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2. Instrumentation. Objectives Level I. Identify two physical properties of light. Define the following wave parameters: amplitude, period, frequency. Define the factors that characterize the energy of a photon. Identify three types of light scatter. Objectives Level I.
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2 Instrumentation
Objectives Level I • Identify two physical properties of light. • Define the following wave parameters: amplitude, period, frequency. • Define the factors that characterize the energy of a photon. • Identify three types of light scatter.
Objectives Level I • List several major instrument components for the following analyzers: • Spectrophotometer • Fluorometer • Nephelometer • Mass spectrometer • Gas chromatograph • Densitometer
Objectives Level I • Identify the significant regions of the electromagnetic spectrum (EMS) from lowest energy to highest energy levels. • List four spectrophotometric function checks. • Define the following terms associated with electrochemical methods: potentiometer, amperometry, coulometry, conductance, resistivity, and voltammetry.
Objectives Level I • Write the Nernst equation. • Write the chemical reactions for the PO2 and PCO2 electrode. • Identify four examples of separation techniques used in the clinical laboratory. • List four examples of transducers used in biosensor devices. • Identify three factors that affect chromatographic resolution.
Objectives Level I • Define the following terms: diffuse reflection, retention time, Rf, fluorescence, and chemiluminescence. • Identify four colligative properties of solutions. • Identify specific analyte(s) that are measured by each device or instrument.
Objectives Level II • Diagram the correct sequence of significant components of a spectrophotometer. • Determine which component of a spectrophotometer malfunctioned, given the failure of a specific photometric function check. • Calculate the concentration of a solution given the absorbance values for tests and standards.
Objectives Level II • Convert between units used to describe wavelengths. • Predict the shape of the line when given the following x-and y-axis parameters: • Absorbance (y) versus concentration (x) on linear graph paper • Percent transmittance (y) versus concentration (x) on linear graph paper
Objectives Level II • Explain the fundamentals principles of selected instruments described in this chapter. • Explain how absorbance and transmittance of light are related. • Calculate wavelength, frequency, and photon energy of electromagnetic radiation (EMR).
Properties of Light • Electromagnetic radiation • Wavelength • Frequency • Amplitude • Period
Figure 2-1 Diagram of a beam of monochromatic, plane-polarized radiation. The electric and magnetic fields are at right angles to one another and direction of travel. Wavelength, , from peak to peak and amplitude A, length of electric waveform at maximum peak height.
Energy of Electromagnetic Radiation • Energy of a photon • Planck’s constant • Frequency • Velocity of light in a vacuum
Figure 2-2 The relationship among frequency, wavelength, and photon energy throughout the electromagnetic spectrum.
Practical Aspects of Light • Beer-Lambert law • Graphs: • Percent transmittance versus concentration • Absorbance versus concentration • Deviation from Beer-Lambert law
Figure 2-3 The attenuation of a beam of monochromatic radiation, P, as it passes through a cuvette with a light path, b, containing an absorbing particle in solution.
Absorption Spectroscopy Beer-Lambert law Also A = 2 – log %T
Figure 2-4 A plot of percent transmittance versus cuvettes 1 through 6 for a series of solutions of identical concentrations and cuvette path length. This illustrates the nonlinearity of the relationship between percent transmittance and concentration.
Figure 2-5 Transmittance and absorbance as a function of concentration. A. %T, linear scale. B. Absorbance, linear scale.
Figure 2-6 A plot of absorbance versus concentration that illustrates deviation from the Beer–Lambert law.
Instrumentation – Spectrometers • Radiant-energy sources • Wavelength selectors • Monochromators • Cuvettes • Radiation transducers
Figure 2-7 Schematic representation of five fundamental components of a spectrophotometer: 1. radiant energy source, 2. wavelength selector, 3. sample holder (cuvette), 4. photodetector, and 5. readout device.
Figure 2-8 Spectral percent transmittance characteristics of a wavelength selector.
Figure 2-9 Spectral percent transmittance characteristics of an interference filter versus an absorption filter.
Figure 2-10 Two types of grating monochromators. A. Echellette-type grating.
Figure 2-10 (continued) Two types of grating monochromators. B. Holographic grating patterned in photoresist.
Figure 2-12 Diagram of a semiconductor diode. (P+ is p-type semiconductor material, N is n-type semiconductor material, E is emitter electrode, and SiO2 is silicon dioxide.
Figure 2-13 Schematic of a single-beam spectrophotometer. Symbol e– represents the electrical signal created by photomultiplier tube.
Tungsten Lamp • Polychromatic light source
Reflectometry • Specular reflectance • Diffuse reflectance • Reflectometers
Figure 2-15 Diagram of a typical reflectometer. A. Polychromatic light source. B. Monochromator. C. Slit. D. Diffuse reflective surface. E. Lens. F. Photodetector. G. Readout device.
Atomic Absorption Spectroscopy • Measure the amount of EMR absorbed by elements in their ground state (Go). • Concentration is directly proportional to absorption.
Hollow Cathode Lamp PMT Detector
Fluorometry • Principles of fluorometry: • Fluorometry involves exciting compounds with EMR (high energy, short wavelength) and detecting emitted EMR (lower energy, longer wavelength). • Concentration is directly proportional to the amount of emitted EMR.
Principles of Fluorometry • Luminescence: Energy exchange process that occurs when valence shell electrons absorb EMR, become excited, and return to energy level lower than their original level • 2 types of luminescence • Excitation requires absorption of radiant energy • Excitation does not require absorption of radiant energy
Types of Luminescence • Excitation requires absorption of radiant energy • Fluorescence: atoms absorb energy at a particular wavelength(excitation), electrons are raised to higher-energy orbitals, electrons release energy as they return to ground state by emitting light energy at a longer wavelength & lower energy
Types of Luminescence • Excitation does not require absorption of radiant energy • Chemiluminescence: process where chemical energy of a reaction produces excited atoms, and upon electron return to ground state, photons of light are emitted • Oxidation of an organic cmpd (dioxetane, luminol, etc.) • Detector: luminometer that contains a PMT
