What is a consequence of placing spots too closely together when spotting a TLC plate?

When spots are placed too closely together on a TLC (Thin Layer Chromatography) plate, one significant consequence is that they can bleed into each other during the development process. As the solvent moves up the plate, it carries the components of the samples along with it. If two spots are too close, the solvent front may cause them to overlap and mix, making it challenging to distinguish between the separated compounds once the TLC run is complete. This bleeding complicates the identification and calculation of Rf values for each component, which are essential for determining the composition of the mixture. As a result, achieving clear enough separation to analyze the distinct components becomes difficult, adversely affecting the reliability of the results.