Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter 5: Analytic Techniques By Julia C. Drees, Matthew S. Petrie, Alan H.B. Wu

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry Photometric instruments measure light intensity without consideration of wavelength. Most instruments use filters (photometers), prisms, or gratings (spectrometers) to select (isolate) a narrow range of incident wavelength. Beer’s Law: Concentration of a substance is directly proportional to right amount of light absorbed or inversely proportional to logarithm of transmitted light. Spectrophotometric Instruments: measure light transmitted by a solution to determine concentration of light-absorbing substance in solution

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Components of a Spectrophotometer –Light source –Monochromators –Sample cell –Photodetectors Spectrophotometer Quality Assurance –Wavelength accuracy –Stray light –Linearity

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Single-beam spectrophotometer

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Atomic Absorption Spectrophotometer –Measures concentration by detecting absorption of electromagnetic radiation by atoms rather than by molecules –Sensitive, precise –Routinely used to measure concentration of trace metals that are not easily excited Flame Photometry –Measures light emitted by excited atoms –Was used to determine concentration of Na, K, or Li –No longer routinely used; replaced by ion-selective electrodes

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Fluorometry –Basic instrumentation Filter fluorometers measure concentrations of solutions that contain fluorescing molecules. Source emits short-wavelength high-energy excitation light. Mechanical attenuator controls light intensity. –Advantages and disadvantage of fluorometry Advantages: greater specificity and sensitivity Disadvantage: very sensitive to environmental changes

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Basic filter fluorometer

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Chemiluminescence –Part of chemical energy generated produces excited intermediates that decay to a ground state with emission of photons. –Unlike fluorescence, no excitation radiation or monochromators are required. –Oxidation reactions of luminol, acridinium esters, & dioxetanes Turbidity and Nephelometry –Turbidity measures concentration of particulate matter in sample. –Nephelometry: similar to turbidity, except different angle of measurement

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Spectrophotometry and Photometry (cont’d) Laser Applications –LASER: Light Amplification by Stimulated Emission of Radiation –Based on interaction of radiant energy and suitably excited atoms or molecules –Laser light is polarized and coherent and has narrow spectral width and small cross-section area with low divergence. –Can serve as source of incident energy in spectrometer or nephelometer –Laser spectrometry can be used for determination of structure, identification of samples, and diagnosis.

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Electrochemistry Galvanic and Electrolytic Cells –Galvanic cells Spontaneous flow of electrons from electrode with lower electron affinity Electrons pass through external meter to cathode, liberating OH - ions; reaction continues until cell is dead. –Electrolytic cells: Current is forced to flow through dead cell by applying external electromotive force E. Half-Cells –Two reactions must be coupled and one reaction compared with other to measure electrochemical activity of half-cell.

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Electrochemistry (cont’d) Ion-Selective Electrodes (ISE): designed to be sensitive toward individual ions pH Electrodes –Indicator electrode –Reference electrode –Liquid junctions –Readout meter –Nernst equation –Calibration –pH combination electrode

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Electrochemistry (cont’d) Gas-Sensing Electrodes –Designed to detect specific gases in solutions –Separated from solution by thin, gas-permeable membrane Enzyme Electrodes –An ISE covered by immobilized enzymes that can catalyze a specific chemical reaction Coulometric Chloridometers and Anodic Stripping Voltammetry –Chloride ISEs have largely replaced coulometric titrations. –Anodic stripping voltammetry was widely used for analysis of lead.

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Electrophoresis Migration of charged solutes/particles in an electrical field Five components: driving force, support medium, buffer, sample, detecting system Procedure –Sample is soaked in hydrated support for 5 minutes. –Support is put into electrophoresis chamber filled with buffer. –Constant voltage or current is applied for a specific time. –Support is removed and placed in fixative or rapidly dried. –Zones are stained with appropriate dye.

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Electrophoresis (cont’d) Support Materials: cellulose acetate, agarose gel, polyacrylamide gel, starch gel Treatment and Application of Sample Detection and Quantitation Electroendosmosis Isoelectric Focusing Capillary Electrophoresis Two-dimensional Electrophoresis

Copyright © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins Osmometry Measures concentration of solute particles in a solution Freezing-point osmometer –Sample in a small tube is lowered into a chamber with cold refrigerant circulating from cooling unit. –Thermistor is immersed in sample. –Wire is used to stir sample until it is cooled to several degrees below its freezing point.