Why is there a negative dip in my HPLC chromatogram?

Because your solvent absorbs UV to some extent, too.

If you constantly see a negative dip, it may come from your sample, diluent, mobile phase, bottle, cap, glassware, column problems etc, that some compound is eluting out but does not absorb UV to the extent that your solvent does.

If there is a difference in composition between the injection solvent and mobile phase, your diluent (eg. water) could have a lower optical density than your mobile phase (eg. methanol) does. The negative dip will appear right before your sample is eluted.


*LC grade means that UV light absorbing impurities have been removed (not that the absolute purity is higher) and absorbance of specified wavelengths is kept within a specified range.

Lost prime

When your instrument prompts an error of a lost prime, it means pressure has dropped to below 25 psi for the last 50 cycles of plunger. Very often, gas bubbles interrupted the solvent path.


First, check that:

1. There is enough solvents in the bottles.
2. All solvent lines are properly connected and not drawing air into the system.
3. All solvents degassed and lines properly primed. It is more effective to prime each line at 100% for 5 min rather than 25/25/25/25 for A/B/C/D.


Other possible causes:

1. Solvent reservoir filters clogged.
2. Bacteria growth in the lines or inline filter and becomes clogged. May be able to clean out with 100% MeOH.
3. Primary inlet check valve failed. Ruby ball can get sticky over time esp if applications involve high ACN content. Ball gets stuck at the close position, so solvents cannot be delivered.


Passivating with 30% Phosphoric acid or 6N of Nitric acid not recommended.
This involves extreme cleaning, takes a lot of time, and unnecessary wearing of parts.