Chapter 23MEASUREMENT INVOLVING LIGHT:PRINCIPLES AND INSTRUMENTATION
LIGHT IS PART OF THE ELECTROMAGNETICSPECTRUM γ rays X rays UV light Visible light IR light Microwave Radio waves
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)
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
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)
ANSWER 400nm is violet. The complementary color of violet is yellow. The solution will look yellow to our eyes.
SPECTROPHOTOMETERY Use of instruments design to measure Transmittance And Absorbance Of light
TRANSMITTANCE t= light transmittance through a sample light transmittance through water blankIt is common to calculate %T%T=t x 100%
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
QUESTIONS If the transmittance is 100%. What is the absorbance? If the transmittance is 0, what is the absorbance?
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
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