questions again

Record: 447

Category: Column Problem
Product: KW
Sample:

Question
I’ve been super busy with course work for the past weeks so halted the HPLC work for a while. Picked it up again yesterday. I had previously done the 0.5M NaCl and 40%MeOH rinse which helped bring down the pressure of the column to below 400psi at 1.0ml/min flow. The Shimadzu people also came in and changed all the tubing. So I did a sample run yesterday, the first trial went pretty well, I was getting smooth and distinct peaks. But just like always, peculiar things followed. For the two following runs, I was only getting a really sharp peak (low magnitude) at the very very beginning and then nothing afterwards even though it was the same sample for all three runs. Then pressure went up again…I’m back to the rinsing with reverse flow. You have any idea of what is going on here? It almost seems like everything tends to get stuck in the column but my protein-polymer conjugate is pretty hydrophilic… Let me know your opinion. Thank you!

Answer
Are you injecting the same sample? meaning no any other treatment to remove the copper or amine? Obviously if you are injecting the same sample, the same phenomena can occur. Also repeated change of mobile phase/cleaning solution can alternate the condition of column, which means it can be very difficult to assume what is really going on inside the column.
Another question, did you try injecting the pullulan standards that we talked about? I would recommend to stay away from your sample for now and try to see if pullulan can be observed, giving you a nice straight calibration curve or not.

 
 
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