Liquid affinity chromatography

Affinity liquid chromatography and chiral separations (enantiomer separations) require similar analyte properties. The solutes may have interactions through hydrogen-bonding, ligand formation, or Coulombic forces with the surface of stationary phase materials or the sites of additives however, the selectivity is controlled by the steric effects of the structures of the analyte molecules and the recognition molecules (chiral selectors). 

Affinity liquid chromatography, 7, 8 Amino acid, 67, 74 Asymmetry 04s), 39... 

T Urano, MW Trucksess, SW Page. Automated affinity liquid chromatography system for on-line isolation, separation, and quantification of aflatoxins in methanol-water extracts of corn or peanuts. J Agric Food Chem 41 1982-1985,1993. 

The method has been applied to study the enantiomer recognition of the protein monoamine oxidase (MAO), which has been used for affinity liquid chromatography. A mutant preparation method has been described using D-amino acid oxidase (DAO). The optimized structure of the docked complex between a protein and a substrate is obtained by performing molecular mechanics calculations, and the data indicates the degree of complex tightness. 

Ion-exchange chromatography involves an electrostatic process which depends on the relative affinities of various types of ions for an immobilised assembly of ions of opposite charge. The stationary phase is an aqueous buffer with a fixed pH or an aqueous mixture of buffers in which the pH is continuously increased or decreased as the separation may require. This form of liquid chromatography can also be performed at high inlet pressures of liquid with increased column performances. 

Lipophilicity represents the affinity of a molecule or a moiety for a lipophilic environment. It is commonly measured by its distribution behavior 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 or thin-layer chromatography system). 

Principles and Characteristics Liquid chromatography is the generic name used to describe any chromatographic procedure in which the mobile phase is a liquid. It may be classified according to the mechanism of retention in adsorption, partition, size-exclusion, affinity and ion-exchange (Scheme 4.4). These mechanisms form the basis for the chromatographic modes of... 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

FIGURE 9.1 Liquid chromatography workflow strategy options in proteomics. (a) bottom-up approach (b) top-down approach (c) selective sample cleanup directly combined with chromatographic separation (d) peptide capture with affinity restricted access material. 

Baker et al. [137] reported that Rhesus monkeys were administered primaquine (6-10.5 mg as the phosphate/kg intravenously) and plasma samples were analyzed by high performance liquid chromatography for the presence of the unchanged drug and the major metabolite, 8-[3-carboxy-l-methylpropylamino)-6-methoxyquinoline. Primaquine had an unusually high affinity for tissue compartments, which produced a rapid initial drop in plasma concentration. Within 15 min, the plasma concentration of the metabolite far exceeded that of primaquine. Thirty-five to eighty-three percent of the primaquine dose was converted to the major metabolite. This metabolite possessed much lower affinity for the tissues compartments than the drug itself. 

Rebif is produced via recombinant DNA technology in a CHO cell line. It displays an identical amino acid sequence to that of native human IFN-P-la and, like the native product, is glycosylated. After cell culture the interferon is purified using a series of chromatographic steps (affinity, ion-exchange, gel-filtration and reverse-phase liquid chromatography). It is formulated as a sterile solution in pre-filled syringes and contains mannitol, HSA, sodium acetate, acetic acid and sodium hydroxide as excipients. It is administered subcutaneously three times weekly. 

Liquid chromatography and especially HPLC is at present a most versatile tool in analytical chemistry. The separation is generally mediated by two phases, a solid one packed into the column and a mobile one, which percolates through this column. Substances are separated due to differences in the affinity towards the two phases. Nowadays different kinds of stationary phases are available for a broad range of analytical problems, including, e.g., reversed phase and ion exchange... 

This chapter focuses on gas-liquid chromatography, in which compounds in a sample are separated based on vapor pressures and differences in affinity for the stationary phase (a high boiling point liquid) versus the gaseous mobile phase. The time between sample injection and detection of the individual compound eluting from the column is called the retention time. Compounds that have limited solubility in the stationary phase will exit the column quickly as a large proportion will remain in the mobile phase. Compounds with polarity similar to that of the stationary phase will have longer retention times and potentially broader peaks, due to increased interaction with the stationary phase. 

Bedair, M., and El Rassl, Z. (2004). Affinity chromatography with monolithic capillary columns I. Polymethacrylate monoliths with Immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography. /. Chromatogr. A 1044, 177-186. 

This phenomenon can be exploited for separation and concentration of solutes. If one solute has certain affinity for the micellar entity in solution then, by altering the conditions of the solution to ensure separation of the micellar solution into two phases, it is possible to separate and concentrate the solute in the surfactant-rich phase. This technique is known as cloud point extraction (CPE) or micelle-mediated extraction (ME). The ratio of the concentrations of the solute in the surfactant-rich phase to that in the dilute phase can exceed 500 with phase volume ratios exceeding 20, which indicates the high efficiency of this technique. Moreover, the surfactant-rich phase is compatible with the micellar and aqueous-organic mobile phases in liquid chromatography and thus facilitates the determination of chemical species by different analytical methods [104]. 

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