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Size Exclusion

Size Exclusion Chromatography (SEC) is the dominant mode of separation for polymers.

It is based on the discrimination of individual sample components by the pores of the packing material. Large sample molecules cannot or can only partially penetrate the pores, whereas smaller molecules can access all or a larger number of pores. 

In SEC, large molecules elute from the column first, smaller molecules elute later, and the smallest molecules that can access all the pores elute last from the column. Size exclusion chromatography is the only mode of chromatography that does not involve interaction with a stationary phase by means of adsorption or partitioning of the solutes.

Particles prepared from organic polymers have traditionally been the most widely used packing materials for size-exclusion chromatography (SEC). The terms SEC, GFC (gel filtration chromatography) and GPC (gel permeation chromatography) all refer to the same chromatographic technique. In GFC an aqueous mobile phase is used, while an organic mobile phase is employed in GPC. The general term SEC covers both uses.

GFC is popular among biochemists for the isolation of protein fractions or for the removal of aggregates in a final polishing step in biotechnology production. GFC is also frequently used for desalting a (protein) sample solution, often to prepare the sample for elution by another chromatographic mode.

GPC plays an important role in the characterization of industrial organic-soluble polymers.

Size Exclusion Chromatography 

 

The TSKgel SW-type columns, first introduced in 1977, are the standard in gel filtration chromatography. GFC is popular among biochemists for the isolation of proteins, for the removal of aggregates, to desalt a protein sample, to separate nucleic acid fractions, or to characterize water-soluble polymers used in food products, paints, pharmaceutical preparations. 

The new TSKgel SW mAb products consist of two specialized columns designed for the separation and analysis of monoclonal antibodies (mAb). Compared to competitive columns, these stainless steel, silica-based TSKgel columns offer reduced lot-to-lot variation, long column life, reduction of unspecified adsorption, and superior recovery of aggregates. 

TSKgel SW, SWXL and SuperSW columns are silica-based GFC columns optimized for the separation of proteins and nucleic acids using an aqueous buffer as mobile phase. A hydrophilic diol-type bonded phase shields the silica surface from interacting with protein samples. Three different pore sizes are available. 

  •  12.5 nm pore size: for analysis of small proteins and peptides 

  •  25 nm pore size: for most protein samples 

  •  45 nm pore size: for very large proteins and nucleic acids 

Compared to competitive HPLC columns, SW-type columns contain more pore volume per unit column volume, which results in higher MW selectivity or better resolution. The TSKgel BioAssist DS column is designed for the desalting and buffer exchange of proteins and polynucleotides at semi-preparative scale. 

Particle sizes for the four SW-type and the BioAssist DS column lines are:
See each of the TSKgel SW-type and the TSKgel BioAssist DS product pages above for more information and representative calibration curves.

TSKgel PW columns are the columns of choice for analyses of water-soluble polymers.  These TSKgel columns are polymer-based gel filtration columns and prepared from polymethacrylate resin.

TSKgel PW columns exhibit a much larger separation range, better linearity of calibration curves, and less adsorption than the TSKgel Gel Filtration Chromatography SW columns, making them an excellent choice for water-soluble polymers. 

While a TSKgel SW column is typically the first column to try for biopolymers, TSKgel PW columns have demonstrated good results for smaller peptides (<1,000 Da), protein aggregates, DNA fragments, and viruses.

  • TSKgel PW columns: for when analysis time is not critical, when sample mass is not limited, to collect fractions, or to obtain maximum number of plates (at the expense of analysis time).

  • TSKgel PWXL columns: high efficiency for optimal resolution, reduced analysis time, and in sample limited applications.

  • TSKgel SuperPW columns: semi-micro columns within series allow wide separation range from high MW polymers to oligomers. SuperMultiporePW series are packed with particles containing a wide range of pore sizes.

See each of the TSKgel PW-type product pages for more information and representative calibration curves.

TSKgel Alpha and TSKgel SuperAW columns were developed for the GPC analysis of polymers of intermediate polarity. As in the TSKgel PW- and PWXL-type resins, the particles in TSKgel Alpha and SuperAW columns have a hydroxylated methacrylate polymer backbone, but they are crosslinked to a higher degree than TSKgel PW- and PWXL-type columns to minimize swelling in polar organic solvents.

Applications include polymers that are soluble in methanol, acetonitrile, DMSO, isopropanol, or THF. These columns can also be used for water-soluble polymers.

TSKgel Super AW and TSKgel Alpha columns feature no or minimal swelling in a wide range of polar organic solvents, including methanol, acetonitrile, DMSO, isopropanol, THF, and HFIP. Accurate MW determination and normal retention of polystrene polymers in DMF solvent can be achieved using either column. System peaks from salts in the eluent elute away from the oligomer of interest.

For high thoughput applications, TSKgel SuperAW columns provide equivalent resolution in half the time compared to TSKgel Alpha columns. 

See each TSKgel SuperAW and TSKgel Alpha column product pages for further information and representative calibration curves.

TSKgel H series GPC columns are recommended for the analysis of organic-soluble polymers and are packed with spherical particles composed of polystyrene crosslinked with divinylbenzene (PS-DVB). This series includes TSKgel HXL, HHR, SuperH, Super HZ, and SuperMultiporeHZ columns. Each line of columns within this series differs in degree of inertness and operating temperature range.

  • TSKgel SuperH and TSKgel SuperHZ columns were developed for high-throughput GPC applications. Both product lines feature particle sizes down to 3 μm, a column length of 15 cm and column diameters of 6 mm ID and 4.6 mm ID.

  • TSKgel HXL and TSKgel HHR columns are conventional GPC columns of 7.8 mm ID x 30 cm, while particles sizes are 5 µm and larger for the larger pore size columns.

  • Tosoh's innovative multi-pore technology, used in TSKgel SuperMultiporeHZ, produces particles that contain a broad range of pore sizes. Columns with an extended linear calibration curve can now be prepared without mixing particles of different pore sizes.

Except for TSKgel SuperMultiporeHZ columns, all other TSKgel H-type column lines contain at least five different pore sizes. Each product line contains columns for the analysis of oligomers to polymers containing molecular weights as high as an estimated 4 x 108Da. All product lines contain a number of linear columns that are prepared by blending particles with different pore sizes.

See each of the TSKgel H-type product pages for more information and representative calibration curves.

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