Wash column 100% MeOH and 100% ACN until to get baseline stable. After that equilibrate with mobile phase ( 97% 0.1% Trifluoroacetic acid (TFA)+3% ACN) for 30 minutes. After that run blank. It shows no peak and then run HPLC grade water as a sample but it always show same peak. Why?
It could be a 'gradient peak' OR a 'refractive index peak'. In any case if it is insignificant it should be ignored unless it interferes with the retention time(s) of your 'molecule of interest(s)'.
Hattie Lae: First, I have no idea what you are trying to do as you provided few method details and the "column wash" method you are using does not follow good chromatography practices. It appears you are using HPLC for the first time without any formal training (which takes many YEARS to learn the basics) and without anyone experienced nearby to help you. *To use HPLC successfully, you need on-site help and assistance (please contact someone at your school for help). This technique can not be learned in a day, week, year.
Now, if you equilibrate any RP HPLC column and mobile phase system on a properly maintained HPLC system, then inject a true "blank" (which would be your MOBILE PHASE solution), you should see a very small "blip" / Peak at the Tzero time. You may have to zoom into the baseline detail to see it, but it will be there as it is caused by the change in pressure resulting from the injection valve changing from the "LOAD" to "Inject" positions. This peak should be VERY SMALL. - - If you inject non-mobile phase into the flow path (i.e. Water), then you should see a much larger peak appear at the Tzero time as the liquid you just injected is DIFFERENT than the mobile phase and a UV/VIS detector will show this change (some of it from RI change, but mostly from differences in UV absorption changes from Water vs Mobile Phase solution). THIS IS NORMAL. Remember, at this stage you have NO HPLC method, no chromatography taking place at all. Both test "samples" are NOT RETAINED on the column. They are eluting out at the void volume only.
- "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
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