Chromatography Forum

Often times, my assay will fail due to peak areas in the blank exceeding the lowest peak area in my cal curve. I flush the sytem with plenty of blanks and allow it to equilbrate for almost an hour and it still has issues.

we have found that this occurs on multiple Agilent 1100's HPLC's in our lab. Any ideas?

Common sense would tell me to flush the system with Elution Buffer at 2ml/min for an hour to "clean the system" but this does not always work. I heard flushing at a low flow rate actually can "clean " the system more efficiently, is this true?


Column: Poros Protein A
needle rinse: HPLC water
Mobile phase A pH 7.4
Mobile Phase B PBS pH 2.6
Flow 2ml/min
Step gradient is just bind and elute 100% A to 100% B

Hi semivoadude

It would be helpful to know if the peak/s in the blank had the same retention time as the main peak in the standards/samples or are they just appearing randomly/non systematically in each blank injection?

Here are my thoughts about the described setup: Water alone is not the best solvent for proteins. Usually some salt (in very low concentration e.g. 0.01 M) is needed in order to salt-in the protein. So your needle rinse is not the best choice. The salt solution mentioned above wouldn’t be my first choice for needle rinse either. Here is a suggestion for needle rinse I would put to a test: 0.04% phosphoric acid in water plus 10 – 20 % isopropanol. As you can guess I am suggesting to you that the problem might be carry-over due to inadequate injector wash.
You don’t mention the phosphate concentration in solvent B, but if it is high e.g. 1 M or something like this, there is the possibility of salting your protein out, due to the abrupt change of solvent from 0 to 100 % B in a single step. At least some of the protein. The salted-out protein can subsequently be dissolved and elute in the following run as carry-over.

The difference in pH between the solvents can often be a very difficult parameter to control and is unadvisable. It can also cause solubility problems.

Finally to your question:

I heard flushing at a low flow rate actually can "clean" the system more efficiently, is this true?

To the best of my knowledge it’s not true. And I can’t think of a rationale for this assumption.

I’m not saying that flow-rate of 2 mL/min is the best choice, and it’s probably not – even more so in ion-exchange mode. But the column diameter should be taken into consideration in order to suggest a different flow rate. So I’ll leave it here.

Best Regards