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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
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