How to determine the optimum separation conditions by using an analytical column.
dear Swagat Mohanty , when scaling up your HPLC or LCMS experiment, even when using a preparative column you are not 100% sure to get the same results, there are a lot of factors that can affect your experiment such as type of column, saturation, and compounds internal interactions, the latter depends largely on the injected sample amount, to make sure you get the best results its better to use a preparative column and respect the injection volume and the analytical parameters, to try to replicate the analytical conditions and have a good séparation. However, you can try to use the analytical column for prep but I don't recommend you to do that because:
- due to the small size of the column the elution pics will be mixed ( like in normal column chromatography a larger and wider column is suitable for purification of big amounts)
- using too much sample may alter the column.
best regards.
Read T. Gu's book on chromatography scale-up. I've implemented his dimensionless parameter approach in the attached spreadsheet. The key is matching the dimensionless parameters between the lab-scale and the larger scale by changing packing porosity, size, column length and diameter within the constraints of allowable pressure drop.
There are several ways to do this. one way is to work in column volumes, as described here:
https://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/HPLC%20to%20Flash.pdf
Although this was written for flash, it applies to HPLC as well. Note that "Dwell volume", also called "gradient delay", isn't accounted for so the calculations are easier.
There are HPLC scale-up calculators on-line:
Supelco HPLC Calculator for Isocratic or Gradient Method Transfer | Sigma-Aldrich (sigmaaldrich.com) There are others available, too.
Another way is to simply calculate a focused gradient from an analytical scouting run. If an isocratic run is required, it is calculated as an intermediate result:
Article An overview of analytical-to-preparative liquid chromatograp...
Kindly visit..
https://www.researchgate.net/publication/334617894_An_overview_of_analytical-to-preparative_liquid_chromatography_method_development
Check ✔️..
https://www.researchgate.net/profile/Luke-Schneider/post/How_could_I_scale_up_a_column_for_preparative_isolation_purification_from_analytical_conditions_in_a_HPLC/attachment/60a9a7805e24cd00015ee275/AS%3A1026429202468865%401621731200469/download/LC+Params+and+Numbers.xlsx
Whereas, Another way is to simply calculate a focused gradient from an analytical scouting run. If an isocratic run is required, it is calculated as an intermediate result
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