Protein sample elutes at the same time with all impurities

[twald]

Hello,
I am using PLRP-S 300A, 5 um column for protein purification (second step purification after NiNTA). Util now I loaded the sample in TEA / 5 % AcN pH 8 and run a gradient elution. Protein of interest ussually eluted at 45 %B (B=95 AcN in H2O). Last week I purified a new batch after 3 months and unfortunatelly all the protein sample elutes at the same B concentration (between 40-50 % B). So I co-elute my protein with impurities and I am unable to get rid of them. Also the chromatogram changed dramatically. Before I have got one single elution peak, now I am getting "growing hills" at once.
Would you help me to fix this? Where is the problem and what should I do?
Thank you very much for all replies.
Tom

[Vlad Orlovsky]

Mixed-mode can provide you with unique selectivity. Look at application for separation of insulins, they defer only in position of two amino acids:
http://www.sielc.com/upload/file/pdf/Pr ... olumns.pdf

let me know if you have questions.

[tom jupille]

Reading between the lines: the separation formerly worked successfully on this column, but no longer works. The question becomes: what's different this time?

Mobile phase is old? wrong pH? Temperature is different?
Column is contaminated? Do you have a procedure for regenerating the column between runs?
Is it possible that you are simply overloading the column this time?
Do you have a check sample that you can run?

[twald]

Hello,

exactly. Last time purification was OK, which means - one nice peak. This time all the sample eluted at the same time (B concentration - between 38-50 % B).
I washed the column O/N with 0,5 M NaOH, water and reequilibrated with 50 mM TEA pH 8 - 5 % AcN. And the result was bad again....
Temp. stable (A/C 19 °C), buffers always fresh, protein load the same...
What do you mean with the check of the sample? SDS-PAGE? I did, the same as always after NiNTA purification....

[twald]

Hello,

exactly. Last time purification was OK, which means - one nice peak. This time all the sample eluted at the same time (B concentration - between 38-50 % B).
I washed the column O/N with 0,5 M NaOH, water and reequilibrated with 50 mM TEA pH 8 - 5 % AcN. And the result was bad again....
Temp. stable (A/C 19 °C), buffers always fresh, protein load the same...
What do you mean with the check of the sample? SDS-PAGE? I did, the same as always after NiNTA purification....

Reading between the lines: the separation formerly worked successfully on this column, but no longer works. The question becomes: what's different this time?

Mobile phase is old? wrong pH? Temperature is different?
Column is contaminated? Do you have a procedure for regenerating the column between runs?
Is it possible that you are simply overloading the column this time?
Do you have a check sample that you can run?

[tom jupille]

No, by "check sample" I mean a sample of known characteristics that you can use to confirm operation of the system ("check" is an adjective here, not a verb; I should have called it a "verification sample"). Something (aside from your present preparation) that you can use to verify the performance of the column.

If you get poor results from your verification sample, then the column may be dead. At that point you have to weigh the cost of spending time to try reviving it versus simply replacing it.

[twald]

No, by "check sample" I mean a sample of known characteristics that you can use to confirm operation of the system ("check" is an adjective here, not a verb; I should have called it a "verification sample"). Something (aside from your present preparation) that you can use to verify the performance of the column.

If you get poor results from your verification sample, then the column may be dead. At that point you have to weigh the cost of spending time to try reviving it versus simply replacing it.

Thanks. I will try BSA Lysozyme mixture and let you know.