Hi everyone,
I've been trying to detect a Peptide Hormone (ca. 9 kDa) by RP-HPLC using H2O + 0,1% TFA and MeOH + 0,1% TFA as mobile phases with a flow rate of 1 mL/min, a C8 column with a diameter of 2,1 mm and 50 mm length and UV-detection at 220 nm. I manage to see the peak of my m-cresol as well as a peak at ~18 min. But when I run a Blank without even injecting any sample, I still get an intense peak at ~18 min (I've repeated this several times). I'm afraid this is residue peptide from previous runs when I didn't manage to elute anything off the column due to using ACN instead of MeOH. Is there a better alternative to cleaning the column than just repeating the run itself several times?
My gradient is
0 - 2,5 min/25% B
17,5 min/80% B
17,5 - 25 min/80% B
and I'm reversing it in a wash program between runs and re-equilibrate the column.
I was thinking of washing the column with H2O/ACN (95:5), ACN:H2O (95:5), MeOH:H2O (95:5), ACN:H2O (95:5) and H2O/ACN (95:5). Does this sound promising?
kind regards
Manuel
You can try using a low concentration of Sodium Bisulfite (BSS) in your mobile phase. This will prevent and cleave any bonding the peptide has with bare silanol groups present on your C8 column.
It is strange that the blank has the same peak height as the sample, about 500 unit. I don't think it is a carry-over.
Interesting capture Narong. Manual, what is your extraction solvent? Is it compatible with your mobile phases? This peak may be due to a difference in the RI or/and UV between your solvents.
I agree with Narong Chamkasem g. If it is a carry over you would expect the intensity to decrease. Try to shorten your gradient (from 25% to 80% in 2min instead of 15) and see if the peak elutes earlier (at 5') and do several runs to see if it deacreases. If the intensity remains I would rather expect it to be in B.
To isolate the issue, I would recommend to run isocratic at 80% just to see the peak first, then inject blank. Or, inject blank first to prove that you don't see anything, then inject std to see the peak, follow by blank to prove that you do or don't have carry-over. This will eliminate the effect of the gradient elution (different issue). The peak you see, may come from the system (mobile phase or something in your extraction solvent.....etc.)
Thank you for your answers so far!
Bruce Neagle This is merely a standard peptide solved in HPLC-Grade water. Therefore it shouldn't interfere with the mobile phases.
Jan Lisec I've tried to steepen the gradient and shorten the time of analysis in order to flush out residue analyte, but didn't get any peaks doing that. I've tried it several times to be honest. Why does it only appear when using that exact method and why is it such an intense signal?
Narong Chamkasem Running isocratic doesn't produce any peaks. Even high yields of ACN or MeOH (80-95%) over a prolonged period of time won't elute anything. Can a peak that high still come from the system?
Try these tests and send me the chromatograms
1 inject air (1 uL) three times
2 inject blank (whatever the injection volume you are using) three times
3 inject 1x protein and 10x protein.
My gut feeling is that the peak you are seeing is not your analyte, but please prove me wrong.
Thank you for your interest in my problem.
Narong Chamkasem, I've tried the protocol you suggested. It seems like my column is clean but now there's no significant signal whatsoever. Am I using to little of my peptide standard? Is it trapped on the column?
Most peptides and proteins cannot be solubilized in water alone and may attach themselves to glass. Thus many biochemists always use PBS (phosphate buffered saline) as a medium or TRIS,
@Manuel. Now we are in the troubleshooting mode. I assume that you injected your samples consecutively. After a few injections of air, the system was at equilibrium as they are all the same after three air injection. My question is between the last issue till now and just before this experiment, did you change the mobile phase or anything that you did not tell us about? To figure out what had happened to you protein, take off the column and inject your protein so see a plug of standard at the solvent front. This will determine if you have standard in your solution and if it is absorbed in the column. You need to change one thing at the time to figure out the problem. Let me know the results.
Bruce Neagle Using a very low concentration (0,1 pmol/L) and without a column, I was able to prove that the detector is able to detect my standard. Therefore it shouldn't fully adsorb on glass surfaces even in such low concentrations. But I'll keep the PBS and TRIS in mind.
Narong Chamkasem Unfortunately there were no changes of mobile phase or anything. I've successfully injected my standard in various concentrations without using the column to see if my detector is able to detect it. I used a delayed injection (at 1 min) and was able to see peaks all around my concentration of interest.
So if the detector is able to see it, why won't it come off the column?!
Afterwards I reinstalled the column and managed to get a good peak at the beginning of the chromatogram (10 pM Std_with column). The following blank and sample both showed a very intense negative Peak at the beginning. Where does this Peak come from and why is it not possible to reproduce peaks?
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