1. Review of Analytical Methods Part 2: Electrochemistry
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
University of Florida Health Science Center/Jacksonville

2. Analytical methods used in clinical chemistry
Spectrophotometry
Electrochemistry
Immunochemistry
Other
Osmometry
Chromatography
Electrophoresis

3. Electrochemistry
Electrochemistry applies to the movement of electrons from one compound to another
The donor of electrons is oxidized
The recipient of electrons is reduced
The direction of flow of electrons from one compound to another is determined by the electrochemical potential

4. Electrochemical potential
Factors that affect electrochemical potential:
Distance/shielding from nucleus
Filled/partially filled orbitals

5. Relative potential Zn Cu Copper is more electronegative than Zinc
When the two metals are connected electrically, current (electrons) will flow spontaneously from Zinc to Copper
Zinc is oxidized; Copper is reduced
Zinc is the anode; Copper is the cathode

6. mV
e-  e-  e- 
Cu2+
Zn2+
Zn0
Cu0
Zn0  Zn2+ + 2e-
Cu2+ + 2e-  Cu0

7. The Nernst Equation Where E = Potential at temperature T
E0 = Standard electrode potential (25ºC, 1.0M)
R = Ideal gas constant
F = Faraday’s constant
n = number of electrons transferred

8. Cu2+ Zn2+ Zn0 Cu0 Elj Zn0  Zn2+ + 2e- E0 = +(-)0.7628 VmV
Cu2+
Zn2+
Zn0
Cu0
Elj
Zn0  Zn2+ + 2e-
E0 = +(-) V
Cu2+ + 2e-  Cu0
E0 = V

9. Electromotive force Ecell = Ecathode + Elj - Eanode
Ecell = ECu(II),Cu + Elj – EZn(II),Zn
Ecell = (+) Elj – (-)0.763
Ecell = (+) Elj
G = -nFEcell

10. Would the reaction occur in the opposite direction?
Ecell = Ecathode + Elj - Eanode
Ecell = EZn(II)Zn + Elj – ECu(II)  Cu
Ecell = (-) Elj – (+)0.340
Ecell = (-) Elj

11. How do we determine standard electrode potentials?
Absolute potential cannot be measured—only the relative potential can be measured
Standard electrode potentials are measured relative to a Reference Electrode
A Reference Electrode should be. . .
Easy to manufacture
Stable

12. The Hydrogen ElectrodemV
Test electrode
H2 gas 
2H+ + 2e-  H2
E0 = 0.0 V

13. The Calomel Electrode Hg2Cl2 + 2e-  2Hg0 + 2Cl- E0 = 0.268VmV
Test electrode
Calomel paste (Hg0/Hg2Cl2)
Saturated KCl
Liquid junction
Hg2Cl2 + 2e-  2Hg0 + 2Cl-
E0 = 0.268V

14. The Silver/Silver Chloride ElectrodemV
Test electrode
Silver wire
Saturated
KCl + AgNO3
Liquid junction
AgCl + e-  Ag0 + Cl-
E0 = 0.222V

15. Ion-selective ElectrodesmV
Ecell = ERef(1) + Elj – ERef(2)
Ref1
Ref2

16. Typical ISE design Ecell  EISM Ecell = ERef(1) + Elj – ERef(2) Ref 1mV
Ecell  EISM
Ecell = ERef(1) + Elj – ERef(2)
Ref 1
Ref 2 +
Ion-selective
membrane

17. Activity and concentration
ISEs do not measure the concentration of an analyte, they measure its activity.
Ionic activity has a specific thermodynamic definition, but for most purposes, it can be regarded as the concentration of free ion in solution.
The activity of an ion is the concentration times the activity coefficient, usually designated by :

18. The activity coefficient
Solutions (and gases) in which none of the components interact are called ideal, and have specific, predictable properties
Deviations from ideal behavior account for the difference between concentration and activity
Dilute solutions exhibit nearly ideal behavior (1)

19. Types of ISE Glass Solid-state Liquid ion-exchange Gas sensors
Various combinations of SiO2 with metal oxides
Solid-state
Involve ionic reaction with a crystalline (or crystal doped) membrane (example: Cl-/AgCl)
Liquid ion-exchange
A carrier compound is dissolved in an inert matrix
Gas sensors
Usually a combination of ISE and gas-permeable membrane

20. pH electrode Shielded connecting cable Non-conducting glass bodymV
Non-conducting
glass body
External
reference
electrode
Internal reference
electrode
H+-responsive
glass membrane

21. pCO2 electrode External reference electrode Electrode assemblymV
External
reference
electrode
NaHCO3/H2O
Electrode
assembly
Gas-permeable
membrane
(silicone rubber)
CO2 + H2O  HCO3- + H+
Flow Cell
CO2(g)

22. NH3 electrode External reference electrode Electrode assemblymV
External
reference
electrode
NH4Cl/H2O
Electrode
assembly
Gas-permeable
membrane
(PTFE)
H2O + NH3  NH4+ + OH-
Flow Cell
NH3(g)

23. Other glass electrodes
Glass electrodes are used to measure Na+
There is some degree of cross-reactivity between H+ and Na+
There are glass electrodes for K+ and NH4+, but these are less useful than other electrode types

24. The Sodium Error (or, direct vs. indirect potentiometry)mV
Na+
Whole
blood
Cells (45%)
Aqueous phase
Lipids, proteins
Plasma
Since potentiometry measures the
activity of the ion at the electrode
surface, the measurement is
independent of the volume of
sample.

25. The Sodium Error (or, direct vs. indirect potentiometry)mV
In indirect potentiometry, the concentration
of ion is diluted to an activity near unity.
Since the concentration will take into
account the original volume and dilution
factor, any excluded volume (lipids, proteins)
introduces an error, which usually is insignificant.
Na+

26. So which is better?
Direct potentiometry gives the true, physiologically active sodium concentration.
However, the reference method for sodium is atomic emission, which measures the total concentration, not the activity, and indirect potentiometry methods are calibrated to agree with AE.
So, to avoid confusion, direct potentiometric methods ordinarily adjust the result to agree with indirect potentiometric (or AE) methods.

27. Then what’s the “sodium error” all about?
When a specimen contains very large amounts of lipid or protein, the dilutional error in indirect potentiometric methods can become significant.
Hyperlipidemia and hyperproteinemia can result in a pseudo-hyponatremia by indirect potentiometry.
Direct potentiometry will reveal the true sodium concentration (activity).

28. Sodium error
Na+
138 mM
Na+
130 mM
Na+
140 mM
Na+
140 mM

29. But. . .why does it only affect sodium?
It doesn’t only affect sodium. It effects any exclusively aqueous component of blood.
The error is more apparent for sodium because the physiological range is so narrow.

30. Solid state chloride electrode
AgCl and Ag2S are pressed into a pellet that forms the liquid junction (ISE membrane)
Cl- ions diffuse into vacancies in the crystal lattice, and change the membrane conductivity

31. Liquid/polymer membrane electrodes
Typically involves an ionophore dissolved in a water-insoluble, viscous solvent
Sometimes called ion-exchange membrane electrodes
The ionophore determines the specificity of the electrode

32. K+ ion-selective electrode
Valinomycin is an antibiotic that has a rigid 3-D structure containing pores with dimensions very close to the un-hydrated radius of the potassium ion. Valinomycin serves as a neutral carrier for K+. K+

33. Ca++ ion selective electrode
di-p-octylphenyl phosphate
PVC membrane
Ca++

34. Ca++ ion selective electrode
Neutral carrier
Inert membrane
Ca++

35. Amperometry
Whereas potentiometric methods measure electrochemical potential, amperometric methods measure the flow of electrical current
Potential (or voltage) is the driving force behind current flow
Current is the amount of electrical flow (electrons) produced in response to an electrical potential

36. Amperometry Current (mA)  Applied potential (V)  Limiting current
Half-wave potential
Applied potential (V) 

37. Amperometry 2•C0 C0 0.5•C0 Current (mA)  Applied potential (V) 
Half-wave potential
Applied potential (V) 

38. Oxygen (pO2) electrode Reference electrode (anode) Platinum wire
-0.65V
Reference electrode
(anode)
Platinum wire
(cathode)
Gas-permeable
membrane O2
Flow cell 

39. Reaction at the platinum electrode
The amount of current (e-) is proportional to the concentration of O2

40. The glucose electrode
Glucose
oxidase
H2O2 + Gluconic acid
Glucose + O2
O2 + 2H-
2e-
(+0.6 V)
O2 electrode
Note that the platinum electrode now carries a positive potential