How to determine whether to use isocratic or step gradient method for HPLC based methods?
Hello Harleen,
I can't give you a definite answer. That depends on the circumstances. For certain applications (ion exchange, RI detection, gel filtration, chiral separations) isocratic separations are usually preferred. Isocratic separations have the advantage that a solvent mixture of constant composition is used. You only need an isocratic pump (price question), the column does not have to be re-equilibrated and routine separations are very stable. However, if you have complex sample mixtures or you want to use UPLC methods, you cannot avoid a gradient method. The advantages here are constant peak width over the complete running time, possibly very short running times and applicable to polar, mid-polar and non-polar compounds. With this method you practically do a splits and can analyze different polar molecules in one run. You probably won't be able to do this under isocratic conditions.
Regards
Markus
Markus Christ gives good advice, but if you are using a reversed phase separation then try the following.
If you only have 1 or 2 substances that you wish to measure then use an isocratic separation with sufficient retention time to keep your analytes clear of each other and interferences such as are usually found at the peak front, usually at least 3 column void volumes. If this leads to peaks that are too wide or poorly resolved then either try a different separation (eg different column chemistry) or a simple gradient.
If you have a more complex mixture, run a simple gradient from 10% organic to 90% organic over 10 minutes or so. If all of your analytes of interest can be found within a single 2 minute window then you should be able to use an isocratic separation.
If the analytes are spread over a wider time window then you may get a better separation using a gradient analysis. You can then use the %B of the first and last peaks as a starting point for your gradient. For example, if your first analyte is at 3 minutes and your last is at 7 minutes then a gradient of 30 - 70% would be a good starting point.
Richard
What is the resolution of your compounds? The only real reason to run a step gradient is to save time by causing the second compound to elute earlier. The time and solvent savings from a step gradient needs to be compared to the time and solvent required for re-equilibrating the column.
The more the number of analytes in the sample and the diverse their polarity are, the higher the nned will be to use a gradient method.
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