After analyzing an eluted fraction from a column via TLC, what should you do next if the desired substances were observed on the plate?

When eluting fractions from a column chromatography and analyzing them via Thin Layer Chromatography (TLC), your next steps should be focused on optimizing the separation of your desired substances. If the desired substances are present on the TLC plate, increasing the eluent's polarity gradually is the correct action.

Increasing the polarity of the eluent can enhance the separation process by allowing compounds that are less polar to migrate faster and separate more effectively from those that are more polar. This could lead to better resolution and purification of your desired substances. It facilitates further elution of compounds that may not have been fully separated or eluted in the initial run.

The other options do not facilitate improved separation or purification. Removing the solvent immediately isn't practical, as it can result in losing the analytes you need for further analysis. Letting the solvent evaporate completely would not aid in further purification and may result in the loss of desired components. Restarting the chromatography process is also counterproductive after TLC analysis has indicated the presence of the desired compounds; it would be more efficient to refine the separation with an adjusted solvent system rather than starting from scratch.

Thus, increasing the eluent's polarity is the most logical step following the identification of desired substances through TLC.