Yukui Zhang

Open tubular capillary electrochromatography with adsorbed stationary phase

A novel preparation method of stationary phase was presented for open tubular capillary electrochromatography (OTCEC). The adsorbed stationary phases were prepared easily by rinse the capillary with a buffer containing cationic surfactant such as cetyltrimethylammonium bromide (CTAB) or basic protein such as lysozyme. After a surfactant bilayer or a protein layer was adsorbed on the capillary wall, the capillary was rinsed again with a running buffer containing no CTAB or protein. The CTAB bilayer was used as a stationary phase for separation of neutral solutes. Five alkyl substituted benzenes were baseline separated within 6 min with theoretical plate number of 223 000/m for benzene and 181 000/m for toluene. The adsorbed lysozyme layer was used as chiral stationary phase for separation of enantiomers. Four amino acids and the drug of mephenytoin were separated within 5 min with a resolution from 1.74 to 2.05.

STUDY OF PHYSICALLY ADSORBED STATIONARY PHASES FOR OPEN TUBULAR CAPILLARY ELECTROCHROMATOGRAPHY

A novel method based on the adsorption of positively charged compounds on the wall of a fused‐silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run‐to‐run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590 000/m and the average one above 250 000/m. Stored at 2—8°C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.

Immobilized iminodiacetic acid (IDA)‐type Cu2+‐chelating membrane affinity chromatography for purification of bovine liver catalase

A metal ion chelating membrane medium based on iminodiacetate-substituted modified short cotton cellulose was examined for the purification of bovine liver catalase (BLC). The effect of buffer pH, chelator surface density, initial concentration of crude enzyme and flow rate on BLC binding efficiency to the copper ion chelating membrane adsorbent were examined. Under the chromatographic conditions chosen, 67.7% recovery of BLC was attained with an overall 4.2-fold increase in specific activity in a single step. After performance of BLC purification, the chelating membrane adsorbent can be easily regenerated by imidazole or EDTA buffer with higher reviving effectiveness with the latter.

Screening and Analysis of Biologically Active Compounds in Angelica sinensis by Molecular Biochromatography

SummaryA novel strategy for the screening and analysis of biologically active compounds in traditional Chinese medicine by molecular biochromatography is proposed. Molecular biochromatography with human serum albumin (HSA) immobilized on silica as stationary phase was used to screen and analyse the bioactive compounds in the typical Chinese medicine ofAngelica sinensis (Oliv.) Diels. Ten peaks showed retention on this column, which is based on their affinity for HSA. Ferulic acid and liguistilide were identified as the principal active components, which agrees very well with the results in the literature. A quality control method was also developed based on the simultaneous determination the concentrations of ferulic acid and liguistilide in solutions ofAngelica sinensis (Oliv.) Diels extracted with water and methanol. It was observed that the concentrations of ferulic acid and liguistilide in solution extracted with methanol were 2 and 53 times higher, respectively, than those with water. It was shown that molecular biochromatography is an effective way of analysing and screening biologically active compounds in traditional Chinese medicine.

Reversed-phase high-performance liquid chromatographic investigation of urinary normal and modified nucleosides of cancer patients

Post-transcriptional modifications in RNA give rise to free modified ribonucleosides circulating in the blood stream and excreted in urine. Due to their abnormal levels in conjunction with several tumor diseases, they have been suggested as possible tumor markers. The developed RP-HPLC method has been applied to analyze the urinary nucleosides in 34 urinary samples from 15 kinds of cancer patients. The statistical analyses showed the urinary nucleoside excretion, especially modified nucleoside levels, in cancer patients were significantly higher than those in normal healthy volunteers. Factor analysis was used to classify the patients with cancer and normal healthy humans. It was found that using 15 urinary nucleoside levels or only five modified nucleoside levels as data vectors the factor analysis plot displayed two almost separate clusters representing each group.

Capillary electrochromatography with a silica column with a dynamically modified cationic surfactant

A novel mode of capillary electrochromatography (CEC), called dynamically modified silica-capillary electrochromatography, is described in this paper. The column packed with bare silica was dynamically modified with long chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added into the mobile phase. CTAB ions were adsorbed onto the surface of bare silica, and the resulted hydrophobic layer on the silica gel was used as the stationary phase. Using the dynamically modified silica column, neutral solutes were separated by CEC. The highest number of theoretical plates obtained was about 71,500/m and the relative standard deviations for t0 and capacity factor of toluene were 4.7% and 4.9% for 20 consecutive runs, respectively. The separation mechanism of neutral solutes and the influence of mobile phase composition on the separation was investigated. The separation of nitrogen-containing solutes was carried out with this mode and the peak tailing of basic solute was effectively eliminated because the adsorption of basic solute on silica was blocked by the preferred adsorption of CTAB.

APPLICATION OF AN ARTIFICIAL NEURAL NETWORK IN CHROMATOGRAPHY-RETENTION BEHAVIOR PREDICTION AND PATTERN RECOGNITION

Abstract Layered feed-forward neural networks are powerful tools particularly suitable for the analysis of nonlinear multivariate data. In this paper, an artificial neural network using improved error back-propagation algorithm has been applied to solve problems in the field of chromatography. In this paper, an artificial neural network has been used in the following two applications: (1) To model retention behavior of 32 solutes in a methanol–tetrahydrofuran–water system and 49 solutes in methanol–acetonitrile–water system as a function of mobile phase compositions in high performance liquid chromatography. The correlation coefficients between the calculated and the experimental capacity factors were all larger than 0.98 for each solute in both the training set and the predicting set. The average deviation for all data points was 8.74% for the tetrahydrofuran-containing system and 7.33% for the acetonitrile-containing system. 2). To classify and predict two groups of different liver and bile diseases using bile acid data analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). The first group includes three classes: healthy persons, choledocholithiasis patients and cholecystolithiasis patients; the total consistent rate of classification was 87%. The second group includes six classes: healthy persons, pancreas cancer patients, hepatoportal high pressure patients, cholelithiasis patients, cholangietic jaundice patients and hepatonecrosis patients; the total consistent rate of classification was 83%. It was shown that artificial neural network possesses considerable potential for retention prediction and pattern recognition based on chromatographic data.

PROTEIN A TANGENTIAL FLOW AFFINITY MEMBRANE CARTRIDGE FOR EXTRACORPOREAL IMMUNOADSORPTION THERAPY

Tangential flow affinity membrane cartridge (TFAMC) is a new model of immunoadsorption therapy for hemoperfusion. Recombinant Protein A was immobilized on the membrane cartridge through Schiff base formation for extracorporeal IgG and immune complex removal from blood. Flow characteristics, immunoadsorption capacity and biocompatibility of protein A TFAMC were studied. The results showed that the pressure drop increased with the increasing flow rate of water, plasma and blood, demonstrating reliable strength of membrane at high flow rate. The adsorption capacities of protein A TFAMC for IgG from human plasma and blood were measured. The cartridge with 139 mg protein A immobilized on the matrix (6 mg protein A/g dry matrix) adsorbed 553 mg IgG (23.8 mg IgG/g dry matrix) from human plasma and 499.4 mg IgG (21.5 mg IgG/g dry matrix) from human blood, respectively. The circulation time had a major influence on IgG adsorption capacity, but the flow rate had little influence. Experiments in vitro and in vivo confirmed that protein A TFAMC mainly adsorbed IgG and little of other plasma proteins, and that blood cell damage was negligible. The extracorporeal circulation system is safe and reliable.

Theoretical study of the separation mechanism of ionizable compounds in capillary electrochromatography

The migration mechanism of ionizable compounds in capillary electrochromatography (CEC) is more complicated than in high perfomance liquid chromatography (HPLC) due to the involvement of electrophoresis and the second chemical equilibrium. The separation mcchanism of ionizable compounds in CEC has been studied theoretically. The electrochromatographic capacity factors of ions (k’) in CEC and in the pressurized CEC are derived by phenomenologicul approach. The influence of pH, voltage, pressure onk’ is discussed. In addition, thek’ of weak acid and weak base are derivcd base on acid-base equilibrium and the influence of pH onk’ is studied theoretically.

Membrane affinity chromatography for analysis and purification of biopolymers

SummaryAffinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only about 2.5 min for analysis at a flow-rate of 1.0 mL min−1. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 mL min−1 within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid. It can also be used for micro-scale purification of biopolymers.