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When doing TLCs to monitor reactions, it is considered good common practice to not only spot the reactants and the reaction mixture (likely after some mini-workup), but also to include a ‘cross-spot’: every other substance (mixture) spotted somewhere on the plate is spotted together on one spot, commonly labelled something like X.

When asking why during my bachelor's thesis, the PhD student supervising me said something that roughly translates to:

You can't be sure that the components don't run differently in the mixture than by themselves.

But in a perfect world, they should. Aside from the obvious case of having a polar reaction solvent that didn't entirely evaporate and skewes the elution mixture on parts of the TLC, are there other reasons why the components of a mixture should have different Rf values when spotted separately?

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    $\begingroup$ I think you have the key point there about the reaction solvent affecting the Rf of your analyte. Using a mini-workup routinely is a good habit to get into. In my experience, the cross spot is primarily useful to distinguish compounds that are very close in polarity. $\endgroup$
    – jerepierre
    Apr 24 '15 at 15:06
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are there other reasons why the components of a mixture should have different Rf values when spotted separately?

One might speculate that two polar reactants form a less polar "adduct". This should however result in an additional spot, rather than in altered $\mathrm{R_f}$ values for the individual compounds.

But this is speculation. Admittedly, I have never used the cross spot method. Neither have I ever observed that spots of the reactants had different $\mathrm{R_f}$ values in the reaction mixture.

On a side note: What always worked for me was:

  • Don't apply spots but bands and overlap them.
  • "Sharpen" the bands on the base line by developping the TLC in methanol or another polar solvent first.

Together, these two tricks allow to destinguish between two compounds with very similar $\mathrm{R_f}$ values.

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