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 ehromogenic 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 KH₂PO₄ and K₂HPO₄, 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 K₂HPO₄. 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. [ABSTRACT FROM AUTHOR]