How reliable is internal standard method in monitoring mechanochemical synthesis? A case study of triphenylmethane in HPLC-UV-MS analysis of hemicucurbit[n]urils†

Quantitative analysis of crude reaction mixtures is essential for the development of new synthetic methodologies and conducting mechanistic studies. While internal standard method is widely used for determining reaction yields in homogeneous solvent-based organic synthesis, its application in mechanochemical synthesis, which often involves heterogeneous mixtures, has not been properly validated. This study showcases applicability of triphenylmethane (TPM) as a solid internal standard in liquid-assisted, multi-component synthesis of homomeric cycHC[8] and mono-biotinylated mixHC[8] eight-membered cyclohexanohemicucurbit[n]urils. A fast and reliable HPLC-UV-MS analytical procedure was developed to determine yields by analyzing crude reaction mixtures, as a prerequisite of applying design-of-experiments optimisation approach. The influence of various parameters, including TPM concentration, reactant mixture weight, milling time, and the type and amount of liquid-assisted grinding additive, on the validity of the analysis was systematically studied. The results indicate that the primary challenge to trustworthy analysis arises from the non-uniform distribution of components. However, this issue can be detected with proper sampling and mitigated by optimising parameters to ensure uniform distribution of the internal standard throughout the reaction mixture. The results could be valuable for ensuring the credibility of ex situ and in situ analytical methods used to track the progress of mechanochemical reactions through single-point measurements.