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Spectrophotometry and Photometry 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. Copyright © 2018 Wolters Kluwer • All Rights Reserved Spectrophotometry and Photometry Spectrophotometry and Photometry (cont.) (cont.) • Components of a Spectrophotometer – Light source – Monochromators – Sample cell – Photodetectors • Spectrophotometer Quality Assurance – Wavelength accuracy – Stray light – Linearity Copyright © 2018 Wolters Kluwer • All Rights Reserved Spectrophotometry and Photometry Spectrophotometry and Photometry (cont.) (cont.) • Single-beam spectrophotometer—see Figure 5.5 Copyright © 2018 Wolters Kluwer • All Rights Reserved Spectrophotometry and Photometry Spectrophotometry and Photometry (cont.) (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 Copyright © 2018 Wolters Kluwer • All Rights Reserved Spectrophotometry and Photometry Spectrophotometry and Photometry (cont.) (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 Copyright © 2018 Wolters Kluwer • All Rights Reserved
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