1.ELECTROCHEMISTRY, ELECTROPHORESIS, AND ISOELECTRIC FOCUSING Presented by: Anthony John DuranAngelica Nhoj Gemora Denfield Jan PamaSiegefred Pue Karen Grace Salao

2. ELECTROCHEMISTRY 3. ELECTROCHEMISTRY Involves measurement of current or voltagegenerated by activity of specific ions. Electrical energyChemical energy Copper and silver nitrateCu AgNO3 2Ag+(aq) + Cu(s)2Ag(s) + Cu2+(aq) 4. 2 TYPES OF ELECTROCHEMICAL CELLS Electrolytic cells nonspontaneouschemical reactions areforced to occur by theinput of electricalenergy. Consist of a containerfor the reaction materialwith electrodesimmersed in thereaction material andconnected to a sourceof direct current. 5. Galvanic orvoltaic cell Spontaneousredox reactionproduce electricalenergy the two halves ofthe redox reactionis separated,requiring electrontransfer to occurthrough anexternal circuit. 6. Electrochemistry includes: Potentiometry Amperometry Coulometry 7. POTENTIOMETRY: GENERAL PRINCIPLES Concentration of ions in solution is calculated fromthe measured potential difference between the twoelectrodes. This type of system includes at least twoelectrodes, identified as an indicator electrode anda reference electrode which act as the cathode andanode respectively. 8. Each electrode is in contact with either the sample(in the case of the indicator electrode) or areference solution ( in the case of the referenceelectrode). This method is made under conditions in whichessentially zero current is flowing through thissystem. 9. The difference is related to the molar concentrationof the solution as expressed by the Nernstequation,E = E- (0.059/z)log (Cred/Cox)Where: E= cell potential measured at 25C E= standard redox potential z= number of electrons involvedCred= molar concentration of the reduced formCox= molar concentration of the oxidized 10. STANDARD REDUCTION POTENTIALS 11. System Components Liquid Junction Reference electrode Indicator or measuring electrode Readout device (Potentiometer) 12. Liquid junction also known as a salt bridge arerequired to complete the circuit between thereference and without contaminating anything.Functions:It allows electrical contact between the twosolutions.It prevents the mixing of the electrode solutions.It maintains the electrical neutrality in each half cellas ions flow into and out of the salt bridge. 13. Reference Electrode- is an electrochemical half-cellthat is used as a fixed reference for the measurementof cell potentials. A half-cell with an accurately known electrode potential,Eref, that is independent of the concentration of theanalyte or any other ions in the solution Always treated as the left-hand electrodeExamples: Normal hydrogen electrode Saturated calomel electrode Ag-AgCl electrode 14. REFERENCE ELECTRODES Calomel electrode- composed of mercury/mercurous chloride; It is dependable but large, bulky, and affected by temperature. Silver/silver chloride- reference electrodes are more compact and handle temperature fluctuations better -- overall better & faster Normal Hydrogen Electrode- consists of a platinized platinum electrode in a 1.228N HCl solution with hydrogen at atmospheric pressure bubbled over the platinum surface. 15. Indicator Electrode- also called the measuringelectrode (platinum wire and carbon rod). It is immersed in a solution of the analyte, developsa potential, Eind that depends on the activity of theanalyte. Is selective in its response It is the other electrochemical half-cell that respondsto changes in the activity of a particular analytespecies in a solution.Example: Ion-Selective Electrodes 16. ION SELECTIVE ELECTRODE Is an indicator electrode that can respond toindividual types of anions or cations, and is one toolthat can be utilized for such a task.Examples: Glass membrane Electrodes Gas-sensing Electrodes 17. pH electrode Selective for thedetection of hydrogenions. The measuring orindicator electrodehas a glassmembrane pH is then determinedfrom potentialbetween the pHelectrode and a 18. PCO2 ELECTRODE Measurement of PCO2 in routine blood gases A modified pH electrode with a CO2 permeable membrane covering the glass membrane surface A bicarbonate buffer separates the membranes Change in pH is proportional to the concentration of dissolved CO2 in the blood 19. COULOMETRY Coulometry is an electrochemical titrationwhere the titrant is electrochemicallygenerated and the endpoint is detected byamperometry. 20. AMPEROMETRY Amperometry- is the measurement of thecurrent flow produced by an oxidation-reduction reaction. A measure of the cell current when thepotential difference between indicator andreference electrodes is controlled. 21. PRINCIPLE In the presence of some conductive buffer. If an electrolyticpotential is applied to the solution through a workingelectrode, then the measured current depends (in part) onthe concentration of the analyte. Measurement of thiscurrent can be used to determine the concentration of theanalyte directly. However, the difficulty is that the measured current dependson several other variables, and it is not always possible tocontrol all of them adequately. This limits the precision ofdirect amperometry. 22. If the potential applied to the working electrode issufficient to reduce the analyte , then theconcentration of analyte close to the workingelectrode will decrease. If the potential applied to the working electrode isgreat enough (an overpotential), then theconcentration of analyte next to the workingelectrode will depend entirely on the rate ofdiffusion. 23. This can be seen in the following equation: Q = It = znFWhere: z = the number of electrons involved in thereaction n = the number of moles of analyte in the sampleWhere: = Faradays constant (96485 C/mol of Felectrons) Q= the electrical charge I= the current t= the time 24. pO2 Gas Electrodes Gas-sensing electrodes that use amperometricor current-sensing electrolytic cell as indicator. They consist of a gas permeable membrane(polypropylene) which allows only dissolvedoxygen to pass through. 25. ADVANTAGES less hazardous process elimination or minimization of polluting byproducts requiring disposal process simplification so that an otherwise multistep chemical route is simplified to one or two steps 26. use of cheaper more readily available startingmaterials the possibility of reaching very high levels ofproduct purity and selectivity 27. DISADVANTAGES requires the use of a solvent to solubilizethe reactants and productsWater is the ideal solvent but too often organicsolvents or co-solvents are required supporting electrolytes to carry the current are veryoften needed Electricity is required in all electrochemical processingwhich may or may not be a critical factor, depending onwhere the process is located. 28. APPLICATION Use of the potential measurements to give directinformation on the activity, or concentration of ananalyte in a sample pH measurements Use of potential measurements to follow the courseof titration, as occurs in a potentiometric titration. Measurement of chloride in body fluids such assweat, urine and CSF. Determination of ascorbic acid or vitamin C 29. INTERFERENCES Errors in ISE measurement can result in any iondetermination if data are not collected for standards andsamples at approximately the same temperature, since theNernst equation that governs the calibration of potentialversus concentration is temperature dependent. Response of an ISE to a non-analyte or an interferent ion inthe sample. 30. Components in certain sample matrices also canchange the sensitivity of an electrode by adsorbingto its surface, thereby blocking access of theanalyte. Sensitivity of the glass pH electrode may bereduced for some electrodes at pH values above 10(i.e. sodium error) because of the interference ofmonovalent cations in high concentrations,especially Na+. In solutions of pH less than 1, low water activitiesalso may give rise to measurement error. 31. ELECTROPHORESIS 32. ELECTROPHORESIS Method of separation and purification Involves migration of charged particles in an electricfield It is suitable for the separation and the quantitation ofproteins in body fluids. Is a tool that is used by clinical laboratoryscientists/medical technologists to separate moleculeprior to molecule identification. 33. GENERAL PRINCIPLES The electrical field is applied to a solution throughoppositely charged electrodes placed in thesolution. An ion then travels through the solution toward theelectrode of opposite charge: positively chargedparticles move to the negatively charged electrode,and negatively charged particles migrate to thepositively charged electrode. 34. The separation of analytes byelectrophoresis has two key requirements: There must be a difference in how analytesinteract with the separation system. The bands or peaks for the analytes must besufficiently narrow to allow them to be resolved. The sample is separated into bands whereeach band has molecules containing similarmobility. 35. FACTORS INFLUENCING MIGRATION OFPARTICLES Net electric charge of the particle Size and shape of the molecules Electric field strength Nature of the supporting medium Temperature of operation 36. COMPONENTS power source with a voltmeter and voltage regulator electrophoresis tank that holds the electrophoresisbufferan anode and a cathode connected with the powersource a glass plate that holds the gel and is submerged intothe electrophoresis buffer a comb which is used to make the sample wells in theagar before it solidifies. 37. TYPES OF ELECTROPHORESIS Moving boundary or frontalelectrophoresis It involves separation of molecules usinghomogenous solution. No distinct zones areformed. The fractionsresolved are those of albumin,, , and globulins. 38. Zonal electrophoresis Involves the use of a support medium. The fractions resolved are albumin, 1, 2, and globulins. The charged particles are placed on a stabilizingmedium which will contain the proteins aftermigration. 39. PAPER ELECTROPHORESIS- It is the form ofelectrophoresis that is carried out on filter paper. Thistechnique is useful for separation of small chargedmolecules such as amino acids and small proteins. FILTER PAPER- It is the stabilizing medium. APPARATUS- Power pack, electrophoretic cell thatcontains electrodes, buffer reservoirs, support forpaper, transparent insulating cover. 40. GEL ELECTROPHORESIS- It is a technique usedfor the separation of Deoxyribonucleic acid,Ribonucleic acid or protein molecules according totheir size and electrical charge using an electriccurrent applied to a gel matrix.What is a gel? Gel is a cross linked polymer whose compositionand porosity is chosen based on the specific weightand porosity of the target molecules.Types of Gel: Agarose gel Polyacrylamide gel 41. Agarose gels Purified agar Afterelectrophoresis,it can be stainedand read in adensitometer Long termstoragepossible 42. PolyacrylamideGel Gels with differentpore sizes can belayered to providegood separation ofmolecules ofdifferent sizes Good resolution 43. TWO-DIMENSIONAL ELECTROPHORESIS- thestandard electrophoretic separation in one directionis followed by SDS-PAGE in the perpendiculardirection. This technique combines the technique IEF (firstdimension), which separates proteins in a mixtureaccording to charge (PI), with the size separationtechnique of SDS-PAGE (second dimension). 44. VIDEO 45. PROCEDURE Serum is applied to thesupport media and theprotein dissolves in thebuffer, giving them anelectric charge A specific amount ofcurrent is applied for aspecific amount of time As the current flowsthrough the media, theelectrically chargedmolecules migrate alongthe supporting media 46. The negatively charged protein molecules migratetowards the oppositely charged electrode. The sample is separated into bands where each bandhas molecules containing similar mobility. Once the medium has been stained and thebackground of the medium support has been cleared,the electrophoretic pattern can be scanned through adensitometer. 47. STAINING OF THE SUPPORTING MEDIUM Staining fixes theprotein to the membraneby denaturing Makes the fractionsvisible Decolorization is used toremove backgroundcolor Each peak in eachcolumn represents adifferent band ofmolecules that migratedtogether 48. ADVANTAGES: Versatility in Identification Accuracy of ResultsDISADVANTAGES: Toxicity Electrophoresis has limited sample analysis Electrophoresis measurements are not precise Only certain molecules can be visualized 49. APPLICATION Specific protein analysis Identification and quantitation of hemoglobin and its subclassesIdentification of monoclonal proteins in either serum or urine.Separationand quantitation of major lipoprotein and lipid classesIsoenzyme analysisWesternblot technique to identify a specific protein.Southernblot techniques to identify specific nucleic acid sequence. 50. INTERFERENCES Sample Contamination Gel Problems Improper Loading Electrical Current Problems Failed Visualization Varied Measuring 51. ISOELECTRIC FOCUSING 52. ISOELECTRIC FOCUSING Involves the migration of proteins in a pH gradient. Addition of acid to the anodic area of theelectrolyte cell and a base to the cathode area. It is the pH where the net charge of the proteinmolecule is zero. Isoelectric focusingrequires solid supportsuch as agarose gel andpolyacrylamide gel. 53. PRINCIPLES Protein in a mixture can be precipitated dependingon its isoeletric point. IEF requires stable pH gradient which can beformed by using mixture of specially designedamphoteric molecules known as ampholytes. When electric field is applied, a pH gradient isestablished, that is negatively charged ampholytesmove towards anode and positively chargedtowards cathode and align themselves according totheir pIs. 54. VIDEO 55. ADVANTAGES IEF offers the following advantages: efficient economic (no sophisticated equipment required) easy (clear, one-dimensional separation principle) fast High capacity and resolution to 0.001 pH unit possible 56. DISADVANTAGE A disadvantage of IEF is that minor bands and aging bands are also seen and may cause confusion in interpretation. 57. APPLICATIONS Useful in measuring serum acid phosphatase isoenzyme. Detects oligoclonal immunoglobulin bands in CSF and isoenzymeof creatine kinase and alkaline phosphatase in serum. Applied in the assay of Acid Phosphatase isoenzyme. General characterization of proteins by pI purity determination ofproteins. Discrimination of caseins Routine clinical analyses 58. THANK YOU