Column stability, reversed-phase HPLC

Reversed-phase HPLC uses a nonpolar stationary phase and a polar mobile phase. The characteristics are operational simplicity, high efficiency, column stability, and ability to analyze simultaneously a broad spectrum of both closely related and widely different compounds. Separation is based on hydrophobicity.33 Findlay et al. provide a comparison of chromatography methods with immunoassays (Table ll.l).27... 

The stability of nimodipine injectable formulations was studied using reversed phase HPLC, which made use of methyltestosterone as an internal standard. A YWG C18 column was eluted with methanol/water (13 7, v/v), and detection was effected at 238 nm. The calibration graph was linear over the range of 5.98-299 pM/L [21],... 

For labelling with temperature, reaction time, pH and molar ratio (peptide/lutetium) were varied with the aim of optimizing labelling efficacy, radiochemical purity and stability. Quahty control procedures included reversed phase HPLC (C18 column) and chromatography on various solvents and/or supports [16.9],... 

The isolation, mass and NMR characterization of the lesions was performed with the dinucleotide model compound d(GpT) since the chromatographic separation of the products of guanine oxidation was followed easily by UV-VIS spectrophotometry (thymine base was not modified by the oxidation) and the very polar oxidized guanine residues could be retained on the reverse phase HPLC column by the dinucleotide structure of the substrate. The oxidation of the guanine moiety of a dinucleoside monophosphate under appropriate conditions afforded the imidazolone (Iz) and the dehydro-guanidinohydantoin derivative 35 in amounts compatible with NMR analysis (261). However, 35 is not stable and was first stabilized in two ways for the... 

Shaw and Wilson (1984) developed a more rapid analytical method using solid phase extraction with reverse phase HPLC. Juice was passed through a C-18 Sep-Pak solid phase extraction cartridge, washed with water, then eluted with acetonitrile. However, the acetonitrile extract was determined not to be stable overnight, even at -16 °C (Shaw 1986). A Brownlee C-18 5-p,m microbore column, 2.1 X 220 mm, or a Perkin-Elmer C-8 5-p,m column, 4.6 X 125 mm, with a 4.6 X 40 mm Brownlee 5-p,m C-8 guard column was used. The mobile phase consisted of an acetonitrile-tetrahydrofuran-water mixture [17.5 17.5 65 (v/v/v)] and a flow rate of 0.2 ml/min was used for the C-18 column. A mixture of 17.5 15 67.5 with a flow rate of 1.5 ml/min was used for the C-8 column. Shaw (1986) listed methanol-acetonitrile-water (26.5 21.5 52) as the optimum mobile phase for C-18 column. This mobile phase was reported to have better baseline stability and freedom from negative peaks. UV detection at 207 nm was used. 

Bonded silicas are fairly stable in an acidic pH range of I to 2. Such conditions are usually applied in the resolution of peptides and proteins by reversed-phase HPLC. Separation of biopolymers is performed in the pH range between 5 and 8 with buffers in ion-exchange and hydrophobic interaction chromatography. The type and concentration of buffer have a remarkable effect on the stability of the bonded silica. For instance, phosphate buffers at pH 7 are not recommended, particularly when separations are performed at elevated temperatures. The lifetime of the column in phosphate will be much shorter than in Tris buffer [31]. Buffers with a pH above 8 should not be applied due to the dissolution of the silica matrix. 

Porous zirconia particles coated with polybutadiene make a reversed-phase HPLC column packing that offers both excellent pressure stability and chemical inertness throughout the entire pH range. Because of the complex surface chemistry of zirconia, mobile phase additives such as phosphate or fluoride are added to facilitate the separa-... 

Another approach to increase HPLC speed is the use of higher temperatures. The viscosity of a typical mobile phase used in reversed-phase separation decreases as the column temperature is increased. This allows an HPLC system to operate at a higher flow rate without suffering too much from increased back pressure. Zirconia-based packing materials provide excellent physical and chemical stability. They have been used successfully for high-throughput bioanalysis at elevated temperatures.9... 

For conventional or normal, in contrast with reversed-phase, LLPC, many materials have been used as the solid support for the stationary liquid. In addition to silica gel, which was the first and is still the most popular material, a variety of other adsorbents that adsorb the polar solvent such as cellulose powder, starch, alumina, and silicic acid have been used. The more recent practice of HPLC has greatly simplified the technique in providing column stability for repeated use and for treatment of large volumes. 

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