Determination of Partition Coefficient of NAD + on Nafion 117 membranes

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 • 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 • Experimental parameters (current study): • pH • NaNO3 concentration • NAD+ concentration • Nafion types (hydrogen form and • sodium form) Molecular structure of Nafion (hydrogen form) A perfluorinatedsulfonic acid polymer

3. Experimental Procedure • Nafion 117 membrane, cut into a desired size • Preparation of External solution: • NAD+ and NaNO3, 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

4. Equilibrium achieved within 22 hours (Nafion, hydrogen form) • [NAD+] partitioning decreased by addition of NaNO3 in external solution UV-Vis spectra of Nafion after immersion in NAD+ containing 0.8 M NaNO3 solution for 22 and 96 hours UV-Vis spectra of Nafion after immersion in NAD+ containing 0.5 M NaNO3 solution for 22 hours

5. EDX Line-scan Analysis for Sodium a. 0.80 M NaNO3 in external solution 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+] b. 0.06 M NaNO3 in external solution c. no NaNO3 in external solution Observation: [Na+]in c. ≤ [Na+]in b. ≤ [Na+]in a. Sodium content in Nafion increased with content of NaNO3 in external solution

6. Partition Coefficients at different [NaNO3] 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 NaNO3) K ≈ 2 (as NaNO3 approaches 0.8 M)

7. Partition Coefficient at pH 7.0 • Experiments conducted with de-protonatedNafion (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-protonatedNafion and equilibrium pH 7 Partition Coefficient (K) ≈ 0.05 (no NaNO3 in solution) Implication: Low NAD+ partition coefficients make the sodium form of Nafion 117 membrane unsuitable for enzyme immobilization

8. Donan Membrane Equilibrium Eq. • Requirements: • Two aqueous compartments separated by a membrane (permeable to water and ions) • Fixed charge in the membrane According to Donan Eq. condition: Electro-neutrality is conserved in each compartment (membrane and external solution) [Donan, F. G. Journal of American Chemical Society1924, 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]

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:

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

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

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