A review of: “HPLC of Biological Compounds-A Laboratory Guide,by William S. Hancock & James T. Sparrow, (Volume 26 of the Chromatographic Science Series,Jack Cazes,Editor), Marcel Dekker,Inc., New York,NY, 1984, 361 pp., £39.75 (US)”.

Abstract

The analytical and preparative separation of the saturated, monoene, diene and triene constituents of cardanol and cardol have been achieved by reverse phase HPLC on a C₁₈ column using gradient elution.     

A Simple Liquid Chromatographic Method for Analysis and Isolation of the Unsaturated Components of Anacardic Acid

Abstract

Good analytical and preparative separation of the saturated, monoene, diene and triene components of anacardic acid have been achieved by reverse phase HPLC on a C₁₈ column by isocratic elution with methanol-aqueous acetic acid.     

Preparative HPLC Separation of Epimeric Guaianolides

Abstract

A technique for the preparative HPLC separation of the C-4 epimeric unstable guaianolides matricin and 4-epimatricin, proazulenes isolated from Artemisia arborescens L., is reported.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

Bile Acids. Lxx. Preparative Separation of Kryptogenin from Companion Sapogenins by High Performance Liquid Chromatography

Abstract

Commercial samples of kryptogenin or its acetate can be purified by preparative high performance liquid chromatography on a PrepPAK-500/Silica cartridge. The free alcohol is separated from accompanying sapogenin with a mixture of chloroform:methanol (50:1), whereas the acetates are separated well with a mixture of methylene chloride:hexane (2:3). The companion sapogenins, diosgenin and yamogenin, 25R- and 25S- isomers, were separated by analytical HPLC with hexane-isopropanol (100:1) or as the acetates with hexane:isopropanol (250:1). Characterization of kryptogenin and yamogenin was completed with ¹H-NMR, IR and MS spectrometry.     

Hydrocarbon Type Separation of Lubricating Base Oil in Multigram Quantity by Preparative HPLC

Abstract

A preparative High Pressure Liquid Chromatography (HPLC) method has been developed to separate lubricating base oil into its three major hydrocarbon fractions: saturates, aromatics, and polars. The results are directly comparable to ASTM Method D2007, hydrocarbon type analysis by gradient elution liquid chromatography. The new method employs a preparative HPLC unit with dual, radially compressed columns consisting of clay and alumina/silica gel columns. Multigram quantities of minor components (1 to 2% by wt.) of a base oil can be isolated for further study.     

Isoelectric Focusing with Immobilized pH Gradients

Abstract

The technique of isoelectric focusing (IEF) via immobilized pH gradients (IPG) was first announced to the scientific community at a meeting of the International Electrophoresis Society in Athens, April 1982, as the result of an intensive collaborative effort [1]. In five years, the technique has been extensively developed in three fundamental aspects: analytical, preparative and as a first dimension of two-dimensional (2-D) maps. The merits and, recently, the flaws of the IPG technique have been evaluated and recognized, so that at the present writing we feel it is ready for successful introduction in most life-science laboratories.     

High-Pressure Liquid Chromatography (HPLC) of Proteins [New Analytical Methods (29)]

The application of high-pressure liquid chromatography (HPLC) to proteins has undergone a dramatic development in recent years. Nowadays its many variants expand the repertoire of high-performance analysis methods available to the protein chemist, which, until now, have been dominated by electrophoretic techniques. The advent of gene technology has resulted in a renaissance of protein chemistry. The new analytical and preparative problems that have thereby emerged are often ideally solved by HPLC methods. HPLC has long since ceased to be solely a laboratory technique; HPLC systems are now being developed for the separation of proteins–particularly those of great pharmaceutical interest – on a 100-g scale. The range of applications of analytical and preparative HPLC will be illustrated by two examples of pharmaceutical importance—insulin and interleukin 2.     

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.     

Linear Multidimensional Liquid Chromatography in Protein Separation

Abstract

The objective of this article is to define, discuss and illustrate the concept of on-line multidimensional liquid chromatography (MDLC) in protein separation. In particular the emphasis of this paper is centered on a special form of on-line MDLC that will be referred to as linear MDLC. Examples of this technique, which involves the coupling of two or more chromatographic columns each employing a different separation mechanism, in both the analytical and preparative mode, are given in order to demonstrate its utility.     

Radiation-induced reactions in poly(1,2,2,2-tetrachloroethyl methacrylate)/monomer mixtures

The work has been aimed at characterizing final products of radiation-induced reactions in polymer/monomer mixtures particularly by analytical and preparative HPLC. Poly(1,2,2,2-tetrachloroethyl methacrylate) (PTCEMA) was used as polymer while p-cumylphenythacrylate (CUPMA) was mainly added as monomer. The substances were irradiated with γ-doses up to 160 kGy. The electron beam doses at an acceleration voltage of 30 kV corresponded to this value.With the HPLC method a CUPMA-grafted PTCEMA and CUPMA oligomers were detected. UV and ¹H-NMR spectra of these separated substances obtained by preparative HPLC confirmed the formation of a graft polymer. IR and Cl-elementary analyses revealed a Cl-substitution in CUPMA oligomers. Evidently, the conversion of unsaturated monomers is decisively determined by the radiation-chemical behaviour of the polymer.     

A High Performance Preparative Procedure for the Isolation of Degradation Products from D-Xylose

Abstract

An improved method for the preparative isolation of 3,8 dihydroxy-2-methylchromone, formed by degradation of D-xylose at 100°C is presented. From the ethyl acetate extractable part of the reaction mixture the chromone was isolated by preparative HPLC in less than one hour using a highly cross-linked dextran gel.     

The Analysis of Putrescine in Plant Samples by Automated Hplc

Abstract

The quantitative analysis of putrescine from plant tissue can be achieved using isocratic reversed-phase high performance liquid chromatography (HPLC). After preliminary extraction and clean up involving an ion exchange purification step, the isolated diamines are derivatised with benzoyl chloride for determination by HPLC. Automation of the HPLC step has led to a considerable saving in time for the total analysis. Potential problems associated with the analytical procedure are described.     

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.     

Mass spectrometry-guided isolation of two new benzoquinoline alkaloids from Macleaya cordata

Two new alkaloids, named 2,3-methylenedioxy-7,10-dimethyl-7,8,9,10-tetrahydro-benzoquinoline (1) and 2,3-methylenedioxy-7,10-dimethyl-8-carboxyl-benzoquinoline (2), were detected primarily from the fruits of Macleaya cordata by their different fragmentation pathways. And then isolation of the two compounds was performed by column chromatography and preparative HPLC under the guiding of mass spectrometry. Finally, their structures were determined by spectroscopic analysis.     

Synthesis and characterisation of a ruthenocenoyl bioconjugate with the cyclic octapeptide octreotate

The reaction of activated ruthenocene carboxylic acid with the resin-bound peptide octreotate yields, after cleavage and purification by preparative HPLC, the first ruthenocenoyl peptide bioconjugate 1. Octreotate is a chemically stabilized analogue of somatostatine. It is a cyclic octapeptide with a disulfide bond and has been previously used for molecular diagnostics due to the fact that somatostatine receptors are over-expressed by a variety of cancer cells. Conjugate 1 was obtained in good yield and purified by preparative HPLC to >95% purity as judged by analytical HPLC. It has been identified by HPLC, IR and mass spectrometry (ESI and MALDI-TOF). The peptide’s NMR signals are assigned by standard 2D methods. In addition, the ¹H NMR spectrum of 1 shows characteristic signals for the metallocene between 5.1 and 4.3 ppm. Compound 1 thus is a new example of tumor-targeted organometallic ruthenium bioconjugates.     

Separation of Bufotalin and Cinobufotalin by Preparative Liquid Chromatography

Abstract

Complete resolution of bufadienolides by traditional column chromatography, or by thin-layer chromatography (TLC), is quite difficult. Separation of various bufadienolide conjugates by high performance liquid chromatography (HPLC) has been described, but not for resolution of the corresponding genins. A preparative HPLC procedure has been developed for resolving the difficultly separable bufotalin (1) and cinobufotalin (2). A Lobar B column packed with LiChromprep Si-60 was used to separate these bufadienolides employing a recycling procedure.     

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%).     

A Low Cost Gram Quantity Preparative Reverse Phase Chromatography System

Abstract

The preparation of a low cost reverse phase preparative liquid chromatography system is described. The described system is capable of providing gram quantity scale-up from analytical reverse phase hplc systems. Load capacity is in the two gram range.     

Preparative argentation reversed-phase high performance liquid chromatography

In this study, a preparative chromatography method named preparative argentation reversed-phase high performance liquid chromatography (Ag-RP-HPLC) was developed by adding silver ion to the mobile phase of preparative HPLC. Firstly, an analytical Ag-RP-HPLC method was developed, and the effects of silver content and acid content in the mobile phase on the column efficiency were studied. Based on the method of linear amplification, a preparative Ag-RP-HPLC technique with optimized separation conditions was developed. The new technique was applied successfully to the separation of the unsaturated aliphatic acid amide isomers contained in Asarum forbesii Maxim. Compared with the commonly used technique of argentation normal phase chromatography, this method with little solvent consumption is simple, fast, efficient, and flexible for the isolation and purification of the unsaturated compounds.