reproducible ghost peaks

[RAquel Hernandez]

I am having a problem with ghost peaks that appear at 5.9 min and 6.2 min. on all my chromatograms. My mobile phase is acetonitrile, water, 1-pentanesulfonic acid, and acetic acid. I am using a C18 reverse phase column. I have already changed the column, bought all new solvents ( including HPLC grade water) and chemicals, and I still seem to get the ghost peaks. I inject only water and I still see the same two ghost peaks, but if I inject only acetonitrile the ghost peaks don't appear on the chromatogram. I have even tried running my samples on a different LC than the one I usually use. I have no clue what it can be. Does anyone have any ideas.

[tom jupille]

Need more information:
- detection wavelength?
- is the separation isocratic or gradient? (details if possible).
- are you adjusting pH? if so, are you putting the pH electrode directly into the mobile phase while you adjust?

[RAquel Hernandez]

The detecting wavelength is 305 nm. The separation is isocratic. The mobile phase is 320 ml of acetonitrile, 680 ml of water, 20 ml of acetic acid, and 0.87 g of 1-pentanesulfonic acid. I combine them and then filter the mobile phase. I don't adjust the pH, but it is approximately 2. The flow rate is 1 ml/min, and each run is 10 minutes. The compound I am intrested in has a retention time of 5.6 minutes. I have done this separation for many months and have never seen the ghost peaks until about a month ago. They appear at 5.9 and 6.2 min and are broad.

[tom jupille]

The fact that it's an isocratic separation eliminates a lot of possibilities.

It sounds like something coming in with the water. You've already checked the primary suspects. Other possibilities:

- something leaching from your filters? (Try making up the same mobile phase without filtering and see if that helps)

- residual detergent on your glassware? (rinse thoroughly with mobile phase and see if that helps)

[RAquel Hernandez]

Okay I will try those two possibilities.

Thank you.

[RAquel Hernandez]

I tried not filtering and rinsing thoroughly with the mobile phase and the ghost peaks are still there.

[Basil]

Hi Raquel !!

Are you using only one chanel ?

Have you tried to use the same HPLC for a different analysis ?. It sounds that something is inside your equipment (in injector part) and it eluates each time when you inject .... Have you cleaned the needle and the reodhyne part ?...

Hope it helps !!

[HW Mueller]

Also:
Did you change anything just before the ghost appeared? Maybe a longer injection loop which you no longer fill with sample, thus possibly co-injecting some air?
Or maybe gunk accumulated on the column and injection of water disturbs/displaces this partially? You still get it after you clean the column? Did you inject the water used in preparing the mobile phase? What happens if you inject acetic or pentanesulfonic?