Spectrophotometry PDF

Spectrophotometry Prepared by: Billy Africa, RMT, MPH  Electromagnetic Energy Radiant energy from short wavelength gamma rays to long wavelength radio waves They are photons of energy traveling in a wavelike manner. The shorter the wavelength, the lighter the electromagnetic ene...

Spectrophotometry Prepared by: Billy Africa, RMT, MPH  Electromagnetic Energy Radiant energy from short wavelength gamma rays to long wavelength radio waves They are photons of energy traveling in a wavelike manner. The shorter the wavelength, the lighter the electromagnetic energy. Types of Electromagnetic energies: Cosmic rays Gamma rays X-rays Ultra-violet (UV) Visible Infrared (IR) Radio, TV, microwave, etc.  Energy – transmitted via electromegnetic waves characterized by frequency and wavelength  Wavelength distance between peaks as light is envisioned to travel in wavelike manner. Kinds of wavelength: – visible spectra – 340 nm – 700 nm – invisible spectra – below 340 nm (ultraviolet region (UV) above 700 (infrared region [IR]) Frequency – number of vibrations of wave motion per second  COLORIMETRY There are two primary considerations in every colorimetric analysis: – quality of the color – intensity of the color  COLORIMETRY Kinds of Colorimetry: Visual Colorimetry – uses the eyes in determining end point Photoelectric Colorimetrty – 2 Categories Spectrophotometry – (Spectrophotometric measurements) Filter photometry – (Photometric measurements)  SPECTROPHOTOMETRY AND FILTER PHOTOMETRY: SPECTROPHOTOMETRY – measurement of light intensity in a much narrower wavelength. It uses a device (prisms and/or gratings) to disperse the source of light into a continuous spectrum. FILTER PHOTOMETRY – measurement of light intensity of multiple wavelengths. It uses filter to isolate part of the spectrum.  BEER’S LAW OR BEER-LAMBERT’S LAW States that the concentration of a substance is directly proportional to the amount of the light absorbed or inversely proportional to the logarithm of transmitted light.  TRANSMITTANCE ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample.  ABSORBANCE the amount of light absorbed Proportional to the inverse log of transmittance Mathematically derived from %T A = 2 – log%T A = -log%T = 1/log%T  A = abc Where: A = absorbance a = molar absorptivity (absorptivity of the compound under standard conditions) b = length of light through the solution (light path) c = concentration of absorbing molecules/solution  Components of Spectrophotometer  LIGHT SOURCE Provides radiant energy in the form of visible or non-visible light that may pass through the monochromator. The light of proper wavelength will be made incident on the analytical cell.  Tungsten Light bulb – commonly used light source in the visible and near infrared region (up to 1,200 nm) LASER (Light Amplification by Stimulated Emission of Radiation) Factors for choosing a light source – Range – Spectral distribution – Source – Stability  Types of Light Source: Tungsten Iodine lamp – produces energy wavelength from 340 to 700 nm (visible region). Quartz Halide lamp – contains small amounts of halogen such as iodine to prevent the decomposition of the vaporized tungsten from the very hot filament. Deuterium Discharge lamp – provides energy source with high output in the UV range (down to 165 nm). Infrared Energy source – used above 800 nm. Examples: Merst glower – an electrically heated rod of rare earth element oxides. Globar – uses silicon carbide Mercury Vapor lamp – exits narrow bands of energy at well defined places in the spectrum (UV and visible) Hollow Cathode lamp – consists of a gas-tight chamber containing anode, a cylindrical cathode, and inert gas such as helium of argon.  Entrance Slit Entrance Slit – minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system Stray light causes systematic error; causes loss of linearity  MONOCHROMATOR Isolate specific wavelength of light. Monochromatic light – light radiation of a single wavelength.  TYPES OF MONOCHROMATOR: Prisms – wedge-shaped pieces of glass, quartz, NaCl, or some other material that allows transmission of light. Disperses white light into a continuous spectrum of colors based on variation of refractive index for different wavelength.  TYPES OF MONOCHROMATOR: Gratings -has small grooves cut at such as angle that each groove behave like a very small prism. Separates white light into various color component. Based on the principle that wavelengths are bent as they pass a sharp corner.  TYPES OF MONOCHROMATOR: Colored filters – made of a glass that absorbs some portion of the electromagnetic spectrum and transmit others. Light energy is absorb by dye compounds on the glass and is dissipated as heat.  TYPES OF MONOCHROMATOR: Interference filter – utilizes the wave character of light to enhance the intensity of the desired wavelength by constructive interference and eliminates others by destructive interference and reflections.  Exit Slit Exit Slit – controls the width of light beam (bandpass; accurate