Dear All,
I am working on HPLC separation of catecholamines with a C-18 column (waters atlantis T3, C-18, 5μm, 4.6x50mm). The mobile phase is Water, Acetonitrile (0 to 25% gradient), all buffered with 5 mM, pH 3, ammonium formate.
My sample will be from supernatant of neural cell cultures, so some protein are present, since this may be a problem, i wanted to know if I could get hints regarding concentrations i could consider acceptable regarding column clogging and/or lifetime.
Performing Bradford assay on my sample, I found a protein concentration of 70 µg/ml.
After protein precipitation using acetonitrile, I had a concentration of 8 µg/ml.
I wrote the manifacturer but basically they didn't give me a value.
Is, at least, one of these two values acceptable?
Thank You,
Cristian Zanetti
I prefer not to inject protein in a sample. I use ethanol to precipitate protein and only inject the supernatant.
I am agree with Omar and wont inject samples that have not been pretreated, which often requires cumbersome column cleaning and regeneration procedures. Therefore, you better do some extractions before analyzing your samples. A quick survey is the treatment of your sample with 30% acetonitrile (slightly more than the highest organic acid concentration in the gradient) to check whether there is any precipitation or not. Alternatively, if you have access to a guard column, which protecting your main C18 column, then you can run your samples directly without any preparation. However, you would still need centrifugation (10000 *g) and most appropriately cleaning the supernatant by passing it through filter membrane or syringe filter (pore size 0.2 μm).
If you follow one of these two procedures then you wont clog the column nor threaten its life-time. Keep in mind that the guard column needs replacement usually after every 200-300 injections depending on the amount of contamination that is present in the samples.
Good Luck
Dear all,
thank you for your kind replies.
Most probably i will precipitate and centrifuge with ACN, followed by typical 0.2µm filtration.
Best Regards
- Badges
- Science method