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Review of Analytical Methods Part 2: Electrochemistry Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology.

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Presentation on theme: "Review of Analytical Methods Part 2: Electrochemistry Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology."— Presentation transcript:

1 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 Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology University of Florida Health Science Center/Jacksonville

2 Analytical methods used in clinical chemistry Spectrophotometry Electrochemistry Immunochemistry Other –Osmometry –Chromatography –Electrophoresis Spectrophotometry Electrochemistry Immunochemistry Other –Osmometry –Chromatography –Electrophoresis

3 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 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

4 Electrochemical 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

5 ZnCu e-e- Relative 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

6 Cu 2+ Zn 2+ Zn 0 Cu 0 Zn 0  Zn e - Cu e -  Cu 0 e -  e -  e -  mV

7 The Nernst Equation WhereE = Potential at temperature T E 0 = Standard electrode potential (25ºC, 1.0M) R = Ideal gas constant F = Faraday’s constant n = number of electrons transferred

8 Cu 2+ Zn 2+ Zn 0 Cu 0 Zn 0  Zn e - E 0 = +(-) V Cu e -  Cu 0 E 0 = V E lj mV

9 Electromotive force E cell = E cathode + E lj - E anode E cell = E Cu(II),Cu + E lj – E Zn(II),Zn E cell = (+) E lj – (-)0.763 E cell = (+) E lj  G = -nFE cell

10 Would the reaction occur in the opposite direction? E cell = E cathode + E lj - E anode E cell = E Zn(II)  Zn + E lj – E Cu(II)  Cu E cell = (-) E lj – (+)0.340 E cell = (-) E lj

11 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

12 The Hydrogen Electrode H 2 gas  2H + + 2e -  H 2 E 0 = 0.0 V mV Test electrode

13 The Calomel Electrode Calomel paste (Hg 0 /Hg 2 Cl 2 ) Saturated KCl Liquid junction mV Test electrode Hg 2 Cl 2 + 2e -  2Hg 0 + 2Cl - E 0 = 0.268V

14 The Silver/Silver Chloride Electrode Silver wire Saturated KCl + AgNO 3 Liquid junction mV Test electrode AgCl + e -  Ag 0 + Cl - E 0 = 0.222V

15 Ion-selective Electrodes Ref 1 Ref 2 mV E cell = E Ref(1) + E lj – E Ref(2)

16 Typical ISE design Ref 1 Ref 2 mV E cell  E ISM Ion-selective membrane E cell = E Ref(1) + E lj – E Ref(2)

17 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  : 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  :

18 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) 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)

19 Types of ISE Glass –Various combinations of SiO 2 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 SiO 2 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

20 pH electrode mV External reference electrode Non-conducting glass body Internal reference electrode H + -responsive glass membrane Shielded connecting cable

21 pCO 2 electrode mV External reference electrode CO 2 (g) Flow Cell Electrode assembly Gas-permeable membrane (silicone rubber) NaHCO 3 /H 2 O CO 2 + H 2 O  HCO H +

22 NH 3 electrode mV External reference electrode NH 3 (g) Flow Cell Electrode assembly Gas-permeable membrane (PTFE) NH 4 Cl/H 2 O H 2 O + NH 3  NH OH -

23 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 NH 4 +, 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 NH 4 +, but these are less useful than other electrode types

24 The Sodium Error (or, direct vs. indirect potentiometry) Na + Whole blood Cells (45%) Aqueous phase Lipids, proteins Plasma mV Since potentiometry measures the activity of the ion at the electrode surface, the measurement is independent of the volume of sample.

25 The Sodium Error (or, direct vs. indirect potentiometry) Na + mV 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.

26 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.

27 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).

28 Sodium error Na mM Na mM Na mM Na mM

29 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.

30 Solid state chloride electrode AgCl and Ag 2 S 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 Ag 2 S 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

31 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

32 K + ion-selective electrode K+K+ 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 +.

33 Ca ++ ion selective electrode Ca ++ di-p-octylphenyl phosphate PVC membrane

34 Ca ++ ion selective electrode Ca ++ Neutral carrierInert membrane

35 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 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

36 Amperometry Current (mA)  Applied potential (V)  Half-wave potential Limiting current

37 Amperometry Current (mA)  Applied potential (V)  Half-wave potential C0C0 0.5C 0 2C 0

38 Gas-permeable membrane Platinum wire (cathode) -0.65V Reference electrode (anode) Oxygen (pO 2 ) electrode Flow cell  O2O2

39 Reaction at the platinum electrode The amount of current (e - ) is proportional to the concentration of O 2

40 The glucose electrode Note that the platinum electrode now carries a positive potential Glucose + O 2 Glucose oxidase H 2 O 2 + Gluconic acid O 2 electrode O 2 + 2H - 2e-2e- (+0.6 V)


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