RESULTS AND DISCUSSION
Experiment 1
Students were assigned to one of two eluting mixtures, butanol/acetic acid/water (4:1:5, upper phase) or a formic acid (formic acid/conc HCl/water, 5:2:3) mixture. These solvents were chosen because of differences in polarities. The more polar formic acid mixture achieves greater separation. Information on similarities and differences between TLC (performed 2 weeks earlier) and paper chromatography were provided in the laboratory manual (Supporting Information). Pure anthocyanins and anthocyanidins have a single wavelength of maximum absorbance (λmax) in the visible region, with a narrow absorbance band range of 80−100 nm. Mixtures may show multiple λmax and/or wider absorbance bands.16,26 Because one anthocyanidin represents many anthocyanins, students analyze only the anthocyanidins with UV−vis spectroscopy using 1:10 to 1:50 dilutions of their hydrolyzed sample with acidic methanol. A typical student spectrum is shown in Figure 2. Students recorded λmax in the visible region and calculate an approximate concentration of anthocyanidins in their berry using a molar absorptivity coefficient of 3.0 × 104 M−1 cm−1 . 16 Students estimated the moles of anthocyanidin/anthocyanin per gram of freeze-dried fruit based on their dilution factor and mass of berry powder. Typical student results are in the range of 3.0 × 10−6 to 2.0 × 10−5 mol/g of berry with raspberry extracts containing less anthocyanin than the other two extracts. Calculations are summarized in the Instructor Notes in Supporting Information. Students pool spectroscopy results and discuss and compare their results with published anthocyanidin content for each berry. The paper chromatography data is pooled both by berry and by eluting solvent to facilitate observations before and after hydrolysis.