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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

substrate NADNADdependent enzyme NADH+products

Partition coefficient values can enable to accurately quantify performance in:

• biofuel cells

• biosensors

• any device operating on immobilized enzymes

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Determination of Partition Coefficient of NAD+ on Nafion 117 membranes

Objective:Determine NAD+ partition coefficient

Molecular structureof Nafion (hydrogen form)A perfluorinated sulfonic acid polymer

Experimental parameters (current study):• pH• NaNO3 concentration• NAD+ concentration• Nafion types (hydrogen form and sodium form)

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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 approximateabsorbance of approximately 16900 M-1 cm-1 at 260 nm

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Equilibrium achieved within 22 hours (Nafion, hydrogen form)

UV-Vis spectra of Nafion after immersion in NAD+ containing 0.8 M NaNO3 solutionfor 22 and 96 hours

UV-Vis spectra of Nafion after immersion in NAD+ containing 0.5 M NaNO3 solutionfor 22 hours

[NAD+] partitioning decreased by addition of NaNO3 in external

solution

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EDX Line-scan Analysis for Sodium

b. 0.06 M NaNO3 in external solution

c. no NaNO3 in external solution

a. 0.80 M NaNO3 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 NaNO3 in external solution

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Partition Coefficients at different [NaNO3] in external solution

Partition coefficient, K,is the slope of thelinear 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)

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Partition Coefficient at pH 7.0Experiments 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 7Partition 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

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Donan Membrane Equilibrium Eq.

i,exte RT ln(ai,ext ) ziFext

i,Nafe RT ln(ai,Naf ) ziFNaf

According to Donan Eq. condition:

i,exte i,Naf

e

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]

ai,ext

ai,Naf

exp[ziFRT

(Naf ext )]

[ai,ext

ai,Naf

]1zi exp[

FRT

(Naf ext )]K

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]

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Calculation of Partition Coefficient (K)

K [C

NAD , Naf

CNAD , ext

]1/1 [C

Na , Naf

CNa , ext

]1/1 [C

H , Naf

CH , ext

]1/1

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 [C

NO3 , Naf

CNO3

, ext

]1/ 1 [C

H2PO4 , Naf

CH2PO4

, ext

]1/ 1 [C

HPO42 , Naf

CHPO4

2 , ext

]1/ 2 [C

OH , Naf

COH , ext

]1/ 1

K{CNa , ext

CNAD , ext

CH , ext

} I

(1 / K){CNO3

−, extC

H2PO4−, ext

COH−, ext

} (1 / K2 )CHPO4

2−, ext

K is determined by applying equilibrium membrane charge balance:

ai Ci

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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

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Main ConclusionsEquilibrium [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 WorkExtend the study to other candidate materials for enzyme immobilization, e.g. chitosan and Tetra Butyl Ammonium Bromide (TBAB) Nafion

Determine partition coefficient of NADH

pH Nafion type [NaNO3] NAD+ partition coefficient

2.25 Hydrogen form none 15

2.25 Hydrogen form 0.8 3

7 Sodium form none 0.05

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NAD+ Activity Coefficient and pH inside Nafion membrane

log(NAD

) 1.173Z 2 Is1 Is

Is is the ionic strength inside Nafion

Z = +1, charge on NAD+

NAD

NAD

Activity coeff. of NAD+

is given by:

Is ciZi2

i

NAD 0.275 = < > = 0.40