Faiz Ali

Molecular imprinted polymers for separation science: A review of reviews

Molecular imprinted polymer is an artificial receptor made by imprinting molecules of a template in a polymer matrix followed by removing the template molecules via thorough washing to give the permanent template grooves. They show favored affinity to the template molecule compared to other molecules, and this property is the basic driving force for such diverse application of this techniques. Such techniques have been increasingly employed in a wide scope of applications such as chromatography, sample pretreatment, purification, catalysts, sensors, and drug delivery, etc., mostly in bioanalytical areas. A major part of them is related to development of new stationary phases and their application in chromatography and sample pretreatment. Embodiments of molecular imprinted polymer materials have been carried out in a variety of forms such as irregularly ground particles, regular spherical particles, nanoparticles, monoliths in a stainless steel or capillary column, open tubular layers in capillaries, surface attached thin layers, membranes, and composites, etc. There have been numerous review articles on molecular imprinted polymer issues. In this special review, the reviews in recent ca. 10 years will be categorized into several subgroups according to specified topics in separation science, and each review in each subgroup will be introduced in the order of date with brief summaries and comments on new developments and different scopes of prospects. Brief summaries of each categories and conclusive future perspectives are also given.

Recent applications of molecular imprinted polymers for enantio-selective recognition.

Molecular imprinted polymer (MIP) techniques have been increasingly used in a variety of fields including chromatography, sample pretreatment, purification, sensors, drug delivery, and catalysts, etc. MIP is a specific artificial receptor that shows favored affinity to the template molecule. The cavities of the template are produced by carrying out polymerization of a reaction mixture followed by eliminating the template molecules by washing. Various forms of MIP materials have been prepared for diverse applications including irregularly ground particles, regular spherical particles, nanoparticles, monoliths in a stainless steel or capillary column, open tubular layers in capillaries, membranes, surface attached thin layers, and composites, etc. When an enantiomer is used as the template, then the resulting MIP can show capability of enantiomeric recognition between the pair of enantiomers. In this review, progresses in applications of enantio-selective recognition by MIPs will be critically reviewed for the recent period since 2007.

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.

Polystyrene bound stationary phase of excellent separation efficiency based on partially sub-2μm silica monolith particles.

Partially sub-2μm porous silica monolith particles have been synthesized by a renovated procedure and modified to polystyrene coated silica particles with excellent separation efficiency when used as chromatographic media. In the procedure of preparing silica monolith particles in this study, subtle control of formulation of the reaction mixture and multi-step heating followed by calcination, without any washing and sieving process, enabled formation of silica particles characterized by proper particle and pore size distribution for high separation efficiency. 3-Chloropropyl trimethoxysilane was used as the halogen terminal spacer to combine the initiator to silica particles. Uniform and thin coating of polystyrene layer on initiator attached silica particles was formed via reversible addition-fragmentation chain transfer (RAFT) polymerization. Micro-columns (1.0mm ID and 300mm length) were packed with the resultant phase and their chromatographic performance was elucidated by HPLC. A mobile phase of 60/40 (v/v) acetonitrile/water containing 0.1% TFA and a flow rate of 15μL/min were found to be the optimized conditions leading to number of theoretical plates close to 50,000 (165,000m(-1)). This is the very first study to get such highly efficient HPLC columns using a silica monolith particulate stationary phase.

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.

Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography

A silica capillary of 50 μm internal diameter and 500 mm length (416 mm effective length) was chemically modified with 4-(trifluoromethoxy) phenyl isocyanate in the presence of dibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with the terminal halogen of the bound ligand to incorporate the initiator moiety, and in situ polymerization was performed using a monomer mixture of styrene, N-phenylacrylamide, and methacrylic acid. The resultant open tubular capillary column immobilized with the copolymer layer was used for the separation of tryptic digest of cytochrome C in capillary electrochromatography. The sample was well eluted and separated into many components. The elution patterns of tryptic digest of cytochrome C were studied with respect to pH and water content in the mobile phase. This preliminary study demonstrates that open tubular capillary electrochromatography columns with a modified copolymer layer composed of proper nonpolar and polar units fabricated by reversible addition-fragmentation transfer polymerization can be useful as separation media for proteomic analysis.

Open tubular capillary electrochromatography with an N‐phenylacrylamide‐styrene copolymer‐based stationary phase for the separation of anomers of glucose and structural isomers of maltotriose

A ligand with a terminal halogen (4-chloromethylphenyl isocyanate) was chemically bound on the inner surface of pretreated silica capillary with 50 μm internal diameter and 58 cm total and 50 cm effective length in the presence of dibutyl tin dichloride as a catalyst through isocyanate-hydroxyl reaction. Attachment of initiator (sodium diethyl dithiocarbamate) to the bound ligand was carried out and followed by in situ polymerization. Reversible addition-fragmentation chain transfer polymerization was used for the immobilization of N-phenylacrylamide-styrene copolymer on the inner surface of capillary column. The resultant open tubular column showed excellent separation performance for derivatized saccharide isomers in capillary electrochromatography. D-Glucose was separated into α- and β-anomers while five structural isomers were separated for derivatized maltotriose with separation efficiency above one million theoretical plates per meter. The effects of pH and acetonitrile composition on the electrochromatographic performance of the derivatized saccharides were studied and the optimized elution condition was found to be 90:10 v/v% acetonitrile/30 mM sodium acetate at pH 6.6. UV absorption at 214 nm was used as detection mode in open tubular capillary electrochromatography separations.

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol-gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end-capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185,000/m) and chromatographic resolution were achieved when the C18 -bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless-steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60,000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C18 modification in addition to partial-monolithic architecture has resulted in a useful stationary phase (C18 -bound silica monolith particles) of low production cost showing excellent chromatographic performance.

Sedimentation assisted preparation of ground particles of silica monolith and their C18 modification resulting in a chromatographic phase of improved separation efficiency

The sedimentation procedure has been adopted in production of ground silica monolith particles to improve chromatographic separation efficiency of the resultant phase. First, silica monolith particles have been successfully prepared in a large scale by a sol-gel process followed by grinding. The particles after calcination were separated by sedimentation into three zones using an Imhoff sedimentation cone. The particles of the bottom zone were derivatized with a C18 ligand and end-capped. The sedimentation process was found to not only eliminate troublesome minute particles but also narrow down the particle size distribution. The resultant phase was packed in glass lined stainless steel micro-columns. The average number of theoretical plates (N) of the columns for a test mixture was 47,000 and 29,300 for the 300 and 150mm columns (1mm internal diameter), corresponding to 157,000/m and 195,000/m, respectively.

C18-bound partially sub-1μm porous silica monolith particles as low cost HPLC-stationary phase of excellent chromatographic performance and fast HPLC analysis

D toxicity observed in pre-/non-clinical animal studies sometimes leads to discontinuation of drug candidates. Understanding the phenomena or backgrounds surrounding toxicological events occurred must be a key element for attrition improvement in research and development (RD therefore, smooth translation of the findings can be made into the clinic. The presentation in this session will review the usefulness of toxicometabolomics technologies which are generally nuclear magnetic resonance (NMR)-based and mass spectrometry (MS)based, and other toxic-Omics technologies. Furthermore, today, newly introduced technology, MS imaging (MSI) is considered applicable in the toxicology field, hence its toxicological usability will be also reviewed.H in the form of chromatin heteroaggregates in cell culture harvests, are becoming recognized as a major challenge in antibody purification. However, recent publications have shown that immunoassays for host cell proteins do not detect histones. In addition, histone standards provided in commercially available histone ELISA kits are not based on authentic CHO histones. This presentation will show how to prepare authentic host cell histone standards, and how they are necessary for accurate quantitation of histone contamination in cell culture harvests and during purification procedures.Q is one of the most important pieces of information when using compounds in testing. It is important that the correct concentration is used when measuring within applications such as monitoring of reactions, protein ligand binding for the measurement of binding constants, dosage, fragment based screening, and monitoring of branching rates of polymers, among others. Such rate limiting steps are self evident in compound library screening where one must verify concentration in a high throughput means. With the improvements in software and NMR instrumentation, it is possible to have on the fly verification where concentration can be a key part of the verification process. Since the submitter has knowledge of the (suggested) structure, a known submission mass and a volume, the calculations for integral accounting and assignment can be output. Concentration is a key aspect because deviations are indicative of compounds purity from other compounds as well as impurities not directly observable by NMR such as salts. Additionally the solubility of the compound and stability are observed due to large changes in calculated versus measured concentration. All important aspects when validating a regular method for development/validation of definitive verification of a compound or mixture of compounds.H Cell-Based Assay (HCA) has attracted great attention due to its ability to be used in the drug discovery-driven research and development required to understand the functions of genes and gene products at the level of the cell. HCA simultaneously measures multiple biomarkers in a single cell with multiplexing fluorescent probes. The complex intracellular responses involved in drug-induced efficacy or cytotoxicity can be observed in organ-specific cells by HCA. Application of HCA to organ-specific cell models provides deeper biological information suitable for better decisions on progressing compounds. Early safety evaluation by HCA reveals the complex cellular responses triggered by potentially harmful molecules in the cells of target organs. Gaining a deep understanding of the mechanisms underlying these cellular toxicological responses is valuable before a series of lead compounds are progressed to time-consuming and expensive animal tests. Despite HCA’s capability, it is not common to simultaneously observe many biomarkers in an intact cell. This is because HCA measurement is dependent on the use of probing materials. Concurrent monitoring of multiple biomarkers is practically limited due to the spectral overlap among probing materials having broad absorption and emission spectrums. Quantum dot-based HCA is capable of supplying cellular imaging at particular wavelengths and each wavelength can be scanned rapidly. This cellular imaging is very advantageous in that it can select particular wavelengths that do not overlap among the probing materials and concurrently monitor a large number of drug targets or biomarkers.The recent rapid developments in microfluidics technologies has enabled the realization of miniaturized laboratories. These Labs-on-a-Chip will play an important role in future medicine, both in point-of-care devices for drug or biomarker monitoring, as well as in early diagnostic devices. We developed a pre-filled ready-to-use capillary electrophoresis platform for measuring ions in blood. It is used to monitor lithium in finger-prick blood of manic-depressive patients, but can also be used for measuring calcium in blood for prevention of milk fever, or for measuring creatinine in blood or sodium in urine for early detection of ESRD. Microfluidics can also be exploited to manipulate and experiment with cells on chip. We have developed a microsystem for sperm analysis and selection for artificial insemination, where we can electrically detect and sort healthy sperm cells. Using microdevices we have been able to electroporate and transfect genes into individual cells, and a microdroplet platform was used for encapsulation of single cells in microdroplets, ordering of these microdroplets and 1:1 fusion of these droplets to form hybridomas. We believe this is a very powerful new tool that can be used for high-throughput single cell experimentation. Apart from diagnostic and cell manipulation devices, microfluidic devices are increasingly used to realise advance disease and organ-models, as illustrated by the blood-brain barrier chip and a blood vessel on a chip to study atherosclerosis. Friday, December 4, 2015, 13:00 Room PH 127 Contact: Dietz Hendrik, dietz@tum.de, phone: 089-289-11615