theory_and_suggestions_for_spec_lab - Spectrophotometric...

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Spectrophotometric Analysis Introduction A wave is often defined as a “vibrating disturbance by which energy is transmitted”. There are three characteristic properties associated with a wave and these are: 1) Wavelength ( λ )- the distance between identical points on successive waves 2) frequency ( ν )- the number of waves that pass through a particular point in a second and 3) amplitude- the height of the peak or trough of a wave. An electromagnetic wave is a type of wave that has associated with it an electrical field and a magnetic field. Light that we can see with our eyes, also known as visible light, is an example of an electromagnetic radiation. You may have come across waves such as radio waves, micro waves, or x-rays which are also examples of electromagnetic radiation. What makes one wave different from another wave is the wavelength and frequency associated with it. The two quantities that describe a particular wave are inversely related to each according to the formula: c = νλ , where c is the speed of light = 3.00 × 10 8 m/s The electromagnetic spectrum (figure 1) is an arrangement of the different kinds of waves in the order of increasing wavelengths (or decreasing frequencies). Figure 1 (Please refer to the BLB text for a color picture, page 201, figure 6.4). As mentioned above, electromagnetic radiation at wavelengths which the human eye can see as colors range from red (longer wavelengths; ~ 700 nanometers) to violet (shorter wavelengths; ~400 nanometers.) and are referred to as the visible region of the electromagnetic spectrum. A solution containing a substance that absorbs light in the visible range of the electromagnetic spectrum will appear colored to the eye. The color that we observe depends on the wavelength of the radiation that substance absorbs. For instance, if you look at a solution of vitamin B 2 (also known as riboflavin), it appears yellow in color. When you examine at what wavelengths of visible light a solution of riboflavin absorbs the most, we find that the solution absorbs maximum light at a wavelength of 450 nm. Gamma ray 10 -2 10 0 10 2 10 4 10 6 10 8 10 10 10 12 X-ray U V Infrared Micro- wave Radio waves Wavelength (nm) 10 20 10 18 10 16 10 14 10 12 10 10 10 8 10 6 Frequency (s -1 ) 400 nm 700 nm
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From figure 1, we find that this wavelength corresponds to a violet-blue color. The molecules of riboflavin therefore are absorbing the violet-blue parts of the visible light, and all the rest of the visible light will be not-absorbed or in other words transmitted. The riboflavin is essentially removing the blue-violet light from the while light and as a result the color you observe for riboflavin is a mixture of the rest of the “un-absorbed” colors. In general the color of a solution that can be observed by the human eye is the
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This note was uploaded on 05/23/2008 for the course BIOL 21 taught by Professor Dahlhoff,elizabeth during the Winter '08 term at Santa Clara.

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theory_and_suggestions_for_spec_lab - Spectrophotometric...

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