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TSK-GEL Affinity Chromatography Columns

The Tosoh Bioscience TSK-GEL Affinity Chromatography (AFC) column line consists of three group-specific stationary phases: ABA-5PW, Boronate-5PW and Chelate-5PW as well as one activated packing material called Tresyl-5PW. Affinity chromatography offers the highest level of specificity and
selectivity in biomolecular separations and purifications. Tosoh Bioscience supplies a full range of products for analytical, preparative and process scale affinity chromatography.

Please click here to see TSK-GEL HIC Column Selection.

TSK-GEL affinity chromatography columns are based on the well-known G5000PW porous resin, which is the basis for high performance size exclusion chromatography columns. The TSKGEL 5PW-type resin is a hydrophilic media with 1,000 Å pores and an estimated protein exclusion limit of 5x106 Da. Tosoh Bioscience's process scale affinity media are based on the 65 µm particle size, semi-rigid Toyopearl HW-65 resin.

Since analytical and semi-preparative columns are made from the same polymer chemistry as the process scale media, seamless scale-up from lab to process scale is achievable. Consult the chapter on bulk media for more information about resins for packing columns to purify medium to large volume samples. Table I on the next page lists the ligand concentration, adsorption capacity and the test analyte used to determine the capacity of each column type.

Please click here to see TSK-GEL Anion Exchange Applications.

Description	ID (mm)	Length (cm)	Particule Size (μm)	Number Theoretical Plates	Flow Rate (mL/min)		Max Pressure Drop (kg/cm2)	Part number
Description	ID (mm)	Length (cm)	Particule Size (μm)	Number Theoretical Plates	Range	Max	Max Pressure Drop (kg/cm2)	Part number
Glass columns
Boronate-5PW, 1000 Å	5.0	5.0	10	≥ 500	0.5-1.0	1.2	20	14449
Chelate-5PW, 1000 Å	5.0	5.0	10	≥ 500	0.5-0.8	1.0	20	14440
Chelate-5PW, 1000 Å	8.0	7.5	10	≥ 1,300	0.5-1.0	1.2	15	14441
Tresyl-5PW, 1000 Å	5.0	5.0	10	≥ 500	0.2-0.8	1.0	10	14457
Phenyl-5PW 1000 Å	8.0	7.5	10	≥ 1,300	0.5-1.0	1.2	10	14458

PEEK Columns
BioAssist Chelate, 1000 Å	7.8	5.0	10	≥ 8,00	0.5-1.0	1.2	10	20022

Stainless steel columns
ABA-5PW, 1000 Å	7.5	7.5	10	≥ 1,300	0.5-1.0	1.2	10	13067
Boronate-5PW, 1000 Å	7.5	7.5	10	≥ 1,300	0.5-1.0	1.2	10	13066
Chelate-5PW, 1000 Å	7.5	7.5	10	≥ 1,300	0.5-1.0	1.2	10	08645
Tresyl-5PW, 1000 Å	6.0	4.0	10		0.2-0.5	1.0	10	14455
Tresyl-5PW, 1000 Å	7.5	7.5	10		0.5-1.0	1.2	10	14456
Guard column products
ABA-5PW				Guardgel Kit for P/N 13067				13127
Boronate-5PW			20.0	Glass Guardgel Kit for P/N 14450 and 14449				14451
Boronate-5PW				Guardgel Kit for P/N 13066				13125

Chelate-5PW			20.0	Glass Guardgel Kit for P/N 14440 and 14441				42156

Bulk packing
Tresyl-5PW, 2 g dry gel*			10					19308

* 1 g is approximately 3.5 mL

Column Selection

TSK-GEL affinity chromatography columns have been developed for purifying peptides, proteins, and nucleic acids. In addition, some columns have been successfully applied to the selective separation of small biomolecules such as nucleosides and catecholamines.
The structures of the functional ligands available from Tosoh Bioscience are shown in Figure 1. The choice of a specific ligand is dictated by the expected interaction between the sample and column bonded phase. For example, the TSKgel Chelate-5PW column will bind high concentrations of Zn2+ ions. If a given protein is known to bind to Zn2+ ions, the Chelate-5PW would be a candidate column for the isolation of that target compound.
Tosoh Bioscience offers AFC columns in both glass and stainless steel formats. Glass columns are available in two formats: 5 mm ID x 5 cm L and 8 mm ID x 7.5 cm L. Stainless steel columns are available as 7.5 mm ID x 7.5 cm L and 6 mm ID x 4 cm L (Tresyl-5PW only). TSKgel Chelate-5PW is also supplied in a semi-preparative size: 21.5 mm ID x 15 cm L. TSKgel BioAssist Chelate is packed in 7.8 mm ID x 5 cm L PEEK hardware. The shipping solvent is distilled water for ABA-5PW and Boronate- 5PW. The Chelate-5PW is shipped in 10 mmol/L acetate buffer, pH 4.5, and the Tresyl-5PW column shipping solvent is acetone. Stainless steel or Pyrex frits are employed in the body of the column end-fittings for the metal and glass columns, respectively. The nominal frit size for stainless steel columns is engraved in the end-fittings and all Pyrex® frits are 10 μm nominal pore size. At the recommended flow rates (see Ordering Information) the pressure drop across a TSK-GEL AFC glass or stainless steel column is less than 20 kg/cm2. Separation columns should be protected with a guard column. Tosoh Bioscience offers a unique Guardgel kit consisting of
guard column hardware and gel packing, allowing the user to repack the guard column as required. Guardgel kits are available for most affinity columns, both glass and stainless steel.
As with all columns used in gradient elution chromatography, affinity columns should be washed with final elution buffer prior to re-equilibration with initial (binding) buffer.


Figure 1	Figure 2

TSK-GEL ABA-5PW The p-aminobenzamidine ligand of the ABA-5PW affinity column mimics a serine protease inhibitor. Proteins are loaded onto the column in an alkaline buffer such as Tris and are desorbed by lowering the pH. The column operates in a pH range of 2-9. Given this ability to bind complex proteins, the applications of ABA-5PW are numerous. Applications for TSKgel ABA-5PW include: trypsin, urokinase, kallikrein, enterokinase, and blood coagulation factors such as thrombin, factor X, and plasminogen activator. Figure 2 compares the isolation of crude trypsin on a TSKgel ABA-5PW column and a purity check of the collected fraction by reversed phase liquid chromatography.

Applications

TSK-GEL BORONATE-5PW

Coupling of m-aminophenyl boronate to the TSK-GEL 5PWtype polymeric support results in a ligand capable of forming a tetrahedral boronate anion under alkaline pH conditions. This anionic structure can bind with 1,2 cis-diol groups such as those found in carbohydrates, carbohydrate-containing compounds, and catecholamines.
Interaction between the boronate anion and the 1,2 cis-diol groups is enhanced in the presence of Mg2+ ions and is inhibited by amine-containing buffers. Adsorption onto the TSKgel Boronate-5PW takes place in basic buffers such as HEPES and morpholine, while desorption takes place in carbohydrate or amine-containing mobile phases like sorbitol or Tris.
Applications for TSKgel Boronate-5PW include: nucleic acids, nucleotides and nucleosides. This affinity column has also been used to isolate catecholamines and other biomolecules containing the 1,2 cis-diol functionality.
Figures 3 & 4 demonstrate the applicability of the TSKgel Boronate-5PW affinity chromatography column for the separation of nucleosides and catecholamines.

Figure 3

Figure 4

TSK-GEL CHELATE-5PW

TSKgel Chelate-5PW utilizes the ability of iminodiacetic acid (IDA) to chelate ions such as Zn2+, Ni2+ and Cu2+. The column is pre-loaded with divalent metal ions by chelation. Peptides and proteins containing histidine residues will normally adsorb to these chelated ions at neutral pH. The retained compounds are then eluted with buffer containing imidazole or glycine.
The key to making successful use of this retention mechanism is the proper selection of metal ions for chelation and the elution buffer to desorb the analytes. In general, Cu2+ interacts better with protein; however, resolution is usually enhanced with Zn2+ ions. A gradient mobile phase containing increasing imidazole or glycine concentrations is used to elute the retained compounds. A decreasing pH gradient can also be used. Glycine, as well as HEPES buffers, will also elute the metallic ion so column regeneration is necessary. Conversely, imidazole in phosphate buffer will extract the metal ions very slowly, avoiding frequent column regeneration.
Applications for TSKgel Chelate-5PW include: immunoglobulins, transferrin, lectins, milk proteins, membrane proteins, and peptides.
In Figure 5, the separation of ribonuclease A (bovine) and transferrin (human) are compared on TSKgel Chelate-5PW columns (glass, 5 mm ID x 5 cm L) containing different metal ions.

Figure 5

TSK-GEL TRESYL-5PW

Unlike other TSK-GEL affinity columns, the TSKgel Tresyl-5PW (tresyl; 2,2,2-trifluoroethanesulfonyl) requires activation with a user-selected ligand containing amino, thiol, phenol, or imidazole groups. The resulting structure is literally a custom affinity ligand with excellent pH stability and minimal ligand loss due to leaching.
TSKgel Tresyl-5PW readily reacts with amino or thiol groups to form stable covalent alkylamines or thioethers. Principal applications for TSKgel Tresyl-5PW include the selective purification of antigens after coupling the appropriate antibody to the solid support. The antibody coupling yield at pH >7.5 is more than 90 %, with the maximum binding occurring at pH 7.5. Antigen adsorption to the antibody ligand is most effective when the antibody concentration is < 2-3 mg/mL of affinity resin. To increase binding capacity, more antibody should be added to the coupling reaction.

However, higher concentrations of antibody can result in steric hindrance, thus lowering the binding capacity of the column. As a general rule, the time required for antibody attachment to
the TSKgel Tresyl-5PW column is directly proportional to the antibody concentration. Small amounts of antibody require about 2 hours to complete the cross-linking reaction, whereas it may take 6-7 hours to fully attach an antibody at the concentration of 10 mg/mL-resin.
Examples of the wide range of applications using TSKgel Tresyl- 5PW include the binding of such ligands as concanavalin A (a lipoprotein lectin that binds to glycoproteins), numerous antibodies and enzymes.

The chromatogram in Figure 6 shows the purification of peroxidase by the concanvalin A ligand coupled to the TSKgel Tresyl-5PW affinity support resin.