Signs you're not equilibriating long enough?

[Jade.Barker]

I'm having some trouble with check solutions failing and I'm trying to investigate all possible avenues. What are the major indications that you need more equilibriation time between injections?
Could this cause an RT shift?

Thanks everyone!

[DR]

Most just run the gradient with a blank injection, see how long after the end of the gradient it takes the baseline to return to zero, add a little bit of time (if your injector turns samples over quickly or at least prefills) - done.

[skunked_once]

The good old standard is 10 column volumes which of course requires knowledge of column void volume and flow rate. You could run a series of tests starting with no equilibration time then increasing equilibration times at regular intervals until your retention times stabilize. Speeding up column flow is one way to reduce the equilibration time if pressure limitations are not a problem.

[Bryan Evans]

I would do a visual inspection of your check standard vials - make sure septum is ok,
and that active has not crashed out of solution.

Sometimes the sample (or standard) solvent is not ideal for an individual component, rather it is chosen
to try and dissolve "everything."

(yes, variable or reduced retention time is an indication your re-equilibration volume is not sufficient)

[Jade.Barker]

Thanks everyone for the quick replies! I'm going to try some of these sugestions and let you know. We changed the sinkers on the mobile lines, and that may have cleared up the RT problem - but it is still back to the drawing board with the check solution.

I havn't seen anything crashed out at the bottom of the vials, and the septums do look punctured... The check solution is mixed up in elution medium which is 65% Acetone and 35% water. The amount of drug is small around 18.5 ug/ml. Aparently this wasn't as much a problem when the elution medium was 90:10 Meoh: H2O...

In your opinion what is the best way to determine if some type of settling or phase separation is happening in the vial? Upon visual inspection I don't see anything funny. I tried putting about 20 ml in a couple of tubes to rest for 24 hours + and I still didn't see any layers. But I do have a suspicion that there could be something like that going on because my bench waste beaker had the tiniest hint of a divide, but that had H20, Acetone and ACN in it. Of course that beaker is very open to evaporation unlike the crimp top HPLC vials.... hmmm.

[Jade.Barker]

A little update, it looks like I had 2 problems. Both drug conversion from one form to another and evaporation... Acidified the std medium and switched to a low volume insert - so far so good... Whew!