextrin as the side group that exhibits the inversion of macromolecular helicity accompanied with a color change is also described.
Miniaturized extraction and separation media have been successfully developed from precisely controlled technologies. In this article, recent developments in these high performance analytical methods, such as miniaturized sample preparation methods and the coupling of these techniques with microscale separation systems, have been reviewed, along with some applications to environmental and biological analysis. The advantage of the miniaturization is not only for the environmental compatibility but also for the developments of the high performance analytical systems. Down-sizing also makes it possible to investigate and introduce various compounds and materials as novel media (such as tailor-made materials and devices) in separation science. As a typical example of the novel miniaturized sample preparation system, the applications of fibrous materials for microcolumn liquid-phase separation methods are described.
This review summarizes recent developments in the field of enantiomer separation by capillary electrochromatography using fritless packed columns. Various enantiomers have been separated by employing fritless packed columns prepared in a fused silica capillary either by the immobilization of chiral packing materials by sintering or sol-gel technology or by in situ polymerization of a mixture containing chiral selectors. The details of the column preparation procedures and the attainable column performance are described.
The enantioselectivity of HPLC chiral stationary phases produced with human serum albumin (HSA) fragments was investigated. An HSA fragment (HSA-FG75) was isolated by size-exclusion chromatography following peptic digestion of HSA. The isolated HSA-FG75 was mainly an N-terminal half peptide with an average molecular weight of about 35000 daltons. The HSA and HSA-FG75 proteins were bound to aminopropylsilica gels activated by N,N′-disuccinimidyl carbonate. Though the HSA-FG75 column showed lower enantioselectivities for all of the racemates tested than the intact HSA column, the enantioseparations of the racemates tested were attained with a shorter analysis time on the HSA-FG75 column. These results are ascribable to removal of the non-specific binding sites of HSA, changes in the globular structure of the HSA fragment and/or changes in the local environment around the binding sites. Further, the HSA-FG75 column was as stable as the intact HSA column for repetitive injection of samples.
Cyclodextrin (CD) polymers were synthesized from the reaction of native CDs with a hexamethylene diisocyanate (HDI) compound in a dried DMF solution. The obtained CD polymer contained a range of 8 - 14% N due to HDI by elemental analysis. The physical and chemical properties of the CD polymers were characterized by IR, solid state 13C NMR, TGA, and DSC, respectively. An HPLC column was prepared using the CD polymer with a carbamate linker by a slurry method. Separation of the phenol isomers was conducted using the CD polymer stationary phase and CD by HPLC. Furthermore, an inclusion complex of the phenol isomer was studied by FT-Raman spectroscopy. From the HPLC and FT-Raman results, inclusion phenomena of o-, m-, and p-nitrophenol onto CD and CD polymers were analyzed.
The imprinted polymers based on a transient complex formation between methacrylic acid and template molecules were prepared by using methacrylic acid and ethylene dimethacrylate as a cross-linking agent. The template molecules used were (R,R)-cyclohexanediamine (1), (S,S)-1,2-diphenylethylenediamine (2) and (S)-1,1′-binaphthyl-2,2′-diamine (3). Another group of templates were those in which the amino group of these templates had been substituted by the hydroxy group: (R,R)-1,2-cyclohexanediol (4) and (S,S)-hydrobenzoin (5). Racemic 2 was separated by the polymer prepared with template 2 (P2) and that with template 1 (P1). Template 2 is larger than template 1 in steric bulkiness, but P1 was effective for the enantiomer separation of racemic 2. P1 was not effective for the separation of racemic 4. Enantioselectivity observed in racemic 2 in P2 was higher than that in racemic 1 in P1. P2 has no definite predetermined shape for solute 1, but it was capable of separating racemic 1. This separation should be thus ascribed to the orientation of at least two carbonyl groups reflecting the conformation of template 2 in P2 cavity. Racemic 5, having the same configuration of the two bulky phenyl groups as that of solute 2, was separated in P2. When the primary amines such as propylamine, cyclohexylamine and 1-adamantanamine were added into the acetic acid-methanol mixures as eluents, both enantioselectivity and retentivity for racemic 2 were enhanced along with the remarkable peak tailing.
A convenient “one step” preparation process of molecularly imprinted overoxidized-polypyrrole (oPPy) colloids by chemical polymerization and their high uptake ability and enantioselectivity are described. Since an oxidizing agent gives a different standard redox potential and rate of polymerization, the property of the resulting oPPy colloid can be controlled by the kind and concentration of the oxidizing agent. At higher concentrations of (NH4)2S2O8 (0.3 M), the overoxidation of PPy colloid automatically occurred. The extraction of L-lactate as a dopant has created a shape-complementary cavity on the surface of the oPPy colloid through overoxidation following polymerization. The oPPy colloid exhibited an excellent selectivity not only on the alanine enantiomer but also on the difference in the side-chain size of amino acids. The uptake of oPPy colloid towards L-alanine over D-alanine was 11.3 µmol/g-colloid against 3.6 µmol/g. The molecularly imprinted cavity can also recognize the existences of the OH or CH3 substituents.
Poly(N-isopropylacrylamide) (PNIPAAm) has the sharpest phase transition of the class of thermo-sensitive N-alkyl acrylamide polymers. We developed a new method of HPLC using packing materials modified with cross-linked poly(N-isopropylacrylamide) (PNIPAAm) hydrogel. A temperature-responsive surface was prepared by polymerization of NIPAAm in the presence of a cross-linker on the silica support. The surface properties and functions of the stationary phases change in response to the external temperature. Therefore it easily changes the interaction of a solute with the surface with a constant aqueous mobile phase. A temperature-responsive elution behavior was observed on the separation of steroids and PTH-amino acids. The method is expected to be applicable to separation in the pharmaceutical and biomedical fields.
The photo-responses of the retention and enantioseparation of several optical isomers were evaluated using an azobenzene-modified γ-cyclodextrin stationary phase (Az γ-CDSP) in micro-HPLC. UV light irradiation induced a decrease in the retention and the chiral selectivity for N-(3,5-dinitrobenzoyl)-1-phenylethylamine (DNBPEA) and N-(3,5-dinitrobenzoyl)-1-(1-naphtylethyl)amine (DNBNEA), while an increase was induced for dansylphenylalanine (DnsPhe) using a mixture of methanol and aqueous phosphate buffer as the mobile phase. No changes in the retention and the enantiomer separation of benzoin were observed with UV light irradiation. The retention behaviors were recovered by visible-light irradiation. It was speculated that the main factor of the change in the retention behavior was a change in the π-π interaction due to the azobenzene moiety of the stationary phase with photo-irradiation. Comparing the retention behavior before and after UV light irradiation, a suitable condition for obtaining a better resolution and enantiomer separation would be chosen using Az γ-CDSP.
Highly cross-linked macroporous polymers were prepared utilizing ethylene dimethacrylate as a cross-linking agent, in the presence or absence of methyl- α-D-glucoside as a kind of template molecule with methacrylic acid as a functional monomer. After the preparation of the polymers, we applied a high temperature to the cross-linked polymers to study the changes of adsorption properties of the polymers for sugar derivatives including the template molecule utilized. Interestingly, the heat treatment up to 250°C afforded improvement of relative adsorption affinity for several sugar derivatives including the template molecule, while heat treatment up to 150°C did not afford those improvements. The detailed studies including polymers prepared using acrylic acid as a functional monomer instead of methacrylic acid prove that temperatures higher than the Tg temperature of the polymer derived from a functional monomer such as methacrylic acid and higher than the melting point (mp) of the sugar template are necessary to afford the observed improvement of relative affinity based on the surface modification effects through the heat treatment to cross-linked polymers.
A low-capacity cation-exchange column was newly developed for the separation of amino acids. A highly cross-linked macro-porous polystyrene-divinylbenzene co-polymer was functionalized by a sulfoacylation reaction. The exchange capacity was controllable at the acylation step. The capacity between 55 and 60 µmol/column was adequate for the practical separations in acceptable retention times. The 5-µm base polymers having average pore diameters smaller than 3 nm gave satisfactory results, and those having 1.5-nm pore was most favorable. Several isocratic elution conditions at different pH values adjusted by phosphate buffer of mM order with or without acetonitrile could provide good separations for individual classes of amino acids, i.e., acidic, neutral, hydrophobic, and basic groups. The results provided fundamental data for constructing gradient elution systems required for the simultaneous separation of protein amino acids.
With the aim of preparing a stationary phase with a high shape-recognition ability for liquid chromatography, a new bonded phase was synthesized by coupling multiply dodecylamino-substituted β-cyclodextrin (β-CD) to 3-glycidoxypropyl-derivatized silica gel. The stationary phase prepared in this way was expected to have increased shape selectivity compared with that of conventional reversed-phase materials, due to solute interactions with the alkyl chain piles built up on the β-CDs bonded to silica. The separation characteristics of the bonded phase were investigated using polycyclic aromatic hydrocarbons (PAHs) with different molecular shapes and compared with those of monomeric ODS and native β-cyclodextrin-bonded phases. The newly developed stationary phase was found to be highly selective for PAHs.
A simple preparation process of alkylamide phase for reversed-phase HPLC (RP-HPLC) is described. The process includes aminopropyltrimethoxysilane firstly reacted with octanoyl chloride, then the intermediate was coupled onto porous silica. The resultant bonded silica has a reproducible ligand surface concentration and homogenous bonded ligand distribution on the porous silica. Characterization of prepared packing was carried out with elemental analysis, solid-state 13C NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes under methanol/water as binary mobile phase. The results showed that the stationary phase have excellent chromatographic properties and can be efficiently used for the separation of basic compounds.
The enantiomeric resolution of N-t-butyloxycarbonyl (N-t-Boc) amino acids D/L isomers by reversed-phase HPLC was investigated using cyclodextrins (CD’s) as chiral selectors for the mobile phase. The use of a low pH (pH<4) for the mobile phase enabled the enantioseparation of N-t-Boc amino acids. The opposite elution order of D/L isomers was observed when hydroxypropyl-derivatized β-CD was used instead of native β-CD. A computer simulation of the enantioseparation showed that the ratio of the retention factors of the chiral selector and the sample determined the elution order and the resolution. When the retention factor of the chiral selector is smaller than that of the sample, an isomer having larger complex formation constant eluted faster. However, when the chiral selector had a larger retention factor than the sample, an opposite elution order of the isomers was obtained. The large difference in the retention factors between the chiral selector and the sample led to good enantiomeric separation.
In this study, a new solid phase microextraction (SPME) fiber based on activated charcoal and polyvinyl chloride (PVC) coated on silver wire is presented. This fiber is very mechanically and thermally stable. Unlike commercial fiber, which is coated on fused silica, this fiber has a metallic base to which the coating adheres very strongly. On the other hand, producing this fiber is very fast, and can be completed in less than 30 min. This compares favorably with the time to make a fiber based on fused silica, which may last several days, due to many steps, such as conditioning in HCl, drying, deactivation, impregnation with a binder and coating. Also, this fiber is stable up to 250°C. It has been successfully used for the analysis of n-alkanes in the gaseous phase and headspace of soil samples after optimization of the experimental parameters by capillary gas chromatography. The relatively high capacity of the sorbent allows 20 - 30% of the analytes to be transferred to a separative system during first extraction.
Chiral linear polymers were prepared by the thermal polymerization of N-acryloyl-L-valine and N-acryloyl-L-alanine derivatives using 3-mercaptopropionic acid (3-MPA) as a radical transfer agent. C-Terminal groups of the derivatives were methyl and tert-butyl esters later removed, and N-methylamide moieties. The N′-methylamide derivative of N-acryloyl-L-valine was copolymerized with its methyl ester at a molar mixing ratio of 1:1. The ester groups were removed to provide anionic linear polymers terminated with carboxylic acid of the amino acid residue. The polymers are thus shown to function as pesudostationary phases that separate enantiomeric solutes in electrokinetic capillary chromatography (EKC). Racemic 3,5-dinitrobenzoylamino isopropyl esters were separated with the polymer derived from N-acryloyl-L-valine esters and with the copolymer from N-acryloyl-L-valine methyl ester and N-acryloyl-L-valine N′-methylamide at pH 7.0. These separations could not be observed at pH 9.0 in migrating solutions containing anionic linear polymers. This pH dependence can be discussed from the standpoint of the microscopic hydrophobicity of the polymers, as assessed from the fluorescence of pyrene adsorbed onto the polymers in water.
Monolithic silica columns prepared from tetramethoxysilane in a fused-silica capillary was octadecylsilylated, and evaluated by capillary electrochromatography. A plate height of 3 - 4 µm was obtained in an 80% acetonitrile-20% aqueous buffer (pH 8) under optimized conditions. An ODS phase having a high surface coverage, prepared by using octadecyldimethylchlorosilane, resulted in slow separation and a low column efficiency due to a slow electroosmotic flow. The results show that faster separation is achieved (i) with an ODS phase with a low surface coverage, (ii) with an ODS phase prepared by using octadecyltrichlorosilane, or (iii) by pressure-assisted CEC utilizing the high permeability of monolithic silica columns.