By Chris Atkinson on Monday, December 18, 2000 - 08:37 am:
HI, I'm using a gradient method to analyze for Retinal and retinyl Acetate (internal std). The gradient is ACN:THF:1%Amm Acetate in HPLC water. SolvA=500:300:200, SolvB=500:450:50. I am looking to detect small amounts of retinal, so I must crank up the attenuation on the chromatogram, which results in a much more evident baseline "hill" (increases in response to gradient going to solvB then declining back to A). Does anyone have suggestions for improving the baseline or does anyone have a suggestion for substituting another solvent for THF. Please help...sincerely a frustrated grad student.
By jclark on Monday, December 18, 2000 - 09:59 am:
The change in your baseline is probably due to increases in the concentration of THF which absorbs strongly in the UV. Try putting identical concentrations of THF in your two mobile phase components and let the gradient increase the concentration of ACN only, keeping the concentration of THF isocratic throughout the run.
By Rajkumar on Monday, December 18, 2000 - 01:36 pm:
Check if a higher wavelength is feasible based on the UV spectra of the compounds.
By bill tindall on Monday, December 18, 2000 - 04:15 pm:
Are you using stabilized THF? If so the hump could be the stabilizer. On the other hand, if you are using unstabilized THF, the hump is probably from the peroxide of THF. Or, if you are at a low UV wavelenght, it is simply the absorbance of the THF, UV cutoff about 210nm with significant absorbance extending to about 300 nm.
About the only way I have ever been able to do high sensitivity work in THf is to do the separation isocratic. is this an option?
By Bryan Wallwork on Tuesday, December 19, 2000 - 03:15 am:
yes, I agree with Bill Tindall:
is it possible to do this separation isocratically, can you use another internal standard? I have found THF excellent for these compounds, together with dichloromethane, on a C18 column... so called non-aqueous reverse phase.
Gradients are only compromises at the best of times; for trace analysis, isocratic elution would be my choice every time.
By beppe on Tuesday, December 19, 2000 - 08:50 am:
If you baseline drift is reproducible, you can try blank substraction (injecting first a blank - or even nothing - and then substracting this signal from any subsequent run).
When done in good conditions, this initial blank can be used for a few hours.
By Chris Atkinson on Wednesday, December 20, 2000 - 07:34 am:
Thanks to everyone that offered suggestions...I'm on my way to try them out! What a great message board this is!!!
-Chris
Posting is currently disabled in this topic. Contact your discussion moderator for more information.