Chaprter 23 spectrophotometer

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  • 1. Chapter 23MEASUREMENT INVOLVING LIGHT:PRINCIPLES AND INSTRUMENTATION
  • 2. LIGHT IS PART OF THE ELECTROMAGNETICSPECTRUM γ rays X rays UV light Visible light IR light Microwave Radio waves
  • 3. UNITS Light progresses in waves (lambda or λ) that produce photons – the basic unit of light Light is measured in nanometers (nm= 10-9 meter)  Visiblelight between 380 and 780nm. Table 23.1 page 405  UV light between 280 and 315nm  Ranges from UV-A (tanning, fluorescence) to UV-B – used to visualize DNA in gels, to UV-C (germicidal lamps)
  • 4. LIGHT AND MATTER Types of interactions of light and matter:  Transmittance (pass through)  Reflection (change direction i.e. mirror)  Scatter - (deflection in many directions , i.e. prism)  Absorbance – energy transferred to the material (i.e. sunlight absorbed by earth) Fig. 23.3 on page 405
  • 5. CONT. White light is a mixture of many wavelength and it is called polychromatic When one color is removed our eyes see the complementary color  If a solution absorb light at 400nm, what will be the color that we see? (table 23.3 page 406)
  • 6. COLOR WHEEL
  • 7. ANSWER 400nm is violet. The complementary color of violet is yellow. The solution will look yellow to our eyes.
  • 8. SPECTROPHOTOMETERY Use of instruments design to measure  Transmittance And  Absorbance Of light
  • 9. TRANSMITTANCE t= light transmittance through a sample light transmittance through water blankIt is common to calculate %T%T=t x 100%
  • 10. ABSORBANCE (A) Represents the amount of light absorbed by a sample Absorbance calculation is based on transmittance Also called optical density or OD A=OD=log10(t) Absorbance does not have units Spectrophotometers convert transmittance values to absorbance
  • 11. QUESTIONS If the transmittance is 100%. What is the absorbance? If the transmittance is 0, what is the absorbance?
  • 12. SPECTROPHOTOMETER Contain  Lightsource  wavelength selector (filter or monochromator)  Sample chamber holds a cuvette. The cuvette dimensions determines the path length  Detector converts light into a readable form. Common detector is photomultiplier  For accuracy t is important to work in the midrange of the of the instrument
  • 13. CUVETTES Made of glass or plastic. Not all transmit/absorb in UV range Quartz glass are commonly used for UV range There are disposable plastic cuvettes suitable for UV UV cuvettes are more expensive
  • 14. SPEC 20