I'm reading a procedure on size exclusion chromatography and it says that the first step is to equilibrate the column with buffer. What does this mean?
What does it mean to equilibrate a column in size exclusion chromatography and why is a buffer used?
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2$\begingroup$ To equilibrate the column with buffer just means to run the column with the buffer only for a while. $\endgroup$– MaxWCommented Nov 9, 2018 at 14:29
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$\begingroup$ Why is this done? $\endgroup$– maybesomedayCommented Nov 11, 2018 at 17:09
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1 Answer
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You want to make sure that your protein does not get damaged in the separation process. The protein travels faster than the small solutes in its solution, and will encounter the small molecules that are on the column, i.e. it will encounter different ionic strength, pH etc. So before you apply the protein solution to the column, you make sure the column is treated with an appropriate solution.
The solution is often called the buffer because it contains pH-controlling buffer substance. You can use size exclusion chromatography to separate by size, but also to switch from one buffer (or more general: one set of small solutes) to another. This is called buffer exchange, and there are short thick columns for that purpose (for separation, you would use a long thin column).
