High-performance liquid chromatography reversed-phase HPLC

Reversed-phase high-performance liquid chromatography (HPLC) column 50 mm x 3.2-mm i.d. with Kromasil 5- um Gig packing High-performance liquid chromatograph coupled to a triple-quadrupole mass spectrometer with an atmospheric pressure chemical ionization (APCI) source Gel permeation chromatograph with a 60 mm x 25-mm i.d. column packed with Bio-Beads SX-3 (50-g)... 

The ELISA can be used as one component of a battery of analyses. Rarely is only one method used in isolation. Other tests include chromatographic methods such as reversed-phase high-performance liquid chromatography (HPLC), size exclusion chromatography, and physical structure analytical methods such as UV spectral analysis, mass spectroscopy, etc. 

The chemical properties of HVA, 5HIAA and 3-MD make them amenable to reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Furthermore, the composition of CSF means that little, if any, sample preparation is required prior to analysis. However, the susceptibility of these metabolites to oxidation means that careful sample collection and storage is required in order to minimise analyte degradation. 

We have followed the pathway of TNT degradation in the consortium using reverse phase high performance liquid chromatography (HPLC) and mass spectroscopy (MS). Analysis of the supernatant in the consortium showed sequential reduction of TNT to TAT however, TAT only occasionally accumulated, and even then only in very low concentrations. In addition, methylphloroglucinol (MPG) and />-cresol transiently appeared in the supernatant. All compounds were identified using HPLC by UV spectra and retention times as compared to authentic standards chromatographed under the same conditions. The mono- and diamino intermediates were also confirmed by the MS results. 

In this unit, methods for reversed-phase high-performance liquid chromatography (HPLC) are described for the analysis of polyphenolics. HPLC analysis can be employed in an easy and fast manner to obtain an accurate elucidation and quantification of individual polyphenolic compounds found in plant-based materials. The separation of each polyphenolic is based on the polarity differences among polyphenolics with structural similarities and uses various combinations of mobile and stationary phases. 

One standard method approach that appears to have wide application in the pharmaceutical industry is the use of reversed-phase high performance liquid chromatography (HPLC) with a wide solvent gradient program. For example, a linear gradient from 95%... 

Hu, J., Leng, X.-F. (1992) Determination of partition coefficients for some pesticides by using reversed-phase high performance liquid chromatography (HPLC). Sepu 10, 344-346. 

The method consists of the complete hydrolysis of a protein or peptide to release its component amino acids. These may be separated by reverse phase high performance liquid chromatography (HPLC), and quantified with fluorometric detection after derivatization with o-phthaldehyde (Larsen and West, 1981). The method serves to determine both the amino acid composition and the total quantity of protein present in the sample. 

Perhaps more troublesome than the moderate yields is the necessity for reversed-phase high-performance liquid chromatography (HPLC) to purify the Fmoc protected amino acid glycosides. It has been reported,23 that the carboxylic acid moiety of an amino acid can be protected as a phenacyl ester and after glycosylation be removed with HOAc/Zn° (Scheme 5.7). Thus, a thioglycosyl donor, having a nonparticipating functionality at C(2), was reacted with the Fmoc protected phenacyl ester... 

Liquid-liquid partitioning is a convenient and often economical method for bioseparations. L. Gu (personal communication, 1999) has shown that an acetonitrile-water system can be used for separation of proteins. This system partitions into two phases under subzero temperatures with the top phase containing more acetonitrile and water. The low temperature and the presence of water in both phases help reduce protein denaturation. An added advantage is that sample solution can be directly applied to reversed-phase high-performance liquid chromatography (HPLC) for further purification. Aqueous liquid-liquid partitioning is likely to remain an attractive choice for the separation of proteins, and exploration of new systems will continue. 

The purity of crude peptide will be analayzed on a reverse-phase high-performance liquid chromatography (HPLC) column. The elution of the peptide is followed by absorbance recorded at 215 nm. The purification on a 10 to 20 mg scale is performed on a larger, preparative column monitored at the less sensitive wavelength of 238 nm. 

---