Measurements involving light –A Basic Principles and Instrumentation Chapter 23

Wave Properties Electromagnetic radiation or radiant energy travels in wave patterns Two important properties of waves are wavelength and frequency Wavelength, l, is the distance between the crest or troughs of two successive waves Frequency, n, is the number of waves passing a given point in one second

Diagram of a Wave

Electromagnetic spectrum

Interaction of light with matter Light may be: transmitted – pass through Reflected – change directions Scattered – deflected in many different directions Absorbed – gives up some or all of its energy to the material

Color Wheel blue lne Bl-green Violet blue red yellow orange

Transmittance and Absorbance of Light Transmittance is the ratio of the amount of light transmitted through the sample to that transmitted by the blank, t t = light through sample/light through blank %T = t x 100 Absorbance is the amount of light not transmitted through the sample, A A = -log(t)

Continued Absorbance is also called optical density Ex: “A260 = 1.6” means that an absorbance of 1.6 is measured at a wavelength of 260 nm. What is the absorbance of a sample having a %T of 23?

Spectrophotometer Schematic Diagram b Light source monochrometer Po sample P detector

Components of a spectrophotometer Light source, bulb or lamp produces light to illuminate the sample Wavelength selector, monochromator, Entrance slit Dispersing element Diffraction gratings Exit slit Sample chamber, holds sample tubes

Continued Path length is the distance that the light travels through the sample or blank, interior dimension of the cuvette Detector senses the light coming from the sample and converts this information into an electric signal Photomultiplier tube Display, meter, or digital readout

Blanks Use the blank to set the spectrophotometer to 100% transmittance Must reset instrument each time the wavelength is changed Blank has everything present except the sample material

Cuvette Is the sample holder Glass and plastic are transparent to visible light but block UV light Quartz glass is transparent to Visible and UV light and is the common choice for cuvettes Must be clean and unscratched

Sample Should be well mixed and homogenous. No air bubbles Solvents must be chosen to eliminate absorbance of light in the region studied Table 23.6 gives details

Spectrophotometer designs Single beam Rugged and useful. Blank must be reset when wavelength is changed Scanning spectrophotometers Capable of rapid scanning through a range of wavelengths and constructing a spectrum Double-beam are popular

Continued Photodiode array detector, PDA, can determine the absorbance of a sample over the entire UV/Vis range, instantaneously Microprocessors and Spectrophotometers Allows even greater flexibility

Performance considerations Calibration Wavelength Accuracy Linearity of response Stray light (reach detector, not interact) Noise Baseline stability Resolution