High-performance Displacement Chromatography Research Articles

Efficient purification of paclitaxel from yews using high-performance displacement chromatography technique

Paclitaxel was purified using high-performance displacement chromatography (HPDC) technique, but not by the mechanism of HPDC. On small scale, paclitaxel was extracted with methanol from dry needles of Taxus canadensis and was enriched by extracting with chloroform after removing water-soluble hydrophilic components and hexane-soluble hydrophobic components. Then, 93–99% purity of paclitaxel was obtained using the HPDC technique. On large scale, taxanes were enriched by solvent partitioning between acetic acid/MeOH/H 2O and hexane and extracted with CH 2Cl 2. Taxanes except paclitaxel were further removed by extracting with methanol–water–trifluoroacetic acid (1.0:98.9:0.1, v/v/v). Applying HPDC technique to water-insoluble substances is problematic as this method requires a highly aqueous solvent system. In order to overcome this incompatibility, a system was set up where paclitaxel, although in low concentration, was extracted by methanol–water–trifluoroacetic acid (10.0:89.9:0.1, v/v/v). Recycling the extracting solvent to ensure minimal volume, the extracted paclitaxel was adsorbed on a C 18 trap column. A C 18 column of 4.6 mm internal diameter was then connected to the trap column. The HPDC technique was thus carried out using an isocratic acetonitrile–water–trifluoroacetic acid (30.0:69.9:0.1, v/v/v) mobile phase consisting of a displacer cetylpyridinium trifluoroacetate (3 mg/mL). Paclitaxel was co-eluted with the displacer and spontaneously crystallized. The crystal (114 mg) showed 99.4% purity and only 10% of paclitaxel in the starting crude extract was lost during the enrichment/purification processes. This large scale purification method was successfully applied to purify paclitaxel from Chinese yew in small scale, suggesting general applicability of the method. This is the first report of purifying a water-insoluble natural product using HPDC technique.

One-step purification of palmatine and its derivative dl-tetrahydropalmatine from Enantia chlorantha using high-performance displacement chromatography

Palmatine and its reduced form, dl-tetrahydropalmatine are a group of isoquinoline alkaloids that have been reported to display a variety of biological and pharmacological activities. Both drugs are hydrophilic and are difficult to be purified by conventional purification methods of natural products. A high-performance displacement chromatography (HPDC) method successfully purified palmatine and its semi-synthetic derivative dl-tetrahydropalmatine from crude extract of the African medicinal plant Enantia chlorantha. The crude extract from the root bark of E. chlorantha was fractionated on an analytical reversed-phase C 18 column by using 0.1% trifluoroacetic acid (TFA) or acetic acid/H 2O as a carrier and cetylpyridinium trifluoroacetate (or acetate) (1.9 mg/mL) in 0.1% TFA (or acetic acid)/H 2O as a displacer. Palmatine was quantitatively purified at >98% purity in the fully developed displacement mode. dl-Tetrahydropalmatine was semi-synthesized by NaBH 4 reduction from crude palmatine and directly purified by HPDC. Both palmatine and dl-tetrahydropalmatine were identified by high-resolution electrospray tandem mass spectrometry, 1H NMR and 13C NMR. This is the first report of one-step HPDC purification of natural and semi-synthetic products from a complex crude extract.

Optimization of parameters of high-performance displacement chromatography for separation of soybean phosphatidylcholine and phosphatidylethanolamine

Hundred milligrams of soybean phospholipids were successfully separated by using high-performance displacement chromatography (HPDC) on a 150 mm × 4.6 mm analytical silica column (3–5 μm packings) with dichloromethane–methanol (9:1, v/v) as carrier and ethanolamine as displacer. From the viewpoint of preparative separation, the effects of loading amount, concentration and flow-rate of displacer on separation efficiency were investigated using throughput and recovery as indices. The parameters were optimized by orthogonal test design and statistical analysis method. Under the optimum conditions, namely displacer concentration being 167 mM, the flow-rate of displacer at 0.2 ml/min and concentration of sample being 211 mg/ml (factual loading amount 211 mg/ml × 0.7 ml = 148 mg), the purity, throughput and recovery of obtained soybean phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were 80.2%, 65.7 mg/h, 70.9% and 90.5%, 272.6 mg/h, 88.3%, respectively. In addition, selections of regenerant and appropriate regeneration condition were also studied.

Separation of phosphatidylcholine and phosphatidylethanolamine by using high-performance displacement chromatography

A binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was successfully separated by high-performance displacement chromatography (HPDC) on an 150 mm×4.6 mm analytical silica column (3–5 μm packing), using dichloromethane–methanol (9:1, v/v) as carrier and ethanolamine as displacer. The effects of displacer concentration, flow-rate, loading amount and the composition of the sample on separation efficiency were studied. Eighty-four milligrams sample (PE:PC 1:1.16) was separated perfectly by using 83 mM ethanolamine (in carrier) as displacer at the flow-rate of 0.1 ml/min. The yields of the pure PE and PC (100% purity) were 94.8% and 87.9%, respectively and the cycle time for a single separation was about 195 min. It was valuable that the optimum loading amount (the allowed maximum of sample loading) was investigated only by using the sample to be simulated the composition of the separated actual one, because the separation efficiency was significantly affected by the composition of the sample. For the same loading amount of 175 mg, the yields of the pure PE and PC were improved greatly from 31.4 and 16.9 to 56.0 and 77.6%, respectively, when the proportion of PE to PC was adjusted from 1:1.16 to 1:4. Furthermore, the separation of PE and PC in an actual sample (soybean phospholipids) was achieved using the proposed HPDC method.

20] Trace analysis peptide mapping by high-performance displacement chromatography

Publisher Summary Peptide mapping, by high-performance liquid chromatography (HPLC), is an indispensable tool for the structural characterization of proteins that is also widely used as a sensitive identity test in the biotechnology industry. This chapter describes the use of high-performance displacement chromatography (HPDC) for high-sensitivity peptide mapping of a recombinant protein and the application of the technique to liquid chromatography-mass spectrometry. The rationale, for adopting high-performance displacement chromatography, for mapping the peptides, formed by tryptic digestion of a protein, is to characterize the minor components of the mixture, whose presence conveys information about the composition of the protein preparation digested as well as about the specificity of the enzymatic digestion and side reactions, occurring in the digestion. Chief among the objectives of examining the “fine structure” in the enzymatic map is to identify the tell-tale peptides, indicating the presence of protein variants in the preparation. These variants can arise from degradative processes, acting on the protein, such as deamidation, oxidation, and proteolysis; from incorrect folding and disulfide rearrangement, from errors in translation by the host cell, from chemical modifications to the protein, caused by processing conditions used, for example, to cleave the target protein from a larger fusion construct, or from the heterogeneity conferred on a glycoprotein, by the covalently linked carbohydrate structures.

Comparison of drug binding interactions on human, rat and rabbit serum albumin using high-performance displacement chromatography

The binding of warfarin, a series of non-steroidal anti-inflammatory drugs and a series of benzodiazepines to rat serum albumin (RatSA) and rabbit serum albumin (RabSA) was compared with their binding to human serum albumin (HSA) using high-performance liquid chromatography on stationary phases based on immobilized albumins. The effect of the addition to the mobile phase of compounds known to bind to HSA at site I (phenylbutazone) or at site II (R- and S-ibuprofen) or at both sites (2,3,5-triiodobenzoic acid) was investigated on all three proteins. The results indicated that for the chiral compounds studied, the stereoselectivity of drug binding was much lower on RatSA than on HSA. On RatSA and RabSA, the benzodiazepine site was not a major binding site for R- and S-ibuprofen. The results indicated the existence of two binding sites for R and S warfarin on RatSA and probably on RabSA. On RatSA, one site is the major stereoselective site and is the major binding site of phenylbutazone and piroxicam. The other one is a major binding site for R- and S-ibuprofen and R- and S-ketoprofen.

Characterization of a tryptic digest by high-performance displacement chromatography and mass spectrometry

High-performance displacement chromatography (HPDC) provides a means of increasing the capacity of a chromatographic column, while maintaining the resolution afforded by high-performance liquid chromatographic (HPLC) instruments. The high capacity and high resolution of HPDC can be exploited in tryptic mapping to facilitate the characterization of a protein preparation. In this manner, minor constituents of the mixture, which may be difficult to isolate by conventional chromatographic methods, can be obtained in sufficient amounts to permit chemical characterization by established techniques. The isolation by HPDC of peptides obtained by digestion of recombinant human growth hormone (rhGH) and the subsequent characterization of the peptides are described. The identification of certain of these peptides revealed information on the specificity of trypsin for the substrate, rhGH, and for autolysis. Fractions from the HPDC tryptic map were collected and analyzed by electrospray ionization mass spectrometry (ESIMS) either directly or following further separation by gradient elution HPLC. Fragment ions observed in the ESI mass spectra facilitated identification of peptides obtained by HPDC tryptic mapping.

High-performance displacement chromatography—mass spectrometry of tryptic peptides of recombinant human growth hormone

The combination of high-performance displacement chromatography with continuous flow fast atom bombardment (FAB)-mass spectrometry (MS) offers a means ov overcoming the sample capacity limitations imposed by the low flow-rates tolerated in microbore systems employed for directly coupled liquid chromatography—MS. Displacement chromatography is performed at high concentrations with the same equipment and columns as typically used in chromatography at low concentrations. By using this mode of chromatography with a soltuion of cetyltrimethylammonium bromide as the displacer, the capacity of a reversed-phase column can be increased 50- to 100-fold for separation of a tryptic digest of biosynthetic human growth hormone. Despite the high load, the use of displacement chromatography allowed high-resolution separation of the complex mixture of eighteen major components. On-line analysis by continuous flow FAB-MS yielded high-quality spectra of these peptides and demonstrated that sharp, single-component bands can be obtained in this separation. Along with the major fragments, the chromatogram showed other peptides originating from protein variants in the sample, from non-specific cleavage in the enzymatic digest or from autolysis of trypsin. On-line analysis also allowed selective ion monitoring of the column effluent for individual pepetides and confirmed the high efficiency and resolution obtained by preparative displacement separations on HPLC columns and equipment.

High-performance displacement chromatography of melanotropins and their derivatives

Mixtures containing derivatives of the biological active peptide ∢-melanocyte stimulating hormone (α-MSH) or β-melanocyte stimulating hormone (β-MSH) were purified by using reversed-phase chromatography in the displacement mode. On a 250 × 4.6 mm octadecyl silica column and with instrumentation used in analytical high-performance liquid chromatography about 30 mg of the peptide mixture were separated by using an aqueous solution of benzyldimethyldodecylamonium bromide as the displacer in a single chromatographic run. The results demonstrate the advantages of displacement over elution in preparative chromatography of peptides on the scale of tens of miligrams that is typical for physiological tissue extracts and in solid-phase peptide synthesis.

High performance displacement chromatography of proteins: Separation of β-Lactoglobulins A and B

β-Lactoglobulins A and B were separated by high performance displacement chromatography on an anion-exchanger column with chondroitin sulfate as the displacer. A sample of 100 mg containing a mixture of the two β-lactoglublins was separated on a column of 75×7.5 mm in a single chromatographic run. The separation process followed the rules of the classical displacement development: the two proteins formed contiguous rectangular bands and their concentrations were dependent on the displacer concentration. The results demonstrate that high performance displacement chromatography is a useful technique for the separation of proteins in preparative amounts with columns and instrumentation typically used in analytical HPLC. Furthermore, it has the potential to become the method of choice in large scale separation of proteins.

High performance displacement chromatography: Calculation and experimental verification of zone development

AbstractEffluent concentration profiles from high performance displacement chromatography were calculated by using the theory of equilibrium chromatography of Helfferich and Klein (1970). The close agreement between predictions of the theory neglecting mass transfer resistances and experimental results demonstrates that conditions close to ideal chromatography are attainable in HPLC. Individual adsorption isotherms, as measured by frontal development, closely followed Langmuirian behavior.

Investigation of operating parameters in high-performance displacement chromatography

The effect of operational parameters of displacement chromatography was examined in the separation of various mixtures such as that of the main hydrolysis products of methylfurylbutyrolactone, a potential anticancer drug, the diastereoisomers benzoyl- d- and benzoyl- l-phenylalanyl- l-alanyl- l-proline, as well as polyethylene glycol homologues containing 1–10 ethylene oxide units. The chromatograph was assembled from modules generally used in analytical high-performance liquid chromatography (HPLC) and the column effluent was analyzed by an on-line HPLC unit at 30-sec intervals. Octadecyl-silica was used throughout as the stationary phase. Derivatives of ethylene glycol and propylene glycol as well as tetrabutylammonium bromide and n-butanol were used as displacers. The throughput was used as the measure of efficiency. In the absence of axial dispersion, for a given separation various displacers are expected to yield the same efficiency if the slope of the operating line is kept the same by appropriate adjustment of displacer concentrations. In practice, however, the optimum slope of the operating line has to be determined experimentally as most available chromatographic systems depart from ideal behavior. The dependence of the throughput on the flow-rate and feed load also indicated the presence of non-equilibrium phenomena and the optimum value of these parameters was established experimentally. In most cases water was used as the carrier solvent but the separation of poorly soluble peptides required the use of hydro-organic carriers. Results obtained with octadecyl-silicas of different origin and a given displacer were found to vary significantly suggesting that even for stationary phases of the same type the selection of displacer requires special consideration. Most experiments were carried out with columns having dimensions customary in analytical HPLC. Increasing the inner diameter of the column did not result in the expected increase in throughput probably due to poor distribution of the sample at the column entrance. Therefore scaling-up the process requires careful engineering of inlet conditions. Throughput can be increased by connecting a small inner diameter column to the outlet of a large diameter preparative column. As theoretical predictions for ideal displacement chromatography do not hold in practice when axial dispersion is significant, optimization of the process requires experimental support. The results obtained in the separation of a variety of mixtures shed light on the most important operational aspects of displacement chromatography and suggest approaches to find optimum conditions. They also point to displacement chromatography as a powerful technique for preparative separations.

High-performance displacement chromatography of nucleic acid fragments in a tandem enzyme reactor—liquid chromatograph system

Separation of the reaction mixture in the ribonuclease T1-catalyzed synthesis of GpU from cyclic GMP in the presence of a large excess of uridine was carried out by displacement chromatography on octadecyl-silica columns with 0.25 M n-butanol in the carrier buffer (50 mM phosphate, pH 3.0) as the displacer. The tandem reaction and separation was performed with an instrument assembled from parts customary in analytical high-performance liquid chromatography (HPLC). The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde coupling and used in a short packed-bed reactor. The conditions of the reaction that reaches equilibrium after approximately half of the cyclic GMP is converted and the results of displacement chromatography were evaluated by HPLC analysis. The synthesis took place in a 2.5 mm long × 4.6 mm I.D. packed-bed reactor used in the recirculating mode and the product was introduced directly into the chromatographic unit consisting of two 4.6 mm I.D. columns, 150 and 250 mm in lengths, in series. The bulk of the uridine separated by frontal chromatography in the first column was withdrawn and displacement development in the second column was used in order to isolate the product from other components. Nearly 100 mg of GpU with a purity of 99.7% could be achieved in a chromatographic experiment of 2.4 h. By returning unreacted starting materials to the reactor the process can be carried out in a continual fashion and the system readily leads itself to automation. The combination of similar enzyme reactors with the liquid chromatograph operated in the displacement mode offers an efficient means for laboratory-scale separation of complex biochemical substances.

High-performance displacement chromatography of corticosteroids : Scouting for displacer and analysis of the effluent by thin-layer chromatography

High-performance displacement chromatography was used for preparative scale separation of corticosterone, deoxycorticosterone and Reichstein's substance S with high-performance liquid chromatographic (HPLC) columns and instrumentation. Thin-layer chromatography (TLC) in the displacement mode was used to find an adequate carrier as well as a suitable displacer and its appropriate concentration. Separation of a mixture containing 60 mg of each component was carried out with a 500 >x 4.6 mm column packed with 5-μm silica gel. The carrier was chloroform and the displacer was 5% (v/v) diethylethanediamine in chloroform. Regeneration of the column was carried out with alcoholic acetic acid-chloroform mixtures and checked by an HPLC test. In order to evaluate the results, fractions of the column effluent were analyzed by TLC and the results were confirmed by HPLC. TLC was found to be a sufficiently accurate and highly convenient tool to determine product distribution upon separation by displacement chromatography.

High-performance displacement chromatography

The potential of the displacement mode of liquid chromatography for preparative-scale separations has been investigated by using microparticulate packed columns and instrumentation typically employed in analytical high-performance liquid chromatography (HPLC). Although the physico-chemical basis of the technique has long been established, its development was handicapped by lack of efficient chromatographic systems and suitable tools to monitor column effluent and gather data required for analysis and design of the displacement chromatographic process and for selection of suitable displacers. The availability of novel solid stationary phases with adequate properties to facilitate rapid sorption kinetics, the possibility of monitoring the effluent of the “fractionator” liquid chromatograph by an auxiliary ”analyzer“ unit as well as the relatively easy evaluation of appropriate adsorption isotherms by using the HPLC equipment, however, provide a basis to advance the displacement mode of liquid chromatography for preparative separations with analytical columns. After a brief review of the principles of the technique the dual chromatographic system used in the experiments for fractionation and analysis is described. Expressions for the efficiency of separation are given and factors affecting the development of the “displacement train” containing the separated feed components are enumerated. The interplay between the nature and concentration of displacer, the column length, amount of feed, flow-rate as well as the nature and concentration of the components of the mixture to be fractionated is discussed qualitatively. Fractionation of mixtures containing phenolic compounds or adenosine and inosine on columns packed with octadecyl-silica bonded phases (reversed-phase chromatography) is used to exemplify the effect of the operating conditions on the afficacy of the separation. The results are related to the adsorption isotherms of the substances that were measured by frontal analysis and found to follow the Langmuir model. The results suggest that upon accumulation of a sufficient amount of data on adsorptivities in chromatographic systems of interest high-performance displacement chromatography offers a promising approach to the use of ubiquitous HPLC equipment for preparative-scale separations with analytical columns.