Chromatography hydrophobic adsorption

Hydrophobic Interaction Chromatography. Hydrophobic interactions of solutes with a stationary phase result in thek adsorption on neutral or mildly hydrophobic stationary phases. The solutes are adsorbed at a high salt concentration, and then desorbed in order of increasing surface hydrophobicity, in a decreasing kosmotrope gradient. This characteristic follows the order of the lyotropic series for the anions ... 

The stationary phase in gel permeation (also called size exclusion) chromatography contains cavities of a defined size distribution, called pores. Analytes larger than the pores are excluded from the pores and pass through the column more rapidly than smaller analytes. There may be secondary effects due to hydrophobic adsorption, ionic interaction, or other interactions between the stationary phase and analyte. Gel permeation and non-ideal interactions in gel permeation are described more fully in Chapter 6. 

The supernatant was subjected to fractional precipitation with ammonium sulfate (step 5) and then with acetone (step 6). PTTH was recovered in the precipitates with 35-55% acetone, while bombyxins were recovered with 55-75% acetone. The 35-55% acetone precipitates were subsequently purified through five steps of conventional chromatography gel filtration on Sephadex G-50 with 0.5M Tris-HCl (pH 8.5) (step 7), anion exchange on DEAE-Sepharose C1-6B with 0.2M sodium acetate (pH 5.2)(step 8), cation exchange on CM-Sepharose C1-6B 0.1-0.5M NaCl in 0.05M sodium acetate (pH 5.2) (step 9), Hydrophobic adsorption on Octyl-Sepharose C1-4B with 4M ammonium acetate, 0.2M ammonium acetate and 40% acetonitrile in 0.2M ammonium acetate (step 10) and gel filtration on Sephadex G-75 with O.OIM phosphate buffer containing 0.2M NaCl and 2% butanol (step 11). 

Hydrophobic adsorption chromatography takes advantage of the hydrophobic properties of substances to be separated and has also found use in biochemistry (Hoftsee Biochem Biophys Res Commun 50 751 1973 ... 

Polarity Adsorption chromatography Reverse-phase chromatography Hydrophobic interaction chromatography... 

Lipoteichoic acid from Streptococcus mutans has been purified by a combination of gel filtration, hydrophobic chromatography, and adsorption on to phospholipid vesicles. ... 

Such characterization investigations could mostly be performed routinely, whereas the purification procedures for each particular enzyme needed to be developed and optimized from the very beginning. With biochemical methods such as a combination of ammonium sulfate fractionation," ion exchange chromatography," gel filtration," adsorption chromatography, hydrophobic interaction chromatography, etc. an entire array of purification methods was available and employed successfully over several years for enzyme purification. 

Liquid chromatography is rather more widely applicable than gas-liquid chromatography and in some forms has the capability to handle larger amounts of substance. All the main forms of LC are used, namely adsorption, partition, ion exchange, and gel filtration, and some newer forms of chromatography including metal-chelate adsorption, hydrophobic adsorption, and, particularly for biochemicals, affinity chromatography. 

Humic acids, a class of naturally occurring organic polyelectrolytes found 1n soil and natural waters, have been the subject of considerable study with Sephadex SEC (34-36). Unfortunately, both electrostatic repulsive effects and hydrophobic adsorption influence the chromatography on Sephadex of these complex polyacids. Since the first effect is diminished and the second increased by added salt, solvents that eliminate non-steric effects have apparently not been identified (36). 

The chemical adsorption of a relatively high molecular weight neutral polymer (poly(succinimide), M = 13000) on aminopropyl-Vydac 101 TP silica gel was applied by Alpert [47, 48] to prepare a reactive composite support for use in cation-exchange [47] and hydrophobic-interaction [48] chromatography of pro-... 

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

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