Transcript Review of Analytical Methods Part 2: Electrochemistry

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
Analytical methods used in
clinical chemistry
•
•
•
•
Spectrophotometry
Electrochemistry
Immunochemistry
Other
– Osmometry
– Chromatography
– Electrophoresis
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
Electrochemical potential
• Factors that affect electrochemical potential:
– Distance/shielding from nucleus
– Filled/partially filled orbitals
Relative potential
Zn
e-
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
mV
e-  e-  e- 
Zn0
Cu0
Zn2+
Cu2+
Zn0  Zn2+ + 2e-
Cu2+ + 2e-  Cu0
The Nernst Equation
RT [Reduced]
2.303RT
[Reduced]
0
EE 
ln
E 
log
nF [Oxidized]
nF
[Oxidized]
0
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
mV
Zn0
Cu0
Zn2+
Cu2+
Elj
Zn0  Zn2+ + 2eE0 = +(-)0.7628 V
Cu2+ + 2e-  Cu0
E0 = +0.3402 V
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
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?
• 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 Electrode
mV
Test electrode
H2 gas 
2H+ + 2e-  H2
E0 = 0.0 V
The Calomel Electrode
mV
Test electrode
Calomel paste (Hg0/Hg2Cl2)
Saturated KCl
Hg2Cl2 + 2e-  2Hg0 + 2ClE0 = 0.268V
Liquid junction
The Silver/Silver Chloride Electrode
mV
Test electrode
Silver wire
Saturated
KCl + AgNO3
AgCl + e-  Ag0 + ClE0 = 0.222V
Liquid junction
Ion-selective Electrodes
mV
Ecell = ERef(1) + Elj – ERef(2)
Ref1
Ref2
Typical ISE design
mV
Ecell = ERef(1) + Elj – ERef(2)
Ecell  EISM
Ref 2
Ref 1
+
+
+
+
+
+
+
+
+
+
+
Ion-selective
membrane
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 :
a[ X ]   [ X ]  m[ X ]



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)
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
pH electrode
Shielded connecting
cable
Non-conducting
glass body
Internal reference
electrode
mV
External
reference
electrode
H+-responsive
glass membrane
pCO2 electrode
mV
Electrode
assembly
Gas-permeable
membrane
(silicone rubber)
NaHCO3/H2O
CO2 + H2O  HCO3- + H+
Flow Cell
CO2(g)
External
reference
electrode
NH3 electrode
mV
Electrode
assembly
Gas-permeable
membrane
(PTFE)
NH4Cl/H2O
H2O + NH3  NH4+ + OH-
Flow Cell
NH3(g)
External
reference
electrode
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
The Sodium Error
(or, direct vs. indirect potentiometry)
mV
Lipids, proteins
Na+
Aqueous phase
Plasma
Cells (45%)
Whole
blood
Since potentiometry measures the
activity of the ion at the electrode
surface, the measurement is
independent of the volume of
sample.
The Sodium Error
(or, direct vs. indirect potentiometry)
mV
Na+
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.
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.
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).
Sodium error
Na+
Na+
138 mM
130 mM
Na+
140 mM
Na+
140 mM
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.
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
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
K+ ion-selective electrode
H
N
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+.
H
O
N
O
O
O
O
O
O
O
N
O
H
H
+
K
O
N
O
O
O
O
O
O
O
N
O
N
H
H
Ca++ ion selective electrode
di-p-octylphenyl phosphate
PVC membrane
H3C
O
O
P
H3C
O
O
-
Ca++
O
O
H3C
P
O
H3C
O
-
Ca++ ion selective electrode
Neutral carrier
Inert membrane
H3C
O
CH3
O
CH3
N
O
O
H3C
O
Ca++
H3C
O
O
O
N
H3C
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
Amperometry
Current (mA) 
Limiting current
Half-wave potential
Applied potential (V) 
Amperometry
2•C0
Current (mA) 
C0
0.5•C0
Half-wave potential
Applied potential (V) 
Oxygen (pO2) electrode
-0.65V
Reference electrode
(anode)
Platinum wire
(cathode)
Gas-permeable
membrane
Flow cell 
O2
Reaction at the platinum
electrode
+
-
O2 + 2H + 2e
Pt
-0.6 V
H2O2
• The amount of current (e-) is proportional to
the concentration of O2
The glucose electrode
Glucose + O2
Glucose
oxidase
H2O2 + Gluconic acid
2e-
O2 + 2H-
O2 electrode
(+0.6 V)
• Note that the platinum electrode now carries
a positive potential