ELECTROCHEMISTRY , CHEMICAL SENSORS & ABG Dr. Smily Pruthi , MD- Biochemistry See video in mobile app – tulip academy of medical sciences
TYPES OF ELECTROCHEMICAL ANALYSIS 1. 2. 3. 4.
POTENTIOMETRY & ISE VOLTAMETRY , AMPEROMETRY & POLAROGRAPHY CONDUCTOMETRY COULOMETRY
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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
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PH METER
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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
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REDOX ELECTRODES • Redox potential is generated as a result of chemical equilibria involving electron transfer reaction Ox form + neReduced 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.
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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
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TYPES OF REDOX ELECTRODES..
• Inert metal electrodes • Platinum & gold are examples • Hydrogen electrode being a special redox electrode for pH measurement.
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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 Website : www.tamsmed.com
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.
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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 Website : www.tamsmed.com
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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 Website : www.tamsmed.com
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.
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POLYMER MEMBRANE ELECTRODES … • PVC membrane ISE • • • •
1-3 wt% ionophore 64 wt% plasticizer 30 wt% PVC < 1wt% additives
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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
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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 Website : www.tamsmed.com
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
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CLARK STYLE OXYGEN SENSOR
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VOLTAMMETRY • External voltage applied • Plot of observed current vs voltage • Less sensitive
AMPEROMETRY • External voltage applied • Fixed voltage applied & resulting current monitored • More sensitive quantitative measure as background charging currents from changing V do not exist
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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 Website : www.tamsmed.com
CONDUCTOMETRY â&#x20AC;˘ Conductometry determines quantity of analyte by measurement of its effect on electrical conductivity of the mixture â&#x20AC;˘ Electrical conductivity - the measure of ability of ions in solution to carry current under the influence of a potential difference
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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)
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LIMITATIONS: • Requires correction for Na & K concentrations • Abnormal protein levels will change plasma conductivity • Preanalytical – insufficient mixing of sample
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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 = nxNxF
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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
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OPTICAL CHEMICAL SENSORS
â&#x20AC;¢ OPTODE= optical sensor used in analytical instruments to measure blood gases & electrolytes
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OPTODES ADVANTAGES OVER ELECTRODES • Ease of miniaturization • Less noise (no transduction wires) • Long term stability • No need for a separate reference electrode
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BIOSENSORS ENZYME BASED BIOSENSOR
AFFINITY BASED BIOSENSOR
• With amperometric detection most common • With potentiometric & conductometric detection • With optical detection Website : www.tamsmed.com
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
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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
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• Other amperometric biosensors reported include
Lactate Creatinine Cholesterol Pyruvate Alanine Glutamate glutamine
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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
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DISADV: â&#x20AC;˘ Nonlinear sensor response at high analyte conc â&#x20AC;˘ Ammonium ions are partially converted to ammonia , which is not sensed by ISE
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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 Website : www.tamsmed.com
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 Website : www.tamsmed.com
ELECTROLYTES
PHYSIOLOGICAL ELECTROLYTES… • Na+ • K+ • Cl• HCO3• Ca 2+ • Mg 2+ • H2PO4 • HPO42• SO42Website : www.tamsmed.com
SPECIMENS FOR ELECTROLYTE DETERMINATIONS….
• Serum • Plasma • Whole blood • Capillary blood • Urine • Feces • Aspirates • Drainages • sweat Website : www.tamsmed.com
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 Website : www.tamsmed.com
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)
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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
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• Recommendation for most reliable K determination: • Collect blood with heparin, to maintain it b/w 25370C • Separate plasma within minutes by high speed centrifugation without cooling
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METHODS OF ANALYSIS OF SODIUM & POTASSIUM • • • •
Atomic absorption spectrophotometry Flame emission spectrophotometry obsolete ISE MOST COMMON Spectrophotometry
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ION SELECTIVE ELECTRODE… • Glass membrane electrodes for Na+ • Liquid ion exchange membranes with valinomycin for K+ • Frequent calibration is a norm for most systems.
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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 Website : www.tamsmed.com
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.
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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
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CHLORIDE. • Major extracellular anion • Most abundant anion • Significant contribution to • Water distribution • Osmotic pressure • Anion-cation balance
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METHODS OF ANALYSIS
• Mercurimetric titration • Spectrophotometry • Coulometric-amperometric titration MOST PRECISE • ISE MOST COMMON
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COULOMETRIC-AMPEROMETRIC TITRATION • Most precise method Principle: • Ag+ ions are generated from a Ag electrode at a constant rate, • Ag+ +ClAgCl (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 Website : www.tamsmed.com
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.
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REFERENCE INTERVALS…
• 98-107mmol/L
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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.
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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
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METHODS FOR DETERMINATION…… • Enzymatic method: • Specimen is first alkalinized to convert all CO2 & carbonic acid to HCO3oxaloactetate • Phosphoenol pyruvate + HCO3• Oxaloacetate + NADH malate + NAD+ • decrease in absorbance of NADH at 340nm is proportional to total CO2 content
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REFERENCE INTERVALS… • Anaerobic samples: • Whole blood venous • Whole blood arterial • Cord blood
22-26 mmol/L 24-29 mmol/L 14-22 mmol/L
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ABGâ&#x20AC;¦. R E F L E C T P A O
2
I S
T H E S A O
O X Y G E N A T I O N , P A R T I A L
2
I S
T H E
G A S
P R E S S U R E A M O U N T
E X C H A N G E , O F
O F
O X Y G E N O X Y G E N
A N D
A C I D - B A S E
D I S S O L V E D B O U N D
T O
I N
B A L A N C E
A R T E R I A L
H E M O G L O B I N
B L O O D
NORMAL VALUES
pH PaO2 PaCO2 HCO3
7.35-7.45 95-100 mmHg 35-45 mmHg 22-26 mmHg
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ABG…. • pH/blood gas: pH (acidity) pCO2 (carbon dioxide tension) pO2 (oxygen tension)
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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) Website : www.tamsmed.com
ELECTROLYTES: • • • •
cK+ (potassium ion concentration) cNa+ (sodium ion concentration) cCa2+ (calcium ion concentration) cCl– (chloride ion concentration)
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METABOLITES: • cGlu (D-glucose concentration) • cLac (L-lactate concentration)
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DETERMINATION OF PCO2, PO2 & PH Specimens: Whole blood Arterial Venous
Arterial vs venous
• pO2 higher by 60-70mmHg • pCO2 lower by 2-8mmHg • pH is generally higher by 0.02-0.05 pH units Website : www.tamsmed.com
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
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PREANALYTICAL ERRORS….. • 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.
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PREANALYTICAL ERRORS….. • 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.
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PREANALYTICAL ERRORS….. • • • • •
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.
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RADIOMETER ABL-800 SERIES
POTENTIOMETRIC MEASURING PRINCIPLES
• pH • pCO2 • Electrolyte electrodes
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PH â&#x20AC;˘ Reference electrode-is made from a Ag rod coated with AgCl to provide the Ag/Ag ions equilibrium and determine the reference potential â&#x20AC;˘ 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.
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• 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.
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â&#x20AC;˘ 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 â&#x20AC;˘ pH calculated by NERNST EQUATION
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PCO2 ELECTRODE â&#x20AC;˘ a combined pH and Ag/AgCl reference electrode mounted in a plastic jacket, which is filled with a bicarbonate electrolyte â&#x20AC;˘ Nernst equation is not directly applied
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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
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â&#x20AC;˘ Cl electrode (E744) is an ionselective electrode whose sensing element is a PVC membrane containing a chloride ion carrier
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AMPEROMETRIC MEASURING PRINCIPLES • pO2 • Glucose • Lactate
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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.
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GLUCOSE AND LACTATE ELECTRODES â&#x20AC;˘ 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. â&#x20AC;˘ 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.
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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
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OXIMETRY PARAMETERS • ctHb(meas) = cO2Hb + cCOHb + cHHb + cMetHb • sO2= conc of oxyHb / oxy+ deoxyHb (effective Hb) • Hct(calc) = 0.0301 × ctHb g / dL
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SUMMARIZING….. • ELECTROCHEMICAL ANALYSIS 1. POTENTIOMETRY & ISE 2. VOLTAMETRY , AMPEROMETRY & POLAROGRAPHY 3. CONDUCTOMETRY 4. COULOMETRY • Chemical sensors • Electrolytes • ABG
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THANKSâ&#x20AC;¦.