1. Determination of Partition Coefficient of NAD + on Nafion 117 membranes Importance: Partitioning process affects actual NAD + and NADH concentrations, available for the enzymatic steps occurring in enzyme immobilized electrodes 1 Partition coefficient values can enable to accurately quantify performance in: biofuel cells biosensors any device operating on immobilized enzymes

2. Determination of Partition Coefficient of NAD + on Nafion 117 membranes Objective: Determine NAD + partition coefficient Molecular structure of Nafion (hydrogen form) A perfluorinated sulfonic acid polymer 2 Experimental parameters (current study): pH NaNO3 concentration NAD + concentration Nafion types (hydrogen form and sodium form)

3. Experimental Procedure Nafion 117 membrane, cut into a desired size Preparation of External solution: NAD + and NaNO 3, dissolved in a 10 mM phosphate buffer solution at a desired pH 7.5 Equilibration: Nafion 117 membranes, soaked in the NAD + solutions for at least 20 hours, to allow electrochemical equilibrium between membrane and external solution [NAD + ] and pH measurements: [NAD + ] in Nafion membranes and external solutions were measured by UV-Vis 0.1 mM [NAD + ] gives an approximate absorbance of approximately 16900 M -1 cm -1 at 260 nm 3

4. Equilibrium achieved within 22 hours (Nafion, hydrogen form) UV-Vis spectra of Nafion after immersion in NAD + containing 0.8 M NaNO 3 solution for 22 and 96 hours UV-Vis spectra of Nafion after immersion in NAD + containing 0.5 M NaNO 3 solution for 22 hours [NAD+] partitioning decreased by addition of NaNO 3 in external solution 4

5. EDX Line-scan Analysis for Sodium b. 0.06 M NaNO 3 in external solution c. no NaNO 3 in external solution a. 0.80 M NaNO 3 in external solution Observation: [Na + ]in c. ≤ [Na + ]in b. ≤ [Na + ]in a. Left: Scanning electron micrographs of the cross-sections of the dry Nafion 117 membranes after partition coefficient study. Scanned lines are shown. Right: EDX along the scanned lines showing sodium content [Na + ] Sodium content in Nafion increased with content of NaNO 3 in external solution 5

6. Partition Coefficients at different [NaNO 3 ] in external solution Partition coefficient, K, is the slope of the linear fit of plot of [NAD + ] in Nafion membrane (as-received) to that inside the external solution As – received Nafion and equilibrium pH 2.25 K ≈ 13 (absence of NaNO 3 ) K ≈ 2 (as NaNO 3 approaches 0.8 M) 6

7. Partition Coefficient at pH 7.0 Experiments conducted with de- protonated Nafion (A and B) Nafion, A - Immersed in In 1 M NaNO3 solution at 75 °C for 18 hours - Rinsing in DI water Nafion, B -Immersed in 1 M NaNO3 solution at 75 °C for 18 hours - heated in DI water at 75 °C for 6 hours De-protonated Nafion and equilibrium pH 7 Partition Coefficient (K) ≈ 0.05 (no NaNO 3 in solution) Implication: Low NAD+ partition coefficients make the sodium form of Nafion 117 membrane unsuitable for enzyme immobilization 7

8. Donan Membrane Equilibrium Eq. According to Donan Eq. condition: Requirements: Two aqueous compartments separated by a membrane (permeable to water and ions) Fixed charge in the membrane Electro-neutrality is conserved in each compartment (membrane and external solution) [Donan, F. G. Journal of American Chemical Society 1924, 1, 73 – 90] Applicability range ph < ≈ 4, because NAD + remains charge neutral above that pH [Moore Jr., C. E.; Underwood, A. L. Anal. Biochem. 1969, 29, 149-153] 8

9. Calculation of Partition Coefficient ( K ) At species concentrations much lower than than the fixed charge concentrations can be replace activities, in order to apply the Donan Membrane Equilibrium Eq. I --- ion exchange capacity = 1.77 M [L. A. Zook and J. Leddy, Analytical Chemistry, 1996, 68, 3793 – 3797] Applying Donan membrane equilibrium eq. for cationic species: Applying Donan membrane equilibrium eq. for anionic species: K is determined by applying equilibrium membrane charge balance: 9

10. Comparison: Calculated Partition Coefficients vs. those measured at various pH pH: Equilibrium pH = 2.25 (measured) for external solution Inside the Nafion membrane, pH ≈ 1 or lower (value not measured) and assumed Observations: Calculated values, assuming unity activity coefficients match Measured partition coefficient value 10

11. Main Conclusions Equilibrium [NAD + ] in Nafion 117 membranes was observed to be already attained at 22 hours Calculated partition coefficient (assuming unity activity coefficient) values match with those measured at low pH. Summary: Ongoing and Future Work Extend the study to other candidate materials for enzyme immobilization, e.g. chitosan and Tetra Butyl Ammonium Bromide (TBAB) Nafion Determine partition coefficient of NADH 11 pHNafion type[NaNO 3 ]NAD + partition coefficient 2.25Hydrogen formnone15 2.25Hydrogen form0.83 7Sodium formnone0.05

12. NAD + Activity Coefficient and pH inside Nafion membrane Is is the ionic strength inside Nafion Z = +1, charge on NAD + Activity coeff. of NAD + is given by: 0.275 = < > = 0.40 12