Size exclusion HPLC

Flow rate Temperature Detection Sample injection Analysis times Sensitivity 

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Size exclusion HPLC has many other common names, such as gel permeation, gel filtration, steric exclusion, molecular sieve chromatography, or gel chromatography. These names all reflect the theoretical mode of action for this type... 

Size-exclusion HPLC (SE-HPLC) separates proteins on the basis of size and shape. As most soluble proteins are globular (i.e. roughly spherical in shape), separation is essentially achieved on the basis of molecular mass in most instances. Commonly used SE-HPLC stationary phases include silica-based supports and cross-linked agarose of defined pore size. Size-exclusion systems are most often used to analyse product for the presence of dimers or higher molecular mass aggregates of itself, as well as proteolysed product variants. 

A method for determination of the aromatic amino acid phenylalanine (45), tyrosine (46) and tryptophan (47) content of peptides at low microgram levels is based on size-exclusion HPLC combined with UVD using a diode array, and data processing of the... 

Fractionation of proteins according to size utilizing cross-linked dextran or polyacrylamide gel columns was first demonstrated by Porath and Flodin 63 in 1959. This technique has become the most widely accepted method for separation and molecular weight determination of hydrophilic and some hydrophobic macromolecules using aqueous buffers with or without organic modifier. While this technique might not be unique in its ability to resolve and separate proteins, it is one additional simple and effective tool in the chemist s armamentarium. The theories behind size-exclusion HPLC and size-exclusion chromatography at low pressure are identical and are described in several publications. 31 34 36 39 44 64 65 ... 

Derivatized silica gel that is used for size-exclusion HPLC contains a proprietary bonded phase that is defined as hydrophilic to minimize nonspecific hydrophobic and ionic interactions. Polymeric supports consist of highly cross-linked agarose beads, with or without bonded dextran or cross-linked copolymers of allyl dextran and AyV -methylenebisacryl-amide. All supports are available with a variety of particle and pore sizes and distribution. 

Note that proteins are now synthesized routinely by stepwise assembly or fragment condensation (see Vol. E22a, Section 4.1 ) 67 68 and chances of product contamination with shorter fragments that may have similar retention times under RP-HPLC should be considered 69 Additionally, size-exclusion HPLC was found to be particularly useful in distinguishing linear from cyclic peptides. 

Molecular Weight Classes Used for the Analysis of the Size Exclusion HPLC Chromatograms of Gelatin... 

All the antibody—toxin conjugates bind in their entirety to the appropriate immunoaffinity HPLC column and are completely separated from free antibody. There is no adverse affect on the integrity of the conjugates as judged by gel electrophoresis, size exclusion HPLC, and assays of cytotoxic potency (5)... 

Table 6 Survey of Commercially Available Prepacked Columns for the Size-Exclusion HPLC of Proteins ... 

GW Welling, S Welling-Wester. Size-exclusion HPLC of proteins. In RWA Oliver, ed. HPLC of Macromolecules, a Practical Approach. Oxford IRL Press, 1989, pp 77-89. 

CE Davis, JB Anderson. Size exclusion/HPLC of heated water soluble bovine and porcine muscle proteins. J Food Sci 49 598-602, 1984. 

The analytical results obtained from size-exclusion HPLC, which was... 

A. M. Zimmer, J. M. Kazikiewicz, S. M. Spies, and S. T. Rosen, Rapid miniaturized chromatography for 11 In labeled monoclonal antibodies comparison to size exclusion HPLC, Nucl. Med. Biol., 15 717(1988). 

Size-exclusion HPLC is particularity useful in either direct pharmacodynamic studies of the radiolabeled product or indirect studies that employ a labeled monoclonal antibody. In order to observe shifts in apparent MW due to noncovalent binding interactions, the mobile phase for these analyses should be a physiological buffer and the ligand size cannot be less than half that of the labeled protein. In cases where complexation may interfer with in vivo targeting, size-exclusion HPLC can be used prior to clinical administration of the potential or existing biotechnology product to establish the most effective regimen or dose. 

Rapid miniaturized chromatography for "In labeled monoclonal antibodies comparison to size exclusion HPLC, Nucl. Med. Biol., 75 717(1988). 

Clearly, all of the related substances, with the exception of hGH dimer, exhibited full biopotency as compared to hGH monomer. Further investigation of hGH dimer demonstrated that traditional immunoassays could not distinguish it from hGH monomer, although an immunoradiometric (IRMA) assay using two monoclonal antibodies could distinguish the two forms (8). As expected, size exclusion HPLC was also able to distinguish monomeric and dimeric forms of hGH. Further studies revealed that the IRMA assay precision was approximately 5-6% RSD, whereas size exclusion HPLC exhibited a precision of 1-2% RSD. Therefore, size exclusion HPLC was selected as the most useful candidate for replacement for the in-vivo bioassay. 

Size exclusion HPLC (e.g., human growth hormone) In-Vitro Bioassays... 

The silica-based TSK-2000 size exclusion HPLC column has excellent separation properties for proteins in MW range 2000-20,000, when 0.1% aqueous TFA is used as eluent (3,10). We have not seen this property when solvents at neutral pH were used or with other size-exclusion HPLC columns (resin-based as well as zirkoniumoxide-based). The use of 0.1% aqueous TFA solution also dramatically increases the recovery of chemokines. Nevertheless, the TSK-2000 column should be used only when other methods of purification cannot be applied. 

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