This document provides an overview of electrochemistry techniques used in clinical chemistry, including potentiometry, voltammetry, coulometry, and conductometry. It describes the basic concepts such as electrochemical cells and electrodes. Potentiometry techniques like ion-selective electrodes and their applications in measuring electrolytes are discussed in detail. Other techniques like amperometry and different types of voltammetry and coulometry are also summarized along with their uses and advantages.

1. ELECTROCHEMISTRY
IN CLINICAL CHEMISTRY
BASIC CONCEPTS, INSTRUMENTATION
AND APPLICATION
By
Dr. BASIL, B – MBBS (Nigeria),
Department of Chemical Pathology/Metabolic Medicine,
Benue State University Teaching Hospital, Makurdi
March, 2015

2. OUTLINE
• Introduction
– Electrochemical Cells
• Potentiometry
– Ion-Selective Electrodes
• Types
• Basic concepts
• Instrumentation
• Preanalytical Considerations
• Quality Control
– Merits and Limitations
– Applications
• Voltametry/Amperometry
• Coulometry
• Conductometry

3. INTRODUCTION
• Electrochemistry involves the measurement of
electrical signals associated with chemical
systems.
• Electrochemical analyses used in the clinical
laboratory include: Potentiometry,
Amperometry/Voltametry, Coulometry and
Conductometry
• The two basic electrochemical cells involved in
these analyses are Galvanic and Electrolytic cells

4. ELECTROCHEMICAL CELLS
• Consist of two half-cells and a salt bridge saturated with
electrolytes, or in a solution which serves as the salt bridge.
• Electrochemical activity of one half-cell cannot be measured,
both must be coupled, and one compared with the other
• To rate a half-cell reaction, a specific electrode reaction is
arbitrarily assigned 0.00V.
• Any other reaction coupled to it is either positive or negative
depending on the relative affinity for electrons
• The electrode defined as 0.00V is the standard hydrogen
electrode: H2 gas at 1atm.

5. • The hydrogen electrode coupled with a Zn half-cell is cathodic,
and with Cu half-cell is anodic
• The potential generated by the hydrogen-gas electrode is used
to rate the electrode potential of metals in 1mol/L solution
GALVANIC CELL:
• There is spontaneous flow of electrons from the electrode with
the lower electron affinity (oxidation e.g silver) as the electrodes
are connected
• The electrons pass through the external meter to the cathode
(reduction), where OH- are liberated
• This reaction continues until one of the chemical components is
depleted, thus, the cell becomes dead and cannot produce
electrical energy to the external meter
ELECTROLYTIC CELL:
• When an external electromotive force, E, is applied > electrons
will flow in the reverse direction
• Thus a Galvanic cell can be built from an Electrolytic cell by
turning off the external E

6. POTENTIOMETRY
• Measurement of the electrical potential difference btw two
electrodes in a galvanic electrochemical cell
• These electrodes – Reference and Indicator(measuring)
electrodes – generate an EMF when the cell current is zero
• The EMF is measured using a potentiometer e.g pH meter –
which does not alter the cell’s composition or draw current
• One of the electrolyte solutions is the sample containing
the analyte(s) of interest – can be replaced by a reference
solution for calibration purposes
• The potential gradients have been classified as
– Redox potentials, Membrane potentials or Diffusion potentials
• The cell can be devised such that all the gradients are kept
constant except one – which is related to the activity a
specific ion of interest
• The activity or EMF generated is then related to the
concentration of the ion (analyte) in the solution (sample).

7. Schematic diagram of an electrochemical cell of potentiometric measurement
Types of potentiometric electrodes used for clinical applications
include:
• Redox electrodes
• Ion-selective electrodes, and
• pCO2 electrodes

8. ION-SELECTIVE ELECTRODES
• Potentiometric methods of analysis involve the direct
measurement of electrical potential due to the activity of free ions
• ISEs are designed to be selectively sensitive toward individual ions
• The potential produced at the membrane-sample-solution
interface is proportional to the log of the ionic activity or
concentration
• Ion-Selective membranes typically consist of glass, crystalline, or
polymeric materials
• The chemical composition of the membrane is designed to achieve
an optimal permselectivity toward the ion of interest
• Other ions exhibit slight interaction with membrane sites and
display some degree of interferences
• Clinically, a correction is required when the interference exceed an
acceptable value
• Measurements with ISE are simple, often rapid, nondestructive,
and applicable to a wide range of concentrations

10. TYPES OF ISE:
• Ion-selective membrane is the heart of the ISE as it controls
the selectivity of the electrode. They include:
– Inert metal electrodes in contact with a Redox couple e.g
Standard Hydrogen electrode
– Metal electrodes participating in a redox reaction, e.g Ag/AgCl
electrode
– and Membrane electrodes
• The membrane can be solid (e.g glass), liquid (e.g ion-
exchange electrodes), or special membrane (e.g compound
electrodes) such as Gas-sensing and Enzyme electrodes
• The ISEs that are commonly used in clinical chemistry are
the membrane (esp Glass and Polymer) electrodes.
GLASS MEMBRANE ELECTRODE:
• Used to measure pH and Na+ and as an internal transducer
for pCO2 sensors
• They are formulated from melts of Si and/or Al-oxide mixed
with other oxides

11. • By varying the glass composition, electrodes with selectivity for
H+, Na+, K+, Li+, Rb+, Cs+, Ag+, Tl+, and NH4+ have been
produced with only those of H+ and Na+ having sufficient
selectivity
• A Glass ISE universally used in the clinical lab is the pH
electrode
The pH Electrode (Prototype):
• Basic components of the pH meter include:
Indicator Electrode:
• Silver wire coated with AgCl immersed into an internal solution
of 0.1mmol/L HCl, and placed into a tube containing special
glass membrane tip
• The membrane is only sensitive to H+ and consist of specific
quantities of Li, Cesium, La, Ba, or Al oxides in silicate
• When placed into the sample solution, H+ move to the tip of
the electrode and produce a potential difference btw internal
solution and the sample solution which is measured as pH
• The glass continually dissolves very slowly from the surface
(lasts for several yrs)

13. • Ionic exchange occurs only in the gel layer – there is
no penetration of H+ through the glass
• It is highly selective for H+, but other cations in high
concentration, esp Na+, can interfere
Reference Electrode:
• Commonly used is the calomel electrode.
• Calomel – a paste of mainly Hg2Cl2 in direct contact
with metallic Hg in an electrolyte solution of KCl
• At constant electrolyte conc. and temp, a stable
voltage is generated at the interphase of the mercury
and its salt
• A filling hole is needed for adding KCl and a tiny
opening at the bottom for completion of electric
contact btw the 2 electrodes
• The liquid junction consist of a fiber or ceramic plug
that allows a small flow of electrolyte filling solution

14. • Hg/HgCl as a reference electrode is
– slow to reach stable voltage following voltage change, and
– unstable above 80oC
• Ag/AgCl as a reference electrode
– can be used at high temp up to 275oC, and
– the AgCl-coated Ag wire make a more compact electrode than Hg
• In measurements where Chloride contamination must be
avoided, Hg-sulfate and K-sulfate reference electrode can be
used
• Reference electrodes generally consist of a metal and its salt,
in contact with a solution containing the same anion.
Liquid Junctions:
• Allows a slow flow of electrolyte from the tip of the reference
electrode, thus, establishing electrical connection btw the
indicator and reference electrode
• A junction potential set up at the boundary btw two different
solutions because of positive and negative ions diffusing
across the boundary at unequal rates

15. This may increase or decrease the potential of the reference
electrode, thus, it should be kept at a minimum reproducible value
when the reference electrode is in solution
Readout Meter:
• Zero potential indicates that both electrodes are generating
same voltage (assuming there is no liquid junction potential)
• Isopotential – temp change has no effect on the response of the
cell
• Achieved by making midscale (pH 7.0) correspond to 0V at all
temps via the use of an internal buffer that alters with response
to temp changes
Nernst equation:
• Describes the EMF generated by H+ at the glass tip
ԑ = ∆pH x (RT ln 10)/F = ∆pH x 0.059V
Where, ԑ = EMF, F = Faraday constant, R = molar gas constant, and T =
temp in kelvin
• As temp increases, H+ activity increases and the potential
generated increases
• Most pH meters are manufactured for greatest accuracy in the
10 – 60oC range

16. pH Combination Electrode:
• Has both indicator and reference electrodes in one
small probe
• Consists of Ag/AgCl internal reference electrode
sealed in a narrow glass cylinder with a pH-sensitive
glass tip
LIQUID MEMBRANE ELECTRODE – (POLYMER
MEMBRANE ISE):
• Porous membrane impregnated with solution (2-
ethylhexyl phosphoric acid dissolved in dioctyl phenyl
phosphate) and mounted at the end of an electrode
body
• This was later modified into a plasticized poly(vinyl
chloride) membrane – Polymer Membrane ISE – it is a
more rugged and convenient to use.

17. • They are the dominant class of potentiometric
electrodes used in modern clinical analysis
instruments
• Calcuim ISE:
– Dioctylphenyl phosphate (an ion-selective carrier)
dissolved in an inert water-insoluble solvent, diffuses
though a porous membrane
– Since the solvent is insoluble in water, test samples cannot
cross the membrane but Ca2+ are exchanged
• Potassium ISE:
– Use antibiotic valinomycin (major breakthrough), which
show great selectivity for K+ over Na+, as the carrier
– The K+ ISE based on valinomycim is extensively used today
for the routine measurement of K+ in blood and urine due
to its wide linear range of over three orders of magnitude

18. • Are recharged every few months to replace the liquid ion
exchanger membrane and the porous membrane
• Interference from other cations, seen as deviation from
linearity, is not apparent at K+ activities >10-4 mol/L
• Other less selective polymembrane ISEs (Mg2+ and Li+) are
subject to interferences from Ca2+/Na+ and Na+
(respectively)
SPECIAL MEMBRANES:
Gas-Sensing Electrodes:
• Similar to pH electrodes but are designed to detect specific
gases (e.g CO2 and NH3) in solutions and are usually
separated from the solution by a thin, gas-permeable
hydrophobic membrane
• CO2 permselectively diffuses into a thin film of NaCO3 soln
• The diffusion changes the pH of the soln and that change is
detected by a pH electrode within it
pCO2 electrode = pH electrode + CO2 membrane

19. • In the NH3 gas electrode, the NaCO3 soln is replaced by
NH4Cl
• Other gas-sensing electrodes function on the basis of
an amperometric principle e.g pO2, Glucose, and
Peroxidase
– Glucose determination is based on reduction in pO2 during
glucose oxidase reaction with glucose and O2
– Peroxidase electrode is dependent on oxidation of H2O2 to
release O2 which is detected
Enzyme Electrodes:
• ISE covered with immobilized enzymes that can
catalyze a specific chemical reaction and the reaction
product detected.
• They include:
– Urease in Urea electrode with an ISE selective for NH3 or
NH4+
– Glucose oxidase used in combination with a pH electrode

20. The pCO2 electrode

21. PRE-ANALYTICAL AND ANALYTICAL
CONSIDERATIONS
SAMPLE COLLECTION:
• Blood Samples:
– Serum or plasma specimen should be collected in heparinised
bottles
– Capillary blood (microsample tubes, capillary tubes or directly
from fingerstick) to some POC devices
– Heparinized whole blood (arterial or venous) for blood gases
should be read promptly
– Samples should be centrifuged unopened and the plasma
separated immediately
– Grossly lipaemic samples should be ultra centrifuged before
analysis
• Urine Samples:
– Assessment of urine sodium, potassium or chloride
– Samples for gases should be layered with oil

22. • Faeces, aspirates and fluids drained from portions of
the GIT can be analyzed for electrolytes
ANALYTICAL VARIATIONS:
• Interferences from undesired ions (most serious
problem limiting ISEs) – no ISE is completely ion
specific
• Sensitive to other ions having similar properties e.g.
Nitrate electrode may have ionic interferences from
perchlorate, iodide, chloride and sulfate
• Blockage of the selective membranes with time by
repeated coating with proteins
• Leakages in the membranes would lead to
reduced/lack of analytical selectivity
• Exposure of electrodes and galvanometer to cold
temperatures would reduce sensitivity

23. QUALITY CONTROL
• Periodic changing of the selective membranes
• Ensure appropriate temperature control protocols
• Ensure adequate and appropriate power supply
• Ensure MANUAL is read thoroughly by all USERS
• All mechanical faults should be promptly reported
to the appropriate channels
• Equipment should be recalibrated periodically by
delegated experts

24. MERITS AND DEMERITS OF POTENTIOMETRY
• Advantages:
– Relatively inexpensive and simple to use and have an extremely
wide range of applications and wide concentration range.
– They can be used very rapidly and easily.
– ISEs can measure both positive and negative ions.
– They are unaffected by sample colour or turbidity.
– Non-destructive: no consumption of analyte.
– Non-contaminating.
– Short response time: in sec. or min. useful in industrial
applications.
• Limitations:
– Precision is rarely better than 1%.
– Electrodes can be affected by proteins or other organic solutes.
– Interference by other ions.
– Electrodes are fragile and have limited shelf life.

25. APPLICATIONS OF POTENTIOMETRY
• Clinical Chemistry
– Ion-selective electrodes are important sensors for clinical
samples because of their selectivity for analytes.
– The most common analytes are electrolytes, such as Na+, K+,
Ca2+,H+, and Cl-, and dissolved gases such as CO2.
• Environmental Chemistry
– For the analysis of CN-, F-, NH3, and NO3
- in water and
wastewater.
• Potentiometric Titrations
– pH electrode used to monitor the change in pH during the
titration.
– For determining the equivalence point of an acid–base titration.
– Possible for acid–base, redox, and precipitation titrations, as
well as for titrations in aqueous and non-aqueous solvents.

26. • Agriculture
– NO3, NH4, Cl, K, Ca, I, CN in soils, plant material,
fertilizers.
• Detergent Manufacture
– Ca, Ba, F for studying effects on water quality
• Food Processing
– NO3, NO2 in meat preservatives
– Salt content of meat, fish, dairy products, fruit juices,
brewing solutions.
– F in drinking water and other drinks.
– Ca in dairy products and beer.
– K in fruit juices and wine making.
– Corrosive effect of NO3 in canned foods.

27. OTHER ELECTROCHEMICAL TECHNIQUES
VOLTAMMETRY:
• Measures current in an electrochemical cell as a function of
the applied potential
• Assortment of electrodes allows for a very specific analysis
of different chemicals
• Types:
– Polarography, Hydrodynamic voltammetry, Stripping
voltammetry, and Amperometry
• Amperometry:
– apply a constant potential to the electrode and measure the
resulting current.
– most often used in the construction of chemical sensors for the
quantitative analysis of single analytes.
– Example is the Clark O2 electrode, which responds to the
concentration of dissolved O2 in solutions such as blood and
water.

28. COULOMETRY:
• Measures electrical charge passing btw two electrodes
• Based on Faraday’s law:
– the total charge passed during an electrolysis is proportional to
the amount of reactants and products in the redox reaction
• If the electrolysis is 100% efficient (i.e only the analyte is
oxidized or reduced), then we can use the total charge or
current to determine the amount of analyte in a sample
• Types:
– In controlled-potential coulometry – a constant potential is
applied and the resulting current is measured as a function of
time
– In controlled-current coulometry – current is held constant and
the time required to completely oxidize or reduce the analyte is
measured
• Gold standard for determination of chloride in serum/plasma
• It is also a mode of transduction for some biosensors
• Interferences: anions with greater affinity for Ag+ e.g Br-

29. CONDUCTOMETRY:
• Determines the quantity of an analyte in a mixture by
measuring its effect on the electrical conductivity of the
mixture
• The current is directly proportional to the solutions
conductance
• Used in the measurement of the volume fraction of
erythrocytes in whole blood (hematocrit)
– They are insulators because of their lipid-based membrane
composition
– Interference: abnormal protein conc and insufficient mixing of
sample will change plasma conductivity
• Applied in the electronic counting of blood cells in
suspension – Coulter principle
– Cells are forced through a tiny orifice flanked by two electrodes
(btw which a constant current is established) which generate
signal (increased resistance) with the passage of cells between
them
– The pulses are amplified and counted

30. REFERENCES
• Tietz Textbook of Clinical Chemistry and
Molecular Diagnostics.
• Clinical Chemistry; Principles, Techniques and
Correlations, 7th ed., by Bishop et al.
• http://chemwiki.ucdavis.edu/Analytical_Chemis
try/Analytical_Chemistry_2.0/11_Electrochemic
al_Methods/11E%3A_Summary_and_Problems
• http://www.cma-science.nl
• www2.vernier.com/booklets/ise.pdf
• http://www.sfu.ca/chemistry/groups/Li/chem2
15/selective.PDF