High-performance liquid chromatography (HPLC) has great contribution in the analysis of the carbohydrates. Various separation modes such as ion exchange chromatography, size exclusion chromatography and partition chromatography have been applied to HPLC of mono- and oligosaccharides. Among them, partition chromatography with a polar stationary phase and aqueous mobile phase, called aqueous normal phase partition (ANP) chromatography, has been used in various fields, and is widely used for the separation of reducing sugars. Currently, this separation mode is considered to be a type of "hydrophilic interaction chromatography (HILIC)". As the use of HILIC mode increases, many novel stationary phases being available for the separation of reducing sugars have been developed. This article focuses on the separation of reducing sugars by ANP (HILIC) mode, and summarizes the traditional and/or recent stationary phases for the separation of reducing sugars. The applicability of various kinds of stationary phases to solid-phase extraction (SPE) of saccharides is also described.
Mass spectrometry (MS) is the most widely used technology for proteome analysis, and MS-based proteomics platforms have identified over 10,000 human proteins in single-state cellular targets. Here, we review recent advances towards complete analysis of the human proteome. The main obstacles are the enormously wide dynamic range and huge complexity of the proteome. New technologies of sample preparation, protein/peptide separation and MS acquisition have been developed to tackle these issues. Pre-fractionation approaches, such as multidimensional separations prior to LC-MS/MS, have been introduced, but the total measurement time increases as the fraction number increases. Alternative approaches using high-resolution LC without pre-fractionation have also been developed, because high sample recovery is expected if the number of sample pretreatment steps is minimized. Although technical and methodological issues remain, human proteome analysis covering 7,000 - 10,000 proteins is currently feasible.
To achieve a tunable molecular sieving in gel electrophoresis, novel hydrogels consisted of a poly(ethylene glycol) based crosslinker and ionic monomers were developed. The gels showed specific shrinking regarding ionic solutions by suppressing an ionic repulsion in the gels as our previous report. Additionally, the intermolecular interactions such as hydrophobic interaction and π-π stacking among the adsorbed solutes also affected the specific shrinking of the gels. These gels were evaluated in gel electrophoresis using several glucans and DNA base pairs to examine the possibility of the molecular sieving. The gel prepared without ionic monomers showed the various separations depending on the differences of crosslinking degree. Also, the gel prepared with acrylic acid provided the various separation patterns of glucans based on the molecular sieving by simply changing pH of the buffer solutions for electrophoresis.
The effect of water content in a mobile phase on chromatographic behavoirs was studied with a monolithc stationary phase of poly(butyl methacrylate-co-ethylene dimethacrylate). A drastic decrease in the separation efficiency was observed when the water content was up to 90%, i.e., 10% acetonitrile (ACN) aqueous solution. Interestingly, the magnitude of the efficiency decrease significantly depended on the retention factor of the analytes, in particular, the separation efficiencies of the slightly retained compounds (0.1 < k < 1, in 10% ACN) were inferior. Furthermore, with a mobile phase of 10% ACN, the acceleration of the flow rate decreased the retention factor of these less retained compounds. The higher flow rate suppressed the interaction in RP-HPLC only when the water rich solution was used. The reduction in the retention factor produced non-theoretical relations in an H-u plot. The change in the monolith structure was also indicated by the variation in the permeability in 10% ACN.
Three types of crown ethers (i.e. 1-aza-15-crown-5-ether, 1-aza-18-crown-6-ether and 2-aminomethyl-18-crown-6-ether) were chemically bonded to silica gels via a simple 2-step reaction and the resulted stationary phases were evaluated for the separation of both anions and cations in capillary ion chromatography. It was found that the chemically bonded stationary phases were most stable and relatively sharper peaks were obtained when a mixture of toluene:acetonitrile = 7:3 was used as the reaction solvent in the second step reaction. The size of crown ether cavity as well as the presence of nitrogen atom within the cavity affected the retention behaviors of the ions. As for the separation of anions, it was found that the eluent cations were trapped on the stationary phases and acted as the anion-exchange site, viz. the anions were separated in the ion-exchange mode. Without the obstruction of nitrogen atom, 2-aminomethyl-18-crown-6-ether could easily form complexes with metal cations, and the retention as well as elution order were dependant on the complex formation constant between crown ether and the metal cations. Eluent with higher pH favored the retention and separation of the metal cations.
A two-dimensional chiral high-performance liquid chromatographic (2D-HPLC) system has been established for the analysis of extraterrestrial amino acids. As the targets, 8 chiral amino acids (alanine (Ala), valine (Val), 2-aminobutyric acid (2AB), norvaline (nVal), N-methylalanine (N-MeAla), isovaline (iVal), 3AB and 3-aminoisobutyric acid (3AIB)) and 5 non-chiral amino acids (glycine (Gly), β-Ala, γ-aminobutyric acid (GABA), sarcosine (Sar) and 2AIB) were selected. These amino acids were tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and non-enantioselectively separated by a capillary monolithic ODS column in the first dimension. The target fractions were automatically introduced into the second dimension and further separated by Pirkle-type enantioselective columns. By using this system, the 2D-HPLC separation of 21 components in small particles of a carbonaceous chondrite (Yamato 791191, Antarctic CM2 meteorite) could be successfully performed, and all of the target amino acids were observed. The D/L ratios of the chiral molecules are almost 50/50 for all of the tested proteinogenic and non-proteinogenic amino acids.