review of analytical methods part 2: electrochemistry roger l. bertholf, ph.d. associate professor...
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Review of Analytical MethodsPart 2: ElectrochemistryReview 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
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
Analytical methods used in clinical chemistry
• Spectrophotometry
• Electrochemistry
• Immunochemistry
• Other– Osmometry– Chromatography– Electrophoresis
• Spectrophotometry
• Electrochemistry
• Immunochemistry
• Other– Osmometry– Chromatography– Electrophoresis
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ElectrochemistryElectrochemistry
• 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
• 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 potentialElectrochemical potential
• Factors that affect electrochemical potential:– Distance/shielding from nucleus– Filled/partially filled orbitals
• Factors that affect electrochemical potential:– Distance/shielding from nucleus– Filled/partially filled orbitals
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Zn Cue-
Relative potentialRelative potential
• 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
• 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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Cu2+Zn2+
Zn0 Cu0
Zn0 Zn2+ + 2e- Cu2+ + 2e- Cu0
e- e- e-
mV
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The Nernst EquationThe Nernst Equation
[Oxidized]
]Reduced[log
303.2
[Oxidized]
]Reduced[ln 00
nF
RTE
nF
RTEE
Where E = Potential at temperature TE0 = Standard electrode potential (25ºC, 1.0M)R = Ideal gas constantF = Faraday’s constantn = number of electrons transferred
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Cu2+Zn2+
Zn0 Cu0
Zn0 Zn2+ + 2e-
E0 = +(-)0.7628 VCu2+ + 2e- Cu0
E0 = +0.3402 V
Elj
mV
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Electromotive forceElectromotive 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?
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?
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
• 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
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The Hydrogen ElectrodeThe Hydrogen Electrode
H2 gas
2H+ + 2e- H2
E0 = 0.0 V
mV
Test electrode
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The Calomel ElectrodeThe Calomel Electrode
Calomel paste (Hg0/Hg2Cl2)
Saturated KCl
Liquid junction
mV
Test electrode
Hg2Cl2 + 2e- 2Hg0 + 2Cl-
E0 = 0.268V
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The Silver/Silver Chloride ElectrodeThe Silver/Silver Chloride Electrode
Silver wire
SaturatedKCl + AgNO3
Liquid junction
mV
Test electrode
AgCl + e- Ag0 + Cl-
E0 = 0.222V
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Ion-selective ElectrodesIon-selective Electrodes
Ref1 Ref2
mV
Ecell = ERef(1) + Elj – ERef(2)
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Typical ISE designTypical ISE design
Ref
1 Ref
2
mV
Ecell EISM
Ion-selectivemembrane++
+
+
+
+
+
+
+
+
+
Ecell = ERef(1) + Elj – ERef(2)
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Activity and concentrationActivity 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 :
• 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 :
][][][ XmXXa
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The activity coefficientThe 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)
• 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 ISETypes 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
• 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 electrodepH electrodemV
Externalreferenceelectrode
Non-conductingglass body
Internal referenceelectrode H+-responsive
glass membrane
Shielded connectingcable
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pCO2 electrodepCO2 electrodemV
Externalreferenceelectrode
CO2(g)
Flow Cell
Electrodeassembly
Gas-permeablemembrane
(silicone rubber)
NaHCO3/H2O
CO2 + H2O HCO3- + H+
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NH3 electrodeNH3 electrodemV
Externalreferenceelectrode
NH3(g)
Flow Cell
Electrodeassembly
Gas-permeablemembrane
(PTFE)
NH4Cl/H2O
H2O + NH3 NH4+ + OH-
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Other glass electrodesOther 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
• 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)
The Sodium Error(or, direct vs. indirect potentiometry)
Na+
Wholeblood
Cells (45%)
Aqueous phase
Lipids, proteins
Plasma
mV
Since potentiometry measures theactivity of the ion at the electrodesurface, the measurement isindependent of the volume of sample.
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The Sodium Error(or, direct vs. indirect potentiometry)
The Sodium Error(or, direct vs. indirect potentiometry)
Na+
mV
In indirect potentiometry, the concentrationof ion is diluted to an activity near unity. Since the concentration will take into account the original volume and dilutionfactor, any excluded volume (lipids, proteins)introduces an error, which usually is insignificant.
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So which is better?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.
• 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 what’s the “sodium error” all about?
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).
• 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 errorSodium error
Na+
140 mMNa+
140 mM
Na+
138 mMNa+
130 mM
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But. . .why does it only affect sodium?
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.
• 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.
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Solid state chloride electrodeSolid 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
• 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
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
• 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 electrodeK+ ion-selective electrode
K+N
OO
O
N O
O
O
N
OO
ON
OO
O
NO
O
O
N
OO
O
H
H
H
H
H
H
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 electrodeCa++ ion selective electrode
Ca++
di-p-octylphenyl phosphate
H3CO
P
OH3C
O
O-
H3CO
P
OH3C
O
O-
PVC membrane
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Ca++ ion selective electrodeCa++ ion selective electrode
Ca++
Neutral carrier
N
O
H3C O CH3
OH3C
OH3C
N
O
H3C O CH3
O
O
Inert membrane
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AmperometryAmperometry
• 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
• 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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AmperometryAmperometryC
urre
nt (
mA
)
Applied potential (V)
Half-wave potential
Limiting current
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AmperometryAmperometryC
urre
nt (
mA
)
Applied potential (V)
Half-wave potential
C0
0.5•C0
2•C0
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Gas-permeablemembrane
Platinum wire(cathode)
-0.65V
Reference electrode(anode)
Oxygen (pO2) electrodeOxygen (pO2) electrode
Flow cell O2
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Reaction at the platinum electrode
Reaction at the platinum electrode
• The amount of current (e-) is proportional to the concentration of O2
• The amount of current (e-) is proportional to the concentration of O2
O2 + 2H+ + 2e- H2O2Pt
-0.6 V
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The glucose electrodeThe glucose electrode
• Note that the platinum electrode now carries a positive potential
• Note that the platinum electrode now carries a positive potential
Glucose + O2
Glucose
oxidaseH2O2 + Gluconic acid
O2 electrode
O2 + 2H-
2e-
(+0.6 V)