Most often the question on loading is “how much can I purify” on a specific column size but once in a while I get the question above – what is the smallest amount I can purify? This is an interesting question, especially since flash chromatography is preparative chromatography technique used to purify large amounts of crude mixtures.
That being said, this is a real situation that occurs, usually for trace components/impurities. My answer always is “it depends on the compound’s detectability” by either UV or ELSD (evaporative light-scattering detector). Compounds with good UV chromophores are easier to detect than those without unless you have an ELSD or UV sensitivity-enhancing software, e.g. Biotage® Spektra software, installed on your flash system.
To maximize UV detection sensitivity, several options can be employed…
- Minimize column size to decrease sample dilution. However, this can negatively impact the separation and lead to impure compounds
- Use UV transparent solvents when purifying by normal-phase, e.g. acetone or methyl t-butyl ether (MTBE), instead of ethyl acetate, especially for compounds with low UV absorbance wavelengths
- Use alternative purification methods, e.g. reversed-phase vs. normal-phase or vice-versa to change separation selectivity and elute the minor product target earlier in the purification
- Use a technique called wavelength-focusing where you utilize your flash system’s PDA UV to select a detection range and maximize sensitivity.
- If none of these work, add/use an ELS detector since it does not require molar absorptivity for sensitivity.
A nice example of utilizing wavelength-focusing with UV transparent solvents is the detection of cholesterol with a UV maximum around 200 nm, Figure 1.
Figure 1. Separation of cholesterol (0.415 mg) by reversed-phase flash chromatography using a Biotage® Selekt flash system and wavelength-focusing.
In this example, a Biotage® Selekt flash system equipped with Biotage® Spektra software was utilized to enhance the sensitivity of cholesterol, a poor UV absorbing compound, at less than 1 mg (0.415 mg). For this example, reversed-phase flash chromatography with a 12-g Biotage Sfär C18 column and 100% methanol was used. Looking at the chromatogram we can see the UV response achieve 100 mAU.
This same approach can be used for other compounds with little UV absorption.
If you would to learn more about this topic and others related to flash chromatography, visit our flash chromatography blog center.