Simultaneous determination of four plant hormones in bananas by molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography.

A highly selective molecularly imprinted solid-phase extraction (MISPE) combined with liquid chromatography-ultraviolet detection was developed for the simultaneous isolation and determination of four plant hormones including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) in banana samples. The new molecularly imprinted microspheres (MIMs) prepared by aqueous suspension polymerization using 3-hydroxy-2-naphthoic acid and 1-methylpiperazine as mimic templates performed with high selectivity and affinity for the four plant hormones, and applied as selective sorbents of solid-phase extraction could effectively eliminate the interferences of the banana matrix. Good linearity was obtained in a range of 0.04-4.00 µg g(-1) and the recoveries of the four plant hormones at three spiked levels ranged from 78.5 to 107.7% with the relative standard deviations (RSD) of less than 4.6%. The developed MISPE-HPLC protocol obviously improved the selectivity and eliminated the effect of template leakage on quantitative analysis, and could be applied for the determination of plant hormones in complicated biological samples.

Macroporous polymer monoliths fabricated by using a metal-organic coordination gel template.

We have found a new way, a metal-organic coordination gel template method, to generate polymer monoliths with an essentially macropore size, in which the pores are organized spontaneously into continuous networks; furthermore, the resulting macroporous monoliths have potential applications in high-throughput and high-efficiency separation of proteins.

Rapid and efficient chiral separation of nateglinide and its L-enantiomer on monolithic molecularly imprinted polymers.

A monolithic molecularly imprinted polymer (monolithic MIP) was designed and prepared for chiral separation of nateglinide and its L-enantiomer. The enantiomers were rapidly separated on this novel monolithic MIP based chiral stationary phase (MIP-CSP), whereas the enantioseparation was not obtained on the non-imprinted polymer (NIP). Chiral recognition was found to be dependent on the stereo structures and the arrangement of functional groups of the imprinted molecule and the cavities on MIP. Thermodynamic data (deltadeltaH and deltadeltaS) obtained by Van't Hoff plots revealed an enthalpy-controlled enantioseparation. The binding capacity was evaluated by frontal analysis. Monolithic nateglinide-MIP had an effective number of binding sites Bt = 41.15 micromol g(-1) with a dissociation constant of Kd = 7.40 mM. The morphological characteristics of the monolithic MIP were investigated by pore analysis and scanning electron microscope (SEM). Results showed that both mesopores and macropores were formed in the monolith. Over all, this study presents a new and practical possibility for providing high rates of mass transfer, fast separations and high efficiencies without the pressure constraints of the traditional bulk molecularly imprinted polymers, through the monolithic MIPs.

Chiral separation of tamsulosin isomers by HPLC using cellulose tris (3,5-dimethhylphenylcarbamate) as a chiral stationary phase.

A high-performance liquid chromatographic (HPLC) method was developed for the chiral separation of an antagonist of alpha1A adrenoceptors, tamsulosin and its S-isomer. Baseline separation of the isomers was achieved within 35 min on a CHIRALCEL OD-RH column with a binary solvent mixture of 50 mmol l(-1) KPF6-acetonitrile (v/v (70:30), pH 5.0) as the optimized mobile phase. The detection limits and quantification limits of both R-isomer and S-isomer were 0.11 and 0.44 ng, respectively. The R.S.D. values of peak-area for the two isomer were 0.42% (of peak-height: 0.77%) for R-isomer and 0.64% (of peak-height:0.92%) for S-isomer (n = 5).

Dispersive solid-phase extraction based on magnetic dummy molecularly imprinted microspheres for selective screening of phthalates in plastic bottled beverages.

A new magnetic dummy molecularly imprinted dispersive solid-phase extraction (MAG-MIM-dSPE) coupled with gas chromatography-FID was developed for selective determination of phthalates in plastic bottled beverages. The new magnetic dummy molecularly imprinted microspheres (MAG-MIM) using diisononyl phthalate as a template mimic were synthesized by coprecipitation coupled with aqueous suspension polymerization and were successfully applied as the adsorbents for MAG-MIM-dSPE to extract and isolate five phthalates from plastic bottled beverages. Validation experiments showed that the MAG-MIM-dSPE method had good linearity at 0.0040-0.40 µg/mL (0.9991-0.9998), good precision (3.1-6.9%), and high recovery (89.5-101.3%), and limits of detection were obtained in a range of 0.53-1.2 µg/L. The presented MAG-MIM-dSPE method combines the quick separation of magnetic particles, special selectivity of MIM, and high extraction efficiency of dSPE, which could potentially be applied to selective screening of phthalates in beverage products.

Preparation of a novel porous poly (trimethylol propane triacrylate-co-ethylene dimethacrylate) monolithic column for highly efficient HPLC separations of small molecules.

A novel poly (trimethylol propane triacrylate-co-ethylene dimethacrylate) [poly (TMPTA-co-EDMA)] monolith was prepared by in situ free-radical polymerization in a 50 mm × 4.6mm i.d. stainless steel column and was investigated for high performance liquid chromatography (HPLC). The porous structure of monolith was optimized by changing the conditions of polymerization. The chemical group of the monolithic column was confirmed by a Fourier transform infrared spectroscopy (FT-IR) method and the morphology of column structure was characterized by scanning electron microscopy (SEM). The mechanical strength and permeability were also studied. Finally, a series of low-molecular-weight organic compounds were utilized to evaluate the retention behaviors of the monolithic column. The result demonstrated that the prepared column exhibited an RP-chromatographic behavior and good separation performance. The method reproducibility was obtained by evaluating the run-to-run and column-to-column with relative standard deviations (RSDs) less than 0.7% (n=6) and 2.9% (n=6), respectively, which indicated that prepared monolithic columns had good reproducibility and stability.

Preparation and characterization of poly(triallyl isocyanurate-co-trimethylolpropane triacrylate) monolith and its applications in the separation of small molecules by liquid chromatography.

A new polymeric monolith was prepared in stainless-steel column and fused-silica capillary, respectively, by atom transfer radical polymerization technique. In the polymerization, triallyl isocyanurate (TAIC) was used as the functional monomer; trimethylolpropane triacrylate (TMPTA) as the crosslinking agent; polyethylene glycol 200 and 1,2-propanediol as the co-porogens; carbon tetrachloride as the initiator and ferrous chloride as the catalyst. The conditions of polymerization were optimized. Morphology of the prepared poly(TAIC-co-TMPTA) monolith was investigated by scanning electron microscopy; pore properties were assayed by mercury porosimetry and nitrogen adsorption. The characterization indicated that the prepared reversed-phase monolith possessed uniform structure, good permeability and mechanical stability. The column was used as the stationary phase of reversed phase high performance liquid chromatography (RP-HPLC) and capillary liquid chromatography (CLC) to separate the mixture of aromatic compounds. The new column performed around 125,000 theoretical plates per meter. The column showed good reproducibility: the relative standard deviation values of the retention factor values for aromatic compounds were less than 1.52% (n=7, column-to-column).

[Applications of polymeric monoliths in separation of bio-macromolecules].

In recent years, the applications of high performance liquid chromatographic polymeric monoliths in the separation of macromolecules have been developed. In the review, the characters and new developments of bio-macromolecules separation by using the polymeric monoliths, combining with the works in our laboratory are summarized. Moreover, related influential reports are referred.

The fabrication of monolithic capillary column based on poly (bisphenol A epoxy vinyl ester resin-co-ethylene glycol dimethacrylate) and its applications for the separation of small molecules in high performance liquid chromatography.

A new polymeric monolith was synthesized in fused-silica capillary by in situ polymerization technique. In the polymerization, bisphenol A epoxy vinyl ester resin (VER) was used as the functional monomer, ethylene glycol dimethacrylate (EDMA) as the crosslinking monomer, 1,4-butanediol, 1-propanol and water as the co-porogens, and azobisisobutyronitrile (AIBN) as the initiator. The conditions of polymerization have been optimized. Morphology of the prepared poly (VER-co-EDMA) monolith was investigated by the scanning electron microscopy (SEM); pore properties were assayed by mercury porosimetry and nitrogen adsorption. The optimized poly (VER-co-EDMA) monolith showed a uniform structure, good permeability and mechanical stability. Then, the column was used as the stationary phase of high performance liquid chromatography (HPLC) to separate the mixture of benzene derivatives. The best column efficiency achieved for phenol was 235790 theoretical plates per meter. Baseline separations of benzene derivatives and halogenated benzene compounds under optimized isocratic mode conditions were achieved with high column efficiency. The column showed good reproducibility: the relative standard deviation (RSD) values based on the retention times (n=3) for run-to-run, column-to-column and batch-to-batch were less than 0.98, 1.68, 5.48%, respectively. Compared with poly (BMA-co-EDMA) monolithic column, the proposed monolith exhibited more efficiency in the separation of small molecules.

On-line solid phase extraction using organic-inorganic hybrid monolithic columns for the determination of trace ß-lactam antibiotics in milk and water samples.

A rapid and simple method with on-line solid phase extraction (SPE) has been developed for the simultaneous determination of beta-lactam antibiotics (BLAs) (amoxicillin, cephradine, and cefazolin sodium) in aquatic environment and milk. The epoxy-based organic-inorganic hybrid monolithic column was used as SPE sorbent to simultaneously monitor three analytes. The morphology of monolithic column and pressure drop across the columns were characterized. Excellent permeability and high selectivity were obtained. The linear range of the standard curve was from 2.0 to 500 ng/mL (r(2)≥0.999). Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 11%. The accuracy and recovery were found to be within the range of 93-103% and 83-105%. The results indicated that the prepared monolithic column could provide excellent reproducibility and implied that the prepared monolith was feasible to be used as an on-line SPE sorbent material.

Dummy molecularly imprinted solid-phase extraction for selective determination of five phthalate esters in plastic bottled functional beverages.

In this paper, a highly selective sample cleanup procedure combing dummy molecular imprinting and solid-phase extraction (DMI-SPE) was developed for the simultaneous isolation and determination of five phthalate esters in plastic bottled beverages. The new imprinted microspheres were synthesized via precipitation polymerization using diisononyl phthalate as a dummy template that showed high selectivity and affinity to the five kinds of phthalate esters and were successfully applied as selective sorbents of DMI-SPE for the simultaneous determination of the phthalate esters from plastic bottled beverages. Good linearity was obtained in a range of 5.0-750.0 µg/L, and the average recoveries of the five phthalate esters at three spiked levels ranged from 84.3 to 96.2% with the relative standard deviations less than 5.49%. The developed extraction protocol eliminated the effect of template leakage on quantitative analysis and could be applied for the determination of phthalate esters in complicated functional beverages products.

On-line simultaneous deproteinization of biological samples and trace enrichment of three dipine series using a poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) monolith.

A porous poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) [poly(NIPAAm-co-EDMA)] monolithic column was prepared by in situ free-radical polymerization. The morphology of monolithic column and pressure drop across the columns were characterized. The results showed excellent permeability and high selectivity. Nifedipine, nitrendipine and nisoldipine were simultaneously selected to validate the extraction efficiency of the prepared monolith both in plasma and urine. The extracted nifedipine, nitrendipine and nisoldipine from plasma and urine samples have been on-line tested quantitatively by using the prepared monolith connected with RP-C18 column. The total analytical run time was 38 min. For all analytes, linear calibration curves were obtained over a range of 2-500 ng/mL with coefficient of correlation>0.997. Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 12%. The accuracy and recovery was found to be in the range of 89-109% and 88-106%. The results indicated that the prepared monolith was feasible to be used as an on-line SPE sorbent material and the method was especially appropriate for multi-analytes monitoring in plasma and urine samples. Finally, the proposed method was successfully applied to simultaneously screen nifedipine, nitrendipine and nisoldipine in plasma.

[Application of molecularly imprinted solid-phase extraction on drug residues in animal source foods].

Due to the superior molecular recognition and good physical and chemical stability, the molecularly imprinted materials have gained more and more concern recently in extrication and separation fields. Using the imprinted materials as adsorbents, the molecularly imprinted solid-phase extraction (MISPE) allows selective extraction of the target molecules and its analogues from complex matrices, and is suitable for complex sample preparation and enrichment processes. This article describes the principle, latest progresses and parameters of molecularly imprinted solid-phase extraction and summarizes its extraction and applications for the determination of drug residues of animal source foods in recent years. Moreover, the shortcomings and future prospects of molecularly imprinted solid-phase extraction are also mentioned.

Immobilization of trypsin on sub-micron skeletal polymer monolith.

A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-α-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-α-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30°C, which is comparable to 24 h digestion in solution at 37°C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.

Preparation of poly(N-isopropylacrylamide)-grafted well-controlled 3D skeletal monolith based on E-51 epoxy resin for protein separation.

A novel type of poly(N-isopropylacrylamide) grafted E-51 epoxy-based monoliths in a 100 mm × 4.6mm I.D. stainless steel column with well-controlled three-dimensional skeletal structures has been prepared and proposed for the separation of proteins. The grafted PNIPAAm chain via surface-initiated atom transfer radical polymerization was successfully performed. The proposed method provided a new route to modify the E-51 epoxy-based monoliths for widening their applications. Meanwhile, the temperature and the salt concentration responses of the grafted monolithic columns were investigated. Under the salt gradient, six proteins were well separated in hydrophobic interaction mode. Moreover, for further confirming the application of the prepared monolith was meaningful for proteome analysis in actual system, the separation of human serum sample was performed.

Investigation of temperature-responsivity and aqueous chromatographic characteristics of a thermo-responsive monolithic column.

A thermo-responsive and macroporous monolithic cryogel was directly prepared by in situ free-radical redox cryo-polymerization in a stainless steel chromatographic column (100 mm × 4.6mm i.d.) using N-isopropylacrylamide (NIPAAm) as functional monomer and PEG-20,000 as porogen at -12°C. The internal morphology of resulting monolithic cryogel was estimated by scanning electron microscopy (SEM). Based on that, a submicron skeleton structure was observed. Besides, the gravimetrically determined rates of swelling/deswelling for thermo-responsive monolithic cryogel were much higher than that of hydrogel adopting the same component proportion via conventional method (25°C for 24h). Simultaneously, a temperature-dependent resolution of steroids was also achieved using only water as a mobile phase. The theoretical plate number of every analyte was more than 2000.

Preparation of a novel hybrid organic-inorganic monolith for the separation of lysozyme by high performance liquid chromatography.

A novel hybrid organic-inorganic monolith for high performance liquid chromatography (HPLC) was firstly developed by atom transfer radical polymerization (ATRP) by a simple and rapid method, in which vinyl ester resin was used as the monomer, natrium bisulfurosum was used both as organic adjunct and coadunate initiator to alter the activity of the free radical in the process of polymerization and then to control the molecular mass. The conditions of polymerization were optimized. The chemical group of the monolith was assayed by infrared spectra method, the morphology of monolithic material was studied by scanning electron microscopy (SEM) and the pore size distribution was determined by a mercury porosimeter. Finally, the monolith was used to separate lysozyme (Lys) from chicken egg white with good resolution and reproducibility that were obtained in a short time (10 min) by HPLC. In addition, the influences of buffer concentration and pH value on elution have been investigated and the hybrid monolith was used to separate benzene and its homologs from the mixture.

Preparation and characterization of temperature-responsive poly(N-isopropylacrylamide-co-N,N'-methylenebisacrylamide) monolith for HPLC.

A temperature-responsive poly(N-isopropylacrylamide-co-N,N'-methylenebisacrylamide) [poly(NIPAAm-co-BIS)] monolith was prepared via a free-radical polymerization technique using an aqueous redox initiator in solution at -12°C. The effect of the % T (total monomer concentration/100 mL) and % C (cross-linker concentration/100 mL) on the visual form was investigated. The effect of the porogen on the pore structure was characterized by SEM. Under the optimum condition, the monolith for HPLC was successfully prepared and its mechanical strength and permeability have been studied. Furthermore, a temperature-dependent resolution of aromatic ketones was achieved using only water as mobile phase. The increasing interaction between solutes and the monolith was observed when temperature increased. The theoretical plate number of every analyte was more than 10(4).

Preparation and characterization of monolithic column by grafting pH-responsive polymer.

A novel modified monolithic column with pH-responsive polymer chains was prepared by grafting methacrylic acid onto the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. The grafting polymerization was achieved in an in situ manner which was performed by pumping methacrylic acid directly through an acidic hydrolysis monolithic column using potassium peroxydisulfate initiated free-radical polymerization. The grafted monolithic column was demonstrated to be the pH-responsive to the pore structure and the chromatographic characterization. The permeability of the column and the retention factors of five benzene homologues decreased due to the conformational changes of the polymer chains when the pH of mobile phase increased from 4.5 to 7.5. Furthermore, the modified monolithic column was used as the pH-responsive stationary phase and exhibited an excellent separation of four basic proteins.

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.