Sample capacity in preparative high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) is a versatile technique in preparative separation and purification of pure compounds from complex matrices. As a preparative chromatography, there is a need to maximize the column production. Based on the plate theory of Van Deemter, the effect of the sample load on the separation was investigated in a preparative HSCCC with a 1000 ml column capacity. The test samples of hydroquinone, pyrocatechol and phenol were separated using a two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (1:1:1:1, v/v/v/v) at different sample loads. The results showed that for the case of HSCCC, the agreement of the effect of sample load on peak height and peak width between the Van Deemter's theory and the experiments is excellent. Furthermore, the factors limiting the mass load, including the resolution between the peaks, the partition isotherm and the solute solubility were also discussed.     

Ultrahigh-speed,ultrahigh-resolution preparative separation of protein biopharmaceuticals using membrane chromatography

This paper discusses ultrahigh-speed, ultrahigh-resolution preparative protein separation using an in-house designed membrane chromatography device. The performance of the membrane chromatography device was systematically compared with an equivalent resin-packed preparative column. Experiments carried out using model proteins showed that membrane chromatography gave more than four times greater resolution than the preparative column, while at the same time being more than 19 times faster. Membrane chromatography was therefore a better option, not only in terms of higher productivity but also in terms of higher product purity. Membrane chromatography was also superior in terms of resolving and presenting tracer impurity peaks in the chromatogram. Experiments carried out using monoclonal antibody samples showed that membrane chromatography was suitable for ultrahigh speed, and ultrahigh resolution fractionation of charge variants. This paper highlights and explains the need for proper device design for enabling the use of membrane chromatography for the efficient purification of protein biopharmaceuticals.     

Continuous purification of a clotting factor IX concentrate and continuous regeneration by preparative annular chromatography

Preparative continuous annular chromatography, a method to separate proteins in a truly continuous manner, was investigated in an industrial environment. Plasma-derived clotting factor IX concentrate was used as model protein. Separation of vitronectin, a common impurity in commercial available factor IX concentrates, from factor IX was studied and compared to conventional packed bed chromatography in batch mode. As sorbent, Toyopearl DEAE 650M was used. Regeneration was performed simultaneously with the purification of factor IX in continuous mode. All required parameters applied for preparative annular chromatography such as feed flow-rate and elution flow-rate were first estimated from experiments on conventional batch columns. Then preparative annular chromatography and conventional packed beds were compared regarding enrichment, purity and productivity. Three different process scenarios, the optimal batch process,the preparative annular chromatography process and the batch process equivalent to the preparative annular chromatography process were investigated. The productivity of the optimal batch process was higher than that of the preparative annular chromatography and batch process equivalent to the preparative annular chromatography process. Therefore the throughput could not be increased by the use of the continuous chromatographic system.     

Separation of ortho-substituted tetraphenylporphyrins by preparative HPLC

Summary A method for the separation of substituted tetraphenylporphyrins by preparative liquid chromatography is reported. The quality of the columns formed by axial compression of various stationary phases is tested.     

Long-acting androgens: Analytical and preparative HPLC of testosterone esters

Procedures are described for the normal phase and reverse phase HPLC analysis of testosterone esters having diverse side chains: examples are given of the gram-scale purification of these compounds using two commercial preparative liquid chromatography and an economical and efficient laboratory-assembled preparative liquid chromatograph.     

Reproducible preparative liquid chromatography columns

The level of reproducibility for sets of preparative liquid chromatography (prep-LC) columns was studied using a self-packing axial compression system. Standard deviations of less than 5% of the mean of the column efficiency and less than 2% of the mean of the retention time are reported for three sets of packed, prep-LC columns.     

Two-dimensional preparative liquid chromatography system for preparative separation of minor amount components from complicated natural products

An on-line comprehensive two-dimensional preparative liquid chromatography system was developed for preparative separation of minor amount components from complicated natural products. Medium-pressure liquid chromatograph (MPLC) was applied as the first dimension and preparative HPLC as the second one, in conjunction with trapping column and makeup pump. The performance of the trapping column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure from the view of medium pressure liquid chromatograph. Satisfactory trapping efficiency can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The instrument operation and the performance of this MPLC × preparative HPLC system were illustrated by gram-scale isolation of crude macro-porous resin enriched water extract of Rheum hotaoense. Automated multi-step preparative separation of 25 compounds, whose structures were identified by MS, ¹H NMR and even by less-sensitive ¹³C NMR, could be achieved in a short period of time using this system, exhibiting great advantages in analytical efficiency and sample treatment capacity compared with conventional methods.     

Experiences with the development and operation of a large-scale preparative gas chroamtograph

Summary A large-scale preparative gas chromatograph containing a 3 m long 100 mm l. D. column was built to investigate the application of preparative gas chromatography for the separation and production of various organic compounds. In this paper some experiences are reported concerning the operation of this large-scale preparative unit. In particular the efficiency of the receivers, the method of sample introduction as well as the stability of column packing were investigated.     

反相液相制备色谱法结合超临界流体制备色谱法分离纯化海风藤中的化合物

建立了基于反相液相制备色谱和超临界流体制备色谱的组合方法,用于分离纯化醇提水沉后石油醚层中的海风藤。首先以甲醇作为改性剂,采用醇提水沉法去除海风藤甲醇提取物中的叶绿素,加入硅藻土后用石油醚回流富集目标成分。选用反相C18制备色谱柱将其分为18个组分,然后将组分在SFC模式下进行制备。选用酰胺色谱柱,以甲醇为改性剂,在柱温30℃、背压15.0 MPa的条件下进行分离。基于反相色谱和超临界流体色谱不同的分离选择性,最后分离得到6个高纯度化合物。该法展示了反相制备色谱和超临界流体制备色谱在海风藤分离纯化方面的优势,特别是超临界流体色谱在天然产物的分析和制备方面的巨大潜力。     

Theoretical aspects of a preparative continuous chromatograph

Summary Equipment which has been used or suggested for preparative scale chromatography is reviewed. Particular attention is paid to one form of equipment for continuous counter-current chromatography which employs a moving mechanical seal. The theoretical effects of practical operating conditions and the mode of operation of this equipment are discussed. The influence of these effects on polymer fractionation is also considered. A theoretical model, based on the equilibrium stage concept, is proposed for the simulation of continuous gel permeation chromatography of a binary feed on such an apparatus. Examples of the results obtained from the computer simulation are given.     

Small-scale preparative supercritical fluid chromatography of cyproterone acetate

Summary A method for the micropreparative fractionation of steroid hormones from process solutions was developed. Using a commercial SFC equipment and raw cyproterone acetate (CPA) as a model sample, we showed the value of SFC as a lab scale purification method and also its potential as an environmentally friendly approach to preparative scale chromatography.     

Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies

Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths' inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 microm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.     

Exploiting pH mismatch in preparative high-performance liquid chromatographic recovery of ertapenem from mother liquor streams

Preparative chromatography was successfully employed to recover ertapenem from mother liquor streams. The recovery process involved concentration of mother liquor stream by evaporation, purification by reversed-phase preparative high-performance liquid chromatography (HPLC), and removal of chromatographic solvents in the recovered fractions by evaporation. HPLC feed was prepared by stripping off the organic solvents from the mother liquor using a wiped-film evaporator. Purification was first carried out on a 25 cm x 0.46 cm analytical column packed with 10-microm Kromasil C8 particles and then scaled up to a 25 cm x 5 cm preparative column. Gram-level recovery of ertapenem with high purity was achieved by exploiting a novel approach based on pH mismatch between the feed and the eluent. Purified ertapenem streams from preparative HPLC runs were combined, evaporated and recycled into the crystallizer for ertapenem isolation.     

Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides

Biologically active peptides synthesized by the solid phase methodology of Merrifield were purified by reversed-phase high-performance liquid chromatography using newly developed preparative radially compressed cartridges fitting Waters Assoc . Prep LC 500 liquid chromatograph. Cartridges were handpacked with Vydac C18, C4 or diphenyl derivatized silicas (pore size 300 A) of different particle sizes (10-20 micron). Large scale purification of gram amounts of gonadotropin releasing hormone analogs (agonist and antagonist) as well as amidated human pancreatic tumor growth hormone releasing factor (a 40-peptide) illustrate the resolutive power of this technique applied to the isolation of more than 300 synthetic peptides in our laboratory over the last two years. Difficult separations were achieved by changing supports (C18, C4, diphenyl) as well as mobile phase composition: (triethylammonium phosphate pH 2.25 or 6.5, 0.1% trifluoroacetic acid, ammonium acetate pH 6.5 and acetonitrile). Protected amino acids and peptides amenable to normal-phase chromatography on Vydac spherical underivatized silica were purified economically by the reversed-phase mode. It is understood that this general, convenient and versatile strategy may be applicable to the preparative scale isolation of any other class of compounds usually separated on reversed-phase high-performance liquid chromatography.     

High-throughput preparative process utilizing three complementary chromatographic purification technologies

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.     

preparative liquid chromatography: History and trends-supplemental remarks

Summary Supplementing the paper of Geeraert and Verzele published in the November 1978 issue of this journal, the controversy between Tswett and Willstätter on preparative liquid chromatography, as well as the pilotscale applications for the isolation of rare earths and pure hydrocarbons are discussed.     

Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds

A fully automated countercurrent chromatography system has been constructed to rapidly screen the commonly used heptane/ethyl acetate/methanol/water solvent system series and translate the results to preparative scale separations. The system utilizes “on‐demand” preparation of the heptane/ethyl acetate/methanol/water solvent system upper and lower phases. Elution‐extrusion countercurrent chromatography was combined with non‐dynamic equilibrium injection reducing the screening time for each heptane/ethyl acetate/methanol/water system to 17 min. The result enabled solvent system development to be reduced to under 2 h. The countercurrent chromatography system was interfaced with a mass spectrometer to allow selective detection of target components in crude medicinal chemistry reaction mixtures. Mass‐directed preparative countercurrent chromatography purification was demonstrated for the first time using a synthetic tetrazole epoxide derived from a routine medicinal chemistry support workflow.     

Excellent combination of counter-current chromatography and preparative high-performance liquid chromatography to separate galactolipids from pumpkin

Galactolipids in the fruits of Cucurbita moschata (pumpkin) could not be completely separated by high-performance liquid chromatography (HPLC). Preparative HPLC was not available for preparing major galactolipid monomers in pumpkin. In the present paper, a combination of high-speed counter-current chromatography (HSCCC) and preparative HPLC was used for preparing the galactolipids. A fraction containing galactolipids (Fr60) from the purification of the n-butanol extract of pumpkin by macro-porous resin column chromatography was first separated by HSCCC to result in three sub-fractions of each containing two galactolipid monomers. The three sub-fractions were further separated by preparative HPLC respectively to yield six galactolipid monomers with purity more than 96%. The method is a good one for preparing galactolipid monomers from plant materials for the studies of bioactivities.     

Improved preparative electrochromatography column design

Improved chromatography column fittings were developed for the efficient and reliable application of an electric field to a preparative chromatography column (a process termed electrochromatography). The improved fittings contained electrodes in close proximity to the column packing media and allowed uniform electric fields to be applied. Membranes in the fittings prevented mixing of the electrode and the column eluent buffers. The membranes prevented gases and electrolytic products generated in the electrode chamber from entering the column eluent buffer. An electrode buffer solution was pumped through the electrode chamber to a large external container. The circulation of buffer through the electrode chamber removed the gases and electrolytic products and ensured a uniform electric field by helping to maintain a constant buffer composition. The membranes prevented macromolecules being separated on the column from coming in contact with the electrodes.     

A quest for the optimal additive in chiral preparative chromatography

Traditionally, the choice of acid/base additives used in chiral preparative chromatography has not been considered very important. However, it was recently demonstrated that strongly adsorbing additives can result in the most unexpected enantiomer band shapes in modern chiral preparative chromatographic systems. In the present study we demonstrate that, depending on the choice of additive, it is actually possible to obtain the following four binary band-shape compositions when a racemic mixture is injected: (i) anti-Langmuir/anti-Langmuir, (ii) anti-Langmuir/Langmuir, (iii) Langmuir/Langmuir and (iv) Langmuir/anti-Langmuir. Further, we made an advanced numerical investigation, in order to ascertain which one of the four band-shape compositions, is the most favourable one in preparative batch chromatography of a racemic mixture. We found that if the target for purification is either the first eluting enantiomer or both ones, the traditional Langmuir/Langmuir band-shape composition should be chosen. But, if only the second eluting enantiomer is to be purified the optimal situation is the anti-Langmuir/Langmuir band-shape composition. Thus, it was concluded that the best choice of additive depends on which enantiomer is of interest and it is useful to perform a thorough additive screening to find the optimal additive, giving the most advantageous peak shape composition and accordingly the best process performance for a particular separation problem.