Faster analysis by reversed-phase high-performance liquid chromatography

Summary Many analysts are not taking full advantage of the high speed possibilities of modern LC. Some analytical procedures reported in the literature, and many in regular use in control laboratories, could be achieved in less time without loss in precision. Some factors which affect retention times are discussed and the advantages and disadvantages of employing shorter column lengths and finer packing materials in reversed-phase HPLC are examined. The effect on efficiency of increased flow rates with 10,5 and 3 m ODS materials is shown. The ability to couple shorter column lengths without loss of efficiency is also demonstrated. This allows a minimum length to be selected that gives adequate resolution. Examples of high speed separations are shown and limitations in state of the art HPLC equipment and chromatographic data systems are discussed briefly.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Porphyrin analysis by reversed-phase high-performance liquid chromatography: biomedical applications

Reversed-phase high-performance liquid chromatography has considerable advantages in porphyrin analysis since many of the extraction and esterification steps required for normal-phase high-performance liquid chromatography can be eliminated. This technique is of increasing importance for the diagnosis of porphyrias and in other clinical and experimental studies of porphyrins.     

Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography

The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Chemometric classification of reversed-phase high-performance liquid chromatography columns

Cluster analysis and principal components analysis have been used to classify nine octadecyl (C18) high-performance liquid chromatography (HPLC) columns into three general groups displaying similar chromatographic behaviour. Principal components analysis was also able to identify the key test compounds on which the classification was most highly dependent. These identifications agreed with the classification and test compound selection by an HPLC specialist. In addition, the chemometric techniques can easily be extended to many more columns and test measurements than could be conveniently examined by a human expert.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

On-column endcapping and derivatization in reversed-phase high-performance liquid chromatography

Summary On-column endcapping and derivatization or regeneration of C₈ and C₁₈ reversed-phase HPLC columns with newly introduced reagents were studied. These treatments can increase column life expectancy by restoring retention times and original chromatographic characteristics of the columns. This is illustrated by examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.     

Separation of cytochromes c by reversed-phase high-performance liquid chromatography

Six kinds of cytochrome c of different origin, i.e., bovine, chicken, dog, horse, rabbit and tuna, were subjected to separation by reversed-phase high-performance liquid chromatography on three commercial packing materials; octadecyl-, octyl- and cyanoalkyl-silicas. The effects of reversed-phase material, mobile phase and temperature on the separation of cytochromes c were examined. The parameters of the mobile phase were the organic modifier, the pH, the salt concentration and additives. Under optimal conditions, five of the six cytochromes c were resolved in 10 min. The relative retention values cannot be explained in terms of the relative lipophilicities of the side-chains of the amino acid residues.     

Separation of pyrimidine derivatives by reversed-phase high-performance liquid chromatography

Chromatographic behavior and separation conditions of pyrimidine derivatives were studied by high-performance liquid chromatography using a reversed-phase column and a multiwave UV detector.     

Models of retention of adamantylamidrazones in reversed-phase high-performance liquid chromatography

Rules governing the chromatographic behavior of some amidrazones of the adamantane series were studied under the conditions of reversed-phase high-performance liquid chromatography. The characteristics of the retention of sorbates in elution by aqueous-acetonitrile phases with various compositions were calculated. Correlations between the structure and physicochemical characteristics of sorbate molecules and their retention were studied.     

Purification of bacilli ribonucleases by reversed-phase high-performance liquid chromatography

A two-step purification method is presented that utilizes the specific chromatographic properties of bacilli extracellular cycling ribonucleases. A double gradient system of elution is used. Initial concentration of the sample followed by reversed-phase HPLC gives high yields (90–95%) of homogeneous, active protein on both analytical and preparative scales. The procedure may be applied to the isolation of ribonucleases from different sources without significant modifications     

Separation of polypeptide antibiotics by reversed-phase high-performance liquid chromatography

Summary The influence of different reversed-phase packings and the addition of acidic modifiers to the mobile phase was observed on the separation of basic and neutral polypeptide antibiotics by gradient elution. A dependence of pore size, coverage, reaction type and endcapping of the packings was not observed. Nevertheless, not all reversed-phase packings were suitable for the separation of polypeptides, especially of basic molecules. The addition of phosphoric or perchloric acid to the mobile phase prevented adsorption of the basic polypeptide antibiotics on the stationary phase.     

Analyses of tetracyline antibiotics by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic (HPLC) separation was developed that is generally applicable to nine commercially important tetracyline antibiotics. The general method uses an isocratic system and mobile phase consisting of 0.001MEDTA, pH 6.6, and methanol and a Vydac C₁₈ reversed-phase column. Quantitation of the particular tetracyline in some commercial preparations is accomplished by adjusting the mobile phase composition. Quantitative assays were developed for small amounts of 4-epitetracycline in tetracycline (TC) preparations, demeclocycline in minocycline preparations and TC in chlortetracycline (chlor-TC) preparations. A fast HPLC assay for potency was also developed for chlor-TC and minocycline in these commercial preparations.     

Analysis of insulin preparations by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic system is described for the rapid and complete separation of bovine and porcine insulin from their readily formed monodesamido derivatives under isocratic conditions in the presence of the ion-pairing agent cetrimide. The system is suitable for the direct analysis of formulations of insulins of mixed bovine and porcine origin, and gives satisfactory results with a number of readily available commercial packings. Human insulin is not resolved from porcine in this system, but an alternative system allows the complete separation of all three insulins and their monodesamido derivatives, although acceptable peak shapes were obtained only on a limited number of packings.