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Gradient Elution and Low Detection Wavelength
Posted: Sun Apr 22, 2007 1:12 pm
by Andreas Karas
I am working with some carboxylic acids and I have set the detection wavelength at 215nm. Also I use CH3CN/H2O as mobile phase with ammonium acetate in both reservoirs so as to keep the concentration of the buffer in the column constant. It is almost impossible for me to run a gradient elution program because the baseline shifts really bad!
Any suggestions would be very useful
Thanks!
Posted: Sun Apr 22, 2007 2:34 pm
by SIELC_Tech
It is impossible to run ammonium acetate at 215 nm even with isocratic method. The cut off point is 230 nm and even at this wavelength you will have big background. I assume you carboxylic acids have low UV activity. You need to move to transparent buffer in order to monitor this; you can try phosphate or sulfate buffers.
Can you provide us with list of acids?
Check our website for analysis of acids with UV/ELSD/LC/MS conditions:
http://www.sielc.com/Applications_By_Detection.html
Posted: Sun Apr 22, 2007 4:27 pm
by Andreas Karas
I am working with citric, malic tartaric and oxalic acid which do have low UV activity. I know about the cut-off points of most buffers, but the choice of ammonium acetate was made in order to run some expirements with LC/MS..Actually, running with isocratic conditions is possible because the baseline is quite stable. Also, I have readen (LC GC Magazine) that keeping the buffer concentration constant it is not much of a problem working at low wavelengths even using buffers such as ammonium acetate...but I guess that this statement is quite theoritical!
What about phosphate solubility at relatively high persentages of CH3CN?
Posted: Sun Apr 22, 2007 6:01 pm
by Uwe Neue
To solve this problem with UV detection is not very promising, but you may have a bit more success at higher wavelengths. The best solution would be to go directly to MS, since your method is supposed to work there anyway. If the available time at the MS is limited, you may need to think through your experiments up front. What column are you using?
Posted: Mon Apr 23, 2007 3:46 am
by tom jupille
If the available time at the MS is limited, you may need to think through your experiments up front.
Following up a bit on Uwe's comment: if your purpose is to "debug" a method that will be used on LC-MS, you might look at either ELSD or CAD detectors. Both of those have similar mobile phase requirements to MS (and are cheaper!)