Chapter 5: Analytic Techniques By Julia C. Drees, Matthew S. Petrie, and 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 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.) Components of a Spectrophotometer Light source Monochromators Sample cell Photodetectors Spectrophotometer Quality Assurance Wavelength accuracy Stray light Linearity

Spectrophotometry and Photometry (cont.) Single-beam spectrophotometer—see Figure 5.5

Spectrophotometry and Photometry (cont.) 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 See Figure 5.11 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.) Fluorometry Basic instrumentation Measure concentrations of solutions that contain fluorescing molecules. 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.) Basic filter fluorometer—see Figure 5.13

Spectrophotometry and Photometry (cont.) Chemiluminescence Part of chemical energy generated produces excited intermediates that decay to a ground state with emission of photons. No excitation radiation or monochromators are required. Involves oxidation reactions of luminol, acridinium esters, and dioxetanes. Turbidity and Nephelometry Turbidity measures concentration of particulate matter in sample. Nephelometry: similar to turbidity, except different angle of measurement. See Figure 5.16

Spectrophotometry and Photometry (cont.) Laser Applications LASER: Light Amplification by Stimulated Emission of Radiation. Based on interaction of radiant energy and suitably excited atoms or molecules. 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.) Ion-Selective Electrodes (ISE): designed to be sensitive toward individual ions pH Electrodes—see Figure 5.18 Indicator electrode Reference electrode Liquid junctions Readout meter Nernst equation Calibration pH combination electrode

Electrochemistry (cont.) 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 See Figure 5.22

Electrophoresis (cont.) 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—see Figure 5.27 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.