Reverse-phase liquid chromatography RP-HPLC

Reversed Phase-Liquid Chromatography (RP-HPLC) Separation of Proteins... 

Peptide mapping is a powerful tool for the analysis of the primary structure of a protein. This method typically takes advantage of one or more specific proteases that cleave the protein into smaller peptides, which are then separated and analyzed by reversed-phase liquid chromatography (RP-HPLC), sometimes in conjunction with mass spectrometry (MS). 

The selectivity of reversed-phase liquid chromatography (RP-LC) columns is known to vary, even columns with the same ligand (e.g., Cjg). Column selectivity can also vary from batch to batch for columns claimed to be equivalent by the manufacturer. For different reasons, it is sometimes necessary to locate a replacement column for a given assay that will provide the same separation as the previous column. In other cases, as in HPLC method development, a column of very different selectivity may be needed - in order to separate peaks that overlap on the original column. For each of these situations, means for measuring and comparing column selectivity are required. Until recently, no such characterization of column selectivity was able to guarantee that two different columns can provide equivalent separation for any sample or separation conditions. 

The aim of the work is investigate possibilities of application of Cartridges Packed DIAPAK for concentrating antibiotics Cefazoline and Levomycetine and analyze them by Reversed Phase High Performance Liquid Chromatography (RP HPLC). 

Lipophilicity represents the affinity of a molecule or a moiety for a lipophilic environment. It is commonly measured by its distribuHon behaviour in a biphasic system, either liquid-liquid (e.g. partition coefficient in 1-octanol/water) or solid-liquid (retention on reversed-phase high-performance liquid chromatography (RP-HPLC) or thin-layer chromatography (TLC) system). 

Reversed-phase high-performance liquid chromatography (RP-HPLC), cholesterol measurement, 456-458 Rhamnogalacturonanas, 735 Rice bran oil, tocopherols/tocotrienols in, 488 (figs.)... 

I de Noni, G de Bernardi, L Pellegrino. Detection of common-wheat (Triticum aestivum) flour in Durum-wheat (Triticum durum) semolina by reverse-phase high-performance liquid chromatography (RP-HPLC) of specific albumins. Food Chem 51 325-329, 1994. 

Reverse-phase liquid chromatography is now virtually the only method used in the analysis of the TG mixtures. The first paper on TG-HPLC analysis was published in 1975 by Pei et al. (81). Triglycerides were separated on a VYDAC reverse-phase (35 - 44 /xm) column and eluted with methanol-water (9 1). Since Pei et al. first applied RP-HPLC to the separation of triacyl-glycerols, a number of reverse-phase systems have been developed as rapid and efficient resolution of complex triacylglycerol mixtures can be achieved. 

An aldehyde was mixed with solid supported triphenylphosphine oxide (3 equiv.), alkyl halide (4 equiv.), and potassium carbonate (4 equiv.) in methanol (2 ml). The mixture was heated at 150° for 5 min. The residue was filtered through a short plug of silica gel and washed. The solution was concentrated and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). 

Experimentally determined retention times or capacity factors (k) generated by reverse phase, usually octadecylsilane (ODS), high performance liquid chromatography (RP-HPLC) have been used widely to estimate Kow values (McDuffie, 1981 Haky and Young, 1984 Sarna, 1984 Doucette and Andren, 1988). More recently, this approach has been used to directly estimate Koc (Vowles and Mantoura, 1987 Hodson and Williams, 1988 Szabo et al., 1990 Kordel et al., 1993 Kordel et al., 1995 Hong et al., 1996). This is not strictly an estimation method because it relies on the acquisition of experimental retention times. 

Synthetic peptides often lack the conformational stability required for a successful drug therefore determination of peptide stability in serum constitutes a powerful and important screening assay for the elimination of unstable peptides in the pipeline of drug development (see Note 1). Peptide stability in serum can rather easily be determined by reverse phase-high-performance liquid chromatography (RP-HPLC) and mass spectroscopy (MS) from both in vitro and in vivo studies. 

Reversed-phase high performance liquid chromatography (RP HPLC) works by eluates partitioning between mobile and stationary phases, and the retention factor log k is proportional to log Kow. Numerous studies have correlated Koc with log k, and Gawlik et al. (1997) list 35 such correlations, most of which relate to specific chemical classes. Note that the correlations vary with the stationary phase used. Some examples are ... 

Equipment and expertise. Synthesis requires dedicated laboratory space and equipment. Protein synthesis is best done with the aid of a peptide synthesizer which is capable of optimal step-wise yields. Purification using reverse-phase high-performance liquid chromatography (RP-HPLC) is an integral part of the procedure (16), so at least one preparative and one analytical HPLC systems is needed. Access to electrospray mass spectrometry is essential. 

The use of cyclodextrins as the mobile phase components which impart stereoselectivity to reversed phase high performance liquid chromatography (RP-HPLC) systems are surveyed. The exemplary separations of structural and geometrical isomers are presented as well as the resolution of some enantiomeric compounds. A simplified scheme of the separation process occurring in RP-HPLC system modified by cyclodextrin is discussed and equations which relate the capacity factors of solutes to cyclodextrin concentration are given. The results are considered in the light of two phenomena influencing separation processes adsorption of inclusion complexes on stationary phase and complexation of solutes in the bulk mobile phase solution. 

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