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What Is Hydrophobic Interaction Chromatography?
Hydrophobic Interaction Chromatography (HIC) is used to separate proteins and peptides on the basis of relative hydrophobicity.
Interactions between hydrophobic groups in water are promoted by the presence of water structuring salts, and at high ionic strengths, hydrophobic residues on the surface of a protein associate strongly with other hydrophobic species (the "salting-out" effect). At very high ionic strengths protein precipitation is observed. However, at intermediate ionic strengths, proteins and peptides may be adsorbed from solution onto hydrophobic surfaces. This adsorption is reversible, and elution is achieved by simply lowering the ionic strength. Consequently, HIC is particularly useful for the purification from high ionic strength biological extracts since binding is performed in the presence of salt, and elution in the absence of salt. The strength of adsorption is determined by a number of factors including the inherent hydrophobicity of the protein, the hydrophobicity of the adsorbent, temperature, and the concentration and type of electrolyte used to promote salting-out. Water structuring salts such as ammonium sulphate are normally used for promoting protein adsorption. Since proteins have very different hydrophobicites, and there is a limit to the amount of salt that can be added, it follows that a series of adsorbents with increasing hydrophobicity are required. This is achieved by increasing the size of the immobilized alkyl/aryl group. Relatively hydrophobic proteins require mildly hydrophobic ligands such as butyl groups (otherwise elution problems are encountered), whereas very hydrophilic proteins require relatively hydrophobic ligÂands such as decyl groups (otherwise excessive salt concentrations may be required for binding).
When to use HIC
The technique may be applied to the isolation, purification and characterisation of most soluble proteins
- 1. Characterisation of Antibodies
- 2. Purification of polypeptides (e.g. glycopeptides, venoms)
- 3. Isolation of proteins from crude extracts (Capacity is several times greater than in RPC)
- 4. Quality control assay using a method complementary to ion-exchange and RPC
- 5. Isolation of integral membrane proteins and their complexes
The PolyLC HIC column series is comprised of three specific chemistries,
PolyPROPYL,
PolyETHYL and
PolyMETHYL Aspartamide,
which together span a wide range of hydrophobicity. Compared to other hydrophobic interaction chemistries, the PolyPROPYL, PolyETHYL and PolyMETHYL Aspartamide brand are extremely responsive to small changes in salt concentration: by approaching binding and loading conditions empirically, you will witness dramatic increases in resolving capacity.
HIC is an extension of reverse phase chromatography (RPC) where the polarity of the mobile phase is increased by the addition of salt. The more polar the solvent, the longer the retention, implying that a partitioning phenomena is occurring. The overriding reason why HIC is done is to avoid denaturing the protein. In RPC, the organic solvents required to elute the protein can denature the sample, or the sample sticks too well and the solid support can denature the sample.
Conditioning new columns
Before you use your column on a day to day basis, these columns should be "conditioned," a process which enhances the conformational sensitivity of the packing for better separations. This dynamic chemistry is stored in organic solvent, and when first presented with an aqueous solvent it undergoes a reversible conformational change, increasing the columns sensitivity to the conformation of the protein. Perform the conditioning process by flushing the column with the following eluents in the sequence described below:
10 column volume (c.v) of water
5 c.v. of a high salt/buffer
5 c.v. of a low salt/buffer
10 c.v. of a high salt/buffer to equilibrate prior to sample loading
Methods development
To best determine how changes in salt concentration will affect your separation, we recommend you start your methods development with the fastest elution conditions and incrementally increase the salt composition of the gradient run-by-run. Simultaneously, the salt concentration of the sample should be proportionally increased by dilution with the high salt solution (solvent A) (minimum 25% sample v/v.)
If using the 4.6 x 20 mm guard column as a methods development column, gradient times should be shortened to 5-8 minutes; the flow rate should remain at the 1.0 ml/minute analytical level. (This way you can accomplish 4-6 experiments in one hour (including flushing and equilibration) speeding up methods development, since a column void volume will take 20 seconds). The semiprep columns, 9.4 mm ID, require flow rates and equilibration volumes 4x that of the analytical columns.
First, make an injection under the low salt strength conditions to assure that the protein will elute. If considerable delay in retention is seen, increase the rate of solubilization and release from the column by lowering the buffer to 10 mM, or use detergent or 1-5% organic solvent in both reservoirs.
If the protein elutes in the void volume then use stepwise increases (i.e. 500 mM, 1.0 M, 1.5 M then 2.0 M salt) of the high salt conditions (solvent A) until the binding profile doesn't change. This will assure sample binding, shorter operation times and minimize the risk of precipitation or irreversible conformational changes of the proteins.
Because proteins are sorted by surface hydrophobicity rather than total contact hydrophobicity, you can change the water of hydration by adding or deleting small amounts of salt to cause subtle changes in their surface hydrophobicity. Structure promoting salts in the Hofmeister series (citrate>tartrate>sulfate> phosphate>chloride) increase the ability of these columns to retain and discriminate between proteins or peptides by affecting the surface hydration and conformation (note that as few as 9 amino acids are necessary to form a beta-pleated sheet and 30 can form an alpha helix.) Using different alkyl chain length columns will then maximize these differences, depending upon the accessibility of the hydrophobic pockets of the proteins.
A dramatic example of this methods development technique working better than a single linear gradient from high salt, was in the isolation of anti-Factor VIII from ascites fluid. It was achieved by determining that the separation of anti-Factor VIII from other blood proteins on the PolyPROPYL Aspartamide column could be achieved if started at 1.0 M salt. If the gradient started at 1.3 M salt they co-eluted at 800-700 mM salt.
If sulfate is not available or the baseline excursions from viscosity changes in the UV detector are unsatisfactory, initial operation with phosphate gradients has been shown to be a good alternative. If the protein is sensitive to sulfate, use of 1.2 M potassium citrate is recommended, but then use 280 nm for UV detection. Alternatively, fluorescence detection (280ex, 330em ) will avoid all baseline changes from impurities or refractive index effects on the UV detector.
Application of the sample should be in the smallest practical volume ( In the "unusual" circumstance where a perfect separation is not obtained on the first injection:
To Increase Retention:
- Raise the temperature.
Change from the ammonium to a sodium salt.
Use a longer chain length (ALKYL) Aspartamide (e.g.pentyl) chemistry.
Increase the salt concentration of the sample.
Increase the salt concentration of the solvent.
Use a better structure promoting salt (Citrate>Tartrate>Sulfate>Phosphate)
To Decrease Retention:
Use a shorter chain length (ALKYL) Aspartamide (e.g. methyl).
Decrease the salt concentration of the solvent.
Use small amounts of sucrose, glycol, or propanol in solvent A&B ( Use of Octyl Glucoside, CHAPS, and CHAPSO work well (below their CMC) although use of hydrophobic detergents is not recommended
Change the pH of the buffer to increase the ionic character of the peptide.
Reversed phase and HIC polarity considerations
The use of these columns as reverse phase columns is possible although the very hydrophilic coating will be weakly retentive relative to a C3 RPC column and they will not tolerate TFA in the mobile phase. The polarity of the peptidic surface chemistry is quite varied as compared to other HIC columns. The PolyETHYL Aspartamide HIC is about 60% as retentive as the PolyPROPYL Aspartamide HIC, and the PolyMETHYL Aspartamide HIC is only 18% as retentive as the PolyPROPYL Aspartamide based on relative retention times.
Selection of a column is quite empirical, although if the protein is soluble without detergents the PolyPROPYL Aspartamide column is probably the best choice.
Instructions for day-to-day use
The following are typical operating conditions. It is recommended that you filter all mobile phases and samples to avoid plugging the inlet frits.
A: 100% 2.0M (NH4)2SO4 + 0.1M K-PO4, pH 6.5
B: 100% 0.1M K-PO4, pH 6.5; 0-100%B, linear, 40 min (15 column volumes), 1.0 ml/min.
1. Flush with 5 c.v. of the high salt buffer
2. Condition with 5 c.v. of the low salt buffer
3. Perform analysis with decreasing salt gradient
4. Flush with 30-40ml H2O at end of day and plug the column ends.
5. Store in H2O for up to 4 days. If the column is to be stored for longer than 4 days, store it in methanol. When it is to be used again after methanol storage, prepare the column as described in "Conditioning new columns" to reproduce earlier results.
A pH range of 3.0-7.0 is recommended since these columns are based on 300Ã… silica, chemically bonded with a polymeric amide (peptide like) chemistry. The capacity of the packing is high (400 mg/gm for batch adsorption) and an analytical column should be able to handle 1-5 mg of protein in a high resolution separation of moderate complexity.
To elute proteins with unusually strong hydrophobic character, detergents or solvent compositions of 50% organic may be used in the gradient without detrimental effects to the packing's surface chemistry. However, to obtain longest column life, exercise care when using organic solvents to prevent precipitation of salts. Do not use TFA or strong acids in the mobile phases on these columns since the peptidic bonded phase could be damaged.
Storage and cleaning
At the end of the day, flush the analytical column with at least 40 ml of distilled water (for guard column use 6 ml and for the semiprep column 120 ml). To remove strongly retained components 1-2 ml DMSO is preferred to 1-2 ml of guanidinium chloride injected in 100-250 µl aliquotes under no salt conditions. If the column is to be left for more than 4 days, storage in 100% methanol is recommended.
Satisfaction guaranteed
If the performance of this column does not meet the specifications of the attached chromatogram upon initial use, or if within 45 days of normal use, the column fails to maintain adequate performance, LCC will replace this product with a new column. We will need to have operating conditions which led to the failure and the column identification number to process your replacement order.
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