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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.
Once 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 one hand on the slope of the desorption curve and on the other hand 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 obrain 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. |
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