Size-exclusion gels

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

Size exclusion (gel filtration or permeation) chromatography (SEC) is suitable for solutes with molecular weights of 2000 or more and is also useful for the preliminary investigation of unknown samples. Separated fractions can then be subjected to one of the other modes of HPLC. Exclusion chromatography is discussed in section 4.3.6. 

Desalting Size-exclusion (gel-permeation) Electrodialysis Dialysis Higher throughput for given size of equipment... 

In size-exclusion (gel permeation) HPLC separations, the retention can be described in terms of volume units (Ve, ) such that... 

Gel Filtration or Size Exclusion. Gel filtration is perhaps the easiest type of chromatography to understand conceptually (Fig. 2-3). It relies on the ability of a given size molecule to enter the uniformly sized pores of a solid support or matrix. Imagine that you have a solution containing two molecules one of 8000 Da and one of 50,000 Da. If this solution is passed through a porous matrix... 

The PEG-hydrazide derivative, 18, is separated and purified by the size exclusion gel chromatography using Sephadex G25m media. 

ASTM D-3593 Test Method for Molecular Weight Averages/Distribu-tion of Certain Polymers by Liquid Size Exclusion (Gel Permeation) Chromatography—GPC Using Universal Calibration (has also been adapted to the investigation of molecular weight distribution in petroleum fractions). 

In this process (Scheme 1) the largomycin complex was separated into three components F-I, F-II and F-III by applying the following procedure in succession (A) gradient extraction with aqueous ammonium sulfate solution, (B) isoelectric point precipitation, and (C) size exclusion gel filtration on Sephadex G-100. 

No single analytical method will provide all the qualitative and quantitative answers during PEG-biopharmaceutical characterization. Size-exclusion/gel-filtration and SDS-PAGE may separate 1-mer from 2-mers and higher molecular weights. However, neither method usually clearly resolves positional isoforms. Ion-exchange or reverse-phase chromatography methods... 

---