Use Of Reversed-phase High-performance Liquid Chromatography Research Articles

Reversed-phase high-performance liquid chromatography of the stable electrophoretic fractions of soil humic acids

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used for the hydrophobicity analysis of soil humic acids and their stable electrophoretic fractions A, B, and C + D preliminarily prepared by the combination of gel permeation chromatography on Sephadex with polyacrylamide gel electrophoresis. In two humic acid preparations of different genesis, the electrophoretic fraction A of the larger molecular size was the most hydrophobic (60–73% of the fraction was irreversibly adsorbed on a hydrophobic reversed-phase (RF) column C18), and the fraction C + D of the smallest molecular size was the most hydrophilic. The fraction B of medium size occupied an intermediate position (33–47% of the fraction was irreversibly adsorbed on the column). The use of RP-HPLC allowed for the first time detecting the hydrophobic electrophoretic fraction A of the largest molecular size mainly composed of aliphatic long-chained hydrocarbon, protein, and carbohydrate fragments in soil humic acids. Data on the degree of hydrophobicity and the earlier obtained physicochemical characteristics of stable electrophoretic fractions are discussed in terms of the supramolecular and macromolecular structure of soil humic acids.

The application of chromatography techniques for the purification process optimization of amide of hydrocortisone-derived cortienic acid and ethyl ester of L-glycine

Soft ('antedrug') glucocorticoids are pharmacologically active compounds which are biotransformed in a predictable and controllable way to inactive and non-toxic metabolites. Amides of cortienic acids (17(-carboxamide derivatives of glucocorticoids) are potential soft drugs with fewer side effects than traditional glucocorticoids. The purification of 17(- carboxamide derivatives of hydrocortisone was explained using the amide of hydrocortisonederived cortienic acid and ethyl ester of L-glycine as an example and performed by use of column chromatography and preparative thin-layer chromatography (TLC). The optimization of purification process was performed employing analytical TLC and reversed-phase highperformance liquid chromatography (RP-HPLC). The mobile phase that enables best chromatographic separation of the amide from impurities on TLC plate (chloroform-methanol (95:5 V/V)) was selected and modified (reduction of polarity and addition of glacial acetic acid) to be used for the column chromatography and preparative TLC purification. It was confirmed by use of RP-HPLC that purification procedures applied in this study resulted in pure (96.2 %) amide.

Identification and Characterization of Isomeric Intermediates in a Catalyst Formation Reaction by Means of Speciation Analysis Using HPLC−ICPMS and HPLC−ESI-MS

Information on chemical speciation is much needed in mechanistic and kinetic studies on catalyst formation processes in pharmaceutical research. Speciation analysis was applied to the identification and quantification of various rhodium species involved in a ligand exchange process leading to formation of catalyst dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate]. Inductively coupled plasma mass spectrometry (ICPMS) was used as an element-specific detector following species separation by reversed-phase high-performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI-MS) was used for species identification and confirmation. A novel interface between the HPLC and ICPMS, which consisted of an eluent splitter, a desolvation unit, and the ICPMS built-in peristaltic pump, enabled the use of RP-HPLC with gradient elution and up to 100% organic components in the LC eluent without organic loading in the plasma. A variety of reaction intermediates were identified and quantified along the pathway to formation of the desired product, including isomeric di-, tri-, and tetrasubstituted species previously believed to be absent. This has provided new insights into the mechanism and kinetics of the reaction. The combination of HPLC-ICPMS and HPLC-ESI-MS has proven to be a valuable tool for the investigation of species evolution in catalyst formation process.

Effects of chronic renal disease on the transport of vitamin A in plasma and urine of dogs.

To determine plasma and urine concentrations of retinol, retinyl esters, retinol-binding protein (RBP), and Tamm-Horsfall protein (THP) in dogs with chronic renal disease (CRD). 17 dogs with naturally developing CRD and 21 healthy control dogs. A diagnosis of CRD was established on the basis of clinical signs, plasma concentrations of creatinine and urea, and results of urinalysis. Concentrations of retinol and retinyl esters were measured by use of reverse-phase high-performance liquid chromatography. Concentrations of RBP and THP were measured by use of sensitive ELISA systems. Dogs with CRD had higher plasma concentrations of retinol, which were not paralleled by differences in plasma concentrations of RBP. Calculated ratio of urinary total vitamin A (sum of concentrations of retinol and retinyl esters to creatinine concentration) and ratio of the concentration of urinary retinyl esters to creatinine concentration did not differ between groups. However, we detected a significantly higher retinol-to-creatinine ratio in the urine of dogs with CRD, which was paralleled by a higher urinary RBP-to-creatinine ratio. Thus, in dogs with CRD, the estimated fractional clearance of total vitamin A, retinol, and RBP was increased. Furthermore, dogs with CRD had a reduced urinary THP-to-creatinine ratio. Results of this study documented that CRD affects the concentrations of retinol in plasma and urine of dogs. Analysis of the data indicates that measurement of urinary RBP and urinary THP concentrations provides valuable information that can be helpful in follow-up monitoring of dogs with CRD.

Phytoplankton pigments designation: an application of RP-HPLC in qualitative and quantitative analysis

An account is given of the use of RP-HPLC (reverse-phase high performance liquid chromatography) analysis of high purity chlorophylls and carotenoids standards originating from reference phytoplankton monocultures. All important chromatography parameters were established: retention time, detection limit, spectrum, wavelength of maximum absorption, calibration curve, response factors. Data were also obtained for pigment mixtures in naturalphytoplankton populations in Spitsbergen and the Gulf of Gdansk. The identity and purity of all pigments were confirmed by co-elution with standards, including canthaxanthin as an internal standard. The data presented enable the qualitative and quantitative analysis of naturally existing phytoplankton pigments at concentrations down to 0.1 μg L-1 to be carried out without the need for standards from commercial sources.

Application of reversed-phase high-performance liquid chromatography and capillary zone electrophoresis to the peptide mapping of pepsin isoenzymes

A combination of reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) was used for the characterization of peptide maps of swine pepsin after its digestion with α-chymotrypsin. Peptide maps obtained by both methods were compared and five selected chromatographic peaks were identified on an electrophoreogram. The different order of peaks found in RP-HPLC compared to CZE confirmed the complementarity of these two methods. More peptide fragments were resolved by RP-HPLC, which was also found to be less sensitive to salt content in peptide mixtures, than by CZE, but only CZE was able to separate and identify phosphorylated and dephosphorylated peptide fragments of swine pepsin digest. CZE peptides faster separation than RP-HPLC, however, the salts have to be removed by ultrafiltration or by RP-HPLC pre-separation prior to CZE analysis. Combined use of RP-HPLC and CZE for peptide mapping makes it possible to distinguish between the phosphorylated and dephosphorylated forms of swine pepsin. This is important from a diagnostic point of view, because pepsin phosphorylation may be associated with gastric cancer.

The presence of low molecular weight germinative substances in Bacillus cereus T spore extract.

An extract from intact spores of Bacillus cereus T having a germination-inducing activity was studied. Two distinct germinative principles were found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non-diffusible. The activity of the former fraction was inhibited by the addition of 1 mM glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system. The diffusible principle maintained the major portion of the activity found in the crude spore extract. By means of high-performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fractions, two fractions (No. 1 and No. 8) were responsible for the germination-inducing activity, but no reconstituted activity was observed unless both fractions No. 1 and No. 8 were added to the assay system. Amino acid analysis of fraction No. 1 revealed that the fraction was rich in free amino acids, especially in alanine. On the other hand, by the use of reverse-phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No. 8 was inosine. Based on these findings, it was suggested that the active substances in fraction No. 1 might be a free amino acid such as L-alanine and/or Ca2+ and a Ca(2+)-binding substance.

Retention data for five ketotrichothecenes in reversed-phase high-performance liquid chromatography with different eluent systems

The effect of the eluent composition (nature of the modifier and its mole fraction in the mobile phase) on the retention of trichothecene mycotoxins of group B (derivatives of 12,13-epoxytrichothecene-9), i.e., desoxynivalenol (DON), nivalenol, DON-3-acetate, DON-15-acetate and 7-desoxy-DON was studied. Ethanol, acetonitrile or tetrahydrofuran were used as the organic component (modifier) of the binary water—organic mobile phases. The retention mechanism and separation selectivity of the trichothecene mycotoxins in reversed-phase high-performance liquid chromatography (RP-HPLC) on Nucleosil C-18 is discussed. Optimum conditions were determined for the separation of the five trichothecene mycotoxins with the use of RP-HPLC under isocratic conditions.

High-performance liquid chromatography in protein sequence determinations

The use of reversed-phase high-performance liquid chromatography (RP-HPLC) HPLC) for the determination of protein sequences is reported. Topics considered include the peptide separation of endoprotease digestion mixtures, the application of HPLC peptide mapping as an efficient system to check the accuracy of an assumed protein sequence, obtained indirectly by DNA sequencing and the use of HPLC for amino acid analysis in the Edman sequence strategy. The use of RP-HPLC for an unconventional sequence strategy is demonstrated; HPLC exopeptidase mapping appears to be particularly useful as a future technique for small terminal sequence analysis. Finally, the coupling of HPLC with fast atom bombardment mass spectrometry is discussed.

Purification of synthetic lipid associating peptides and the monitoring of the deformylation of N in-formyltryptophan by reversed-phase high-performance liquid chromatography

A series of five synthetic lipid associating peptides have been purified by reversed-phase high-performance liquid chromatography (HPLC). Volatile mobile phases were used to allow facile isolation of the purified peptide. These consisted of either 0.1 M ammonium bicarbonate, pH 7.9, or 0.5 M ammonium formate, pH 6.3, with gradients of 2-propanol and acetonitrile. The columns used were either Radial-Pak C 18 or Radial-Pak CN. The recovery of the purified peptides was usually in excess of 95%. In addition, this chromatographic system allowed the facile purification of the product isolated from the removal of the N in-formyl protecting group from tryptophan. The use of reversed-phase HPLC has allowed an improvement in both the purity and yield of purified material when compared with the results of low-pressure gel filtration and ion-exchange chromatography. The use of guanidine hydrochloride in the injection mixture and the presence of organic solvents in the mobile phase have allowed the minimization of aggregation of the lipid associating peptides with a consequent improvement in the chromatographic properties of these peptides.

The Purification of Prostatic Acid Phosphatase from Seminal Plasma by Reverse Phase High Pressure Liquid Chromatography

Abstract We report here the rapid isolation of PAP directly from dialyzed human seminal plasma by reverse phase high performance liquid chromatography (RP-HPLC). The recovery of PAP of high specific activity was found to be dependent on the treatment of the fractions after separation. The collection of fractions into a stabilizing medium was essential to the preservation of the specific activity of the enzyme. This finding extends the use of RP-HPLC to the purification of active enzymes from complex biological matrixes.

Fractionation of Gibberellins in Plant Extracts by Reverse Phase High Performance Liquid Chromatography

In studies on endogenous plant gibberellins (GAs), reverse phase (Bondapak C(18)) high performance liquid chromatography (HPLC) has proved to be a useful method for the fractionation of plant extracts. The behavior of 18 authentic GAs in such a chromatographic system is described. The main factors determining chromatographic behavior are the degree and the position of hydroxylation of the GA. Generally, dihydroxylated GAs elute before monohydroxylated GAs, whereas 13-hydroxylated GAs elute before 3-hydroxylated GAs. The number of carboxyl groups and the degree of saturation of the A-ring have little effect. For 20-carbon GAs, the oxidation state at C-20 is only relevant insofar as GAs having a methyl group at this position elute later than those with other groups (lactone, aldehyde, or carboxyl).As an illustration of the use of reverse phase HPLC, the endogenous GAs of immature seeds of Pharbitis nil L., strain "Violet," were reinvestigated. The presence of gibberellins A(3), A(5), A(17), A(20), and A(29) was confirmed by gas-liquid chromatography-mass spectrometry. In addition, two other GAs, A(19) and A(44), were also identified in extracts of this material.