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column efficiency
Posted: Tue Mar 22, 2011 9:23 am
by archie
Hi all...
I have questions about column efficiency.
When I use acetonitrile and methanol as mobile phase, the efficiency is about 2000,
but when I introduce water to the mobile phase, the efficiency fell to 500, and my analyte peak became short and wide..
How to increase the efficiency so I can get narrow and tall peak? I have to use water in mobile phase to separate my analyte from the matrix. The concentration of water I used is about 15%.
Thanks for your help..
Re: column efficiency
Posted: Tue Mar 22, 2011 9:53 am
by Gerhard Kratz
It would be helpful to know what column you are using, RP, HILIC or which one.
Re: column efficiency
Posted: Tue Mar 22, 2011 3:49 pm
by tom jupille
Also the column dimensions, packing particle size, flow rate, injection volume, and diluent. All of those have an effect.
Re: column efficiency
Posted: Tue Mar 22, 2011 7:30 pm
by JGK
Are you using a sysyem capable of running gradients?
Re: column efficiency
Posted: Wed Mar 23, 2011 5:30 am
by archie
@ Gerhard & Tom:
I used RP column, Zorbax Eclipse XDB C18 150mm x 4.6, 5 micron.
I use 1.2 ml/minute flow rate, 20 micron injection volume and acetonitrile as solvent.
I use metahnol :water = 85% : 15% as mobile phase, Uv detection at 210nm, and 40°C column temperature.
I have changed the Zorbax column with Hypersil ODS, 125mm x 4.0, 5 micron, but the efficiency still low.
@ JGK:
Yes, it is possible to run gradient analysis
Re: column efficiency
Posted: Wed Mar 23, 2011 4:26 pm
by tom jupille
First of all, even 2000 plates is low for this column. A 150 x 4.6 mm / 5 micron column should be able to generate 5-10,000 plates for small molecules. If nothing else, you should check your system for exessive extra-column volume (too large a tubing diameter; excessively long tubing; poorly assembled fittings).
Second, you did not tell us the approximate retention time of your analyte. If it is weakly retained (on that column at 1.2 mL/min, the dead time is around 1.2 minutes, give or take 15%) then plate count measurements are unreliable. To get reasonable results, you should measure on a peak whose k' is > 1 (i.e., > 2x the dead time).
Third, peak shape problems (including excessive broadening) are reasonably common when the diluent is stronger than the mobile phase (in your case, 100% ACN as the diluent vs. 85% MeOH in the mobile phase. For best results, it's a good idea for the diluent to match the mobile phase.