Chromatography Forum

Hi, I met some problem with setting up the standard curve.

The response are not stable, high RSD sometime, and some points are far away from the curve
I think it may caused from my sample preparation process.

1) This might get a bit more discussion if posted under the sample prep discussions.

2) We do not knowwhat kind of compund you are analyzing, other than it appears to be an aldehyde or ketone. If it is a small molecule, there is risk of loss in the evaporation in the evaporation step.

I am not sure what CSS is, but it appears that you have something that does not go into solution in MTBE. It could be possible that the standard can be adsorbed on or disolved in the material that does not disolve in MTBE?

In some cases, the formation of a derivative with hydroxyalamine-HCl works better when using pyridine as the solvent.

But, not knowing what compund and matrix we are dealing with, it becomes difficult to give specific answers.

One thing I would suggest is to optimize the transfer step after LLE. Because MTBE is lighter than water/CSS, you can store sample in freezer for 20 min and then simply decant MTBE layer to a new tube for the drying step. I am concerned that the yellow drop/particle during hydroxylamine hydrochloride derivatization step may come from the aqueous layer from LLE.

If you have already implemented this process, then disregard this suggestion.

Thanks for your suggestion. I will try freezing sample 20min after adding MTBE. It's steroid in Serum.

I've tried freeze the sample after extraction with MTBE, then pour out the supernatant directly. But still, after dried with N2, there are still yellowish dots at the bottom. Even I use methanol to dissolve it, there were some brown particles I could see.

I think it's from my extraction part. The extraction is not clean.

Is this a common problem? Any suggestion?


Thanks!

I have worked with CSS and MTBE extraction but I have never seen those yellow residues. What if you don't spike your standards? Will you still see those yellow residues?

If yes, maybe try different source of CSS?