Separation of polyesters by gradient reversed-phase high-performance liquid chromatography on a 1.5 microm non-porous column

Efficient separation of polyesters composed of a large number of individual oligomers was achieved on a 1.5 microm "non-porous" octadecylsilyl (ODS) silica support by gradient high-performance reversed-phase liquid chromatography (gRP-HPLC) with a mobile phase of acetonitrile, aqueous trifluoroacetic acid (0.2%) and tetrahydrofuran at ambient temperature and signal monitoring by UV absorption at 280 nm. Substantial signal splitting of oligomers in the low molecular weight (Mr) region is indicative that separation not only occurs with respect to molecular weight distribution (MWD) but also to chemical composition distribution (CCD) and functionality type distribution (FTD). Although separation according to CCD and FTD decreases with increasing number of oligomers, co-elution of species with identical number of repeat units but differing in either structure of repeat units or end-groups can be assumed from the relatively broad signals succeeding the aforementioned peaks showing at least partial resolution. Despite the observation that high Mr oligomers elute as sharp signals, the preceding observations suggest that each of these peaks presumably composes of more than one individual component. The polyester oligomers are eluted in the range of increasing Mr and therefore, either separation according to MWD or CCD/FTD was at least achieved for the low Mr sample constituents. Some principal mechanistic aspects of separation are discussed and adsorption seems to play the dominant role. The detection limit, defined as that sample amount yielding an unequivocal recognition on the base of its characteristic chromatographic fingerprint pattern was about 5,000 ppm for the pair Alftalat 3258 - Alftalat 3352 and 10,000 ppm for the pair Crylcoat 430 - Crylcoat 801.     

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 beta-chain variants, 7 alpha-chain variants, 9 gamma-chain variants, and 4 variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the delta/non-alpha ratios allow the detection of beta-thalassemic conditions in situations where the quantitation of hemoglobin A2 by other procedures is impossible or complicated.     

Separation of porphyrin isomers by high-performance liquid chromatography

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.     

Separation of pyrethroid enantiomers by chiral high-performance liquid chromatography

The separation of enantiomers of pyrethroid insecticides has been systematically studied using a commercially available Pirkle type 1-A chiral-phase high-performance liquid chromatography column. Useful resolution was obtained for compounds with a variety of acid and alcohol moieties, and containing one to four chiral centres. The chromatographic behaviour of the diastereomers of some of these insecticides on a cyano-bonded column was also examined.     

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.     

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.     

Separation of potential flavoring compounds by high-performance liquid chromatography

High-performance liquid chromatography separated successively and quantitatively the food flavoring agents pyrimidines, purines and nucleosides, followed by nucleotides, then by polyphenols and finally by pyrazines with a reversed-phase octadecylsilica (μBondapak C₁₈) column and various proportions of methanol, water, acetic acid and tetrabutylammonium phosphate (PIC A). The polar solvent (solvent A) was water—acetic acid—PIC A (97.5:1.5:1.0) and the relatively non-polar solvent (solvent B) was methanol—acetic acid—PIC A (97.5:1.5:1.0). Purines, pyrimidines, and nucleosides were eluted with solvent A. Nucleotides were eluted with a mixture of solvents A and B (9:1). Polyphenols were separated with a gradient starting at 10% solvent B and finishing at 25% solvent B, and finally the pyrazines were removed successively from the column with a gradient starting at 25% solvent B and finishing at 45% solvent B. The resolution and reproducibility were excellent for more than 50 compounds. By this method beverages could be analyzed directly, without solvent extraction, for flavoring compounds.     

Separation of tubulin subunits by reversed-phase high-performance liquid chromatography

When properly solubilized with trifluoroacetic acid (TFA), alpha- and beta-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters muBondapak C18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the beta-tubulin subunit eluting before the alpha. Column temperature above ambient improve both the resolution and the yield; less polar solvent systems do not. Tubulins not freely soluble in aqueous TFA may be solubilized in 6 M guanidine-hydrochloric acid with no change in retention time. Other columns with shorter carbon chain lengths and larger pore size produce a single, unresolved tubulin peak. Reversed-phase HPLC analysis provides an independent comparative evaluation of organelle-specific tubulins, with characteristic retention time differences observed between homologous ciliary and flagellar outer doublet tubulin subunits and also between them and their cytoplasmic counterparts.     

Separation of pantothenic acid derivatives by reversed-phase high-performance liquid chromatography

A variant of the use of reversed-phase high-performance liquid chromatography is described which permits the separation of pantothenic acid derivatives. The stationary phase used was a Bondapak-C₁₈ (4.1 × 250 mm column; 4.6 × 50 nm precolumn). Elution was performed in the isocratic regime using as mobile phase 20 M potassium phosphate buffer (pH 5.0)-methanol (91.5:8.5). The rate of elution was 1 ml/min. Retention times in the column for phosphopantothenate, pantothenate, phosphopantetheine, CoA, and dephosphoCoA were about 3.5, 6, 10.5, 16, and 42 min, respectively. This method, with radioactive detection, can be used for the analysis of pantothenic acid derivatives in liver extracts. One hour after white rats had been injected with [¹⁴C]pantothenic acid, the abovementioned components (with the exception of dephosphoCoA) contained the label in a ratio of 4:18:54:24.Institute of Biochemistry, Academy of Sciences of the Belorussian SSR, Grodno. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 855–858, November–December, 1988.     

Separation of retinyl esters by non-aqueous reversed-phase high-performance liquid chromatography

Rapid separation and sensitive quantitation of vitamin A esters can be achieved by use of an acetonitrile-dichloromethane (80:20) mobile phase with a 5-microns C18 column (15 cm X 4.6 mm) and absorbance detection at 325 nm. Either a Waters Resolve or a Rainin Microsorb column was used satisfactorily. Retinyl palmitate is eluted at about 7 min (capacity factor, k' = 5.5) at a flow-rate of 1.5 ml/min; retinyl palmitate and retinyl oleate, which are usually difficult to separate, are well resolved (resolution 1.2). Sensitivity (at a signal-to-noise ratio of 10:1) is 8 pmol retinyl palmitate (equivalent to 2.5 ng retinol). Quantitation of total retinyl esters is identical to that determined by a gradient high-performance liquid chromatographic technique over the range 30-1000 ng retinyl esters. Retinyl ester peaks in rat liver extracts were identified by their characteristic light absorption spectra, susceptibility to saponification, and by co-chromatography with authentic standards. Nine vitamin A ester peaks were identified and quantitated in rat liver extracts. A 10-microns Whatman Partisil 10/25 ODS-2 column was used with the same mobile phase to obtain partial resolution of retinyl esters (resolution 1.05 between retinyl oleate and retinyl palmitate; k' = 11.0 for retinyl palmitate) and improved retention for retinol (k' = 2.5, compared with k' = 0.6 for retinol on the 5-microns column).     

Separation of pantothenic acid derivatives by reversed-phase high-performance liquid chromatography

A variant of the use of reversed-phase high-performance liquid chromatography is described which permits the separation of pantothenic acid derivatives. The stationary phase used was a μBondapak-C₁₈ (4.1 × 250 mm column; 4.6 × 50 nm precolumn). Elution was performed in the isocratic regime using as mobile phase 20 M potassium phosphate buffer (pH 5.0)-methanol (91.5:8.5). The rate of elution was 1 ml/min. Retention times in the column for phosphopantothenate, pantothenate, phosphopantetheine, CoA, and dephosphoCoA were about 3.5, 6, 10.5, 16, and 42 min, respectively. This method, with radioactive detection, can be used for the analysis of pantothenic acid derivatives in liver extracts. One hour after white rats had been injected with [¹⁴C]pantothenic acid, the abovementioned components (with the exception of dephosphoCoA) contained the label in a ratio of 4:18:54:24. Institute of Biochemistry, Academy of Sciences of the Belorussian SSR, Grodno. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 855–858, November–December, 1988.     

Separation of stilbenes by capillary electrophoresis and high-performance liquid chromatography

Stilbenes, fluorescence whitening agents (FWAs), are usually added to cleaning agents in household and in industry. Capillary electrophoresis (CE) was often applied to separate various compounds simultaneously for its multinomial advantages. In this paper, we established analytical methods of six diaminostilbenes with CE and ion-pair chromatography (IPC). The optimum mobile phase for IPC was 11.78 mM tetrabutylammonium hydrogen sulfate (TBA) aqueous and acetonitrile. An IPC method has been developed for simple and direct separation for diaminostilbenes, anionic substances, with TBA as ion-pair reagent. Satisfactory linear ranges (7.0 x 10(-3) approximately 3.0 x 10 microg/mL), correlation coefficients (0.9992-0.9999), and detection limits (6-13 ng/mL) were obtained. Separations were also performed by capillary zone electrophoresis (CZE) using a buffer consisting of Tris (pH 10.1), n-tetradecyltrimethylammonium bromide (TTAB) and acetonitrile. A linear range of 5.0 x 10(-1) - 4.0 x 10 microg/mL, correlation coefficients between 0.9975 and 0.9998, and detection limits between 337 and 446 ng/mL were obtained. In particular, the separation of a pair of similar compounds (mass difference of 2) was achieved by addition of TTAB. The optimum analytical methods of CE and high-performance liquid chromatography (HPLC) were applied to commercial household with direct analysis and standard addition. No significant bias were shown between them by t-test at 95% confidence level.