Transcript LWW PPT Slide Template Master

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
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Sample cell
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Photodetectors
• Spectrophotometer Quality Assurance
–
Wavelength accuracy
–
Stray light
–
Linearity
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Spectrophotometry and Photometry
(cont’d)
• Single-beam spectrophotometer
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Spectrophotometry and Photometry
(cont’d)
• Atomic Absorption Spectrophotometer
–
Measures concentration by detecting absorption of
electromagnetic radiation by atoms rather than by molecules
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Sensitive, precise
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Routinely used to measure concentration of trace metals that are
not easily excited
• Flame Photometry
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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
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Spectrophotometry and Photometry
(cont’d)
• Basic filter fluorometer
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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
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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.
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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.
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Electrochemistry (cont’d)
• Ion-Selective Electrodes (ISE): designed to be sensitive
toward individual ions
• pH Electrodes
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Indicator electrode
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Reference electrode
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Liquid junctions
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Readout meter
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Nernst equation
–
Calibration
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pH combination electrode
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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.
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Electrophoresis
• Migration of charged solutes/particles in an electrical field
• Five components: driving force, support medium, buffer,
sample, detecting system
• Procedure
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Sample is soaked in hydrated support for 5 minutes.
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Support is put into electrophoresis chamber filled with buffer.
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Constant voltage or current is applied for a specific time.
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Support is removed and placed in fixative or rapidly dried.
–
Zones are stained with appropriate dye.
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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
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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.
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