Column chromatography hydrophobic

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

The main toxic pore forming component of P. marmoratus secretion, named pardaxin, was isolated by liquid column chromatography (5). Originally two toxic (5) polypeptides, Pardaxin I and II, were isolated. However, their primary sequences have been found to be identical (6) therefore, the two components most probably represent different aggregates of one polypeptide. This finding is in contrast to the secretion of P. pavonicuSj which contains three toxic polypeptides (8). Pardaxin is a single chain, acidic, amphipathic, hydrophobic polypeptide, composed of 33 amino acids and with a mass around 3500 daltons (5,6). The primary sequence is (6) NHj-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Ile-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu-COOH. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

Hydrophobic interaction chromatography (HIC) is a column chromatography technique which can determine particle hydrophobicity by interaction with a hydrophobic gel matrix [142,149,150]. Hydrophilic particles pass through the column without interaction, whereas particles with increased hydrophobicity show a retarded elution and are retained by the column. Hydrophobicity measurements are used to determine the hydrophobicity of nanoparticulate carriers and correlate this to their in vivo biodistribution [10, 149]. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

It has also been proposed that the presence of endophytes can alter the usual suite of secondary metabolites of plants. For example, whilst Murraya spp. have been reported to produce indole and carbazole alkaloids, the Brazilian M. paniculata did not [25]. An endophytic Eupenicillium sp. was isolated from surface-sterilized leaf material of the Brazilian M. paniculata and subsequently cultured on white corn. The Eupenicillium sp. produced hydrophobic spiroquinazoline alkaloids that were separated using silica gel column chromatography and preparative gel-filtration... 

Hjerten and his research group [41] introduced monolithic columns based on acrylamides as chromatographic separation media in the late 1980s. Xie et al. [42] prepared rigid porous polyacrylamide-co-butylmethacrylate-co-A,A -methylene-bis-acrylamide monolithic column for hydrophobic interaction chromatography. They also prepared polyacrylamide-co-At, M-methylene-bis-acrylamide monolithic rods and smdied the effect of polymerization conditions on rods morphology. 

Reverse-phase column chromatography using HPLC can be used to separate these compounds according to their hydrophobicities. However, the reported solvent systems contain salt. The TC-CCC system has an advantage in that lipids can be separated without the use of salted buffer. We plan to monitor the eluted lipids from... 

In reversed-phase (RP) chromatography, a relatively nonpolar support phase binds the complexes (species) being separated, and these are moved by a polar solvent phase (water, alcohols, acetonitrile, or mixtures). Generally, as predictable for RP columns, more hydrophobic contacts on the exterior of a metal complex favor column binding, which retards migration. Examples for RP separations are given in the next paragraphs. 

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