Chromatography Forum

Hi,

My sample is a super low dose vitamin D5 and my method has a sensitivity issue with UV detector. I've tried using 100ul injection volume, increased smp concentraion, and used the wavelength that has the maximum absorption, but my peak area is only 5. Does anyone know if Vitamin D5 have fluorephore? or can be derived so that fluorosence detector can be used? Or any other idea at all?

Thanks.

Sensitivity boils down to signal/noise. So:
How tall is your peak (in absorbance units)?
How much noise are you seeing (also in absorbance uits)?

Is a longer pathlength flow cell available for your UV detector?

One also very important question is how much sample you have. If you have "unlimited" amount you can try for an increased injection volume (1 ml?) in combination with precolumn trappning of your analyte. You will need some new hardware though (new syringe, loop, column switch).

Due to the budget, we don't have the choice of changing a bigger UV cell pathway like 2cm, or larger loop, I only have 25mg of drug for the whole development. And that's very stressful! I am pulling my hair.

So approach the problem by decreasing your baseline noise. That doesn't cost you any sample.

So approach the problem by decreasing your baseline noise. That doesn't cost you any sample.

Hi,

How shall I decrease baseline noise? Could you give me a list that I could try? I am already using a 3um column. Thanks.

See these two articles from John Dolan on decreasing baseline noise and increasing the signal-to-noise ratio.

http://chromatographyonline.findanalyti ... rticle.pdf

http://chromatographyonline.findanalyti ... ?id=660930