150 likes | 160 Views
Chapter 5: Analytic Techniques. By Julia C. Drees, Matthew S. Petrie, Alan H.B. Wu. Spectrophotometry and Photometry. Photometric instruments measure light intensity without consideration of wavelength.
E N D
Chapter 5: Analytic Techniques By Julia C. Drees, Matthew S. Petrie, Alan H.B. Wu
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
Spectrophotometry and Photometry (cont’d) • Components of a Spectrophotometer • Light source • Monochromators • Sample cell • Photodetectors • Spectrophotometer Quality Assurance • Wavelength accuracy • Stray light • Linearity
Spectrophotometry and Photometry (cont’d) • Single-beam spectrophotometer
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
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
Spectrophotometry and Photometry (cont’d) • Basic filter fluorometer
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
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.
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.
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
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.
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.
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
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.