Chromatography Forum

Hello all,

I am running many protein A HPLC runs for several different projects, and I am encountering a small problem that I am curious if anyone has any helpful tips.

After my runs are complete I am analyzing my data and noticing that I am having protein showing up in my cleaning solution. I have dont many cleanings with 10% methanol, column conditioning for two hours prior to each run, staggered my samples in my method to not run for than six at a time, made new lots of cleaning solution, ensured I wasnt using the same pipette tip twice on anything, performed needle washes and purged the injector. I am getting roughly 0.100 mg/mL in my data analysis of my cleaning solution. Does anyone have any idea on how to stop this from occurring?

My method set is set up as:

Purge inj
Column Conditioning (2 hours at 1ml/min)
Clean Soln
Blank
0.100 Control
Standards (eight standards ranging from 0.0125-0.300 mg/mL)
Clean Soln
Blank
0.100 Control
Samples (six total)
Clean Soln
Blank

Sincerely,

Nick

"Notes on Cleaning bound Protein from RP HPLC columns"; https://hplctips.blogspot.com/2016/10/n ... om-rp.html

Thank you! I found out that it wasnt protein in my cleaning solution, but rather it was a bad lot of ethyl alcohol that was causing some absorbance and a large blog on my chromatogram.