Problem with C18M 4E-RP column

Record: 575

Category: Column Problem
Product: C18
Sample:

Question
I have a problem with my column and I need some advice. The column was working fine for me until recently, when one of lab members used it to run ~15-20 samples. This person did not filter out the proteins from the enzyme assay and loaded the entire enzyme assay mixture. (Usually I filter my enzyme assay reactions through a 0.5 ml Amicon YM-10 centrifugal membrane filter and load the flow through).

Now the column pressure is shooting up too high (at 0.6 ml/min, which is what I previously ran at) and the high-pressure limit (which I set to 2800 psi) is cutting off the run. I tried to clean the column with ~50ml of 75% methanol, but the pressure still shoots up too high and the machine cuts off. Is there a method for cleaning to remove the protein contaminates? If so can you please send me some information?

Answer
You may try to introduce 0.3 M NaCl solution for removing protein.
First flush the column with water then switch to NaCl solution.

When you clean the column, reduce the flow rate to <0.3 mL/min or lower to stay within the maximum pressure limit. You may also increase the temperature to ~60oC. Please monitor the pressure and let me know if this reduces the back pressure. If not, we need to think about other washing solvent. And please do not leave NaCl solution in the column for long time, it may cause rust on the column housing. Need to be flushed with water after this solvent.     Additional Comments

Download or Print PDF