where pKa is of the 'mixed' type (Section 4C, Supporting information). The pH was observed to drop from ca. 7.1 to ca. 5.1 over 24 h following inoculation, with the pH being higher at the colonocyte level compared to the mucin and commensal levels (Figure ). Spatially resolved metabolic crosstalk. To probe the origin of the pH gradient across the depth of the NMR tube, we carried out metabolic profiling against a set of SCFAsformate, propionate, butyrate, lactate, and succinate at the top (25-35 mm from the base of the NMR tube), middle (15-25 mm) and bottom (5-15 mm) regions of the sample. There were no differences in the concentration of formate, lactate, and succinate across the depth of the sample. However, the concentration of butyrate and propionate was different across the three regions of the NMR tube and followed the order of top > middle > bottom (Figure ). To probe whether these changes in SCFA concentration at different depths were driven by the commensal bacteria, colonocyte metabolism or a combination of both, three sets of controls were carried out (Figure ). There were no significant differences in the concentration of propionate and butyrate at different depths when colonocytes were fixed (cells -/bacteria + , Figure ). There was no SCFA production when commensals were filtered out prior to the inoculation (cells + /bacteria -, Figure ). When bacteria were filtered out and propionate and butyrate were supplemented in the media, the concentration of butyrate decreased the fastest nearer the colonocytes (Figure , right), compared to the middle and top regions of the NMR tube. These data suggest colonocytes preferentially utilise butyrate over other SCFAs produced by commensal bacteria, which is also likely to drive the higher pH at the colonocyte level. Together these data highlight the importance of applying spatially resolved NMR techniques for mapping out distance-dependent dynamic exchange on a molecular level, providing details that would be missed by conventional analysis.