I am using isocratic HPLC (UV detection) to quantify chloride, dextrose, and acetic acid using 0.01 N H2SO4 as mobile phase. Method is well established and has worked well for many years. All of a sudden, after a column reconditioning, the chromatography is awful. The first peak, chloride, developed a huge tail and now the following peak, dextrose, is not resolvable. I've changed mix filter, guard column, and column. Does anyone have any further advice for troubleshooting this method?

Need more details:

- from the mobile phase, I would surmise you're using a cation exchange column in the H+ form for the separation. What, exactly, did you do to "recondition" the column?

- I interpret your post to say that the problem continues even after you replace the analytical column with a new one. Is this correct?

- What wavelength are you using?

After all that, I'll go out on a limb and speculate: if the problem cropped up abruptly after your disconnected/connected your column to recondition it, and if the problem persists with a new column, the most likely cause is a mis-connected fitting or excessively large piece of tubing between injector and column or between column and detector.

-- Tom Jupille / LC Resources Inc.