Run failure - concentration increase over time

I've been thrown back into the world of HPLC after I thought I'd left it behind 8 years ago!

The company I work for has 1 HPLC system and the two methods used on it were transferred from an external company. It is used for determine levels of glutaraldehyde and formaldehyde in separate solutions.

We use DNPH to derivatise the glutaraldehyde and formaldehyde in the sample solutions and are sometimes finding that the concentration increases over the course of the run ~ 6-8 hours.

Does anyone have any experience of this? What can we look at to stop this happening?


Mobile phase: acetonitrile and water

Gradient elution with on-line mixing

Column: C8, 4.6 x 150mm

Diluent: 50:50 acetonitrile and water


Thank you!

  • It sounds like a carryover.


    Try to run a blank injection after every sample. If this doesn't help try to wash your column with a different mobile phase which clean it of.

    You have to see if 40:60 ACN/H2O helps. After it you do a blank run to equilibrate the column to its starter conditions.


    If you have the problem that the concentration increase over a few days, then try to put a wash program after every sequence.

    For example 10/90 ACN/Water for like 1 hr. Just write a method which runs after each run and uncheck "save Data or put Signals off"

    After this 95/5 ACN Water. So u retain the storage conditions.


    Good Luck

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