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To clean and regenerate a normal phase column flush it with the following volumes of solvent. Ion exchange phases can be either anion or cation exchangers (WAX, SIX, WAX, and SAX). To clean and regenerate an ion exchange column flush it with the following volumes of solvent.
To clean and regenerate a normal phase column flush it with the following volumes of solvent. Ion exchange phases can be either anion or cation exchangers (WAX, SIX, WAX, and SAX). To clean and regenerate an ion exchange column flush it with the following volumes of solvent.
In your case wash the column with 70% water. 15% methanol. 15% acetonitrile. Divert the column fluent to waste not to contaminate your detector(s). Wash the column slowly over to 100% methanol and wash for at least 15 minutes. Wash the column over to 100% acetonitrile and wash for at least 15 minutes.
Connect only the inlet end of the new column to the LC system, and connect spare waste or connecting tubing to the column outlet and allow the column flush effluent to flow to waste. Flush the column with at least 10 column volumes of 100% methanol or 100% acetonitrilewhichever is more convenient.
”column volume” is a bit ambiguous. The bed volume (volume of resin in the column) is easy: V = 1/4 * (pi * dc^2 * L) where DC is the radius of the column (half the diameter) and L is the length. The “void volume” (Vm = the amount of mobile phase in the column) is a bit more complex.
After the column has been flushed, it is usually best to store the column in the flushing solvent (acetonitrile or methanol) until it is used again. To re-equilibrate to normal operating conditions, equilibrating with 1020 column volumes of mobile phase usually is sufficient.
Column Equilibrating A buffer that is compatible with the protein of interest and the resin of choice is passed over the column. Prior to sample application, the resin is therefore equilibrated in a buffer of high ionic strength.
To equilibrate the column with buffer just means to run the column with the buffer only for a while.
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