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| Why is it important to have the correct pH/salt concentration of the sample? |
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If the salt concentration of the sample is at or above the salt concentration necessary for elution, the sample will not bind and will wash through the column.
The sample buffer condition is particularly crucial for larger sample volumes (e.g. sample volume greater than the column volume), as the column will effectively be equilibrated in the sample buffer during sample application. For small sample volumes (less than ca.10% column volume), the buffering capacity of the mobile phase can compensate for variations in the sample buffer, assuming the sample buffer is not drastically different in pH, ionic strength or buffering capacity.
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Working in the correct pH range, a buffer concentration of 20 - 50 mM will give sufficient buffering capacity. Higher buffer concentrations in the starting buffer can inhibit binding. |
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