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Effect of eluent composition
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 optimising parameter. |
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