HELP! Peak areas getting smaller during validation!

[cody84]

Ok after a bunch of work on the injector and a recalibration I was about to perform validation. Last friday I ran a test injection of standard and sample. Got perfect results. Today I ran test injection of both again just to be sure before weighing all of my samples and my results for the sample are low again.

CPG, I have been trying to implement a wash after the peak elutes in each injection, any advice as to the best gradient to run and for how long approx? My run is 12 mins, but can be extended to re-equilibrate between injections. Right now I will try injecting 5uL instead of 10 and see how that affects everything.

Only my sample peak areas are small, the standard is perfectly fine, so it must be something in the matrix affecting the active in the column.

[cody84]

I ran 100% ACN through the column all night and just equilibrated now with the 58% acetic and 42% ACN and now the pressure is at 2000 psi....down from 2500! Something must have came out! Trying a standard and sample again now, with 5 uL injection and a gradient up to 100% ACN after the peak.

[Antje_Traub]

Did you try a new column?? Do you use a crude catcher? We also use KINETEX columns and the shelf life is not so long! (I assume because of the small particles)

[Klaus I.]

Sorry Cody84, I am not sure that I really have understand all the discribed details. When Areas become lower with the time, you have usually a stabilitiy problem in the sample solvent

If your sample can reacts with water, please be carefully with methanol. With a freshly opened bottle of methanol you will never observe degaradation of your compound. But after few days you will perhaps have a degradation even when the amount of water is really low (about 0.3 %; I have observed this with real samples)

Sample solvent stability is also difficult, when you compare e.g a drug-product (with many ions, which have also influence to the pH) with an API.

Maybe you can try a other sample solvent?

[cody84]

It was the sample preparation. There must have been a solubility issue in the methanol. I switched to IPA as diluent and my validation is running great as we speak! Thanks for all of your help!

[Klaus I.]

It was the sample preparation. There must have been a solubility issue in the methanol. I switched to IPA as diluent and my validation is running great as we speak! Thanks for all of your help!

Was it really the methanol or the water solved in methanol? Also IPA can contain some water or not. Check this before you must make a complete validation again. In general 'solubility problems' are often used as a lame excuse.

Take the oldest bottle of IPA in your lab and examine the water content Make you test with this IPA again. Are there differences? Alternativily you can also mix some water to the solvent.

[cody84]

Actually I had the SAME issues with peak areas when I tried the mobile phase as well as aqueous diluent. IPA has the perfect polarity to dissolve this sample and my validation run is complete and has passed everything.

Also we only use high purity IPA, MeOH and ACN here and I am well aware of the hygroscopic properties of these as I had to monitor water content of MeOH every day at my previous job.

[hplctroubleshooting]

Actually I had the SAME issues with peak areas when I tried the mobile phase as well as aqueous diluent. IPA has the perfect polarity to dissolve this sample and my validation run is complete and has passed everything.

Also we only use high purity IPA, MeOH and ACN here and I am well aware of the hygroscopic properties of these as I had to monitor water content of MeOH every day at my previous job.

I'm kinda having the same issue myself , the technician came over and discovered that the top of the vials were wet after an injection ensued which led him to believe that there was something wrong with the injection valve . He replaced the valve and it worked for a day and then the problem manifested itself once again. Perhaps it wasn't an injection valve issue - where the flow isn't diverted when making the injections - so he placed an empty vial and made an injection on the spot where he discovered that it hadn't become wet. Today , he replaced the seal pack and injection needle and the areas returned , in a ball park sense , to normal values ... there was variance .... plan to clean out the column today and test it tommorrow with repeat injections of a standard.

[Klaus I.]

It was the sample preparation. There must have been a solubility issue in the methanol. I switched to IPA as diluent and my validation is running great as we speak! Thanks for all of your help!

You used a cooled autosampler and you have observed crumbs in your sample vial after few hours?

[cody84]

No, there was no crumbs