Hydrophobic Interaction Chromatography (HIC) is used for the separation and purification of biological samples, primarily proteins. Components separate based on differences in the hydrophobic nature of the part of the molecule near the surface. This is similar in principle to the separation mechanism for standard reversed phase HPLC, but the density of the bonded phase groups on the surface of the column packing is much less, and hence HIC involves quite weak interactions. The attraction of the sample to the stationary phase is therefore much less than with HPLC, and hence quite weak eluents are used.
Hydrophobic parts of a molecule repel water. So in aqueous solution, molecules such as proteins which contain hydrophobic components will generate a repulsion between these groups and the water molecules. This repulsion leads to a higher energy state, and causes the water molecules to form a more ordered layer at the interface, just as they do at the surface with air. Allowing the molecules to interact with an HIC column reduces the size of the interface where the repulsion occurs, and hence reduces the energy state. Binding to the column is therefore an energetically favourable situation.
Use of salts in the eluent. The hydrophobic interaction is often so weak that it would not even occur in pure water. However by adding a salt to the eluent, the polarity of the water is increased, thus making adsorption more favourable for the hydrophobic part of the molecule. Sometimes quite high salt concentrations are required (ca 1M) but normally 0.02-0.05M is enough.
The most commonly used salt is ammonium sulphate, although others are occasionally used. Different salts give rise to a greater hydrophobic interaction than others, allowing a measure of fine-tuning.
Elution of sample components. This is achieved easily by progressively reducing the salt concentration until components elute in order of increasing hydrophobic interaction.
Principles of Desorption of samples from the column. As the salt concentration is decreased, a point is reached where the sample component starts to partitiion between the stationary phase and the mobile phase, and therefore starts to move down the column. The rate of migration down the column is proportional to the percentage of the sample in the mobile phase at a given time. The greater the partition in the mobile phase, the faster the band moves, until the sample is totally in the mobile phase and has no further interaction with the column.
There is therefore a salt concentration range from the point where the sample just starts to partition with the eluent (and hence starts to move) down to the point where the partition is 100% in the eluent, and no further interaction with the column occurs. This concentration range is known as the Partition Zone.
Flow rate is not especially important as long as it is not too fast, because otherwise mass transfer does not have time to occur, and horrendous band-broadening will occur.
Gradient Elution is normally used with HIC. Because we are running a decreasing salt concentration gradient, it is almost always a binary gradient, where solvent A is just a higher concentration of solvent B, both being an aqueous salt (such as ammonium sulphate.) The concentration of solvent A will be selected to be significantly higher than the beginning of the partition zone of the least hydrophobic component of the sample.
As the gradient progresses and the salt concentration drops it approaches the beginning of the partition zone. When this point is reached, sample partition with the eleunt starts, and the sample begins to migrate down the column. As the eluent concentration continues to drop, hydrophobic interaction decreases, and the migration speed of the sample increases until it reaches the linear velocity of the eluent. This continues until the peak is eluted from the column.
Gradient concentration range. It is important to select carefully the starting point of the gradient. If the salt concentration is not high enough, the sample does not bind to the top bed of the column, and broad, poorly separated peaks result. However if the concentration is too high, all impurities also bind to the column, possibly then interfering with the peaks of interest during elution.
Gradient slope. Once the salt concentration range needed to elute all the components has been established, it may be necessary to reduce the slope of the gradient (ie to increase the gradient run time) to achieve resolution. Hydrophobic Interaction is a weak phenomenon, and it may be that all sample components will have quite similar hydrophicities. In this situation, a longer gradient time is required to elute the components sequentially and with good resolution.
It is important that all components are fully desorbed from the stationary phase before they leave the column, if reproducible results are to be achieved. For really shallow gradients, this may require a longer column. However HIC columns are normally quite short (1-10cm) and total desorption is usually possible within this distance. An unnecessarily long column simply gives rise to band broadening.
Salt choice. For some protein species, the use of too high a salt concentration causes precipitation on the column (exactly as per "salting out" of solution). Sometimes there is not a great difference between the salt concentration required to achieve adsorption and that which gives rise to precipitation. In this situation, a weaker salt is required to increase the concentration gap, or a more strongly binding column is required, (eg. phenyl.) As a rough guide:
Na2SO4 > K2SO4 > (NH4)2SO4 > NaCl > NH4Cl > NaBr
Column Choice. Although the hydrophobic interaction is weak, some proteins have a relatively high hydrophobicity, and can bind quite strongly to an HIC column. In extreme cases, even 0% buffer is not enough to cause elution and it is necessary to us an organic solvent such as methanol mixed with water to achieve elution. Hence although a strongly adsorbing column is recommended for most applications, it is useful to know of weaker options when needed.
In order of decreasing hydrophobic interaction, columns are:
Phenyl > C8 > C4 > Isopropyl
Temperature. Hydrophobic Interaction increases with temperature. In the interests of not denaturing the proteins it is normal not to work much above ambient. However the effect is very marked if the samples are stored in an autosampler tray at 4oC! In this situation, very little interaction is observed, and the peaks elute unretained with the solvent front.
pH. Separations are generally robust to pH changes in the region of 4-8, but outside this range, unpredictable results are obtained. Hence most HIC separations are carried out within the range pH4-8.
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