M 2+ •EDTA Binding Affinities: A Modern Experiment in Thermodynamics for the Physical Chemistry Laboratory Leah C. O’Brien,* ,† Hannah B. Root, † Chin-Chuan Wei, † Drake Jensen, † Nahid Shabestary, † Cristina De Meo, † and Douglas J. Eder ‡ † Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, Illinois 62026-1652, United States ‡ Emeritus, Southern Illinois University Edwardsville, Edwardsville Illinois 62026-2224, United States * S Supporting Information ABSTRACT: Isothermal titration calorimetry was used to experimen- tally determine thermodynamic values for the ethylenediaminetetraacetic acid (EDTA)(aq) + M 2+ (aq) reactions (M 2+ = Ca 2+ and Mg 2+ ). Students showed that for reactions in a N-(2-hydroxyethyl)piperazine-N′- ethanesulfonic acid (HEPES) buffer (pH = 7.4), the Mg 2+ + EDTA reaction was endothermic, while Ca 2+ binding to EDTA was exothermic. EDTA is triply ionized at pH 7.4 and therefore must shed a proton to the buffer prior to chelating the M 2+ ion; thus the observed reaction enthalpies are strongly dependent on pH and buffer ionization enthalpy. KEYWORDS: Upper-Divion Undergraduate, Physical Chemistry, Aqueous Solution Chemistry, Biophysical Chemistry, Calorimetry/Thermochemistry, Metals, Thermodynamics, Hands-On Learning/Manipulatives, Laboratory Instruction ■ INTRODUCTION TO ISOTHERMAL TITRATION CALORIMETRY Isothermal titration calorimetry (ITC) is an experimental technique where the heat of reaction is measured for sequential injections of a titrant into the titrand contained within an adiabatic chamber. ITC was first described, to the best of our knowledge, in 1968 by H.D. Johnston. 1,2 ITC gained in popularity in the 1970s, where cell volumes were on the order of 10-100 mL. In 1989, Wiseman et al. 3 reported the development of an ultrasensitive titration calorimeter, and this instrument was the forerunner of the modern nano-ITCs designed for small-scale reactions with volumes ≤ 1 mL using mM and nM solutions. 4 Nano-ITC has found many applications in biochemistry and pharmaceutical research. Protein-protein, protein-drug, protein-DNA, and protein- ligand interactions are examples of intermolecular interactions that can be studied by ITC. 3-14 Despite the explosion of nano- ITC in physical biochemistry research and in the pharmaceut- ical industry in the past decade, only a few articles related to this important analytical method have appeared in the science education literature. Three papers from the Journal of Chemical Education 15-17 were identified that describe ITC undergraduate experiments. The 2001 JCE paper 15 describes an isothermal heat conduction calorimeter and offers several ideas for undergraduate experiments. The 2008 and 2011 JCE papers 16,17 describe the construction of a low-cost, homemade isothermal calorimeter and a [Ba 2+ + 18-crown-6] binding experiment. These experiments show the great variety in applications of calorimetry at the mL scale. However, the equipment described in these papers does not offer the ultrasensitivity of the modern nano-ITC systems that is needed for enzyme binding studies, which are normally conducted at the sub-mL scale. Schematic diagrams for the nano-ITC apparatus can be found online (e.g., see refs 18-21). Briefly, a reference cell and a sample cell are submerged in an adiabatic chamber, as shown in Figure 1. One reagent is contained in the sample cell, and the other reagent is loaded into a syringe injector. Both reagents are in an identical buffer solution to minimize mixing/dilution enthalpy. In a standard ITC experiment, the automatic injector introduces a known amount of solution into the sample cell using a programmable series of small injections. A sensitive thermocouple on each cell activates feedback electronics, which will increase or decrease the electrical current to the sample cell and maintain the cells at the exact same temperature difference. A graph of μW versus time produces the ITC isotherm. ΔH is obtained by integrating the area under the curve, and both exothermic and endothermic reactions can be monitored. Each titration peak is integrated separately, and these values are used to create the sigmoid-shaped curve of the integrated isotherm. The equilibrium binding constant K can be determined by the severity of the sigmoid curve, and the mole ratio n can be determined using the inflection point of the sigmoid curve. The experimentally determined ΔH° and K° from the ITC analyses can be used to calculate ΔG° and ΔS° based on the well-known Laboratory Experiment pubs.acs.org/jchemeduc © XXXX American Chemical Society and Division of Chemical Education, Inc. A DOI: 10.1021/acs.jchemed.5b00159 J. Chem. Educ. XXXX, XXX, XXX-XXX