Gengliang Yang

Preparation of poly(N-isopropylacrylamide)-grafted polymer monolith for hydrophobic interaction chromatography of proteins

Poly(N-isopropylacrylamide)-grafted polymer monolith has been achieved using a surface-initiated atom transfer radical polymerization grafting polymerization within the pores of poly(chloromethylstyrene-divinylbenzene) macroporous monolith contained in a 100 mm x 4.6mm I.D. stainless steel column. The grafted-poly(N-isopropylacrylamide) on the surface of the grafted monolith that was used as chromatographic stationary phase showed a response to the variation of temperatures and/or salt concentrations. This study focus on its salt concentration responsive property and it has been revealed that the hydrophobicity of the grafted monolith can be adjusted by changing salt concentrations in the range of 0.05-2.0 mol/L. A variety of salts including sodium sulfate, ammonium sulfate and sodium chloride exhibited different effects on the alteration of hydrophobicity of the grafted monolith, and the effect of the salts was in the order of sodium sulfate>ammonium sulfate>sodium chloride. Based on this response to salt concentrations, the grafted monolith was applied in hydrophobic interaction chromatography of proteins, and the base-line separation of a six proteins mixture consisting of cytochrome c, myoglobin, ribonuclease A, bovine serum albumin, ovalbumin and thyroglobulin bovine was achieved by a salt gradient elution.

Determination of nicardipine and amlodipine in human plasma using on-line solid-phase extraction with a monolithic weak cation-exchange column

An on-line solid-phase extraction (SPE)-HPLC method was developed for simultaneous screening of nicardipine and amlodipine in human plasma. A short monolithic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [p(GMA-EDMA)]-based weak cation-exchange (WCX) column was prepared and employed as the selective extraction sorbent, which exhibited good permeability and biocompatibility. During the on-line SPE protocol, high-abundance proteins (human serum albumin, immunoglobulin G, immunoglobulin A and transferrin) and most matrixes in plasma were fast removed while nicardipine and amlodipine were effectively trapped on this monolithic column. Furthermore, the monolithic WCX sorbent could be continuously reused more than 300 times without obvious changes in analytes extraction and proteins cleanup. The proposed method was linear over a range of 0.5-50.0 ng mL(-1) for both analytes with a linear regression coefficient greater than 0.998, and the limit of detection (LOD) for each analyte was 0.2 ng mL(-1). Validation assays also demonstrated acceptable precision and adequate recovery for simultaneous quantitative screening of nicardipine and amlodipine in human plasma. Real plasma samples from hypertensive patients receiving a dosing of 5mg antagonists were examined by using the proposed method. Results indicated that the on-line SPE-HPLC method could be applied for simultaneously monitoring of nicardipine and amlodipine in clinical plasma samples.

p-tert-Butylcalix[8]arene-bonded silica monoliths for liquid chromatography.

Monolithic silica columns have inspired considerable research interests in the separation science because of their unique properties in permeability, mass transfer, efficiency and throughput. In this paper, a chemically p-tert-butylcalix[8]arene-bonded silica monolith was prepared as the promising candidate for versatile LC separations. Micrometer-sized macropores and nanometer-sized mesopores in this derivatized silica monolith reduce the diffusion path length and provide both low backpressure and high column efficiencies, leading to high-speed and high-throughput separations. Since p-tert-butylcalix[8]arene possesses a pi-donors cavity composed of benzene rings while polycyclic aromatic hydrocarbons, anthraquinones, phenol regio isomers and fullerenes are pi-systems with appreciable electron affinity, they may have a chance to get involved in forming host-guest inclusion complexes through non-covalent interactions, e.g. hydrophobic and pi-pi interactions. Compared with RP-18e, the prepared calixarene-bonded monolith exhibited better selectivity to molecules which contains more pi-electrons and more condensed cyclic moieties. The column efficiency was about 22,000 plates/m. The calixarene-bonded monolith also showed its good performances in separation of fullerenes and dihydropyridines, indicating a promising approach for purification of fullerenes with high purity from the carbon soot.

Chiral Separation and Enantiomeric Purity Determination of Pazufloxacin Mesilate by HPLC Using Chiral Mobile Phase Additives

Abstract In this work, a chiral separation method based on an ODS column and chiral mobile phase additives (CMPA) for a new fluoroquinolones drug pazufloxacin mesilate [(−)‐(S)‐10‐(1‐amino‐cyclopropyl)‐9‐fluoro‐3‐methyl‐7‐oxo‐2,3‐dihydro‐7H‐pyrido(1,2,3‐de)‐1,4‐benzoxazine‐6‐carboxylic acid mesilate] and its R‐(+)‐enantiomer was developed. The chiral separation was performed on a Shimadzu CLC‐ODS column (150 × 6.3 mm, 10 µm), methanol–phenylalanine solution (containing 8 mmol L−1 L‐phenylalanine and 6.3 mmol L−1 copper sulfate, adjusted to pH = 3.5 with sodium hydroxide solution) (30:70) was used as the chiral mobile phase and detected at 332 nm. Under the above conditions, the two enantiomers could be baseline separated with a resolution 1.9 and the chiral purity was quantitatively determined. Calibration graphs were constructed in the range of 2.48–248 µg mL−1 for S‐(−)‐ and 2.52–252 µg mL−1 for R‐(+)‐enantiomer, respectively. The linear correlation equations were: Y = 1.38 × 105 X − 130.9 (r = 0.9998) for S‐(−)‐ and Y = 1.26 × 105 X − 60.6 (r = 0.9998) for R‐(+) enantiomer, respectively. The detection limits were 1.2 ng for S‐(−)‐ and 1.5 ng for R‐(+)‐ enantiomer(obtained by S/N = 3), respectively. The RSD of the method was below 1.5% (n = 5).