Bong Kyo Seo: Based on your previous question and this one, it appears you have not had any formal training in liquid chromatography yet so I strongly suggest you contact an experienced chromatographer at your place of work (or if not available, have them hire one or send the sample out) to assist you with your questions. You need many years of on-site basic HPLC training before using these analytical tools. Please be sure to dedicate some time to reading one of the classic texts on Liquid Chromatography too (i.e. "Introduction to Modern Liquid Chromatography). Professional training coupled to lots of hands-on time with an experienced user will answer many of your questions. That is the best way to learn.

Some general comments: The method info you included is incomplete and does not appear to be fully developed. Why are you running a 4.6mm ID C18 column at 0.25 mL/min? This is not a linear flow rate (for particles >2.5u, 1.0 mL/min would be). Your LC-MS (TOF) method runs to a mobile phase of pure 100% MeOH. Again, why are you doing this? Try not to run a gradient to 100% organic phase (95% might be a better max to use). "Water" is not a blank for your method. A real "blank" would be the initial mobile phase composition (i.e. 5% 'A' / 95% 'B').

You wrote: "Would this be normal or acceptable or is this actually a C18 column bleed? - - Not "column bleed". Your mobile phase composition contains additives in one bottle (HFIP/TEA) which decrease over time (gradient analysis) so you would expect the detector used to show a change in output because you are changing the mobile phase composition over time (normal). You are changing to pure methanol over time, resulting in instability. Develop a proper method first to retain the sample which follows good chromatography fundamentals and then it will makes sense to evaluate the data and observations made. Right now, you do not have an acceptable method so it is too early to scrutinize it.

BTW: A word of caution about using HFIP. It is not compatible with many brands and models of HPLC vacuum degasser modules which use Teflon AF membranes. Contamination of the flow path can result.

William Letter Thank you so much for your answer. Your last paragraph helped me a ton as when I added HFIP/TEA to MPB (MeOH), the baseline drift went away mostly. I have to use HFIP/TEA as my analyte is an oligonucleotide, and the instrument is dedicated to analyzing oligos so I am not too worried about TEA contamination in positive mode. At first I didn't add the additives to MPB (pure MeOH) to save the reagents and you also mentioned on your previous answer that I should add the additives to the aqueous phase (but I did not catch the next part of your answer that adding to both "may help minimize some of the signal changes as composition changes over time" so it's my bad).

As for the flow rate, I analyzed my analyte under different flow rates and found that 0.25 mL/min produced the highest area and better peak shape. In fact, I did see somewhere that for LC/MS high flow rate may not be good as lower flow rate allows for better desolvation in ESI. I did know for my column 1 mL/min would be the default parameter.

100% MeOH gradient was used in my hope to remove any carryover, and I also asked the column vendor and they did say my column's compatible with 100% organic solvent. But with your advice, I may need to further optimize/test my method.

In any case, please let me know if there's something incorrect on what I've said above. I will make sure to go over the classic texts that you've mentioned as well. Thank you so much for your input!

Your comments imply that your method may be invalid. Could you provide us with the peak retention time(s) using your above method? A chromatogram, with scales shown would also be very useful to see too.

Lower flow rates are preferred with most types of MS detectors (some can be run at 1.00 mL/min, depends on the method). However, in all cases, the HPLC column used must be sized properly for the flow rate and method used. Your 4.6mm ID column is the wrong size for your flow rate of 0.25 mL/min and points out the need for training before using an LC-MS system for analysis. A 2.1mm ID column would be scientifically appropriate for flow rates ~ 250 ul/min.

*BTW: I think it is great that you are trying to learn these techniques, but please be aware that it takes many years of professional industrial experience/training just to acquire a basic level of training in HPLC (emphasis on 'basic'). As you are new to this area, please start by learning the basic fundamentals of chromatography before using the HPLC to analyze samples (text books can help to learn these concepts when starting out). Please have someone with HPLC experience and training help you with this project. Data acquired using poor quality methods leads to poor quality conclusions.