1. Measurements involving light –A Basic Principles and Instrumentation
Chapter 23

2. 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

3. Diagram of a Wave

4. Electromagnetic spectrum

5. 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

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

7. 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)

8. 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?

9. Spectrophotometer Schematic Diagramb
Light source
monochrometer Po
sample P
detector

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

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