Size chromatography

The peak fractions of protein eluting at 0.2 M NaCl are individually evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (see Note 10). Pooled fractions should be >90% pure and not be contaminated with proteins of similar molecular weight. Otherwise, the contaminants will still be present after sizing chromatography. 

Our method employs small particle size chromatography (<2 Lim), which allows improved chromatographic resolution and inherently narrower peak widths. Typically peak widths are around 0.1 min and the coefficient of variation for retention times consistently around 1% (14). Identification of each opiate drug is based on an accurate relative retention time, proto-nated molecule of a precursor ion, a minimum of two selective fragment ions, and relative ratios of these ions as compared to an authentic standard from the same run. Isotopically labeled standards are used for each compound in quantitative applications. Internal standards for screening purposes are limited to one or two per experiment for practical reasons and to make the interpretation less complicated. 

Wei G T, Liu F K and Wang C R C 1999 Shape separation of nanometre gold particles by size-exclusion chromatography Anal. Chem. in press... 

In most inorganic chromatography, resins of 100 to 200 mesh size are suitable difficult separations may require 200 to 400 mesh resins. A flow rate of 1 mL cm min is often satisfactory. With HPEC columns, the flow rate in long columns of fine adsorbent can be increased by applying pressure. 

Schematics showing the basis of separation in (a) adsorption chromatography, (b) partition chromatography, (c) ion-exchange chromatography, (d) size-exciusion chromatography, and (e) eiectrophoresis. For the separations in (a), (b), and (d) the soiute represented by the soiid circie ( ) is the more strongiy retained.

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

A form of liquid chromatography in which the stationary phase is a porous material and in which separations are based on the size of the solutes. 

In size-exclusion chromatography, the smallest solute that can be separated from other solutes all smaller solutes elute together. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Calibration curve for the determination of formula weight by size-exclusion chromatography. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

A series of polyvinylpyridine standards of different molecular weight were analyzed by size-exclusion chromatography, yielding the following results. 

SI units stands for Systeme International d Unites. These are the internationally agreed on units for measurements, (p. 12) size-exclusion chromatography a separation method in which a mixture passes through a bed of porous particles, with smaller particles taking longer to pass through the bed due to their ability to move into the porous structure, (p. 206)... 

These factors make it necessary to reduce the amount of solvent vapor entering the flame to as low a level as possible and to make any droplets or particulates entering the flame as small and of as uniform a droplet size as possible. Desolvation chambers are designed to optimize these factors so as to maintain a near-constant efficiency of ionization and to flatten out fluctuations in droplet size from the nebulizer. Droplets of less than 10 pm in diameter are preferred. For flow rates of less than about 10 pl/min issuing from micro- or nanobore liquid chromatography columns, a desolvation chamber is unlikely to be needed. 

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