I have been testing my new hydroxyapatite column with water HPLC system by injecting 900ul BSA (1mg/ml), the flow rate is 1ml/min. The column vol is 12ml. I used 40ml of 10mM tribasic potassium phosphate (pH7.1) to wash the column after injecting sample, following with a 80min gradient from 10mM-800mM of potassium phosphate. After the gradient I use 800mM to continue eluting for 10min. However, the peak of the BSA appears at around 70min, so even after the 10min 800mM solution, the right half of the peak just started, so I had to stop the flow.
Then I ran several rounds trying to washing out the left BSA out of the column by using either 1M phosphate solution or even using 1M NaCl in 500mM phosphate solution for at least one hour, however, I got no luck, nothing come out.
I did try another gradient from 10mM-1M phosphate solution for 20min and following with 1M phosphate solution, I did got a high peak after 20min, but I got a trailing tail, which would take forever to reach to the basal line, so I had to stop that.
Right now, I already stop the flow and take the column out of the system with BSA in the column, so I hope it would not do too much harm to the column?
I also would like to ask if anyone have rich experience with HTP column and would give me suggestions on how to elute the BSA out of it? Is gradient the best way or isocratic with high salt? If gradient is best, what is the optimal gradient I should use(i.e. time and salt conc.)?Do I need to run the low salt buffer everytime when I start the gradient? Another question is does tribasic potassium phosphate affect elution since I saw people usually use monobasic potassium phosphate?
How did prepare your column before loading sample? How did you prepare your loading sample? Lots of details are not clear, I am not sure how to give some helpful suggestions. Good luck.
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