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

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

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

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

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

Cu2+Zn2+

Zn0 Cu0

Zn0 Zn2+ + 2e- Cu2+ + 2e- Cu0

e- e- e-

mV

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

Cu2+Zn2+

Zn0 Cu0

Zn0 Zn2+ + 2e-

E0 = +(-)0.7628 VCu2+ + 2e- Cu0

E0 = +0.3402 V

Elj

mV

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

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

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

The Hydrogen ElectrodeThe Hydrogen Electrode

H2 gas

2H+ + 2e- H2

E0 = 0.0 V

mV

Test electrode

The Calomel ElectrodeThe Calomel Electrode

Calomel paste (Hg0/Hg2Cl2)

Saturated KCl

Liquid junction

mV

Test electrode

Hg2Cl2 + 2e- 2Hg0 + 2Cl-

E0 = 0.268V

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

Ion-selective ElectrodesIon-selective Electrodes

Ref1 Ref2

mV

Ecell = ERef(1) + Elj – ERef(2)

Typical ISE designTypical ISE design

Ref

1 Ref

2

mV

Ecell EISM

Ion-selectivemembrane++

+

+

+

+

+

+

+

+

+

Ecell = ERef(1) + Elj – ERef(2)

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

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)

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

pH electrodepH electrodemV

Externalreferenceelectrode

Non-conductingglass body

Internal referenceelectrode H+-responsive

glass membrane

Shielded connectingcable

pCO2 electrodepCO2 electrodemV

Externalreferenceelectrode

CO2(g)

Flow Cell

Electrodeassembly

Gas-permeablemembrane

(silicone rubber)

NaHCO3/H2O

CO2 + H2O HCO3- + H+

NH3 electrodeNH3 electrodemV

Externalreferenceelectrode

NH3(g)

Flow Cell

Electrodeassembly

Gas-permeablemembrane

(PTFE)

NH4Cl/H2O

H2O + NH3 NH4+ + OH-

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

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.

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.

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.

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).

Sodium errorSodium error

Na+

140 mMNa+

140 mM

Na+

138 mMNa+

130 mM

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.

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

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

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+.

Ca++ ion selective electrodeCa++ ion selective electrode

Ca++

di-p-octylphenyl phosphate

H3CO

P

OH3C

O

O-

H3CO

P

OH3C

O

O-

PVC membrane

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

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

AmperometryAmperometryC

urre

nt (

mA

)

Applied potential (V)

Half-wave potential

Limiting current

AmperometryAmperometryC

urre

nt (

mA

)

Applied potential (V)

Half-wave potential

C0

0.5•C0

2•C0

Gas-permeablemembrane

Platinum wire(cathode)

-0.65V

Reference electrode(anode)

Oxygen (pO2) electrodeOxygen (pO2) electrode

Flow cell O2

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

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)