Interaction Chromatography Research Articles

Synthesis and characterization of polystyrene-b-polyisoprene-b-poly(methylmethacrylate) triblock copolymer

Polystyrene-b-polyisoprene-b-poly(methylmethacrylate) (PS-b-PI-b-PMMA) triblock copolymer was synthesized by sequential anionic polymerization. The as-synthesized triblock copolymer contains side products of inadvertently terminated PS and PS-b-PI precursors. The side products can be effectively removed by semi-prep scale liquid chromatography using multiple injection method to obtain pure PS-b-PI-b-PMMA. It was found that the removed side product contains another polymer species, coupled PS-b-PI, which could not be recognized by size exclusion chromatography (SEC) analysis since the elution peak of the coupled product overlaps with that of PS-b-PI-b-PMMA. This study demonstrates that the size based separation of SEC is often not good enough to characterize complex polymers precisely and interaction chromatography can render unique advantage over SEC analysis.

ChemInform Abstract: Isolation and Purification of Proteoglycans

Purification of a protein typically involves development of a quantitative assay to track protein integrity (e.g. enzyme activity) during subsequent isolation steps. The generalized procedure involves choosing the source of the protein, defining extraction conditions, developing bulk purification methods followed by refined, more selective methods. The purification of proteoglycans is often complicated by a) limited source quantities, b) necessity of chaotropic solvents for efficient extraction, c) their large molecular size and d) lack of defined functions to enable purity (i.e. activity, conformation) to be assessed. Because the usual goal of proteoglycan purification is physical characterization (intact molecular weight, core protein and glycosaminoglycan class and size), the problems of a suitable assay and/or native conformation are avoided. The ‘assay’ for tracking proteoglycan isolation typically utilizes uronic acid content or radiolabel incorporation as a marker. Once extracted from their cellular/extracellular environment, proteoglycans can be isolated by density gradient centrifugation and/or column chromatography techniques. Recent advances in the composition of chromatographic supports have enabled the application of ion-exchange, gel permeation, hydrophobic interaction and affinity chromatography resins using efficient high-pressure liquid chromatography to proteoglycan purification.

NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity.

BackgroundNAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.ResultsHydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.ConclusionsThe results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.

Liquid chromatography at the critical condition: Thermodynamic significance and influence of pore size

AbstractLiquid chromatography at the critical condition (LCCC) is a high performance liquid chromatography (HPLC) technique that lies between size exclusion chromatography and adsorption‐based interaction chromatography, where the elution of polymers becomes independent of polymer molecular weight. At LCCC, the balance between the entropic exclusion and the enthalpic adsorption interactions between polymers and stationary phases results in the simultaneous HPLC elution of polymers regardless of molecular weight. Using C18‐bonded silica chromatographic columns with 5 μm particle size and different average pore size (diameter = 300 Å, 120 Å, 100 Å, and 50 Å), we report (1) the thermodynamic significance of LCCC conditions and (2) the influence of column pore size on the determination of critical conditions for linear polymer chains. Specifically, we used mixtures of monodisperse polystyrene samples ranging in molecular weight from 162 to 371,100 g/mol and controlled the temperature of the HPLC columns at a fixed composition of a mobile phase consisting of 57(v/v)% methylene chloride and 43(v/v)% acetonitrile. It was found that, at the fixed mobile phase composition, the temperature of LCCC (TLCCC) is higher for C18‐bonded chromatographic columns with larger average pore size. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 2533–2540, 2009

Discriminating Among Co‐monomer Sequence Distributions in Random Copolymers Using Interaction Chromatography

Interaction chromatography has been employed to validate that adsorption of poly[styrene-co-(4-bromostyrene)] (PBr(x) S) random copolymers, where x denotes the mole fraction of 4-bromostyrene (4-BrS) in PBr(x) S in solution depends on the average number of adsorptive segments, the type of adsorbing substrate, and on the co-monomer sequence distribution in PBr(x) S.

Evaluation of Chromatographic Methods for Drug Products Containing Polar and Non-Polar Molecules Using Reversed Phase, Hydrophilic Interaction, and Ion Exchange Chromatography

In recent years the pharmaceutical industry has developed an increasing variety of complex double or triple combination drug therapies. Method development for these combination drug products is particularly challenging when the analytes have significantly different polarities, often requiring multiple chromatographic methods for each active component. An alternative to the commonly used practice of developing multiple reversed phase chromatographic methods for analyzing small molecules in combination drug products is discussed herein. Ion exchange chromatography offers many advantages to other chromatographic techniques for the analysis of combination products if one of the molecules is highly polar (log P < 0) and the other moderately polar to non-polar (log P ≥ 0). As the primary mode of retention for ion exchange chromatography is based upon the charge of the molecule, the hydrophobicity of the molecule does not play as large of a part in the retention mechanism. As long as all of the molecules being analyzed are ionizable, retention on an ion exchange column should be possible. The analysis of polar and non-polar molecules simultaneously using ion exchange chromatography is a unique process, and the approach of developing accurate and robust analytical methods for analysis of all active pharmaceutical ingredients in a combination product by a single fast ion exchange method differs from the industry standard of using reverse phase methodology first. Six well characterized pharmaceuticals with varying degrees of polarity (metformin HCl, isoniazid, sodium nitroprusside, timolol maleate, naproxen sodium, and clonidine hydrochloride) have been chromatographically evaluated using RP, HILIC, and ion exchange chromatography. In addition, a well characterized pharmaceutical product, Metaglip™ Tablets, containing the highly polar molecule metformin HCl and the moderately non-polar glipizide, has been analyzed using a cation exchange (CE) HPLC method and compared to chromatography and results generated using RP-HPLC and hydrophilic interaction (HILIC)-HPLC.

Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic- interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine into the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein-cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high-molecular-mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesin-coated slides. These results indicate that the pollen TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes.

THE MICELLIZATION OF PLURONICS (PEO-PPO-PEO TRIBLOCK COPOLYMERS)

The micellization of several Pluronics (PEO-PPO-PEO triblock copolymers) was investigated by Differential Scanning Calorimetry (DSC). These copolymers were also characterized and purified by liquid chromatography. The PEO/PPO compositions of Pluronics were determined by 'H-NMR. The thermograms from DSC showed a pre micellization shoulder for L81 and L121 solutions. The effects of temperature, PO/EO ratios and impurities on the micellization of copolymers were investigated. The origin of the shoulders in DSC thermograms was shown to be due to the most hydrophobic components in the copolymers, which could be removed by hexane extraction. Low molecular weight fractions of the copolymers were separated by interaction chromatography. The low molecular weight fractions did not contribute to the micellization of the copolymers.

Novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography, an excellent orthogonal system for practical analysis

A novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography (2D-RPLC/HILIC) system is developed with the introduction of the click β-cyclodextrin (β-CD) stationary phase. The offline system shows excellent abilities for the separation of polar and medium-polarity components in traditional Chinese medicine (TCM). It facilitates us not only to separate components that cannot be resolved by uni-dimensional chromatography, but also to achieve much more efficient detection of components with low abundance. The orthogonality of this new system is excellent. A simple geometric approach is developed to characterize the practical orthogonality of 2D-LC in the analysis of complex unknown samples. The mathematical characterization results are in good accordance with the experimental findings, and both demonstrate that the proposed 2D-RPLC/HILIC system is a powerful tool for the separation of polar and medium-polarity complex samples. This system may have a great potential for the separation of peptides and metabolomic compounds with similar polarity.

Unusual Sensitivity of Closed-Loop Phase Behavior to Chain Size and Distribution

An anionically polymerized polystyrene-block-poly(n-pentyl methacrylate) (PS-b-PnPMA) was fractionated by interaction chromatography (IC), and the closed-loop phase behavior of the mother block copolymer and its fractions with different chemical compositions was investigated by depolarized light scattering and polarized optical microscopy. With increasing elution time the average chain size (or the average molecular weight) of the block copolymer decreased slightly, and the average chemical composition changed steadily from a PS volume fraction of 0.583 to 0.403, thereby producing five fractions collected over the different ranges of elution time. This is caused by the difference of relative interaction strength between the stationary phase and the PnPMA chains of PS-b-PnPMA, in which PnPMA chains tend to be adsorbed onto the stationary phase while PS chains behave oppositely. Over the range of accessible temperatures studied (110−250 °C) a variety of phase transitions were observed, including fully ordered state, a closed-loop (consisting of the lower disorder-to-order transition and upper order-to-disorder transition), and fully disordered state. The original, unfractionated block copolymer was disordered over the entire temperature range. The variety of observed phase transitions obtained from the fractionated block copolymers is assumed to be governed by the sensitivity of the closed-loop phase behavior to average chain size, average chemical composition, and the compositional broadness.

Preparation and Characterization of Cyclic Polystyrenes

Macrocyclic polystyrenes with various molecular weight were prepared by the reaction of linear polystyrene having two end vinyl groups and potassium naphthalenide as a coupling agent. Intermolecular side reactions produced higher molecular weight polycondensates and undesirable chain termination reactions also produced linear precursors in addition to the designed molecules. In order to isolate cyclic polymers from the ring closure reaction mixtures, preparative size exclusion chromatography (SEC) method was employed. The purity of SEC-fractionated cyclic polystyrenes was rigorously examined by liquid chromatography at the critical condition (LCCC) and interaction chromatography (IC). After SEC-fractionation, we obtained large amount of highly pure cyclic polymers, though high molecular weight cyclic polymers contain very small amount of linear precursor (<5%). The purity and isotope effect on reversed-phase liquid chromatography (RPLC) were also rigorously investigated by preparing a deuterated cyclic polystyrene.

Polymer characterization by interaction chromatography

AbstractLiquid chromatography (LC) is a powerful tool for the characterization of synthetic polymers, that are inherently heterogeneous in molecular weight, chain architecture, chemical composition, and microstructure. Of different versions of the LC methods, size exclusion chromatography (SEC) is most commonly used for the molecular weight distribution analysis. SEC separates the polymer molecules according to the size of a polymer chain, a well‐defined function of molecular weight for linear homopolymers. The same, however, cannot be said of nonlinear polymers or copolymers. Hence, SEC is ill suited for and inefficient in separating the molecules in terms of chemical heterogeneity, such as differences in chemical composition of copolymers, tacticity, and functionality. For these purposes, another chromatographic method called interaction chromatography (IC) is found as a better tool because its separation mechanism is sensitive to the chemical nature of the molecules. The IC separation utilizes the enthalpic interactions to vary adsorption or partition of solute molecules to the stationary phase. Thus, it is used to separate polymers in terms of their chemical composition distribution or functionality. Further, the IC method has been shown to give rise to much higher resolution over SEC in separating polymers by molecular weight. We present here our recent progress in polymer characterization with this method. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1591‐1607, 2005

Effects of Poloxamer Inhomogeneities on Micellization in Water

Pluronic P85 (poloxamer 235), which is a symmetrical PEO−PPO−PEO triblock copolymer of poly(ethylene oxide) and poly(propylene oxide), has been investigated by differential scanning calorimetry and chromatography. Interaction chromatography at critical conditions for PEO was used for chromatographic characterization and preparative separation of P85 according to PPO block lengths. The results show that the P85 sample is heterogeneous with respect to chemical composition and molar mass. P85 contains 12 wt % lower size impurities, which do not contribute to the main micellization transition. The shapes and widths of the micellization peaks observed in P85 thermograms are influenced by the distribution of PPO block lengths in P85, as shown from measurements on chromatographic fractions. PPO block lengths of the main P85 fraction follow a Poisson distribution closely. The characteristic temperatures of micellization, sphere-to-rod, and phase separation transitions observed in thermograms are influenced by polydispersity.

Production and characterization of recombinant guamerin, an elastase-specific inhibitor, in the methylotrophic yeast Pichia pastoris.

The elastase-specific inhibitor, guamerin, was expressed and secreted into a culture medium using the methylotrophic yeast Pichia pastoris, and the resulting recombinant guamerin was purified from the culture media using a two-step procedure composed of a hydrophobic interaction and reverse-phase chromatography. Up to 90 g/L of dry cell weight, the guamerin-producing recombinant P. pastoris was cultivated and guamerin was secreted into the culture medium at a level of 0.69 g/L. The recombinant guamerin was highly purified (>98%) with a recovery yield of 68%. Analyses of the purified guamerin revealed the same N-terminal amino acid sequence, amino acid composition, and molecular mass as found in the native leech protein. The recombinant guamerin exhibited the tight binding to porcine pancreatic elastase. Furthermore, the recombinant guamerin did not produce a humoral immune response in mice.

Mixed-mode anion-cation exchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometry as an alternative to reversed phase for small molecule drug discovery

Within pharmaceutical drug discovery, significant needs currently exist for the analysis and purification of structurally diverse samples prior to or immediately following high-throughput screening. These processes are required to facilitate rapid and accurate biological profiling, structural determination, and resupply of new drug candidates. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) for both analytical and preparative applications has become the small molecule separation/detection tool of choice for meeting many of these needs. However, the separation selectivity provided by RP-HPLC has been limited to the hydrophobicity-based resolution of relatively nonpolar sample components, and for high-throughput drug discovery applications, no sufficient alternative procedures have been identified. In this investigation, a mixed-mode anion-cation exchange/hydrophilic interaction chromatography (ACE-HILIC) method has been developed to provide both direct compatibility with ESI-MS and evaporative light-scattering detection (ELSD) and separation selectivity highly orthogonal to RP-HPLC. The technique employed silica-based small-pore weak ion exchange resins eluted with a combined aqueous and pH gradient. A diverse set of dipeptide probes was employed for the elucidation of the relative contributions of three retention mechanisms. ACE-HILIC-ESI-MS-ELSD should prove useful for the analysis and purification of compounds from both biological (e.g., natural products) and synthetic (e.g., combinatorial chemistry) sources of molecular diversity.

Resolution and Retention of Proteins near Isoelectric Points in Ion-Exchange Chromatography. Molecular Recognition in Electrostatic Interaction Chromatography

Both molecular recognition and transport phenomena play important roles in very high resolution ion-exchange (electrostatic interaction) chromatography (IEC) of proteins. A fast and simple method for obtaining important information on molecular recognition, peak retention (and the number of binding sites), and peak resolution of proteins from linear gradient elution experiments (salt concentration is increased linearly at a fixed mobile phase pH) is described. The proposed method was verified with β-lactoglobulin A and B (LgA, LgB) as model proteins. The gradient elution experimental data were obtained over a wide range of mobile phase pH with various types of IEC media. The number of binding sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI (= 5.1–5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached the pI in anion-exchange chromatography columns where the number of adsorption site values are small(ca. 2–3). The two proteins were not separated even with efficient cation-exchange chromatography columns. The resolution and the retention near the pI (pH 5.2) did not significantly depend on sample loading compared with those at pH 6.0.

Purification and properties of a lipase fromPenicillium chrysogenum isolated from industrial wastes

A fungal lipase was isolated from the wastes of industrial cultures of Penicillium chrysogenum employed in antibiotics production. Lipase activity was only detected in solid aggregates present in the protein concentrates. To extract the lipase, Triton X-100 was the most suitable surfactant. The lipase was further purified by hydrophobic interaction and anion exchange chromatography. A molecular mass of approx 40 kDa and a pI of approx 3.8 were estimated. Optimum pH and temperature were 7.9–8.1 and 45 °C respectively. Although its activity in the hydrolysis of lipids and esters is not very high, the lipase from P chrysogenum proved to be notably efficient in several synthetic reactions in non-aqueous media. © 2000 Society of Chemical Industry

Properties and substrate specificities of an extracellular lipase purified from Ophiostoma piceae

An extracellular lipase produced by the sapstaining fungus Ophiostoma piceae 387N in a liquid medium was purified to homogeneity using ammonium sulphate and acetone fractionation, hydrophobic interaction and anion exchange chromatography. The overall purification based on lipase activity was 5200-fold with a yield of 26%. The molecular mass of the lipase was 35kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and 37 kDa, as measured by size exclusion chromatography. The purified enzyme was resolved as three bands at pI values of 4.3, 4.1 and 3.8 in IEF (isoelectric focusing) gels. Lipolytic stain demonstrated that all three bands were lipolytically active. The N-terminal amino acid sequence was determined asD1-V2-S3-V4-T5-T6-T7-D8-I9-D10-A11-L12-A13-F14-F15-T16-Q17-W18-A19-G20 . The lipase was shown to be glycosylated, containing 10.1% carbohydrate. The lipase was stable between pH 4 and pH 8 and at temperatures below 40°C. The lipase activity had a pH optimum of approximately 5 and a temperature optimum of 30°C. The enzyme activity was not influenced by N-ethylmaleimide, β-mercaptoethanol or dithiothreitol, was enhanced by Ca2+ or Mn2+, but was severely inhibited by Hg2+, Fe3+, butyric acid, caproic acid, diethyl pyrocarbonate, and diethyl p-nitrophenyl phosphate. The lipase hydrolysed mainly triglycerides, although some activity was measured on waxes and cholesteryl esters. It belongs to a group of 1 (3) positional specific lipases. It showed little activity for substrates with short chain fatty acids (C2–C6), but demonstrated high specificity for substrates with intermediate and long chain fatty acid residues (C10–C18).

Characterization of Binary Polymer Mixtures by Simultaneous Size Exclusion Chromatography and Interaction Chromatography

It is shown that polystyrene/poly(methyl methacrylate) blends can be separated with a good resolution in terms of their chemical nature and molecular weight simultaneously

Biochemical properties of 1-aminocyclopropane-1-carboxylateN-malonyltransferase activity from early growing embryonic axes of chick-pea (Cicer arietinumL.) seeds

In the present work, certain biochemical characteristics of the enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase (ACC N-MTase) which is responsible for the malonylation of 1-aminocyclopropane-1-carboxylate (ACC) in chickpea (Cicer arietinum) are described. Phosphate buffer was the most appropriate buffer with regard to enzyme stability and, therefore, ACC N-MTase was extracted, assayed and purified in the presence of this buffer. ACC N-MTase was partially purified approximately 900-fold from embryonic axes of chick-pea seeds using ammonium sulphate precipitation, hydrophobic interaction and molecular filtration chromatography. By gel filtration chromatography on Superose-12, the molecular mass of the enzyme was estimated to be 54±4 kDa. ACC N-MTase had an optimal pH and temperature of 7.5 and 40°C, respectively, as well as a K m for ACC and malonyl-CoA of 400 μM and 90 μM, respectively. D-Phenylalanine was a competitive inhibitor of ACC N-MTase with respect to ACC (K i of 720 μM), whereas co-enzyme A was a competitive product inhibitor with respect to malonyl-CoA (K i of 300 μM) and a non-competitive inhibitor with respect to ACC (K i of 600 μM). Under optimal assay conditions, ACC N-MTase was strongly inhibited by (a) divalent [Zn 2+ > Mg 2+ > Cu 2+ >> Co 2+ > (NH 4 ) 2+ > Fe 2+ ] and monovalent metal cations (Li + > Na + > K + ), without activity being detected in the presence of Hg 2+ , and (b) PCMB or mersalic acid, suggesting that sulphydryl group(s) are involved at the active site of the enzyme.