Use of a laboratory exercise on molar absorptivity to promote student skepticism

Skepticism, when promoted as a desirable trait amongst undergraduate students, can effect a marked increase in their cognitive development. We will describe a biochemistry laboratory exercise that can be utilized to promote student skepticism. The individual exercises in our laboratory course are made more relevant by providing a thematic thread, β‐galactosidase, that links concepts in one exercise with those in latter exercises. The substrate for the continuous assay of β‐galactosidase is the chromogenic 2‐nitrophenyl‐β‐D‐galactopyranoside that produces 2‐nitrophenol (2‐NP). In one of the laboratory exercises, students prepare a series of ten differently buffered solutions by using KH 2 PO 4 and K 2 HPO 4 , mixed with 50 μM 2‐NP, to determine the wavelength of maximum absorption for the protonated and deprotonated form of each molecule. After determining the pH of the individual solution they determine the pKa value for 2‐NP. The molar absorptivity of 2‐NP is then determined, by preparing different dilutions of 5mM 2‐NP in a solution of K 2 HPO 4 . The average molar absorptivity value for 2‐NP determined by 38 students in one semester was 3560 ± 1060 at 420 nm, at a pH of 9.2. Students are encouraged to discuss that apparent discrepancies in the published molar absorptivity values for 2‐NP (2150 to 21,300) at almost the same pH and at 420 nm with their own molar absorptivity values for 2‐NP.