Styrene-N-phenylacrylamide co-polymer modified silica monolith particles with an optimized mixing ratio of monomers as a new stationary phase for the separation of peptides in high performance liquid chromatography.

A stationary phase was prepared by chemical derivatization of the support particles with a layer of copolymer composed of styrene and N-phenyl acrylamide. Silica monolith particles of ca. 2.6 µm (volume-based average) have been prepared as the support particles by sol-gel reaction followed by differential sedimentation. The particles were reacted with 3-chloropropyl trimethoxysilane followed by sodium diethyldithiocarbamate to introduce an initiator moiety. Then, the copolymer layer was immobilized via reversible addition-fragmentation transfer polymerization. The resultant phase was packed in glass-lined stainless-steel micro-columns (1 x 150 mm) and evaluated for the separation of a mixture composed of five peptides (Trp-Gly, Thr-Tyr-Ser, angiotensin I, isotocin and bradykinin). The effect of monomer mixing ratio (styrene versus N-phenyl acrylamide) on the chromatographic separation efficiency of the stationary phase was examined. A number of theoretical plates (N) as high as 33 600 plates/column (224 000 plates/m, 4.46 µm plate height) was achieved using the column packed with the optimized stationary phase. The column-to-column reproducibility based on three columns packed with three different batches of stationary phase was found satisfactory in separation efficiency, retention factor, and asymmetry factor.

An optimized mixed-mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high-performance liquid chromatography.

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1-[3-(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow-bore glass-lined stainless-steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr-Tyr-Ser, Val-Ala-Pro-Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.

Catalyst assisted synthesis of initiator attached silica monolith particles via isocyanate-hydroxyl reaction for production of polystyrene bound chromatographic stationary phase of excellent separation efficiency.

Dibutyltin dichloride (DBTDC) was used as a catalyst to chemically bind 4-chloromehtylphenylisocynate (4-CPI) to porous monolithic silica particles via isocyanate-hydroxyl reaction, and the reaction product was reacted with sodium diethyldithiocarbamate (SDDC) to yield initiator attached silica monolith particles. Reversible addition-fragmentation transfer (RAFT) polymerization was taken place on them to result in polystyrene attached silica particles that showed excellent separation efficiency when packed in a chromatographic column (1.0 mm × 300 mm). The numbers of theoretical plates (N) of 56,500 is better than those of any commercially available HPLC or UHPLC column yet.

Comprehensive overview of recent preparation and application trends of various open tubular capillary columns in separation science.

Open tubular (OT) capillary columns have been increasingly used in a variety of fields of separation science such as CEC, LC, and SPE. Especially their application in CEC has attracted a lot of attention for their outstanding separation performance. Various forms of OT stationary phase materials have been employed such as in-situ prepared polymers, molecular imprinted polymers (MIPs), brush ligands, host ligands, block copolymers, aptamers, carbon nanotubes, polysaccharides, proteins, tentacles, nanoparticles, monoliths, and polyelectrolyte multi-layers. They have been prepared either in the chemically bound format or physically adsorbed format. Sol-gel technologies and nanoparticles have been sometimes involved in their preparation. There have been also some unique miscellaneous studies, for example, adopting preferentially adsorbed mobile phase components as stationary phases. In this review, recent progresses since mostly 2007 will be critically discussed in detail with some summarized descriptions for the work before the date.