I am running a gradient of ACN and Water in 4.6 x 250 mm C18 RP column. Column temp is 25 degrees and flow rate is 2 ml per min. I go from 50:50 (Water:ACN) to 1:99 (Water:ACN) over 10 min. I ofter observe that at the starting of my run, the real time absorbance first rises then drops to negative region and maintains stability. However this only happens once or twice between runs.what could be the reason. I have attached a pic for your reference.
You might have "ghost peaks". They are difficult to troubleshoot as they have several causes. Some examples are carryover from previous runs and contaminants in the mobile phase.
https://www.linkedin.com/pulse/troubleshooting-how-remove-ghost-peaks-raymond-wong/
Your HPLC system and also the method used have not been setup properly (based on your chromatograms). You would need to include ALL of the basic HPLC parameter settings used to for us to help further, but the chromatograms you present do not match your descriptions of what is occurring. As you are using HPLC for the time, please enlist the assistance of a local professional chromatographer before starting your project. Many basic items need to be checked and you need help to do this. For example:
- The HPLC needs to be checked to make sure the two channels used are fully primed, degassed and operating normally.
- The flow rate for your 4.6 x 250mm column (3-10um ?) should probably be 1mL/min, not 2mL/min.
- The gradient run should be adjusted to end at 95% ACN (not 99%) and at 1mL/min maybe end it at 20 minutes, not 10 min(too fast with that column).
- The large baseline shift at zero to 0.5 minutes is abnormal and indicates that the HPLC system may not be: primed/not equilibrated/overloaded with sample volume/void present etc. It is not being operated correctly (a training issue).
- The baseline should be stable at the start of the run and not significantly change until Tzero has been reached (at 1mL/min flow, this would be ~ 2.7 minutes for your column). After Tzero, the compositional change in the mobile phase caused by the gradient will result in a change in the baseline signal (normal) and will be most pronounced at the lowest wavelengths using ACN (214nm = Large change; 280nm very small baseline change). *During this time your sample may be retained, then eluted off the column. It takes most people five years of full time training to learn the basics of HPLC. Do you have 5 years available? I bet you do not. Request the help of a chromatographer who has learned these basics and you will make more progress on your project. Ask your teacher for help.
- Your compound may not absorb at 254nm. *Note how you have a signal at 214nm, but none at 254nm.
in addition to what @William Letter mentions, at the end of the run, make the mobile phase return to the initial condition (50/50) through a gradient of a couple of minutes
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