Modeling and prediction of the mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monoliths

n-Octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monoliths were prepared for the rapid screening and determination of puerarin content of a crude extract Radix puerariae. The mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified monoliths were investigated using a variety of solvent systems, chromatographic evaluation and molecular dynamics (MDs) modeling. The equilibrated conformations between cross-linked polymers and target molecules were obtained from MD modeling. Both the polymer skeleton and functional groups played important roles in the recognition process. The cross-linker formed a structural network skeleton, in which recognition cavities were formed surrounded by functional groups. The polymer network structures provided good interaction access for isoflavones. The active groups recognized isoflavones by both intermolecular hydrogen bonding and hydrophobic interaction. The interaction energies and retention factors between polymers and target molecules were also evaluated and compared. A higher value of interaction energy corresponded to a higher value of retention factor. The potential of using modeling technology for predicting the chromatographic performances of target molecules was explored.     

Preparation of novel β-cyclodextrin functionalized monolith and its application in chiral separation

A novel β-cyclodextrin (β-CD) functionalized organic polymer monolith was prepared by covalently bonding ethylenediamine-β-CD (EDA-β-CD) to poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monolith via ring opening reaction of epoxy groups. SEM characterization was performed to confirm the homogeneity of the monolithic polymer. The resulting monolith was then characterized by DSC and XPS elemental analysis to study the thermal stability of the monolith, and to prove the successful immobilization of β-CD on the polymer substrate. The β-CD ligand density of 0.68 mmol g^?1 was obtained for the modified monolith, indicating the high reactivity and efficiency of the EDA-β-CD modifier. The ethylenediamine-β-CD functionalized monoliths were used for the chiral separation of ibuprofen racemic mixture and showed promising results.     

Application of poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith microextraction coupled with high performance liquid chromatography to the determination of polycyclic aromatic hydrocarbons in smoked meat products

This paper presents a study of the synthesis of a polymer monolith column and its application to the analysis of PAHs in smoked meat products. A poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith capillary has been successfully prepared with in situ polymerization method. The polymer monolith microextraction combined with HPLC determinations is employed for the analysis of naphthalene, biphenyl, phenanthrene, and anthracene. Various parameters affecting the extraction efficiency have been investigated and optimized. Under the optimum experimental conditions, the method provides an acceptable linearity (2-10,000 μg/L), low limits of detection (1.4-2.0 μg/L), and good precision (intraday relative standard deviations<4.1%, interday relative standard deviations<5.7%). When applied to the determination of the four PAHs in smoked meat samples, recoveries are obtained in the range of 86.6-101.5%.     

A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.     

"Molded" rods of macroporous polymer for preparative separations of biological products

A continuous rod of porous poly(glycidy1 methacrylate-co-ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 16-mm-i.d. glass column. The epoxide groups of the rod have been modified in situ by their reaction with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydroxypropyl functionalities that are required for the ion-exchange chromatographic mode. The bimodal pore size distribution curve typical for other molded separation media also prevail for the preparative-size rod. The column has been used successfully for the chromatographic separation of a mixture of standard proteins and yeast enzymes. The column exhibits a dynamic capacity that exceeds 420 mg of bovine serum albumin at a flow velocity of 60 cm/h. (c) 1995 John Wiley & Sons, Inc.     

Immobilization of trypsin onto "molded" macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) rods and use of the conjugates as bioreactors and for affinity chromatography

Trypsin immobilization onto continuous "molded" rods of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and some applications of the conjugate have been studied. The rods polymerized within a tubular mold (chromatographic column), were treated in situ with ethylenediamine, activated with glutaraldehyde and finally modified with trypsin. The performance of the trypsin-modified rods was evaluated and compared to that of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, modified with the same enzyme. Overall the enzyme-modified rods performed substantially better than the corresponding beads. In particular, the performance of the molded supports as enzymatic reactors or as chromatographic media benefits greatly from the enhanced mass transfer that is characteristic of the molded rod at high flow rates. (c) 1996 John Wiley & Sons, Inc.     

Hydrazide-functionalized poly(2-hydroxyethyl methacrylate) microspheres for immobilization of horseradish peroxidase

Nonporous cross-linked poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (poly(HEMA-co-EDMA)) microspheres were prepared by dispersion polymerization of HEMA and EDMA. The polymerization was performed in toluene/2-methylpropan-1-ol in the presence of cellulose acetate butyrate as a steric stabilizer and dibenzoyl peroxide initiator. The particle size may be increased by decreasing the toluene/2-methylpropan-1-ol ratio and by increasing polymerization temperature. Adipohydrazide was attached to the microspheres activated with 2,4,6-trichloro-1,3,5-triazine. After periodate oxidation of its carbohydrate moieties, horseradish peroxidase was coupled to the hydrazide-functionalized poly(HEMA-co-EDMA) microparticles up to 7.3 microgram of enzyme/g of carrier without a significant loss of its activity. Immobilized peroxidase was found to be stable, retaining more than 97% of its initial activity when stored for 23 days after the preparation.     

Development of rigid bidisperse porous microspheres for high-speed protein chromatography

Development of a high-performance stationary phase is an essential demand for high-speed separation of proteins by liquid chromatography. Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and a mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores, that is, micropores (10-90 nm) and macropores (180-4000 nm). Furthermore, the biporous medium possesses specific surface area as high as 91.3 m(2)/g. Because of the presence of the macropores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was found to be as high as 54.6 mg/g wet bead at 300 cm/h, approximately 63.2% of its static capacity. In addition, the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000 cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.     

Matrine-imprinted monolithic stationary phase for extraction and purification of matrine from Sophorae flavescentis Ait

A matrine-imprinted monolithic stationary phase (MIP monolith) was prepared by in situ polymerization for extraction and purification of matrine from Sophorae flavescentis Ait. Matrine was used as the template molecule, methacrylic acid as the function monomer, ethylene glycol dimethacrylate as the cross-linking agent, and toluene and dodecanol as the porogenic solvents. Scanning electron microscope study revealed that a monolithic structure with mesopores and 36 μm diameter nodules was obtained. The molecular recognition process and the effect of varying chromatographic conditions on separation were examined by high-performance liquid chromatography (HPLC). Hydrogen bonding, electrostatic, hydrophobic interactions and the molecular shape matching in MIP monolith cavities were proposed to be responsible for the recognition mechanism. The use of MIP monolith as a solid-phase extraction (SPE) sorbent for extraction and purification of matrine from S. flavescentis Ait was investigated. The extraction yield was 89.2% (for 3.0 mmol l(-1) matrine) with enrichment factor 29.     

Simultaneous determination of 5-hydroxyindoles and catechols from urine using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

To make analytes amenable for fluorescence (FL) detection, polymer monolith microextraction (PMME) coupled to high-performance liquid chromatography with FL detection was developed for the simultaneous determination of catechols and 5-hydroxyindoleamines (5-HIAs) from urine samples. In this method, a two-step pre-column derivatization method was employed to derivatize the analytes and a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary column was used as the extraction medium for PMME. The conditions for the derivatization and subsequent extraction of 5-HIAs and catechols derivatives were optimized. Using our optimum conditions, the detection limit of the target analytes were 0.11–21 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations less than 12% and a recovery of higher than 82%. In this study, we show how our proposed method can be used as a rapid sensitive technique for the determination of catechols and 5-HIAs from urine samples.     

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50-5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.     

One-step purification of YLLIP2 isoforms from Candida sp. 99–125 by polyethyleneimine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith

The extracellular lipase Yarrowia lipolytica (YLLIP2) crude extract was efficiently separated and purified from Candida sp. 99–125 by one-step ion-exchange chromatography on polyethyleneimine (PEI) functionalized monolithic columns. The preparative conditions for the functionalization of monoliths were optimized, including PEI molecular mass, PEI concentration, modification time and temperature. The monolithic skeleton was prepared in situ by polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with a volume ratio of 8:2. Heptane was used as the porogen. PEI 30 kDa with the concentration of 10% (v/v) was applied for the modification of the monolith at 55 °C for 12 h. Lipase (EC.3.1.1.3) from Candida sp. 99–125 was separated to four isoforms (isoform A, isoform B, isoform C and isoform D). As analyzed on non-denaturing PAGE and MALDI-TOF–MS, the four isoforms are homogenous and have the same molecular mass of approximate 38 kDa. The monoliths can afford direct crude lipase loading without increasing too much back pressure, which explores the great potential of the application of monoliths for one-single step fast separation and purification of complicated proteins.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

Polymer-immobilized fluorinase: Recyclable catalyst for fluorination reactions

Polymer-immobilized fluorinase for the synthesis of 5′-fluoro-5′-deoxyadenosine (FDA) from S-adenosyl-l-methionine (SAM) and fluoride ion in aqueous media is described. The optimal composition of the poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) and the heterogeneous catalytic reaction conditions were developed to yield FDA in 49% within 150 min. In PET radiochemistry, using [¹⁸F]fluoride ion in [¹⁸O]H₂O obtained from the cyclotron, [¹⁸F]FDA was synthesized with 68% fluorination efficiency. The immobilized fluorinase was recycled for up to four runs with 80% of catalytic activity in the final cycle.     

The detection of radical scavenging compounds in crude extract of borage (Borago officinalis L.) by using an on-line HPLC-DPPH method

The rapid evaluation of antioxidant activity of crude borage (Borago officinalis L.) extract was determined by using DPPH free radical method. This borage extract resulted in a rapid decrease of the absorbance and showed very high hydrogen-donating capacity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. A new HPLC-DPPH on-line method was applied for a screening of several radical scavenging components in this borage extract as well as for quantitative analysis. This on-line HPLC-DPPH method was developed using a methanolic solution of DPPH-stable radical. The HPLC-separated analytes reacted post-column with the DPPH solution in methanol. The induced bleaching was detected as a negative peak photometrically at 515 nm. The separation of antioxidative components was carried out by gradient HPLC with mobile-phase composition ranging from 2% to 80% acetonitrile with 2% acetic acid in water, UV detection was carried out at 280 nm. The HPLC analysis of borage extract revealed the presence of several radical scavenging components in the borage extract. The results obtained from the chromatograms suggest that some compounds present in the extract possess high radical quenching ability. The dominant antioxidative compound in the crude extract of borage leaves was identified as rosmarinic acid.     

Determination of nitric oxide in hydrophytes using poly(methacrylic acid-ethylene glycol dimethacrylate) monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

Nitric oxide (NO) is a bioactive molecule that has recently emerged as a cellular messenger in numerous physiological processes in plants. A novel high-performance liquid chromatography (HPLC) method combined with poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) is developed for sensitive determination of NO in hydrophytes. NO is derivatized using a fluorescent probe, 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene (DAMBO), and then the derivatives are extracted with PMME and analyzed by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of NO derivatives are optimized in detail. The detection limit (S/N=3) of NO is determined to be 2x10(-12)mol L(-1). Close correlation coefficient and excellent method reproducibility are obtained for the analyte over a linear range of 9x10(-11)-4.5x10(-8)mol L(-1). The inter- and intraday relative standard deviations (R.S.D.s) are less than 5%. The proposed method is successfully applied to the determination of NO levels in hydrophytes samples.     

Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography

Hydroxymethyl methacrylate-based monolithic columns for separation of oligonucleotides by capillary liquid chromatography (CLC) were prepared. We optimized composition of the polymerization mixture, which contained the monomer mixture consisting of N-(hydroxymethyl) methacrylamide (HMMAA) and ethylene dimethacrylate (EDMA), and the porogenic system composed of propane-1-ol, butane-1,4-diol and α, α′-azoisobutyronitrile (AIBN) as initiator. Separations of oligonucleotides were performed in HILIC (hydrophilic-interaction) mode using 100 mM triethylamine acetate (TEAA) in acetonitrile and in water as eluents. The influence of steepness of the mobile phase gradient on separation of the oligonucleotides was evaluated as well as the reproducibility of HMMAA monolith preparation.     

Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification

The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40–120 μm) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 μmol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.     

In vitro monitoring of nanogram levels of puerarin in human urine using flow injection chemiluminescence

A rapid and sensitive chemiluminescence (CL) determination of puerarin with the flow injection technique was presented. It was found that puerarin could enhance the CL generated from luminol-KIO(4) system significantly. The increment of CL intensity was linear over the concentration of puerarin ranging from 0.3 to 100.0 ng mL(-1) (r(2)=0.9963), offering a detection limit as low as 0.1 ng mL(-1) (3 sigma). At a flow rate of 2.0 mL min(-1), one analysis cycle, including sampling and washing, could be accomplished in 20 s with a relative standard deviation of less than 5.0%. The experimental results demonstrated that the excretive amounts of puerarin reached its maximum in 3 h after taking 250 mL green drink containing 275 mg puerarin, and the puerarin excretive ratio during 24 h was 9.70% in the body of volunteers.     

Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography

Hydroxymethyl methacrylate-based monolithic columns for separation of oligonucleotides by capillary liquid chromatography (CLC) were prepared. We optimized composition of the polymerization mixture, which contained the monomer mixture consisting of N-(hydroxymethyl) methacrylamide (HMMAA) and ethylene dimethacrylate (EDMA), and the porogenic system composed of propane-1-ol, butane-1,4-diol and alpha, alpha'-azoisobutyronitrile (AIBN) as initiator. Separations of oligonucleotides were performed in HILIC (hydrophilic-interaction) mode using 100 mM triethylamine acetate (TEAA) in acetonitrile and in water as eluents. The influence of steepness of the mobile phase gradient on separation of the oligonucleotides was evaluated as well as the reproducibility of HMMAA monolith preparation.