Could anyone please give merits and demerits of each of the elution mode in LC-MS. Also, what is the minimum column volumes (mL) of mobile phase that should be passed through a column during the isocratic elution.
Eg. 100 X 2.1 mm, particle size of 3.0 µm : What should be minimum runtime on this column?
It depends on the column. You can read these data in the column sheet enclosed with the column.
Isocratic LC is preffered in chiral separation where the stationary phase is sensitive to the mobile phase composition and slowly adapts to it. In analysis of small molecules it is preffered to work with gradient LC, especially with complex matrices. In front all ions go out to the waste (if you have switching valve which is a great benefit), and at the and the protein residues are retained and washed from the column by high percentage of organic phase.
With a few exceptions (chiral as mentioned above), gradients are useful when one doesn't know when the compound(s) of interest will elute. Isocratic mode is useful when one does know when the compound does elute, or when more resolution may be required.
A gradient covers all of the polarity ranges of the solvent system so most compounds will elute during some point of the gradient. An isocratic system may have one compound elute fairly early, and another very late which extends the run time; a gradient could reduce the run time while still providing adequate resolution.
The minimum run time for your column depends on your sample and when it elutes. The absolute minimum run time will be whatever the void volume for this column is, but that's hardly chromatography. Elution at 2 to 10 times the void volume for an isocratic run is much better because the compound and impurities have been able to interact with the column.
Dear Jack,
Thanks for elaboration. Actually I was confused as a general review of analytical LC-MS/MS methods suggests that isocratic is preferred over gradient. However as I had asked, I was not able to put a logical explanation to run-time which most of the researchers have used. Eg. for a 100 X 2.1 mm column with a void volume of 0.4 ml, approximately 10 minutes of run-time would be ideal at a flowrate of 200 microL per min. However I was amazed to see most of them with a run-time of 4-5 minute ( which translates to 1 ml or 4-5 times).
Thanks again for clarification.
do not forget that if you run samples in isocratic modes you need to be sure that there is no sample accumulation inside your column that will slowly change retention of your compounds on your compounds.
At least what is good with gradient mode is you wash well your column so you reduce carryover... It always improve the quality of the analysis...
Dr. Bertrand is correct; often analysis are run isocratically with a gradient at the end to wash off retained material that isn't relevant to the experiment.
When adding a gradient at the end and resetting back to the isocratic conditions, there may be extra equilibration needed between runs. However, by washing the column, it reduces greatly the chance of seeing a peak carried over from a previous injection/run.
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