Arterial blood gases

ELECTROCHEMISTRY , CHEMICAL
SENSORS & ABG
Dr. Smily Pruthi , MD- Biochemistry
See video in mobile app –
tulip academy of medical sciences

TYPES OF ELECTROCHEMICAL
ANALYSIS
1. POTENTIOMETRY & ISE
2. VOLTAMETRY , AMPEROMETRY &
POLAROGRAPHY
3. CONDUCTOMETRY
4. COULOMETRY
Website : www.tamsmed.com

POTENTIOMETRY & ISE
• Measurement of electrical potential difference between two
electrodes in an electrochemical cell when the cell current is
zero.
• Widely used clinically for the measurement of pH,PCO2
& electrolytes.
• The common pH meter is a special type

PH METER

TYPES OF ELECTRODES USED IN
POTENTIOMETRY
• Redox electrodes
Inert metal electrodes
Metal electrodes participating in Redox Reactions
• Ion selective electrodes
Glass
Polymer type
• PCO2 electrodes

REDOX ELECTRODES
• Redox potential is generated as a result of chemical
equilibria involving electron transfer reaction
Ox form + ne- Reduced form
• Electrode potential for a redox couple is defined as the
couple’s potential measured with respect to standard
hydrogen electrode, which is set to zero.

NERNST EQUATION……
• E = EO – N/n Χ log a Red/aOx
= E0 – 0.0592/n Χ log a Red/aOx
N is Nernst factor,
N = R Χ T Χ In 10 / F

TYPES OF REDOX ELECTRODES..
• Inert metal electrodes
• Platinum & gold are examples
• Hydrogen electrode being a special redox electrode for pH
measurement.

METAL ELECTRODES PARTICIPATING IN
REDOX REACTIONS..
• Silver-silver chloride
electrode is an eg.
• It can be used both as an
internal or external
reference electrode.
• A porous membrane is
employed to separate
conc. KCl from sample
solution.
– Mechanical barrier
– Diffusional barrier

METAL ELECTRODES PARTICIPATING IN
REDOX REACTIONS..
• Calomel electrode:
• Consists of mercury
covered by a layer of
calomel (Hg2Cl2), which is in
contact with electrolyte
solution containing Cl-
• Used as reference electrode
for pH measurements using
glass pH electrodes.

ION SELECTIVE ELECTRODES…
• The ion selective
membrane is the heart of
an ISE as it controls the
selectivity of the
electrode.
• Membrane potential α log
of ionic activity or ion
conc.
• Types:
• Glass electrode
• Polymer membrane
electrode

GLASS MEMBRANE ELECTRODE…..
• Used to measure pH & Na+
• By varying glass composition,
specific ion selectivity can be
generated.
• H+ selective glass:
• 72% SiO2,
• 22% Na2O
• 6% CaO
• Na+ selective glass:
• 71% SiO2
• 11% Na2O
• 18% Al2O2

POLYMER MEMBRANE ELECTRODES
• Predominant class of potentiometric electrodes
• A major breakthrough was the discovery that
valinomycin incorporated into organic liquid
membrane or PVC membranes resulted in a sensor
with high selectivity for K+ ions
• High selectivity for carbonate ions can be achieved
using a neutral carrier ionophore possessing
trifluoroacetophenone groups.

POLYMER MEMBRANE ELECTRODES …
• PVC membrane ISE
• 1-3 wt% ionophore
• 64 wt% plasticizer
• 30 wt% PVC
• < 1wt% additives

TYPES OF POLYMER ELECTRODES:
• Based on mechanism :
Charge associated ion exchanger ISE e.g. Ca
Charge dissociated ion exchanger e.g. Cl (interference
from salicylate, thiocyanate & heparin)
Neutral ionophore e.g. K -ISE based on valinomycin

ELECTRODES FOR PCO2
(SEVERINGHAUS TYPE PCO2 ELECTRODE)
• First pCO2 electrode was
developed in 1950’s by
Stow & Severinghaus
• Used a glass pH electrode
as an internal element in a
potentiometric cell for
measurement of pCO2
• Widely used in modern
blood gas analyzers
• PVC membrane based
electrodes are also
available

VOLTAMETRY /AMPEROMETRY
• Principle:
• voltametric & amperometric methods are based on
electrolytic electrochemical cells in which an external
voltage is applied to a polarizable working electrode.
• Applications:
• Clarke style amperometric oxygen sensor

CLARK STYLE OXYGEN SENSOR

VOLTAMMETRY AMPEROMETRY
• External voltage
applied
• Plot of observed current
vs voltage
• Less sensitive
• External voltage
applied
• Fixed voltage applied &
resulting current
monitored
• More sensitive
quantitative measure as
background charging
currents from changing
V do not exist

POLAROGRAPHY
• Polarography is a subclass of voltammetry where
the working electrode is a dropping mercury electrode (DME)
• DME- wide cathodic ranges and renewable surfaces
• two electrodes, one polarizable and one unpolarizable
• polarizable is formed by mercury regularly dropping from
a capillary tube
• category of linear-sweep voltammetry where the electrode
potential is altered in a linear fashion from the initial potential
to the final potential
• current vs. potential = sigmoidal shape

CONDUCTOMETRY
• Conductometry determines quantity of analyte by
measurement of its effect on electrical conductivity of the
mixture
• Electrical conductivity - the measure of ability of ions in
solution to carry current under the influence of a potential
difference

CONDUCTOMETRY= USES:
• Measurement of hematocrit
• As transduction mechanism for some biosensors
• Electronic counting of blood cells in suspension
(COULTER PRINCIPLE conductivity of blood cells is
lower than that of a salt solution used as a suspension
medium)

LIMITATIONS:
• Requires correction for Na & K concentrations
• Abnormal protein levels will change plasma conductivity
• Preanalytical – insufficient mixing of sample

COULOMETRY
• Measures the electrical charge passing between 2
electrodes in an electrochemical cell
• Amount of charge is directly proportional to oxidation or
reduction of an electroactive substance at one of the
electrode
• Q = n x N x F

COULOMETRY
• USES:
• Gold standard for measurement of chloride in serum/
plasma
• As mode of transduction in certain biosensors
• ADV: measures absolute amount of substance
• Interference: other anions in sample like bromide

OPTICAL CHEMICAL SENSORS
• OPTODE= optical sensor used in analytical instruments
to measure blood gases & electrolytes

OPTODES ADVANTAGES OVER
ELECTRODES
• Ease of miniaturization
• Less noise (no transduction wires)
• Long term stability
• No need for a separate reference electrode

BIOSENSORS
ENZYME BASED AFFINITY BASED
BIOSENSOR BIOSENSOR
• With amperometric detection  most
common
• With potentiometric & conductometric
detection
• With optical detection

ENZYME BISOSENSORS WITH AMPEROMETRIC
DETECTION
• Clark & lyons developed the first amperometric biosensor which was
used for measuring glucose based on immobilizing glucose oxidase
on the surface of an amperometric pO2 sensor
• Amperometric detection by oxidation of H2O2 is the detection
mechanism

ENZYME IMMOBILIZATION
1. Simple entrapment
2. Cross linking enz with an inert protein (bovine serum
albumin)
3. Adsorption of enzyme to electrode surface
4. Covalent binding to insoluble carrier e.g. nylon, glass
5. Bulk modification of an electrode material, mixing enz
with carbon paste, which serves as both enz
immobilization matrix & electroactive surface

• Other amperometric biosensors reported include
Lactate
Creatinine
Cholesterol
Pyruvate
Alanine
Glutamate
glutamine

WITH POTENTIOMETRIC &
CONDUCTOMETRIC DETECTION
• ISE can be used as transducer in potententiometric
biosensors
• An example is a biosensor for urea based on a
polymembrane ISE for ammonium ion
• Enz urease is immobilized on the surface of ISE

DISADV:
• Nonlinear sensor response at high analyte conc
• Ammonium ions are partially converted to ammonia ,
which is not sensed by ISE

WITH OPTICAL DETECTION
• Absorbance , fluorescence and luminescence can be the
modes of detection
• A sensitive indicator is co-immobilized with an enzyme at the
end of a fibre optic probe
• Prepared for many anlytes:
Glucose
cholesterol
Bilirubin
 pH change resulting from enzyme catalyzed reactions has
also been measured optically (use fluorescein pH sensitive
indicator) e.g. using glucose oxidase enzyme , a pH optode
has been employed to follow the formation of gluconic acid

AFFINITY SENSORS
• Immobilized biological recognition element is a binding
protein, antibody or oligonucleotide with high binding affinity &
high specificity towards the analyte
• ADVANTAGES:
Increased speed & convenience
Cover the full range of analyte
Limits the reversibility
• But affinity sensors based on optical, electrochemical or other
transduction modes are typically SINGLE USE DEVICES

PHYSIOLOGICAL ELECTROLYTES…
• Na+
• K+
• Cl-
• HCO3
-
• Ca 2+
• Mg 2+
• H2PO4
-
• HPO4
2-
• SO4
2-

SPECIMENS FOR ELECTROLYTE DETERMINATIONS….
• Serum
• Plasma
• Whole blood
• Capillary blood
• Urine
• Feces
• Aspirates
• Drainages
• sweat

SODIUM
• Major cation of ECF
• Central role in maintaining the osmotic pressure & normal
distribution of water
• Kidneys are the major regulators of amount of Na in the body
• Specimen:
• Serum/plasma/urine
• Stored at 2-40C
• Hemolysis does not significantly affect Na levels
Reference intervals
• Serum: 135-145 mmol/L
• Urine: 40-220 mmol/day

POTASSIUM
• Major intracellular cation
• Na K ATPase pump is critical in maintaining the
intracellular conc. of K
• Nerve impulse transmission
• Skeletal & cardiac muscle contraction
• Pump gets slowed
• Depletion of metabolic substrates like glucose for ATP
production
• Competition for ATP between pump & other energy requiring
activities
• Cellular metabolism is slowed (refrigeration)

SPECIMENS
• Plasma is preferred
• As breakdown of platelets during coagulation can serum K levels
by 0.2-0.5mmol/L
• Should not be refrigerated
• ⁭ K levels
• Not stored at R.T.
• ↓ K levels
• Skeletal muscle activity
• Markedly K levels
• ⁭ TLC K levels

• Recommendation for most reliable K determination:
• Collect blood with heparin, to maintain it b/w 25-370C
• Separate plasma within minutes by high speed
centrifugation without cooling

METHODS OF ANALYSIS OF SODIUM &
POTASSIUM
• Atomic absorption spectrophotometry
• Flame emission spectrophotometry  obsolete
• ISE  MOST COMMON
• Spectrophotometry

ION SELECTIVE ELECTRODE…
• Glass membrane electrodes for Na+
• Liquid ion exchange membranes with valinomycin for K+
• Frequent calibration is a norm for most systems.

TYPES OF ISE METHODS
• Indirect
• Sample is introduced into measuring chamber after mixing
with a rather large volume of diluent
• Dilution is necessary,
• to present a small amount of sample to a large electrode &
• to minimize concentration of proteins at electrode surface
• Olympus AU2700, Cobas 6000
• Direct
• Sample is presented to electrodes without dilution
• Miniaturization of electrodes
• Radiometer ABL series

ERRORS…..
• Lack of selectivity
• Many Cl- electrodes lack selectivity against other
halide ions
• Repeated protein coating of ion selective membrane
• Contamination of the membrane or salt bridge by
competing/reacting ions.

ERRORS…..
• Electrolyte exclusion effect:
• Exclusion of electrolytes from the fraction of total
plasma volume that is occupied by solids.
• Solvent displacing effect of lipids & proteins in
hyperlipidemic/hyperproteinemic samples result in
falsely decreased values
• Indirect ISE, flame photometry

CHLORIDE.
• Major extracellular anion
• Most abundant anion
• Significant contribution to
• Water distribution
• Osmotic pressure
• Anion-cation balance

METHODS OF ANALYSIS
• Mercurimetric titration
• Spectrophotometry
• Coulometric-amperometric titration  MOST PRECISE
• ISE  MOST
COMMON

COULOMETRIC-AMPEROMETRIC TITRATION
• Most precise method
Principle:
• Ag+ ions are generated from a Ag electrode at a
constant rate,
• Ag+ +Cl- AgCl (INSOLUBLE)
• After a stoichiometric point is reached, excess Ag+ in
mixture triggers shutdown of Ag+ generation system.
• A timing device records the elapsed time.
• Disadvantage:
• high maintenance

ION SELECTIVE ELECTRODE
• Polymeric membranes incorporating quaternary
ammonium salt anion exchanger are used
• Disadvantages:
• Membrane instability
• Lot to lot inconsistency in selectivity to other ions.

REFERENCE INTERVALS…
• 98-107mmol/L

BICARBONATE (TOTAL CO2)…
• Specimen: serum or heparinized plasma
• Venous/capillary blood
• Most accurately determined when assay is done
immediately after opening the tube
• Ambient air contains far less CO2 than does plasma & gaseous
dissolved CO2 will escape from specimen into air with a
consequent decrease in CO2 values of upto 6mmol/L in course of
1 hour.

METHODS FOR DETERMINATION……
• Manometric
• Automated methods:
• Electrode based
• Enzymatic
• Indirect electrode based method:
• Released gaseous CO2 after acidification is determined by a
PCO2 electrode
• Direct ISE methods are not frequently used
• As they lack in specificity

METHODS FOR DETERMINATION……
• Enzymatic method:
• Specimen is first alkalinized to convert all CO2 & carbonic acid to
HCO3
-
• Phosphoenol pyruvate + HCO3
- oxaloactetate
• Oxaloacetate + NADH malate + NAD+
• decrease in absorbance of NADH at 340nm is proportional to
total CO2 content

REFERENCE INTERVALS…
• Anaerobic samples:
• Whole blood venous 22-26 mmol/L
• Whole blood arterial 24-29 mmol/L
• Cord blood 14-22 mmol/L

R E F L E C T O X Y G E N A T I O N , G A S E X C H A N G E , A N D A C I D - B A S E B A L A N C E
P A O 2 I S T H E P A R T I A L P R E S S U R E O F O X Y G E N D I S S O L V E D I N A R T E R I A L B L O O D
S A O 2 I S T H E A M O U N T O F O X Y G E N B O U N D T O H E M O G L O B I N
ABG….

NORMAL VALUES
pH 7.35-7.45
PaO2 95-100
mmHg
PaCO2 35-45
mmHg
HCO3 22-26
mmHg

ABG….
• pH/blood gas:
pH (acidity)
pCO2 (carbon dioxide tension)
pO2 (oxygen tension)

OXIMETRY:
 ctHb (total hemoglobin concentration)
 sO2 (oxygen saturation)
 FO2Hb (fraction of oxyhemoglobin in total
hemoglobin)
 FCOHb (fraction of carboxyhemoglobin in total
hemoglobin)
 FHHb (fraction of deoxyhemoglobin in total
hemoglobin)
 FMetHb (fraction of methemoglobin in total
hemoglobin)
 FHbF (fraction of fetal hemoglobin)

ELECTROLYTES:
• cK+ (potassium ion concentration)
• cNa+ (sodium ion concentration)
• cCa2+ (calcium ion concentration)
• cCl– (chloride ion concentration)

METABOLITES:
• cGlu (D-glucose concentration)
• cLac (L-lactate concentration)

DETERMINATION OF PCO2, PO2 & PH
Arterial vs venous
• pO2 higher by 60-70mmHg
• pCO2 lower by 2-8mmHg
• pH is generally higher by 0.02-0.05
pH units
Specimens:
Whole blood
Arterial
Venous

PREANALYTICAL ERRORS…..
• Arterial sample:
• No tourniquet is used
• No pull is applied
• Venous sample:
• Specimen to be taken after release of tourniquet, without
clenching of fists.
• Preservative: lyophilized heparin anticoagulant
• Dry vs liquid heparin
• When liquid heparin is used, it has to be used in a
concentration of 0.05mg heparin/ml blood

• An increase in ratio of heparin to blood can have marked
effect on measured pCO2 & parameters calculated from
it.
• Before analysis proper mixing of sample by simple but
vigorous rolling of syringe between palms is adequate to
establish proper homogeneity whether liquid or
lyophilized heparin is used.

• Anaerobic technique:
• No or minimal exposure to air
• On exposure to air
• CO2 content & pCO2 of blood sample will decrease
• Increase in pH
• pO2 will increase
• Syringe should be held tip up, a drop of blood ejected
from it & a tightly fitting cap placed on tip.

• Transport & analysis of specimens should be prompt
• pH of blood 0.04-0.08 pH unit/hr
• pCO2 of blood 5mmHg/hr
• pO2 of blood 5-10mmHg/hr
• Analysis should be performed within half an hour to
prevent adverse effects.

POTENTIOMETRIC MEASURING PRINCIPLES
• pH
• pCO2
• Electrolyte electrodes

PH
• Reference electrode-is made from a Ag rod coated with
AgCl to provide the Ag/Ag ions equilibrium and
determine the reference potential
• pH electrode (E777) is a pH-sensitive glass electrode.
The pH-sensitive glass membrane is located at the tip
and seals the inner buffer solution with a constant and
known pH.

• The membrane consists of three separate membranes:
• Membrane Function
• Inner To limit diffusion through the
membrane and stabilize the whole membrane system
• Middle To prevent protein interference.
• Outer To reduce the interchange of sample
or Rinse Solution and HCOONa solution.

• A difference in the ion exchange on either side of the
membrane occurs if proton concentration (and therefore
pH) is unequal on both sides. The number of positive
and negative ions is no longer equal, so the potential
difference across the membrane changes
• pH calculated by NERNST EQUATION

PCO2 ELECTRODE
• a combined pH and Ag/AgCl reference electrode
mounted in a plastic jacket, which is filled with a
bicarbonate electrolyte
• Nernst equation is not directly applied

ELECTROLYTE ELECTRODES
• The K electrode (E722) is an ionselective electrode
whose sensing element is a PVC membrane containing
a potassium-neutral ion carrier
• The Na electrode (E755) is an ionselective electrode
whose sensing element is a Na+sensitive ceramic pin
contained in the tip of the jacket
• Ca electrode (E733) is an ionselective electrode whose
sensing element is a PVC membrane containing a
calcium-neutral ion carrier

• Cl electrode (E744) is an ionselective electrode whose
sensing element is a PVC membrane containing a
chloride ion carrier

AMPEROMETRIC MEASURING PRINCIPLES
• pO2
• Glucose
• Lactate

PO2 ELECTRODE
• The pO2 electrode is an amperometric electrode which
consists of a silver anode,
• platinum cathode and Ag/AgCl reference band, all
protected by an electrode jacket
• which is filled with electrolyte solution. At the tip of the
electrode jacket an
• oxygen-permeable membrane protects the Pt cathode
from protein contamination
• and is covered on the inner side with Pt-black.

GLUCOSE AND LACTATE ELECTRODES
• The electrode consists of a silver cathode and a platinum
anode. The electrode is protected by an electrode jacket
filled with electrolyte solution and a multilayer membrane
mounted at the tip.
• The membrane consisting of three layers:
1. outer membrane layer - permeable to
glucose/lactate.
2. middle - enzyme layer.
3. inner membrane layer- permeable to H2O2.

OPTICAL MEASURING PRINCIPLES
• ctHb Concentration of total hemoglobin
• sO2 Oxygen saturation
• FO2Hb Fraction of oxyhemoglobin
• FCOHb Fraction of carboxyhemoglobin
• FHHb Fraction of deoxyhemoglobin
• FMetHb Fraction of methemoglobin
• FHbF Fraction of fetal hemoglobin
• Hematocrit (Hct) is also available as a derived parameter

OXIMETRY PARAMETERS
• ctHb(meas) = cO2Hb + cCOHb + cHHb + cMetHb
• sO2= conc of oxyHb / oxy+ deoxyHb (effective Hb)
• Hct(calc) = 0.0301 × ctHb
g / dL

SUMMARIZING…..
• ELECTROCHEMICAL ANALYSIS
1. POTENTIOMETRY & ISE
2. VOLTAMETRY , AMPEROMETRY &
POLAROGRAPHY
3. CONDUCTOMETRY
4. COULOMETRY
• Chemical sensors
• Electrolytes
• ABG

Arterial blood gases

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