[Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins].

A partially filled monolith was prepared by in situ polymerization, and then carrier ampholytes (CAs, pH 3-10) were immobilized on its surface. For effective utilization of capillary isoelectric focusing (cIEF) with the monolithic immobilized pH gradient (M-IPG), a new online platform was established by the introducing of an eight-way injection valve, a three-way valve and a cross-shaped unit. Besides, a capillary coated with hydroxypropyl cellulose (HPC capillary) was prepared and used to determine the isoelectric points (pI) of trastuzumab and etanercept. In parallel, using the newly built capillary isoelectric focusing platform, the pI values of trastuzumab and etanercept were measured with the M-IPG column, and compared with the results obtained using the HPC capillary. It was found that these two cIEF columns can be effectively used to separate proteins and determine the pI values of monoclonal antibodies and fusion proteins in protein drugs. Moreover, the measured pI values were consistent with those estimated using the HPC capillary.

Capillary isoelectric focusing with free or immobilized pH gradient in silica particles packed column.

The use of immobilized pH gradient (IPG) capillary isoelectric focusing (CIEF) was confirmed to be possible with packed capillaries. The success of this experiment was due to two key factors: first, the use of surface-confined atom transfer radical polymerization method led to an increase in active reaction sites on the surface of silica particles; second, the subsequent free radical reaction caused carrier ampholytes (CAs) to bond faster and firmer. Based on this scheme, both CIEF with free pH gradient and CIEF with IPG were performed in capillaries packed with 3 µm modified silica particles. In our online CIEF-UV platform, both reversible and irreversible adsorption of proteins was shown to be negligible. Four proteins were focused: cytochrome c (pI 10.2), myoglobin (pI 7.3), carbonic anhydrase (pI 5.9) and trypsin inhibitor (pI 4.5). The comparison of the two CIEF columns showed that the time required for focusing in the packed capillary with IPG is only increased by a factor of 1.5 compared to the packed capillary, giving complete focusing in less than 12 min at 400 V/cm. With the increment of the electric field (the maximum at 600 V/cm), the run time was continually decreasing in these packed capillaries while the peak shape was improving. The four proteins (pH 4.5-10.2) could be successfully separated in our online CIEF platform. Moreover, for the newly online CIEF platform, pressure-driven mobilization without an applied electric field was achieved in the packed capillary with immobilized pH gradient.

Epinephrine-modified methacrylate monolithic column with fumed silica nanoparticle for pressurized capillary electrochromatography.

Via the ring-opening reaction of epoxy groups with epinephrine, a novel epinephrine functionalized polymethacrylate monolith with fumed silica nanoparticles has been fabricated for pressurized capillary electrochromatography. The preparation of epinephrine-modified monoliths has been optimized. In addition, morphology, electroosmotic flow, separation mechanism and column performance have been studied. The internal structure of the monolithic stationary phase was more uniform and the column efficiency increased after the incorporation of nanoparticles. With this column, satisfactory separation capability of aromatic compounds and alkaloids has been achieved and the column efficiency for naphthalene reached 138 696 plates/m. As for the real sample, 3 alkaloids were separated in Huanglian Shangqing capsules, a Chinese traditional medicine.

[Preparation and characterization of a novel hydrophilic stationary phase and its application in pressurized capillary electrochromatography].

Silica particles having different sizes were synthesized by a modified Stöber method. The reaction temperature, stirring rate, and time conditions were optimized. Finally, 580 nm monodispersed silica particles were synthesized. Moreover, a novel zwitterionic stationary phase with strong hydrophilicity was prepared based on a "thiolene" click reaction between cysteine and the silica particles modified vinyl group (Cys-vinyltrichlorosilane (VTMS)-SiO2). A column packed with submicron cysteine-bonded silica was prepared by the slurry packing method. The hydrophilic mechanism was revealed by the separation of toluene, acrylamide, and thiourea using different ratios of acetonitrile. The effects of applied voltage, buffer concentration, and buffer pH on the separation ability of the capillary column packed with 580 nm Cys-VTMS-SiO2 were examined to achieve optimal results. In addition, the run-to-run and day-to-day reproducibilities in terms of retention time and peak area for the above mentioned compounds were both 2.0%. The good batch-to-batch reproducibility indicated that the preparation method was suitable for the fabricated column. The column used in the pressurized capillary electrochromatography (pCEC) system demonstrated efficient and fast separation of a mixture consisting of nucleosides, phenols, amines, and peptides after the optimization of the separation condition. The proposed method shows good potential for the separation of polar and hydrophilic compounds.

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform.

In the presence of methanol and n-decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3-10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M-IPG). In addition, an online self-built platform for protein separation was established on account of the introduction of a cross-shaped unit and two short-off valves. In this platform, a cross-shaped unit was not only used to join the M-IPG column and a six-way injection valve (1.5 µL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short-off valve in the tee unit or cross-shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7-proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M-IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A1c have been successfully resolved with the newly developed M-IPG column.

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) for isoelectric focusing of amino acids and proteins.

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) was prepared in two steps. First, a partially filled porous layer open tubular capillary column containing active epoxy groups was synthesized by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide. Then, an aqueous solution of commercial carrier ampholytes (CAs, Pharmalyte) was focused in the prepared polymer column. With the column immobilized by Pharmalyte 4.5-6.0 (narrow pH range) or Pharmalyte 3.0-10.0 (wide pH range), proteins with known pIs were separated and a good linear correlation between pI values and migration times was obtained. Meanwhile, resolution was improved compared to the traditional gradient with a wide pH range. A mixture of low molecular weight amino acids was separated with the narrow pH range PLOT-IPG column; from the obtained results, the resolution of our new column was calculated to be at most 0.09 pH unit. In addition, human serum proteins were analyzed with the newly developed wide and narrow pH range PLOT-IPG columns; its sensitivity and resolution in the narrow pH range one were better than capillary electrophoresis.

Recent advances in microscale separation.

This review focuses on the recent advances on microscale separation technology, particularly on the new development on column and detection technologies in electrophoretic and chromatographic separation modes (i.e., CE, CEC, cLC, etc.). Representative examples illustrate also applications on the small molecules, peptides, proteins, polysaccharides, chiral and so on. 246 relevant articles are cited covering the literature published from 2013 to May 2017.

Polyprenylated acylphloroglucinols from Hypericum scabrum.

Fourteen phloroglucinols, named hyperciumoxide A-N, and a known compound were isolated from air-dried aerial parts of Hypericum scabrum. The structures of these compounds were deduced on the basis of extensive 1D- and 2D-NMR experiments. Hepatoprotective properties against D-galactosamine-induced HL-7702 cell damage of isolated compounds were evaluated. Meanwhile, these compounds were also tested for antidepressant activity by inhibiting reuptake of tritiated serotonin ([3H]-5-HT) and Noradrenalinet ([3H]-NE) in rat brain synaptosomes.

Two new prenylated phloroglucinol derivatives from Hypericum scabrum.

Two new prenylated phloroglucinol derivatives (1-2), and a known compound furohyperforim isomer 2 (3), were isolated from the aerial parts of Hypericum scabrum. Their structures were elucidated by various spectroscopic methods, including MS, IR, UV, and NMR.

Two new polyprenylated acylphloroglucinols from Hypericum scabrum.

Two new polyprenylated acylphloroglucinols, (1S,32R,5S,6R,7R)-6-((R)-3,4-di-hydroxy-4-methylpentyl)-2-(2-hydroxypropan-2-yl)-7-isobutyryl-6-methyl-5,9-bis(3-methylbut-2-en-1-yl)-4,5,6,7-tetrahydro-2H-32,7-methanocycloocta[b]furan-8,10(3H)-dione (1) and (4R,5R,7R)-4-((R)-3,4-dihydroxy-4-methylpentyl)-2,2,4-trimethyl-5,7-bis(3-methyl-but-2-en-1-yl)-7-(5-methylhex-4-enoyl)-4,5,6,7-tetrahydrobenzofuran-3(2H)-one (2) were isolated from Hypericum scabrum. The structures were elucidated by means of spectroscopic methods, including MS, IR, NMR, OR, and CD.