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TSK-GEL HIC Columns |
Hydrophobic Interaction Chromatography (HIC) is based on the
interaction between hydrophobic groups on a protein and a
hydrophobic ligand on the solid support. HIC offers a distinct
advantage for easily denatured proteins; it can be run using
moderate concentrations of ammonium sulfate, which favors the
stability of many proteins.
The binding of proteins to a hydrophobic matrix is affected by a
number of factors including (1) the type of ligand, (2) the ligand
density on the solid support, (3) the backbone material of the
matrix, (4) the hydrophobic nature of the protein, and (5) the type
of salt used. All of these factors help to make HIC a powerful
technique for the separation of biomolecules.
Tosoh Bioscience offers three different HIC column types in
analytical format: TSKgel Phenyl-5PW, Ether-5PW and Butyl
NPR. TSKgel Phenyl-5PW and Ether-5PW are also available in
preparative column formats.
Please click here to see TSK-GEL HIC Column Selection. |
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- Ether, phenyl, and butyl functionalities are available.
- TSKgel Ether-5PW and Phenyl-5PW columns are available in
a 2 mm ID format.
- Large 1000 Å pore size of the base matrix accommodates proteins
up to 5 x 106 Da.
- Polymeric resin is chemically and physically stable to changes
in pH and ionic strength and compatible with a variety of
organic solvents.
- High binding capacity is achieved for TSK-GEL 5PW-type HIC
packing materials.
- Nonporous resins (NPR) allow fast analysis for quality control
or process monitoring
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| Please click here to see TSK-GEL Anion Exchange Applications. |
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| Please click here to see TSK-GEL HIC Column Stability. |
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Description |
ID (mm) |
Length (cm) |
Particule Size (μm) |
Number Theoretical Plates |
Flow Rate (mL/min)
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Max Pressure Drop (kg/cm2)
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Part # |
| Range |
Max |
| Glass columns |
Ether-5PW
1000 Å |
5.0 |
5.0 |
10 |
≥ 600 |
0.5-0.8 |
1.0 |
20 |
14013 |
Ether-5PW
1000 Å |
8.0 |
7.5 |
10 |
≥ 1,000 |
0.5-1.0 |
1.2 |
20 |
14014 |
Ether-5PW
1000 Å |
20.0 |
15.0 |
13 |
≥ 3,000 |
4.0-6.0 |
8.0 |
20 |
14016 |
| Phenyl-5PW 1000 Å |
5.0 |
5.0 |
10 |
≥ 600 |
0.5-0.8 |
1.0 |
20 |
13063 |
| Phenyl-5PW 1000 Å |
8.0 |
7.5 |
10 |
≥ 1,000 |
0.5-1.0 |
1.2 |
20 |
08804 |
| Phenyl-5PW 1000 Å |
20.0 |
15.0 |
13 |
≥ 3,000 |
4.0-6.0 |
8.0 |
20 |
14018 |
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| PEEK Columns |
| BioAssist Phenyl, 1000 Å |
7.8 |
5.0 |
10 |
≥ 1,000 |
0.5-1.0 |
1.2 |
200 |
20023 |
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| Stainless steel columns |
Ether-5PW
1000 Å |
2.0 |
7.5 |
10 |
≥ 1,000 |
0.05-0.10 |
0.12 |
15 |
18760 |
Ether-5PW
1000 Å |
7.5 |
7.5 |
10 |
≥ 1,000 |
0.5-1.0 |
1.2 |
15 |
08641 |
Ether-5PW
1000 Å |
21.5 |
15.0 |
13 |
≥ 3,000 |
4.0-6.0 |
8.0 |
25 |
08642 |
Ether-5PW
1000 Å |
55.0 |
20.0 |
20 |
≥ 1,500 |
20.0-40.0 |
50.0 |
4 |
16255 |
Ether-5PW
1000 Å |
2.0 |
7.5 |
10 |
≥ 1,000 |
0.05-0.10 |
0.12 |
15 |
18759 |
Ether-5PW
1000 Å |
7.5 |
7.5 |
10 |
≥ 1,000 |
0.5-1.0 |
1.2 |
15 |
07573 |
Ether-5PW
1000 Å |
21.5 |
15.0 |
13 |
≥ 3,000 |
4.0-6.0 |
8.0 |
25 |
07656 |
Ether-5PW
1000 Å |
55.0 |
20.0 |
20 |
≥ 1,500 |
20.0-40.0 |
50.0 |
4 |
07938 |
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| Butyl-NPR, nonporous |
4.6 |
3.5 |
2.5 |
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0.5 - 1.0 |
1.2 |
200 |
14947 |
| Guard column products |
| Ether-5PW |
2.0 |
1.0 |
10 |
Guard cartridge for P/N 18760 |
42156 |
| Ether-5PW |
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|
20.0 |
Guardgel Kit, Glass for P/N 14013 and 14014 |
14025 |
| Ether-5PW |
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|
20.0 |
Guardgel Kit for P/N 08641 |
08643 |
| Ether-5PW |
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|
20.0 |
Prep Guardgel Kit for P/N 08642 |
16091 |
| Ether-5PW |
20.0 |
2.0 |
13.0 |
Guard column, Glass for P/N 14015 |
14470 |
| Ether-5PW |
45.0 |
5.0 |
20 |
Guard cartridge for P/N 16255 |
16253 |
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| Phenyl-5PW |
2.0 |
1.0 |
10 |
Guard cartridge for P/N 18759 |
42156 |
| Phenyl-5PW |
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|
20.0 |
Guardgel Kit, Glass for P/N 08804 and 13063 |
14025 |
| Phenyl-5PW |
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|
20.0 |
Guardgel Kit for P/N 07573 |
08643 |
| Phenyl-5PW |
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|
20.0 |
Prep Guardgel Kit for P/N 07656 |
16091 |
| Phenyl-5PW |
20.0 |
2.0 |
13.0 |
Guard column, Glass for P/N 16255 |
14470 |
| Phenyl-5PW |
45.0 |
5.0 |
20 |
Guard cartridge for P/N 07938 |
16253 |
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| Guard cartridge holder(2.0mm ID, 1.5cm L) |
For all 2mm ID guard cartridges |
19308 |
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| Column Selection |
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The HIC packing materials are based on the polymeric TSKgel G5000PW size exclusion resin (a hydrophilic gel with an estimated protein exclusion limit of 5,000,000 Da) which is then derivatized
with oligoethylene-glycol (Ether-5PW) or phenyl (Phenyl-5PW) groups. Columns, depending on diameter, are packed with 10, 13 or 20 μm particles. TSKgel Ether-5PW is less hydrophobic than TSKgel Phenyl-5PW. It displays weaker interaction and thus shorter retention times compared to Phenyl-5PW, as shown in Figure 1.
TSKgel Ether- 5PW is the best choice for the separation of very hydrophobic proteins such as membrane proteins or monoclonal antibodies. The TSKgel Phenyl-5PW columns were the first commercially available, polymer-based columns for high performance HIC.
These columns have been instrumental to the increase in popularity of this technique for analytical, preparative, and process scale separations of biopolymers. |
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Figure 1 |
Figure 2 compares the separation of standard proteins on the Ether, Phenyl, and Butyl supports under similar operating conditions. The base material of TSKgel Butyl-NPR is of the same chemical composition as the G5000PW base material used to prepare Phenyl-5PW and Ether-5PW. The difference between the two packings is that the G5000PW packing is porous, whereas the base material of the TSKgel Butyl-NPR column consists of spherical 2.5 μm nonporous particles. Nonporous resins (NPR) are typically used for high-speed analytical applications.
See Figure 3 for the structure of the HIC resins. TSKgel Butyl-NPR is the least hydrophobic among the three TSK-GEL HIC columns and requires a higher salt concentration for binding. TSKgel Butyl-NPR columns provide fast and quantitative HIC, because smaller particles provide higher efficiency. By packing the 2.5 μm nonporous resin particles into shorter columns, typical analysis times are reduced to less than 10 minutes. Pore diffusion is often the rate-limiting step in the overall mass transport of large biomolecules through a porous column. Eliminating the pores provides higher resolution at higher flow rates. Another benefit of NPR resins is excellent mass recovery, allowing quantitation down to nanogram levels. These properties make TSKgel Butyl-NPR the preferred choice for process monitoring and quality control. TSK-GEL HIC columns are compatible with water-soluble organic solvents at concentration below 50 % (20 % for Butyl-NPR). |
 Figure 2
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Figure 3 |
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| Chemical stability |
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| TSK-GEL 5PW-type HIC columns are physically and chemically
stable in water-soluble organic solvents (at < 50% methanol,
ethanol, ACN, DMF, DMSO or < 30 % chloroform). Change the
solvent gradually by reducing the flow rate (preferably with
a gradient) because rapid change may cause degradation of
column efficiency. Note: When changing to an organic solvent,
reduce the salt concentration to prevent precipitation of the salt
on the column. Also, chaotropic agents (urea, SDS, etc.) will
reduce the adsorption of biomolecules; therefore, use low levels
of these agents (<2 mol/L). |
| The addition of organic solvents or chaotropic agents in the final buffer can improve separations. However, relative elution positions may change. Therefore, add chaotropic agent and organic solvent in small quantities. See Figure 5 for the effect of chaotropic agents and organic solvents on the HIC separation of two different samples. Polymer-based columns are stable when cleaning at alkaline pH. All TSK-GEL HIC columns can be routinely operated from pH 2-12. Table I shows that the phenyl groups on the TSKgel Phenyl- 5PW are stable for more than 10 days upon exposure to 0.5 mol/L NaOH or 0.5 mol/L acetic acid. |
 Figure 4
Table1
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Figure 5 |
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| Applications |
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Monoclonal Antibodies
Monoclonal antibodies (mAbs) play a part in many research, diagnostic, and therapeutic applications. Monoclonal antibodies are generally the most hydrophobic proteins in ascites fluid and
cell culture supernatant. Figure 6 shows typical results from the screening of two mAbs.
Antibiotics
The TSKgel Ether-5PW column was used to determine the relative purity of the antibiotic components C-1027 and C-1027- AG as shown in Figure 7. Antibiotic C-1027 is composed of a protein consisting of many hydrophobic and hydroxyamino acids with a non-protein chromophore. Antibiotic C-1027-AG is composed of the hydrophobic and hydroxyamino acids without the chromophore.
Human serum
Figure 8 displays the excellent recovery of albumin when 16 mL of human serum was purified on a 55 mm ID preparative TSKgel Ether-5PW column. |
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Figure 6 |
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Figure 7 |
Figure 8 |
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Recovery of biological activity for milligram to sub-gram amounts
of enzymes eluted from TSKgel Phenyl-5PW columns is shown
in table 2. In all cases, at least 80 % of the enzymatic
activity was recovered.
RNAs
Figure 9 illustrates the separation of 16S and 23S ribosomal
RNA on a TSKgel Phenyl-5PW column. The approximate
molecular weights of these RNAs are 560,000 and 1,100,000 Da,
respectively.
Proteins
Figure 10 compares the resolution of standard proteins on
analytical and preparative TSKgel Phenyl-5PW columns.
Different flow rates compensated for the change in particle size
and column dimensions. High resolution was obtained on both
columns. |
 Table 2 |
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Figure 9 |
Figure 10 |
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Proteins
Although loading capacity is limited on NPR columns, small scale
separation of proteins is possible.
Almost identical separations were obtained at sample loads
from 25 μg up to 100 μg in the separation of a crude sample of
phosphoglucose isomerase as shown in Figure 11. |
Figure 12 shows the separation of Fab and Fc fragments of an
antibody on TSKgel Butyl-NPR. The appearance of additional Fc
fragments is due to the oxidation of methionine residues by 0.10%
t-butylhydroperoxide (tBHP). The numbers above the Fc peaks
correspond to the number of oxidized residues in each fragment. |
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Figure 11 |
Figure 12 |
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