Gel filtration pdf

In addition, the high mechanical strength of BEH particles enables a reduction in particle size to 1. Superdex is a series of size-exclusion chromatographic media consisting of a composite base matrix of dextran covalently attached to highly cross-linked agarose. Its relatively low nonspecific interaction generally permits high recovery of biological material, such as proteins. They are also stable to repeated autoclaving and to exposure to abrasive chemicals and detergents such as SDS, urea, and guanidine hydrochloride.

These types of media are typically available both as small beads, which are optimal for micropreparative and analytical runs, as well as larger beads which are more suitable for larger scale preparative work. The high rigidity of the Superdex media allows for viscous eluents to be run at relatively high flow rates. Nonspecific interactions are negligible when using buffers with ionic strengths in the range 0.

For example, Superdex Increase has a broad fractionation range that allows separation of a large variety of proteins Mr 10,—, Da with an optimized resolution for the antibody molecular weight range of Mr ,—, Agarose-based support matrices are alkali tolerant and, therefore, may be cleaned using sodium hydroxide up to 1 M concentration.

This type of cleaning is highly efficient, prolongs column life and minimizes the risk for carry-over between different runs. The support material in these media are polymers of silica.

Silica gel, made from sodium silicate, is a granular, vitreous, porous mineral that can be processed into either granular or beaded form. As a desiccant, it has an average pore size of 2. As a chromatographic material, silica has the advantage that it has a larger pore volume and narrower pore size distribution. Hence, it often gives sharper peaks and better resolution in certain cases.

However, larger pore size silica particles may be brittle and fragile, and subject to collapse at high pressures. In addition, only a limited range of pH values may be utilized with these media. Because silica dissolves quite readily above pH values of 7—8, it is not possible to exceed pH 7. In general, silica has been found to give the best results for the separation of small proteins, ranging from 10, to 1,, Da.

Compared to other types of HPLC columns, silica-based SEC columns often claim to contain more pore volume per unit column volume, which would result in higher MW selectivity and better resolution. Some SEC media are hydroxylated meth-acrylic polymer resins e. They vary from the typical Toyopearl HW beads by having a higher degree of cross-linking such cross-linking is often necessitated by the higher flow pressures required when using smaller particles for chromatographic separations.

Since separation efficiency, peak width and resolutionare all improved with this smaller particle size, these matrices are regarded as higher performing chromatography resins. TSKgel based resins may be used with high flow rates and are designed to withstand operating pressures up to 20 bar. For this reason, gel-filtration chromatography has an inherent low sample-handling capacity and, accordingly, should be performed late in a purification procedure when the numbers of different molecules in a sample are relatively low.

The concentration of sample that can be applied to the column will be limited by the viscosity of the sample which increases with sample concentration relative to the eluent. Maximum resolutionin gel-filtration chromatography is obtained with long columns. The ratio of column diameter to length can range from up to As gel-filtration chromatography separates molecules only on the basis of their relative sizes, the technique is effectively independent of the type of eluent used.

Elution conditions pH, essential ions, cofactors, protease inhibitors etc. However, the ionic strength of the eluent should be high enough to minimize protein-matrix and protein—protein associations by electrostatic or van der Waals interactions. The addition of 0. Low flow rates offer maximum resolutionduring gel-filtration chromatography since flow rate and resolution are inversely related.

Unfortunately, low flow rates mean longer separation times. Therefore, a compromise between desired resolution and speed must be decided upon.

Most gel-filtration matrices can be cleaned with 0. One of the principal advantages of gel-filtration chromatography is that separation can be performed under conditions specifically designed to maintain the stability and activity of the molecule of interest without compromising resolution. Absence of a molecule-matrix binding step also prevents unnecessary damage to fragile molecules, ensuring that gel-filtration separations generally give high recoveries of activity.

This technique, however, is not without its disadvantages. When separating proteins by gel-filtration chromatography, for example, proteolysis becomes an increasing problem, since the target protein frequently becomes the abundant substrate for proteases also present in the mixture, consequently reducing recovery of activity.

Because of the large size of gel-filtration columns, large volumes of eluent are usually required for their operation, often creating excessive running costs. Gel filtration also has an inherent low resolutioncompared to other chromatographic techniques because none of the molecules are retained by the column and nonideal flow occurs around the beads. In addition, this technique has a low sample-handling capacity dictated by the need to optimize resolution. Despite these disadvantages, gel-filtration chromatography still occupies a key position in the field of biomolecule separation because of its simplicity, reliability, versatility, and ease of scale-up.

A brief overview of its main applications is given below. Because of its unique mode of separation, gel-filtration chromatography has been used successfully in the purification of literally thousands of proteins and peptides from various sources.

These range from therapeutic proteins and peptides, which together constitute a multibillion euro world-wide market, to enzymes and proteins for industrial applications; some examples are outlined below.

Recombinant human granulocyte colony stimulating factor rhG-CSF was refolded from inclusion bodies in high yield, with great suppression of aggregates formation, by urea-gradient size-exclusion chromatography on a Superdex 75 column [ 4 ].

Luteinizing hormone LH was purified fold from crude pituitary extract by gel filtration on two Sephacryl S columns. The method exploited differential binding of LH in the crude extract to blue dextran for the first chromatography step.

Before the second step, addition of high salt released LH from the blue dextran, enabling effective purification [ 6 ]. Fusion ferritin heavy-chain ferritin plus light-chain ferritin has also been purified by urea-gradient gel filtration. In this case, fusion ferritin solubilized from inclusion bodies with 4 M urea was applied to the column. Refolding enhancers were included in the urea-diluent buffer subsequently applied to the column to produce properly folded fusion ferritin multimers [ 7 ].

Among food-use proteins, hen egg lysozyme has been successfully refolded using both acrylamide- and dextran-based gel columns Sephacryl S and Superdex 75, respectively [ 9 ]. Gel filtration has also proven useful for the purification of the whey proteins alpha-lactalbumin and beta-lactoglobulin from aqueous two-phase systems [ 10 ].

Protein engineeringtechniques enable the design of self-assembling multimeric protein cages for applications in nanotechnology [ 11 ]. Grove et al. Size-exclusion reaction chromatography SERC permits one to control the extent of a reaction such as PEGylation that alters molecular size and to separate reactants and products. In SERC, injection of reactants onto a size-exclusion chromatography column forms a moving reaction zone.

Reactants and products partition differently within the mobile phase leading to different flow rates through the column. Thus, products are removed selectively from the reaction zone, shortening their residence time in the reaction zone and separating them into the downstream section of the column. The principle was successfully demonstrated using two model proteins, alpha-lactalbumin and beta-lactoglobulin [ 13 ].

Gel-filtration chromatography has for many years been used to separate various nucleic acid species such as DNA, RNA, and tRNA as well as their constituent bases, adenine, guanine, thymine, cytosine, and uracil.

Plasmid DNA can also be purified by gel filtration [ 15 ], although modern commercial kits often use a centrifugal spin column format for greater convenience.

Limonta et al. Krober et al. Overall productivity of the SMB process was estimated to be up to 3. A Sephacryl S SF proved to be effective and economical in the purification of recombinant Bombyx mori nucleopolyhedrosis virus displaying human pro renin receptor [ 19 ]. Sephacryl S gel-filtration chromatography gave more effective purification of turkey coronavirus from infected turkey embryos than did use of a sucrose gradient [ 20 ].

The presence of bacterialendotoxin is unacceptable in injectable recombinant biologicals, since endotoxin in the bloodstream can induce a pyrogenic response. Good manufacturing practice GMP will effectively remove endotoxin, but preclinical biologics may be produced under non-GMP conditions.

London et al. Endotoxins typically form aggregates, which may be quite large. A Superdex size-exclusion column 1. Absolute size-exclusion chromatography ASEC is a technique that couples a dynamic light scattering DLS instrument to a size-exclusion chromatography system for absolute size measurements of proteins and other macromolecules as they elute from the chromatographic system.

Dynamic light scattering DLS; also known as photon correlation spectroscopy or quasi-elastic light scattering is a technique that uses light scattering patterns usually from a laser source to determine the size distribution profile of small particles in suspension, or of polymers such as proteins in solution [ 22 ]. DLS can also be used to probe the behavior of complex fluids such as concentrated polymer solutions. The sizes of the macromolecules are measured as they elute into the flow cell of the DLS instrument from the size-exclusion column.

It should be noted that the technique measures the hydrodynamic size of the molecules or particles and not their molecular weights. For proteins, a Mark—Houwink type of calculation can be used to estimate the molecular weight from the hydrodynamic size [ 23 ].

Batch DLS is quick and simple to perform. Using SEC, the proteins and protein oligomers are separated, allowing oligomeric resolution. ASEC can also be used for aggregation studies: although the aggregate concentration may not be calculated, the size of the aggregate will be measured, being limited only by the maximum size eluting from the SEC columns.

Limitations of ASEC include flow rate, concentration, and precision. Because a correlation function requires anywhere from 3 to 7 s to properly build, only a limited number of data points can be collected across the peak. Gel-filtration chromatographyis an excellent alternative to SDS-PAGE for the determination of relative molecular masses of proteins, since the elution volume of a globular protein is linearly related to the logarithm of its molecular weight [ 24 ].

When a protein of unknown molecular weight is applied to the same column and eluted under the same conditions, one can use the elution volume of the protein to determine its molecular weight from the calibration curve. By selecting a matrix pore size which completely excludes all of the larger molecules in a sample from the internal bead volume, but which allows very small molecules to enter this volume easily, one can effect a group separation in a single, rapid gel-filtration step which would traditionally require dialysis for up to 24 h to achieve.

Group separation can be used, for example, to effect buffer exchanges within samples, for desalting of labile samples prior to concentration and lyophilization, to remove phenol from nucleic acid preparations and to remove inhibitors from enzymes see , for example, [ 25 ].

Despite its disadvantages of sample dilution and the need for a low ratio of sample volume to column volume, gel filtration remains a popular separation method due to its versatility, the wide range of matrices commercially available and the mild conditions of operation. Useful handbooks on both gel-filtration and other chromatography techniques are available through the GE Healthcare Lifesciences website www.

National Center for Biotechnology Information , U. Protein Chromatography. Published online May Cummins , 4 and Brendan F. Philip M. Brendan F. Author information Copyright and License information Disclaimer. Corresponding author. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source.

Abstract Gel-filtration chromatography is a versatile method that permits the effective separation of biological molecules in high yield. Key words: Gel-filtration chromatography, Gel permeation, Gel exclusion, Size exclusion, Molecular sieve, Operating conditions, Separations, Molecular mass estimation, Size-exclusion reaction chromatography.

Introduction Gel-filtration chromatography is a form of partition chromatography used to separate molecules of different molecular sizes. Selection of Operating Conditions Various factors should be considered when designing a gel-filtration system. Explore Magazines. Editors' Picks All magazines. Explore Podcasts All podcasts. Difficulty Beginner Intermediate Advanced.

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