UNIT: Electromagnetic Radiation and Photometric Equipment

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1 UNIT: Electromagnetic Radiation and Photometric Equipment 3photo.wpd Task Instrumentation I To review the theory of electromagnetic radiation and the principle and use of common laboratory instruments using electromagnetic radiation. Objectives Upon completion of this exercise, the student will be able to: 1. State the wave and particle theory of electromagnetic radiation. 2. Know what colors correspond to visible wavelengths. 3. Identify the regions of the electromagnetic spectrum occupied by gamma, x-rays, UV, visible, IR, and microwaves indicating relative wavelength, frequency, and energy. 4. Know basic principles of how instruments determine results. 5. List and compare the principles/applications of different photometric equipment. Principle 1. Properties of light and radiant energy. Radiant energy behaves as if it has electric and magnetic fields and is found as discrete bundles of energy (photons) traveling in wave form (particle and wave theories). The more energy contained, the more frequent the wave and therefore, the shorter the wavelength. -9 Wavelengths are measured in nanometers (nm) or 10 meters. 2. Interactions of light and matter. A. Absorption (absorption spectroscopy UV, visible, infra-red, atomic) When an atom, ion, or molecule absorbs a photon, the additional energy results in an alteration of state (it becomes excited). Depending on the individual species, this may mean that a valence electron has been put into a higher energy level, or that the vibration or rotation of covalent bonds of the molecule have been changed. In order for a ray of radiation to be absorbed it must: 1. have the same frequency of the rotational or vibrational frequency in the molecules it strikes, and; 2. be able to give up energy to the molecule it strikes. MLAB Clinical Chemistry Lab Manual B 15

2 Energy Frequency Wavelength (nm) Type of Radiant Energy Reflects Absorbs <0.1 gamma non-visible x-rays non-visible <380 ultra-violet UV visible green-yellow yellow red blue green-blue green >750 infra-red (heat) non-visible IR 7 continuous >25 x 10 radiowaves (uwaves) non-visible violet blue green yellow orange red The energy absorbed is distributed throughout the structure. The absorption pattern of complex organic molecules will be the cumulative sum of the absorption of all of the individual covalent bonds as a result of the energy's distribution throughout the molecule. As the energy is released from the atoms, molecules, or incandescent solids, an emission spectra is formed. B. Emission spectra are of three types. 1. Line spectrum is light of one wavelength formed as the result of excited electrons of atoms returning to their ground state. The atom emits the same wavelengths as those absorbed by the atom. 2. Band spectrum is a group(s) of wavelengths formed from the return of excited electrons in molecules returning to their ground state. An array of wavelengths are emitted as the process is complicated by the internal energy of vibration and rotation (all substances except rarefied gas and atoms). 3. Continuous spectrum is a very broad and continuous group of wavelengths emitted by incandescent solids (tungsten lamps). Electromagnetic radiation in its various forms is measured in common laboratory instruments: A. Absorption of EM radiation 1. atomic absorption 2. spectrophotometry B. Emission of EM radiation 1. flame photometer 2. fluorometer 3. scintillation counter B 16 MLAB Clinical Chemistry Lab Manual

3 Transmission and Absorbance Instruments Fluorescence and Instruments Nephelometers Reflectance Spectrophotometers (diagram above left), as found in many modern instruments such as UA dipstick readers and Kodac s Ectachem: A, light source; B, ; C, filter or wavelength selector; D, collimating lens or ; E, test surface; F, collimating lens or ; G, detector; H, readout device. MLAB Clinical Chemistry Lab Manual B 17

4 Equipment Clinical Applications Principle Colorimetry/ photometry Spectrophotometry Reflectance Spectrophotometry Turbidimetry any procedure measuring color development or loss Same as above lipids, CSF protein, serum proteins, amylase absorption/transmission of light through a colored solution providing a means of measuring light absorbing particles visible change in Abs. between nm UV change in Abs. at 340nm of NAD NADH Filtered light is focused on a flat test surface. Some of the light is absorbed, the remainder is reflected onto a photodetector. The absorption of light by chromophores (reflection density) is inversely related to the intensity of reflected light. light transmission through a turbid solution Energy Source tungsten (deuterium, Hg, He) tungsten/uv (deuterium, Hg, He) tungsten tungsten/uv filter (interferenc e filter) prism or diffraction grating Nephelometry degree of light scattered UV filter fluorometer Fluorometry Emission Flame Photometry Atomic Absorption Spectrometry Densitometry (very basic photometer) drugs, hormones, intermediary metabolites electrolytes, trace minerals (Na, K, Li) Ca, Mg, Cu, Mn, Fe, Pb, Hg, etc. Hb, protein electrophoresis, CPK, LDH isoenzymes absorbs high intensity UV and emits longer wavelength visible light (lower energy) The energy absorbed as heat is released in the form of light energy of a wavelength specific for the element proportional to its concentration A hot flame releases metallic atoms from molecules. The ground state metallic atoms absorb monochromatic light, generated by a hollow cathode tube, proportional to their concentration photometer scans a colored strip Refractometry TP, specific gravity light bends with concentration; refractive index Scintillation Counters a. gamma b. beta (liquid) drug assays, hormones immunoassays (TSH, T3, T4, B, Folate) 12 EM radiation counts directly proportional to concentration. Sample in liquid phosphor. Fluorescence converted into longer wavelength filter filter UV source 2 filters/prism Primary exciter filter Secondary fluorescenc e filter hot flame hollow cathode tube of measured metal specific filter for Na, K, Li prism or diffraction grating Sample Holder varies paper, plastic strip, dry film or slide Monochromater burneraspirator burneraspirator tungsten/uv filter stage tungsten none stage atomic nucleus none B 18 MLAB Clinical Chemistry Lab Manual

5 Study Questions Name Instructions: Legibly write your answers in the space provided. Unless otherwise indicated, each question is worth one point. 1. Describe / define wavelength. 2. In clinical laboratory work, wavelength is most commonly measured in what unit? 3. The discrete bundles of energy emission in the electromagnetic spectrum are called. 4. Ultraviolet light has shorter / longer (circle one) wavelength than visible light and would therefore have higher / lower (circle one) energy than visible light. 5. Gamma rays have a very short wavelength. You would, therefore, expect them to have higher / lower (circle one) frequency and energy. 6. What type of emission spectra is expected from: a. atoms b. molecules c. solids 7. According to information presented in this lab, briefly summarize what happens when electromagnetic radiation is absorbed by an atom or molecule. (2 points) 8. A spectrophotometer and an atomic absorption spectrophotometer can be grouped together as they measure light. 9. A flame photometer and scintillation counter (gamma counter) measure light/radiation. 10. From the Table of Photometric Equipment included in the lab, list two (2) types of chemistry equipment that measure change in the electromagnetic radiation. MLAB Clinical Chemistry Lab Manual B 19

6 After reviewing the lab, provide a brief explanation of the principles of the following in your own words. (2 points each) 11. photometry 12. emission flame photometry 13. atomic absorption spectrophotometry 14. reflectance photometry fluorometry - B 20 MLAB Clinical Chemistry Lab Manual

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