Question

When performing a flash column chromatography, it is advised to select an eluent in which the desired compound has an RFof about 0.25in TLC. However, if I want to separate and isolate all the compounds present in the mixture, or I do not know which is the desired product, it is advised to use an eluent gradient for the column. But what eluent should be selected to start with? Maybe an eluent in which the compound with the highestRF(least polar in direct phase chromatography) has RF=0.25? What should I consider when performing such chromatography? I do a gradient from hexane to ethyl acetate until everything is separated. Is there a more efficient method that saves time and solvents?

Answer 1

When isolating all compounds in a mixture via flash column chromatography, start with an eluent where the most non-polar compound has an Rf of 0.25. Utilizing an eluent gradient can efficiently separate unknown mixtures. For greater efficiency, UHPLC and monolithic columns can be employed to save time and solvents.

Step-by-step explanation:

When performing flash column chromatography and looking to separate and isolate all compounds in a mixture, the selection of the starting eluent is crucial. Considerations for the initial eluent include starting with a mobile phase where the most non-polar compound in direct phase chromatography shows an Rf of about 0.25 in TLC.

This will help prevent the most non-polar compounds from eluting too fast and co-eluting with the solvent front. An eluent gradient is beneficial when multiple compounds need to be separated, especially when their properties aren't well known in advance.

Using an eluent gradient from non-polar to more polar solvents (e.g., hexane to ethyl acetate) is a common approach. However, this method can sometimes be time-consuming and use excessive solvents.

Advancements such as ultra-high pressure liquid chromatography (UHPLC) or monolithic columns have been developed to increase separation efficiency, speed, and sensitivity. UHPLC, for instance, uses high pressures and columns packed with small particles, resulting in better separation quality and reduced analysis time.

For maximizing separation efficiency, it's essential to consider the affinity of the compounds to the hydrophobic stationary phase versus the mobile phase, and adjust the gradient accordingly. As a starting point for the gradient, a standard analytical, reverse-phase HPLC column equilibrated with a predominantly non-polar solvent, like 98% methanol, can be optimal before gradually increasing the polarity of the mobile phase.