Preparative high-performance liquid chromatography with reversed phase packed glass columns

An efficient system for preparative reversed-phase separations with packed glass columns is described. The advantage of this system is the use of relatively simple and inexpensive equipment. Column performance, load capacity, effect of the feed volume and the feed concentration on peak broadening are shown. The influence of the selectivity and the capacity factors on column load have been measured. The effect of the column dimensions is demonstrated by means of practical applications. The loading capacity of a column depends on the thermodynamic proporties of the separation system used. It is therefore not expedient in preparative chromatography to correlate the loading capacity of a column by means of grams dissolved per grams of adsorbent.     

High Performance Liquid Chromatography of Low Molecular Weight Proteins on a Non-Ionic Macroreticular Polystyrene Resin

Abstract

A high performance liquid chromatography system is presented for analytical and preparative separation of proteins. The method utilizes a macroreticular polystyrene resin having no specific functional groups, and proteins are eluted by the use of linear gradient of acetonitrile (20% ?75%, v/v) in 0.1% (v/v) trifluoroacetic acid. In this standard elution system, twenty proteins having a molecular weight of 4,200–58,000 and an iso-electric point of 3.9–11.0 have been chromatographed successfully within 80 min. The method allows a rapid, sensitive, and high resolution separation of relatively low molecular weight proteins, where the isolated proteins can be used for subsequent biochemical determinations.     

Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis

This feasibility study deals with the use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, to the separations and isolations of glycoforms of recombinant human erythropoietin (rhEPO). The preparative CITP separations were monitored by capillary zone electrophoresis (CZE) with a hydrodynamically closed separation unit. Such a CZE system, suppressing fluctuations of the migration data linked with fluctuations of EOF and hydrodynamic flow, made possible to evaluate and compare the preparative CITP separations performed within a longer time frame. Preparative CITP, carried out in the separation unit with coupled columns of enhanced sample loadability, separating 100 microg of rhEPO in a run lasting ca. 30 min, gave the production rate higher than 55 ng/s for the rhEPO glycoforms. The preparative separations included valve isolations of the glycoforms from the ITP stack into four or six fractions. Such numbers of the fractions corresponded to typical numbers of the major glycoform peaks as resolved in CZE of rhEPO. With respect to close effective mobilities of the glycoforms and a multicomponent nature of rhEPO, the fractions contained mixtures of glycoforms with the dominant glycoforms enriched 10-100-fold, relative to the original rhEPO sample.     

Plant Hormone Analysis by Countercurrent Chromatography

Abstract

Countercurrent chromatography (CCC) has been successfully applied for the separation of plant hormones; namely, indole auxins, gibberellins, cytokinins and abscisic acid. In our present study three different types of CCC devices were evaluated for their performance in separation of plant hormones with a special emphasis on analysis and purification of abscisic acid (ABA). A large-scale preparative CCC apparatus consisting of a slowly rotating coil assembly was used for preliminary separations of ABA from a large volume of crude plant extracts. The toroidal coil planet centrifuge (CPC) for analytical-scale separations was subsequently applied for purification of ABA, the final confirmation being obtained by HPLC and combined gas chromatographic-mass spectrometric method. This two-step procedure utilizing preparative CCC and toroidal CPC was successfully applied for determination of ABA content in several plant tissues. A recently introduced high-speed CCC apparatus was tested for semipreparative separation of ABA and indole-3-acetic acid. The method yielded high peak resolution within 2 hours.     

Fast Affinity Chromatography Using Small Particle Silica-Based Packing Materials

Summary

Affinity chromatography is one of the most powerful techniques for the purification of biologically active proteins available (for review see [1]). The ability of this method to purify proteins is based on highly specific, selective or characteristic interactions with immobilized ligands. Several advantages over traditional soft gel affinity supports have been observed with the use of small particle silica based materials for high performance affinity chromatography. These include greatly improved mass transfer properties which allow separations that are not always practical in the low performance mode, greatly reduced equilibration and isolation times, high available ligand densities, small elution volumes, excellent recovery of very small quantities of protein and high dynamic capacities. The criteria for developing a general, derivatizable, high performance support for high performance affinity chromatography are discussed. The step-by-step examination of these criteria and experimental evidence for determining parameters such as ligand density, non-specific adsorption and column life time for such a system are described. Chromatographic results are shown for preparative separations of (i) receptor proteins, (ii) antibodies and (iii) active enzymes.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

Preparative-scale fractionation by isoelectric trapping under nondenaturing conditions: separation of egg white protein isoforms on a modified Gradiflow unit

pH-biased isoelectric trapping was used to separate proteins from egg white at the preparative level (80 mg), into discrete protein fractions based on isoelectric point. The problems of isoelectric precipitation that are common for the separation of complex protein mixtures under isoelectric conditions were mitigated by using single-component isoelectric buffers within the sample separation compartments. This combined with the mild process conditions of the Gradiflow unit that was modified for binary isoelectric trapping separations, ensured that biological activity was maintained. This was verified by measurement of the trypsin protease inhibitory activity of the extract and separated fractions. Furthermore, the high resolving power of this system under preparative conditions was demonstrated by separation of three protein isoforms using isoelectric membranes with differences of 0.025 pH units from each other.     

Protein Separation and Enzyme Purification by Preparative Capillary Isotachophoresis

The use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, for separation and fast purification of the enzyme uridine diphosphate galactopyranose mutase (UGM) from the cell extract of Escherichia coli overproducing the recombinant enzyme is presented in this feasibility study. UGM is required to produce galactofuranose for the cell wall biosynthesis of many pathogenic microorganisms and represents a very attractive candidate for the development of new antimicrobial drugs. CITP separations were carried out under slightly alkaline pH conditions (8.7), in which UGM enzyme is negatively charged. Significantly simplified proteinous matrix isolated in several fractions by employed preparative CITP procedure with the aid of properly selected discrete spacers was subsequently confirmed by SDS PAGE with Coomassie staining. It was shown that preparative CITP is very effective tool for fast purification of the target enzyme from other proteinous matrix constituents when purification and isolation step lasted 20 min. The enzymatic activity of UGM was confirmed in the sample after the preparative CITP purification step, which is a crucial requirement for further biochemical applications.     

Separations of enantiomers by preparative capillary isotachophoresis.

The use of capillary isotachophoresis (ITP), operating in a discontinuous fractionation mode, for preparative separations of enantiomers of chiral compounds was studied. The ITP separations were carried out in the column-coupling configuration of the separation unit provided with the preseparation column of a 1.0 mm ID and the trapping column of a 0.8 mm ID. Such a configuration of the CE separation unit offers several working regimes suitable to preparative separations of enantiomers. 2,4-Dinitrophenyl-DL-norleucine (DNP-Norleu) was employed as a model analyte in our experiments with beta-cyclodextrin serving in the electrolyte solutions as a chiral selector. The preparative separations lasting about 20 min were evaluated by ITP and (more often) by capillary zone electrophoresis (CZE). It was found that one preparative run provided up to 14 microg of pure DNP-Norleu enantiomers. This corresponded to a 75 times higher production rate of ITP relative to a maximum value of this parameter as estimated for preparative CZE runs in cylindrical capillaries (0.5 pmol/s). About 75% of the DNP-Norleu enantiomers loaded into the preparative equipment could be recovered in pure enantiomer fractions. Contiguous natures of the zones in the ITP stack and adsorption losses of the enantiomers in the isolation step were found to set practical limits for a further enhancement of the recovery rates in the isolation of pure enantiomers.     

High-Performance Hydrophobic Interaction Chromatography of Proteins on TSKgel Phenyl-5PW Preparative Column

Abstract

TSKgel Phenyl-5PW preparative column of 200 × 55 mm I.D. was evaluated with respect to resolution, sample loading capacity and applications to the purification of enzymes. The preparative column provided similar separations as analytical column (75 × 7.5 mm I.D.) and 150 × 21.5 mm I.D. preparative column. The sample loading capacity was 200 – 1000 mg depending on the sample. If the slight decrease in resolution is acceptable, much more samples could be applied. Lipoxidase, phosphoglucose isomerase and lactate dehydrogenase could be purified to a great extent with high recovery of activity (more than 80%).     

Orthogonal analytical screening for liquid chromatography-mass spectrometry method development and preparative scale-up

An analytical HPLC-MS screening methodology has been developed to improve preparative RP-HPLC-MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical separations, none of them met our needs for the optimization of preparative conditions. Our screening protocol is based on searching among several orthogonal conditions to find the optimum preparative separation. Five different buffer conditions, from low to high pH, two organic solvents, acetonitrile and methanol, and five stationary phases of different polarities and characteristics were used. The orthogonality of the system was demonstrated using both, a standard mixture and mixtures from synthesis. To carry out the screening one of the analytical "open access" HPLC-MS systems was modified to perform the analytical screening while maintaining the open-access functionality for synthesis reaction monitoring. A software tool for automated sample programming and data reporting was also developed.     

Large-Scale Preparative Counter-Current Chromatography with a Coil Planet Centrifuge

Abstract

Development of the large-scale preparative countercurrent chromatographic schemes has been continued by increasing the diameter of the separation column. A 0.55 cm i.d. FEP tube was coaxially coiled around the holder (7.5 cm, 10 cm or 15 cm in diameter) of a horizontal flow-through coil planet centrifuge (15 cm revolutional radius). Performance of each column was evaluated on the separation of dinitrophenyl amino acid samples with a two-phase solvent system composed of chloroform, acetic acid, and 0.1N hydrochloric acid (2:2:1) by using both aqueous and nonaqueous phases as the mobile phase. Experiments with the short preliminary columns (114 ml capacity) revealed that the hydrodynamic distribution of the two solvent phases was sensitively affected by the helical diameter of the column. However, by choosing the proper elution mode of the mobile phase, satisfactory results were obtained with the helical diameters of 7.5 cm and 15 cm at a high flow rate of 500 ml/h under a moderate revolutional speed of 300 rpm. With the long coiled columns (750 ml capacity), the preparative capability of the present scheme was successfully demonstrated on separations of the 1g-quantity sample mixture under optimized operational conditions. Overall results indicated that the sample-loading capacity of the present scheme can be further increased by the use of longer and/or larger-diameter columns.     

Reverse Phase High Performance Liquid Chromatography of Human Bence Jones Proteins

Abstract

A high performance liquid chromatography system is presented for analytical and preparative separations of human Bence Jones proteins. The method utilizes 5–7 um macroreticular polystylene resin with bonded hydroxymethyl functional groups, and the proteins are eluted with a linear gradient of an increasing concentration of acetonitorile(10–60%, V/V) in 0.1 % (V/V) trifluoroacetic acid, pH 2.1. By this elution condition, seven X type Bence Jones proteins with molecular weights of 23,600 (monomer)-47,000(dimers) daltons(216–434 amino acids) were eluted within 80 min with the yields of 78%–98%. The method allows a rapid and sharp elution of Bence Jones proteins.     

Multidimensional preparative liquid chromatography to isolate flavonoids from bergamot juice and evaluation of their anti‐inflammatory potential

Important objectives of a high‐performance liquid chromatography preparative process are: purity of products isolated, yield, and throughput. The multidimensional preparative liquid chromatography method used in this work was developed mainly to increase the throughput; moreover purity and yield are increased thanks to the automated collection of the molecules based on the intensity of a signal generated from the mass spectrometer detector, in this way only a specific product can be targeted. This preparative system allowed, in few analyses both in the first and second dimensions, the isolation of eight pure compounds present at very different concentration in the original sample with high purity (>95%) and yield, which showed how the system is efficient and versatile. Pure molecules were used to validate the analytical method and to test the anti‐inflammatory and antiproliferative potential of flavonoids. The contemporary presence, in bergamot juice, of all the flavonoids together increases the anti‐inflammatory effect with respect to the single compound alone.     

Generic method for systematic phase selection and method development of biochromatographic processes. Part I. Selection of a suitable cation-exchanger for the purification of a pharmaceutical protein

Even if the first protein therapeutics are now for more than 20 years on the market the selection of suitable adsorbents for the preparative downstream processing (DSP) of these biomolecules as well as the method development towards process conditions are still based mainly on 'trial and error'. Therefore, theses processes are not perfectly efficient, but indeed very time consuming and laborious. In this study a novel systematic method is introduced to find a suitable adsorbent (not necessarily the best one) with appropriate separation parameters for a specific separation with reduced effort. Following this strategy, the adsorbents must first be packed into columns under preparative conditions and then characterized completely with regard to, e.g. pressure drop, k'-values, plate heights (HETP curves), selectivity and capacity by using test substances, which are similar in their characteristics (molecular mass, size, charge distribution, hydrophobicity) to the target proteins. With the database once determined, a preselection of most suitable adsorbents including separation parameters is made regarding chromatographic and also economical properties. After this, preparative experiments must be conducted with a reduced number of adsorbents to figure out the individual influence of side components. This approach is demonstrated for the separation of an exemplary industrial protein mixture using cation-exchange chromatography (CEX). Characterization of different weak CEX-adsorbents is illustrated. After comparing these phases with each other, a first preselection and a prediction of suitable adsorbents is made. In the following preparative separation conditions (load, velocity, gradient) are determined for the preparative separations using the database and results of some additional experiments. The final comparison of separation performance in preparative scale confirms this selection and so the applicability of the new method.     

Applications of High Performance Size Exclusion Chromatography To The Analysis of Oligomers and Small Molecules

Abstract

Size exclusion chromatography of small molecules has been investigated with high efficiency columns of crosslinked polystyrene. Performance evaluations and calibrations have been determined in toluene and in tetrahydrofuran, and the effect of the solvents on solute retention is discussed. Oligomer separations of samples obtained in the thermal degradation of several polymeric materials are reported, and the utility of the technique for polymer degradation studies is clearly evidentiated.     

Rapid Sara Seprations by High Performance Liqid Chromatography

Abstract

This paper presents both rapid analytical and preparative high performance liquid chromatographic (HPLC) techniques for separating liquid-fuel type materials into saturates, aromatics, resins, and asphaltenes (SARA). The preparative method, an adaptation of a technique developed by Jewell, et. al. (1, 4), significantly decreases analysis time. The analytical technique utilizes HPLC to achieve the same separations in less time.     

Large volume injection for preparative supercritical fluid chromatography

Summary A large volume injection system for preparative supercritical fluid chromatography is described. The method which is based on the solvent venting technique coupled with dilution of the sample solution consists of three steps. The first step is continuous dilution of the sample solution with liquid carbon dioxide at a controlled flow rate. The second step is solvent removal and solute trapping in a packed trap column. Combination of these two steps results in efficient solvent removal and the volume of sample which can be injected in a single injection becomes virtually unlimited. The third step is transfer and re-concentration of the solutes from the trap column on to the separation column with the pressures of both columns controlled independently; the final step is the separation. With this method, mass overloading behavior has been investigated and preparative separations performed.     

Gram Quantity Separation of DNP (Dinitrophenyl) Amino Acids with Multi-Layer Coil Countercurrent Chromatography (CCC)

Abstract

The efforts have been successfully made to extend the preparative capability of the high-speed CCC scheme with a multi-layer large capacity coiled column. The apparatus is a table top model of a horizontal flow-through coil planet centrifuge which produces a synchronous planetary motion of the column holder. The separation column was prepared from a single piece of 70 m long, 2.6 mm i.d., PTFE tubing coiled around the spool-shaped holder to form multiple layers of the coil with a total capacity of about 400 ml. The performance of the apparatus was assessed with a standard set of DNP amino acid samples and a two-phase solvent system composed of chloroform, acetic acid and 0.1N HCl (2:2:1). Preparative capability of the method was evaluated in terms of the retention level of the stationary phase and peak resolution for various sample size ranging from 0.05g to 2g. The effects of sample volume, sample concentration and the choice of the sample diluent on the separation were studied. The results indicated that both the retention level and the peak resolution tend to decrease with the increase of the sample volume applied at a given concentration. For separation of 1 gram quantity, best results were obtained by applying the sample dissolved in a small volume (10 ml) consisting of equal amounts of the two phases. Overall results indicate that the present scheme is capable of efficient separation for gram quantity of samples in a short period of time. The preparative capability may be further increased by the use of a larger-diameter and/or longer coil.