What is Hydrophobic interaction chromatography?

Hydrophobic interaction chromatography (HIC) is a liquid chromatography technique that separates biomolecules according to hydrophobicity.

Although most hydrophobic amino acids are buried in the interior of globular proteins, some of them are exposed on the protein surface. These can interact with hydrophobic ligands on the HIC gel. The amount of exposed hydrophobic amino acids differs between proteins and so does the ability of proteins to interact with HIC gels.

HIC is complementary to ion exchange (IEX) and gel filtration (GF) and high overall resolutions are obtained when HIC is combined with other LC techniques.
Since HIC is an adsorptive technique, gradient or step elution can be applied and the sample elutes in concentrated form.

High salt concentrations are needed to adsorb the sample to the HIC gel. This makes it especially well-suited as a capture step after ammonium sulfate precipitation and as a next step after IEX, since no or very simple inter-step conditioning will be necessary in these cases.
However, the high salt necessary for binding may sometimes cause precipitation of some sample components including the target protein. If so, a ligand with stronger hydrophobicity has to be chosen.

This picture shows hydrophobic and hydrophilic parts on the surface of lysozyme. The most hydrophobic parts are dark red, the less hydrophobic lighter red. The most hydrophilic parts are shown in dark blue, while the less hydrophilic parts are lighter blue.

The Separation Mechanism

Before going into details with the separation mechanism of HIC there is a need to define terms like hydrophilicity and hydrohobicity, and to explain the phenomenon called hydrophobic interaction. Definitions Hydrophilic means "loves water" and is assigned to molecules that readily interact with water through dipole-dipole interaction or by hydrogen bonding. Hydrophobic means "hates water" and is assigned to molecules that do not interact with water. There is no sharp boundary between hydrophilic and hydrophobic but rather a continuum of hydrophobicity (or hydrophilicity for that matter) as illustrated in Figure 1.1. Fig 1.1. The ability to "love water" resides in the possibility of interacting with water molecules through dipole-dipole interaction or by hydrogen bonding. In HIC sample molecules (in most cases proteins) are made to interact with hydrophobic ligands grafted on a hydrophilic matrix. There is no general answer to how a protein surface interacts with hydrophobic ligands. The perhaps most widely accepted, however, explains the hydrophobic interaction as an entropy-driven mechanism (Ref. Porath et al. 1973, Nature 245: 465). Hydrophobic interaction Consider what happens when you prepare a salad dressing. You basically mix oil, water and herbs. When you shake this mixture a lot of small droplets of oil are formed and suspended in the water. If you have done your shaking well the suspension will last for some time, but will eventually go back to the state with two separate phases, one water phase and one oil phase. If you follow this return to two separate phases closely you will notice that it starts with the joining of small droplets to form larger and larger ones. Fig 1.2. Oil droplets dispersed in water will join to form larger and larger droplets, which eventually form two continuous phases over time. Water is an exceptionally good solvent for polar substances but not for non-polar ones like oil and fat. These solubility properties stem from water being a strong dipole that, moreover, can form hydrogen bonds with molecules containing HX groups (X = O; F; N or Cl). In liquid water a majority of the water molecules occur in clusters due to hydrogen bonding between them selves. One water molecule can bind to four others (Fig 1.3). .Although the half-life of water clusters is very short, the net effect is a very strong cohesion between the water molecules reflected example by its high boiling point. Fig 1.3. The solubilizing properties of water resides in its ability to interact with dipoles and to form hydrogen bonds with HX-containing molecules. At an air-water interface, water molecules arrange themselves into a strong shell of highly ordered structure. The possibility to form hydrogen bonds is here no longer balanced but is dominated by the liquid side of the interface. This gives rise to the ordered structure and manifests itself in a strong surface tension. Anything that influences the stability of the water shell will also affect the surface tension. Fig 1.4. Surface tension at the air-water interface stems from a shell of highly ordered water. In contrast, bulk water is organized in short-lived clusters by hydrogen bonding. A rise in temperature will lead to smaller clusters and a higher exchange rate of water molecules between them. When a hydrophobic substance is immersed in water something analogous to the surface tension phenomenon happens. The water molecules cannot "wet" the surface of the hydrophobic substance. Instead they will form a highly ordered shell, around the hydrophobic substance, caused by the lack of possibility to form hydrogen bonds in all directions. Minimizing the extent of this shell will lead to a decrease in the number of ordered water molecules which represents a thermodynamically more favourable situation by an increase in entropy (DS). Consequently hydrophobic surfaces combine to accomplish this. Fig 1.5. Hydrophobic surfaces in water are surrounded by a shell of highly ordered water. The system strives to minimize the total area of such shells in order to gain in entropy and forces hydrophobic substances to merge. This phenomenon is called hydrophobic interaction and depends on the behaviour of the water molecules rather than on direct attraction between the hydrophobic molecules. Turning back to the oil droplets of the dressing, we now have an explanation for their tendency to combine. Proteins carry both hydrophilic and hydrophobic areas on their surfaces and at high concentrations of certain salts they precipitate (salting out) the main cause is enforced hydrophobic interaction. Fig 1.6. Salting out is (at least in part) a result of the capability of certain salts to enhance hydrophobic interaction. Adsorption HIC media contain ligands that can combine with hydrophobic surfaces of proteins. In pure water this hydrophobic effect is too weak to cause interaction between either ligand and proteins, or between the proteins themselves. However, certain salts enhance hydrophobic interactions and adding such salts brings about adsorption to HIC media. Fig 1.7. HIC deals with the relation between water shells around hydrophobic surfaces, with bulk water clusters and salts enhancing hydrophobic interaction. The following salts strengthen hydrophobic interaction in the order indicated: Na2SO4 > K2SO4 > (NH4)2SO4 > NaCl > NH4Cl > NaBr > NaSCN Ammonium sulfate is the salt commonly used to control adsorption in HIC. The sample is applied and adsorbed at high salt concentrations (~ 1M). To bring about selective desorption, the salt concentration is then lowered gradually and the sample components elute in order of hydrophobicity. Fig 1.8. The adsorption of hydrophobic molecules is a reversible reaction whose equilibrium is controlled by the salt concentration. Desorption curves The adsorption reaction is a dynamic equilibrium between free and adsorbed molecules. It can be described in terms of a desorption curve obtained by plotting the relative amount of free sample molecules as a function of the salt concentration as shown in Figure 1.9. Fig 1.9. The desorption curve reflects the distribution of the sample between the mobile and the stationary phase. Within the partition zone this distribution varies as a function of the salt concentration and the elution velocity varies accordingly. (Desorption curves have little practical value and are used here only to demonstrate the working principles of IEX.) In a column experiment all transport of a sample down the column is carried out by the mobile phase (the eluent) and acts only on the molecules present in the mobile phase. When a sample travels down the column, its velocity is proportional to the part of sample molecules present in the mobile phase. The desorption curve thus represents the velocity of a sample zone as a function on the salt concentration. The salt concentration interval corresponding to the desorption curve will be referred to as the partition zone. The position of the desorption curve along the salt concentration axis is governed by the net hydrophobicity of the sample. An increase of the net hydrophobicity will shift it to the right and a decrease to the left (Fig 1.10). Fig 1.10. The desorption curve is shifted to the right with increasing net hydrophobicity. Within the partition zone the sample will move down the column by way of a continuous re-partitioning mechanism. The transport of the molecules present in the mobile phase creates an uneven distribution of “stationary” and “mobile” sample molecules in that the concentration profile in the mobile phase will always be slightly ahead of that in the stationary phase. The partitioning mechanism strives to correct this, resulting in a mass transfer of sample molecules from the mobile phase to the stationary phase at the front of the sample zone and a mass transfer in the opposite direction at the rear end of the zone (Fig 1.11). Fig 1.11. Mass transfer directions in a travelling sample zone. Blue arrows indicate net transport direction between mobile and stationary phases. This positional discrepancy in concentration profiles causes broadening of the sample zone and is a consequence of the chromatographic process itself i.e. the continuous re-partitioning. Although always present, the extent of this type of zone broadening depends on: Mass transfer rate. Mobile phase flow rate. To minimize zone broadening, mass transfer must be given enough time for equilibration (Fig 1.12). Fig 1.12. The flow rate must be balanced against the mass transfer rate or sample zones will broaden excessively due to incomplete mass transfer. Arrow length indicates flow rate.

Elution modes Gradient elution The most frequently used elution mode in high resolution applications of HIC is gradient elution. Gradients of the salt are formed by mixing two eluents, one containing a high concentration of the salt (buffer A) and one containing a low concentration of this salt (buffer B). But for their salt contents, the two eluents are identical. Chromatography systems usually control the gradient formation by the use of two pumps, one for buffer A and one for buffer B Figure 2.1. Fig 2.1. The gradient liquid chromatography (LC) system needs a gradient forming device i.e. an extra pump to control "buffer B". To understand how gradient elution works, consider a protein with the desorption curve shown in Figure 2.2. Fig 2.2. In gradient elution the salt concentration sweeps the partition zone and causes the sample component to accelerate from zero speed to that of the mobile phase. At 100% desorption the sample component has reached a fixed position in the gradient. The protein is applied to the column at a salt concentration well above that of the partition zone and is thus adsorbed at the top of the column. When the gradient starts, it will remain fully adsorbed until the salt concentration enters the partition zone. As the salt concentration passes through the partition zone, the protein becomes increasingly desorbed and moves down the column with a velocity reflecting its relative desorption. When the salt concentration leaves the partition zone, all protein molecules are fully desorbed. The protein now moves with the same velocity as the eluent and its position within the gradient becomes fixed. The time (and thus the travel distance) needed to reach this fixed position depends on the slope of the desorption curve and on the slope of the gradient. The steeper the desorption curve, the shorter the distance travel required to reach the fixed position in the gradient. The shallower the gradient, the longer the distance travel required to reach the fixed position in the gradient. Now consider four proteins with desorption curves as shown in Figure 2.3. Fig 2.3. Gradient elution will sort the sample components according to their respective 100 %-desorption points. A gradient embracing all the partition zones of the respective proteins will separate all four proteins in one run, provided the column is long enough to allow all four proteins to reach their respective final positions in the gradient. In contrast to isocratic elution the volume of the sample applied will not influence the final results. Since the gradient starts at a salt concentration well above that of the partition zones of all the proteins, these will all adsorb at the top of the column during sample application. When eluted, they will all appear as narrow zones, regardless the original sample volume. The distance between the peaks is controlled by the slope of the gradient (Fig 2.4). Fig 2.4. Resolution can be increased by lowering the gradient slope. The shallower the gradient, the larger the distance between the peaks. However, the sample peaks broaden as a result of the re-partitioning mechanism when the salt concentration passes through the partition zone and the longer the distance traveled to reach full desorption, the broader the peaks. The gain in distance between the peaks is larger than the peak-broadening effect and the net result is that resolution increases with decreased gradient slopes. To obtain reproducible results it is important that all gradient-eluted components have reached 100% desorption before they leave the column. The column should be long enough to allow this and the shallower the gradient, the longer the column needed. Protein desorption curves are normally quite steep and gradient volumes of 20 column volumes and more are used without any problems. Once all sample components have reached their final positions in the gradient no further increase in separation is possible. On the contrary peaks will broaden due to diffusion if the column is excessively long (Fig 2.5). Fig 2.5. The column should be long enough to allow the sample proteins to reach their final positions in the gradient. However, excessively long columns will only contribute to peak-broadening. Step elution Another way of using desorption curves is to avoid the partition zone by going directly from fully adsorbed conditions to fully desorbed conditions (Fig 2.6). The salt concentration during sample application is set low enough to leave most of the weaker binding contaminants unadsorbed, while the target component and those with higher net hydrophobicities than the target component will adsorb. Fig 2.6. Step elution is a binding/non-binding technique used to concentrate and to reduce the complexity of crude samples. The salt concentration is then abruptly decreased to a level just enough to fully desorb the target component. This type of group separation will not provide the high resolution obtained with gradients. Sample components with overlapping desorption curves will in part co-elute with the target protein. Being a binding/non-binding technique rather than based on continuous re-partitioning, zone broadening will not be dependent of mass transfer and several times larger sample loads are accepted. Column dimensions are less important and both larger bead sizes and higher flow rates may be used without any detrimental effects on the separation.

Explanation of symbols
Symbolic representation of a section of a HIC bead.	Slightly hydrophobic sample component.	Reasonable hydrophobic sample component.	Quite hydrophobic sample component.	Highly hydrophobic contaminant.

1. Equilibration. Buffer A is pumped through the column until conditions match those of buffer A.	2. Sample application and wash. Sample is added and adsorbed and non-adsorbed components are washed out.	3. Gradient elution. Gradient is started and adsorbed sample components are eluted in order of increasing hydrophobicity. Elution order:			4. Regeneration. Hydrophobic contaminants still remaining are washed out.

Effect of choice of ligand

The possibility to select between ligands of different hydrophobicities is important, since this offers a way to control the salt concentration necessary to adsorb a certain protein. With low-hydrophobicity ligands the difference between adsorption and precipitation may sometimes be so small that certain proteins may partially precipitate under binding conditions (Fig 4.1). Fig 4.1 Selecting the appropriate HIC ligand is often a balance between strong enough binding and precipitation. On the other hand, quite hydrophobic proteins may bind sufficiently strongly to high-hydrophobicity ligands that harsh, potentially denaturing conditions (e.g. organic solvents) are needed to desorbe them. Listed below are the most commonly used ligands in order of binding strength: Ether < Isopropyl < Butyl < Octyl < Phenyl The difference between the first four ligands is quantitative rather than qualitative. The phenyl ligand, however, offers another type of selectivity, mainly because of its possibility to form additional bonds. Figure 4.2 demonstrates some effects of varying the ligand used. Fig 4.2 Varying the type of HIC ligand has a profound effect on the separation.

Salt By adjusting the salt concentration used to bind the sample molecules, it is possible to control which of the sample proteins that bind and which are just washed through during sample application (Fig 5.1). Fig 5.1. The start level of the gradient concentration is quite important for the results. In the left chromatogram the salt concentration was not enough to fully bind the target protein (arrow). In the center chromatogram the target protein elutes within the gradient as a sharp peak. From a purification point of view the start level of the gradient concentration in the right chromatogram is less advantageous, since sample contaminants also bind an elute within the gradient. Ideally all proteins less hydrophobic than the target protein should remain unadsorbed and appear in the flow-through fraction, while the target protein should bind strongly enough to elute within the gradient. Eluent pH The pH of the eluent certainly influences the elution behaviour of proteins in HIC. Pronounced effects, however, are normally seen only at rather extreme pH values outside the stability window of most proteins (Fig 5.2). Fig 5.2. Separation results are definitely influenced by the running pH. In the interval pH 4 - 8, however, the effect is rather small. Thus, for standard protein purification by HIC, eluent pH is normally ignored as an optimizing parameter.

Unlike most other LC techniques, in HIC binding strength increases with temperature. The fact that bulk water occurs as loosely associated, temperature sensitive clusters in dynamic equilibrium with “free” water molecules probably explains this. Increasing the temperature shifts the equilibrium towards “free” water, thereby increasing the difference in entropy between bulk water and the shell of ordered water at the hydrophobic surface. The gain in entropy upon minimizing the exposure of hydrophobic surfaces thus increases with temperature, which strengthens the hydrophobic interaction. Fig 6.1. The bulk water cluster sizes and turn over rates increase with temperature, a fact that strengthens hydrophobic interaction. From a practical point of view this means that the eluent, the sample and the chromatography system all should hold the same temperature or unexpected results may be experienced. In Figure 6.2 several column volumes of a sample at 4o C were applied to the column at room temperature. This caused the target protein to stay unadsorbed and to appear in the flow-through fraction. After having raised the sample temperature to equal that of the chromatography system (i.e. room temperature) the target protein binds and elutes as expected. Fig 6.2. The temperature of the sample must equal that of the chromatography system or results may deviate from those expected. The experiment in Figure 6.3 shows that temperature can be used to control the experiment in much the same way as with the salt concentration of the binding buffer. Moving from room temperature (23o C) to cold room temperature (4o C) required an elevation of the ammonium sulfate concentration in the binding buffer from 1.25 M to 1.55 M to restore the chromatogram. Fig 6.3. By adjusting the salt concentration of buffer A the temperature effect can be fully compensated for.

Hydrophobic interaction chromatography in practice

Use of hydrophobic interaction chromatography

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Experimental

	High resolution mode (gradient elution)	Group separation mode (step elution)
HIC medium	Use media with 10-30 mm beads for purification work.	Use media with 30 mm beads or larger to allow higher flow rates.
Column	Typical column lengths are 1-10 cm. Short columns may prohibit the use of very shallow gradients (large gradient volumes).	Column length is less important. Short and "fat" columns, however, will allow higher flow rates.
Eluents	Buffers in the pH range 4-8 are compatible with the stability of most proteins. The pH in this range has little influence on the selectivity Gradient salt can be varied quite widely. However most experiments are run with ammonium sulfate. A buffer concentration of 20-50 mM is usually sufficient.	Buffers in the pH range 4-8 are compatible with the stability of most proteins. The pH in this range has little influence on the selectivity Gradient salt can be varied quite widely. However most experiments are run with ammonium sulfate. A buffer concentration of 20-50 mM is usually sufficient.
Sample volume	With a properly designed gradient the target molecule will bind and concentrate at the top of the column. The sample volume is thus not important.	Sample volume is not important since the sample will bind to the column.
Sample amount	5-10 % of the total loading capacity of the column used can be applied without loss of resolution.	Around 40 % of the total loading capacity of the column used can be applied.

Buffer preparation tips
• Adjust pH of start (buffer A) after the salt has been added.
• Adjust buffer pH at the temperature intended for the experiment.
• Always test that target protein is stable in start buffer!

Sample preparation tips
• Filter or spin the sample to remove any particulate matter.
• Especially with large sample volumes, adjust sample pH and salt content to match those of start buffer.
• Buffer exchange on Sephadex™ G-25 is a rapid and mild way to adjust sample conditions.
• Especially with large sample volumes, adjust sample temperature to match that of start buffer A.



Arriving at optimal results

Choice of HIC medium	Optimising resolution
First test the stability of the target protein in ammonium sulfate up to 1.4 M. Test binding and recovery using HiTrap™ HIC test kit or individual resource HIC columns. Adjust ammonium sulphate of "Buffer A" to make the target protein elute late in the gradient. If necessary use a larger column applying the condition worked out above.	Screen the media (HIC ligands) for proper selectivity Optimise type and concentration of salt during adsorption. Select the steepest gradient providing acceptable resolution. If sample binding is unsatisfactory, temperature can be used to adjust it (decrease binding by lowering temp. and increase by elevating temp.).

Hydrophobic interaction chromatography profile

Working principle	Separates molecules according to hydrophobicity.
Used for	Sample concentration Removal of hydrophobic contaminants. Separation of complex samples.
Suitable stage in a purification protocol	Capture: Intermediate: Polishing: (Number of stars indicate suitability.)
Separation characteristics	Resolution High Load capacity - General separation: High - Group separation: Very High - Sample concentration: Very High . Speed - General separation: High - Group separation: High - Sample concentration: High . Gentleness Moderate to High Mass yield. Moderate to High
Main optimizing parameter	Choice of ligand.
Special features	Complementary to IEX. Can be used directly after ammonium sulphate precipitation. More gentle to proteins than RPC.

Hydrophobic Interaction Chromatography

Handbooks

Hydrophobic Interaction and Reversed Phase Chromatography pdf
Protein Purification Handbook pdf