Presentation on theme: "Review of Analytical Methods Part 2: Electrochemistry"— Presentation transcript:
1 Review of Analytical Methods Part 2: Electrochemistry Roger L. Bertholf, Ph.D.Associate Professor of PathologyChief of Clinical Chemistry & ToxicologyUniversity of Florida Health Science Center/Jacksonville
2 Analytical methods used in clinical chemistry SpectrophotometryElectrochemistryImmunochemistryOtherOsmometryChromatographyElectrophoresis
3 ElectrochemistryElectrochemistry applies to the movement of electrons from one compound to anotherThe donor of electrons is oxidizedThe recipient of electrons is reducedThe 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 nucleusFilled/partially filled orbitals
5 Relative potential Zn Cu Copper is more electronegative than Zinc When the two metals are connected electrically, current (electrons) will flow spontaneously from Zinc to CopperZinc is oxidized; Copper is reducedZinc is the anode; Copper is the cathode
10 Would the reaction occur in the opposite direction? Ecell = Ecathode + Elj - EanodeEcell = EZn(II)Zn + Elj – ECu(II) CuEcell = (-) Elj – (+)0.340Ecell = (-) Elj
11 How do we determine standard electrode potentials? Absolute potential cannot be measured—only the relative potential can be measuredStandard electrode potentials are measured relative to a Reference ElectrodeA Reference Electrode should be. . .Easy to manufactureStable
12 The Hydrogen Electrode mVTest electrodeH2 gas 2H+ + 2e- H2E0 = 0.0 V
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 :
18 The activity coefficient Solutions (and gases) in which none of the components interact are called ideal, and have specific, predictable propertiesDeviations from ideal behavior account for the difference between concentration and activityDilute solutions exhibit nearly ideal behavior (1)
19 Types of ISE Glass Solid-state Liquid ion-exchange Gas sensors Various combinations of SiO2 with metal oxidesSolid-stateInvolve ionic reaction with a crystalline (or crystal doped) membrane (example: Cl-/AgCl)Liquid ion-exchangeA carrier compound is dissolved in an inert matrixGas sensorsUsually a combination of ISE and gas-permeable membrane
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 NH4+, but these are less useful than other electrode types
24 The Sodium Error (or, direct vs. indirect potentiometry) mVNa+WholebloodCells (45%)Aqueous phaseLipids, proteinsPlasmaSince potentiometry measures theactivity of the ion at the electrodesurface, the measurement isindependent of the volume ofsample.
25 The Sodium Error (or, direct vs. indirect potentiometry) mVIn indirect potentiometry, the concentrationof ion is diluted to an activity near unity.Since the concentration will take intoaccount the original volume and dilutionfactor, any excluded volume (lipids, proteins)introduces an error, which usually is insignificant.Na+
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.
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).
28 Sodium errorNa+138 mMNa+130 mMNa+140 mMNa+140 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.
30 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
31 Liquid/polymer membrane electrodes Typically involves an ionophore dissolved in a water-insoluble, viscous solventSometimes called ion-exchange membrane electrodesThe ionophore determines the specificity of the electrode
32 K+ ion-selective electrode 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+.K+
33 Ca++ ion selective electrode di-p-octylphenyl phosphatePVC membraneCa++
34 Ca++ ion selective electrode Neutral carrierInert membraneCa++
35 AmperometryWhereas potentiometric methods measure electrochemical potential, amperometric methods measure the flow of electrical currentPotential (or voltage) is the driving force behind current flowCurrent is the amount of electrical flow (electrons) produced in response to an electrical potential
36 Amperometry Current (mA) Applied potential (V) Limiting current Half-wave potentialApplied potential (V)