Hi,
I am brand new to HPLC and my training so far has been very limited. I have developed a method that gives me consistent success at detecting my peptide of interest (PTH) at fairly high concentrations (0.4mg/mL), however these exact peaks and slopes (https://imgur.com/a/G1PuqQS) are always present in all of my runs. When using lower concentrations of PTH these peaks/slopes become problematic when trying to determine my PTH peak.
The attached image is from a blank run.
My mobile phase A is ddH2O with 0.1% TFA and my mobile phase B is ACN with 0.1% TFA. I am also using a C4 column due to availability, but a believe a C18 column would be more appropriate. Could column be playing a role?
My current method is:
0min (2% B)
30min (35% B)
30.1min (2%B)
50min (2%B)
Once again, I get the same baseline present in all of my runs. How do I achieve a flat baseline for my runs?
Attached Image:
https://imgur.com/a/G1PuqQS
To quote Rod's law, the less you tell me the more I cannot help you. You provided us no information about your detector: type, wavelength if UV, etc. I assume from the chromatogram that it is UV, and from your analyte (peptide) that the wavelength is very short (maybe 210?). Given these assumptions, the entire world absorbs down there and you have to be very careful with your reagent selection as far as purity goes. You appear to be seeing systematic peaks; these are typically from your solvents, your pumping system, your injection system (including sample vials), etc. You can move these, as Soleya points out, by changing your gradient. I would try a couple of different gradients and see if I couldn't move my analyte of interest away from these peaks, and then I would also try other batches of reagents to see if I couldn't get a cleaner system.
Hi!
The reason of your problem is in the mobile phases mixing and not in the column.
I would advice you to optimize the mobile phases and the gradient. It is always better making phase A (0.1%TFA in water) with a small portion of AcN and B (0.1%TFA in AcN) with a small portion of water. In general TFA acid mobile phase generate higher drift so the gradient must be very smooth. Most of the drifts in your chromatogram are after 25min where the composition of mobile phase changes remarkable.
Good luck!
I can't access the image (Imgur blocked by work server). But this sounds like "ghost peaks". These could come from the solvent, or contamination in the injector. Ghost peaks sometimes also come from previously injected samples, where late retained peaks elute during later runs.
-Make sure the column is washed before storing it; depending on the impurities, it may need a wash with the strong solvent at the end of the run.
-Make sure the solvents used are clean and high quality. Make sure the water is fresh
-Try to get someone at your university to help as you are new at this.They can help you troubleshoot the ghost peaks.
To quote Rod's law, the less you tell me the more I cannot help you. You provided us no information about your detector: type, wavelength if UV, etc. I assume from the chromatogram that it is UV, and from your analyte (peptide) that the wavelength is very short (maybe 210?). Given these assumptions, the entire world absorbs down there and you have to be very careful with your reagent selection as far as purity goes. You appear to be seeing systematic peaks; these are typically from your solvents, your pumping system, your injection system (including sample vials), etc. You can move these, as Soleya points out, by changing your gradient. I would try a couple of different gradients and see if I couldn't move my analyte of interest away from these peaks, and then I would also try other batches of reagents to see if I couldn't get a cleaner system.
As others have mentioned more information would better help us, help you. I does sound like it is mobile phase related. A contaminant in the water appears to be eluting with the ACN gradient. If Mobile Phase A was pH adjusted then it could be buffer residues from your pH probe, or just poor water quality. I assume ddH2O stands for double distilled. This is not adequate for low UV wavelength HPLC work. Carbon adsorption, ion exchange, and UV treated following distillation or reverse osmosis is generally required.
Most probable the ghost peak you are seeing in the chromatogram comes from contaminants in the TFA, Water or Acetonitrile. Try to use TFA gradient quality (it comes in ampules of 1 mL) and check water purity. Run a blank gradient and see if the peaks comes in a reproducible way. If it is the case are contaminant from the reagent, water or solvent. Choose also a different Lot of ACN and try to use ACN gradient quality (like Hypersolv)
As Mark commented above, you have not provided us with any of the basic information needed to review the problem. This is complicated by the fact, as you say, " I am brand new to HPLC ". New users will not even know which types of information are required to answer such questions so you really need to have someone at your school (with professional training) assist you. We can only speculate and point out the obvious problems, but you need years of professional training to do this on your own. So save yourself some time, and find someone to work with at your school who can run this analysis for you while you learn a few things during the process. If you take the time to research the topic at the same time, you will start to better understand what questions to ask and what type of information is important to understand the issues presented too.
General comments from what is shown in your chromatogram of the "blank".
(1) Your method runs to 30 minutes, but your chromatogram shows peak eluting to ~ 35 minutes. You then reverse it before the peaks have eluted. This makes no sense. Set up the gradient to gradually ramp up to the desired max % composition, THEN HOLD IT THERE for a period of time and when everything has eluted off the column, STOP the analysis. Do not return to initial conditions during the analysis run. Flushing the column (which you are currently NOT doing, but need to start doing) and then equilbrate the column back in the initial mobile phase should be a second step, separate from the analysis. Please USE a wash method between runs (wash methods use a mobile phase which is stronger than the one you use during the analysis).
(2) Purchase and use FRESH TFA in your mobile phase. Do not buy the big bottle of TFA and then use it for the rest of the year(s). TFA degrades quickly and the result is lots of extra peaks, esp in the low UV region. Save yourself the time and buy the tiny vials, as Jose suggested above. That is what we use in professional chromatography laboratories. The small vials cost more, but we waste less time having to troubleshoot and re-run samples (and time is money), so in the long run they save us far more money overall.
If you have drifting in your chromatogram plus ghost peaks (peaks appearing in the baseline during a gradient or isocratic run) you have to change the reagents you are using especially the HPLC solvents. Try to use gradient quality solvents (for instance HiPerSolv for gradient) and if using buffers try to change the supplier of the reagents. Ghost peaks are a result from 2 two sources of contamination
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