Peaks larger then expected

[leadazide]

Now I have tried the following.

I have run 3 standards (8 ppm riboflavin in 20 mM acetate buffer)

The first one was frozen in a vail and then placed in the autosampler

The second was frozen in an eppendorf tube then transfered to a vail that was placed in the autosampler

The third was transfer directly to a vail and placed in the autosampler

Each standard was run 3 times.

What i saw was a HUGE peak (2800 LU )for the first run on the standard frozen in a vail and on the next 2 runs on that vail the peak got smaller(1100 LU and 670 LU) but still to big.

The other 2 standards had peaks that were of the same size (605 +/-2 LU)on all 3 runs of each standard...

Something strange happens in the vail when a sample/standard is frozen in the vail and then run directly from the same vail.

Now i'm going to try to freeze a standard in a vail and when I'm going to run it change the alu cap on the vail to see it the peaks are caused by internal pressure in the vail. I can't see that the pressure should be able to influece the peak size this much but I'm running out of ideas!

JM: I see no other peaks on my chromatogram so I don't know. I don't think I have an injection problem since I can run other samples very reliably.. Just as long as they haven't been frozen in a vail...

[bert]

I am very curious for the results; must be your injector or HW Mueller is right and you have problems with precipitation/complex formation or something like that. Good luck and let us know!

Regards Bert

[HW Mueller]

Maybe you are really using a vail instead of a vial....that would explain things. Just a poke.
Seriously: Maybe your vial which was in the refridg had a precipitate which your autosampler kanulae picked up and then injected it? In the others you inadvertendly removed the precipitates, wholly or partially, before injection? Try a very soluble INST.

[leadazide]

Having worked on the problem now for some time now I have finally found the reason for the large peaks...

By upscaling the amounts of standard I froze I noticed that the riboflavin was up concentated in certain areals in the sample. And in the vials there isn't enough space for sample to be proberbly mixed using a vortex mixer.

The problem was that I was working with so small amounts of sample and standards that we werbn't able to see this up concentration of riboflavin..

Such a simple thing...

Thank you everybody for your help and support!