Robustness

[agen]

I'm working on an HPLC technique using a C18 column, buffer Phosphate pH=3, Methanol (90:10), Temp 25ºC, 326 nm. The drug is Cromolyn sodium. The retention time changes drastically, from analysis to analysis, taking the aqueous and organic phases from differents channels of my pump. Could anybody help me with this? I'm rather lost about finding an explanation to the phenomena!
Thank you in advance.

[skunked_once]

It may be that the solvent proportioning of the different channels is not equal. You could test the flow rate of each channel using both of the components of the mobile phase to check out this possibility.

[tom jupille]

Try premixing the mobile phase and running that on only one channel (or on each channel in turn). If the problem persists, then you have exonerated the proportioning system and your poor robustness is due to something else (temperature? pH?).

If the problem goes away, then you have a proportioning problem. Best way to check is to do a gradient step-test (you can find a brief description under "Diagnostics" in the Troubleshooting Wizard on our web site: http://www.lcresources.com/resources/TSWiz/hs370.htm

You should compare the results to the manufacturer's spec for your system. If it's out of spec it needs to be fixed. If it's within spec, then your method is simply non-robust with respect to organic solvent concentration. The easiest solution in that case may simply be to specify pre-mixed mobile phase.

[Bruce Hamilton]

Assuming that the problem is your pump's mixing, the above suggestions should solve the problem.

If they don't, then consider the sample load and buffer concentration. Ensure that your sample is at a suitable pH, and that your mobile phase buffer has sufficient capacity to ensure the analyte is always in the same ionic form.

Please keep having fun,

Bruce Hamilton.