Isocratic elution
In isocratic elution the chromatographic conditions are kept constant throughout the entire experiment. This makes the basic construction of an isocratic chromatography system rather simple (Fig 2.1).
Fig 2.1 The necessary components of an isocratic chromatography system.
The isocratic experiment works within the partition zones for the sample components to be separated.
In other words, each sample component moves down the column with its own specific and constant velocity, as set by the organic solvent concentration of the eluent.
The longer the column the larger the distance between the peaks.
However, the sample zones broaden at the same time. Fortunately, the distance between two peaks increases in direct proportion to column length, while the peaks broaden in proportion to the square root of the column length. A longer column will thus always provide better resolution when run under isocratic conditions. However, one need to increase the column length four times to double the resolution.
Figure 2.2 below illustrates the effect of varying the salt concentration in isocratic experiments.
Fig 2.2. The chromatograms show the effect of varying the organic solvent concentration in isocratic experiments. Note that no concentration is capable of eluting all four components in the same run.
Gradient elution
The most frequently used elution mode in high resolution protein/peptide applications of RPC is gradient elution.
Gradients of organic solvents are formed by mixing two eluents, one containing no or a low concentration of the organic solvent (buffer A) and one containing a high concentration of this solvent in water (buffer B). But for their organic solvent 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 (Fig 2.3).
Fig 2.3. The gradient system needs a gradient forming
device i.e. an extra pump to control "buffer B".
To understand how gradient elution works, consider a peptide with the desorption curve shown in Figure 2.4.
Fig 2.4. In gradient elution the organic solvent 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 peptide is applied to the column at an organic solvent concentration well below the partition zone and is thus adsorbed at the top of the column. When the gradient starts, it will remain fully adsorbed until the organic solvent concentration reaches the lower end of the partition zone.
As the concentration passes through the partition zone, the peptide becomes increasingly desorbed and move down the column with a velocity reflecting its relative desorption.
When the organic solvent concentration reaches the upper end of the partition zone, all peptide molecules are desorbed. The peptide now moves with the same velocity as the eluent and its position within the gradient becomes fixed.
The time (and thus the distance of travel) 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 of travel needed to reach the fixed position in the gradient.
The shallower the gradient, the longer the
distance of travel needed to reach the fixed position in the gradient.
Now consider the four peptides used to exemplify isocratic elution above.
As pointed out, no isocratic conditions could separate all four peptides in one single run.
A gradient embracing all the partition zones of the respective peptides will, however, separate all four peptide in one run, provided the column is long enough to allow all four peptide to reach their respective final positions in the gradient (Fig 2.5).
Fig 2.5. Gradient elution will sort the sample components
according to their respective 100 %- desorption points.
Another important difference compared to isocratic elution is that the volume of the sample applied will not influence the final results.
Since the gradient starts at an organic solvent concentration well below the partition zones of all the peptides, these will all adsorb at the top of the column during sample application. When eluted they will all appear as narrow zones, regardless of the original sample volume.
In contrast to isocratic elution, gradient elution does not require a lot of test runs to find a suitable elution strength.
The distance between the peaks is controlled by the slope of the gradient (Fig 2.6).
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Fig 2.6. Resolution can be increased by lowering the gradient slope.
The shallower the gradient, greater the distance between the peaks.
However, as a result of the re-partitioning mechanism the sample peaks broaden when the gradient passes through the partition zone and the longer the travel distance to reach full desorption, the broader the peaks.
The gain in distance between the peaks is, however, 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.
Peptide and 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 2.7).
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Fig 2.7. The column should be long enough to allow the sample components
to reach their final positions in the gradient. However, excessively long
columns will only contribute to peakbroadening. |
