Review of Analytical Methods Part 2: Electrochemistry

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Review of Analytical MethodsPart 2
Electrochemistry

• Roger L. Bertholf, Ph.D.
• Associate Professor of Pathology
• Chief of Clinical Chemistry Toxicology
• University of Florida Health Science
  Center/Jacksonville

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Analytical methods used in clinical chemistry

• Spectrophotometry
• Electrochemistry
• Immunochemistry
• Other
• Osmometry
• Chromatography
• Electrophoresis

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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

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Electrochemical potential

• Factors that affect electrochemical potential
• Distance/shielding from nucleus
• Filled/partially filled orbitals

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Relative potential
Zn
Cu
e-

• 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

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e- ? e- ? e- ?
Zn0 ? Zn2 2e-
Cu2 2e- ? Cu0
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The Nernst Equation
Where E Potential at temperature T E0
Standard electrode potential (25ºC, 1.0M) R
Ideal gas constant F Faradays constant n
number of electrons transferred
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Zn0 ? Zn2 2e- E0 (-)0.7628 V
Cu2 2e- ? Cu0 E0 0.3402 V
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Electromotive force
Ecell Ecathode Elj - Eanode
Ecell ECu(II),Cu Elj EZn(II),Zn
Ecell ()0.340 Elj (-)0.763
Ecell ()1.103 Elj
?G -nFEcell
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Would the reaction occur in the opposite
direction?
Ecell Ecathode Elj - Eanode
Ecell EZn(II)?Zn Elj ECu(II) ? Cu
Ecell (-)0.763 Elj ()0.340
Ecell (-)1.103 Elj
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How do we determine standard electrode potentials?

• Absolute potential cannot be measuredonly 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

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The Hydrogen Electrode
H2 gas ?
2H 2e- ? H2 E0 0.0 V
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The Calomel Electrode
Hg2Cl2 2e- ? 2Hg0 2Cl- E0 0.268V
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The Silver/Silver Chloride Electrode
AgCl e- ? Ag0 Cl- E0 0.222V
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Ion-selective Electrodes
Ecell ERef(1) Elj ERef(2)
Ref1
Ref2
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Typical ISE design
Ecell ERef(1) Elj ERef(2)
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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
  ?

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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)

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Types of ISE

• Glass
• 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

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pH electrode
Shielded connecting cable
External reference electrode
Non-conducting glass body
Internal reference electrode
H-responsive glass membrane
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pCO2 electrode
Electrode assembly
Gas-permeable membrane (silicone rubber)
CO2 H2O ? HCO3- H
Flow Cell
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NH3 electrode
Electrode assembly
Gas-permeable membrane (PTFE)
H2O NH3 ? NH4 OH-
Flow Cell
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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

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The Sodium Error(or, direct vs. indirect
potentiometry)
Since potentiometry measures the activity of the
ion at the electrode surface, the measurement
is independent of the volume of sample.
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The Sodium Error(or, direct vs. indirect
potentiometry)
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.
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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.

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Then whats 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).

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Sodium error
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But. . .why does it only affect sodium?

• It doesnt 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.

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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

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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

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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.
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Ca ion selective electrode
PVC membrane
Ca
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Ca ion selective electrode
Inert membrane
Ca
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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

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Amperometry
Limiting current
Current (mA) ?
Half-wave potential
Applied potential (V) ?
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Amperometry
Current (mA) ?
Half-wave potential
Applied potential (V) ?
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Oxygen (pO2) electrode
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Reaction at the platinum electrode

• The amount of current (e-) is proportional to the
  concentration of O2

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The glucose electrode
Glucose O2

• Note that the platinum electrode now carries a
  positive potential