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Introduction to Spectrophotometry

Introduction to Spectrophotometry. Properties of Light. Electromagnetic radiation moves in waves. Electromagnetic Spectrum. Electromagnetic Spectrum. Colors & Wavelengths. Visible Light. What is Colorimetry?. The solutions of many compounds have characteristic colors.

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Introduction to Spectrophotometry

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  1. Introduction to Spectrophotometry

  2. Properties of Light • Electromagnetic radiation moves in waves

  3. Electromagnetic Spectrum

  4. Electromagnetic Spectrum

  5. Colors & Wavelengths Visible Light

  6. What is Colorimetry? • The solutions of many compounds have characteristic colors. • The intensity of such a color is proportional to the concentration of the compound.

  7. What are Spectroscopy and Spectrophotometry?? • Light can either be transmitted or absorbed by dissolved substances • Presence & concentration of dissolved substances is analyzed by passing light through the sample • Spectroscopes measure electromagnetic emission • Spectrophotometers measure electromagnetic absorption

  8. Instruments of Measurement • Two most common: • Visible Spectrophotometer • Spect 20, Spect 88 • Atomic-Absorption Spectrophotometer

  9. Instruments of Measurement • What do visible spectrophotometers measure? • Amount of light absorbed by the dissolved substance • Qualitative • Quantitative

  10. Absorption of Light • White light • All colors • Polychromatic light

  11. Absorption of Light • Monochromatic light • Light of one color Red light is absorbed by the green solution

  12. The Spectrophotometer

  13. Definitions & Symbols • Intensity (I) • Transmittance (T) • It’s also referred to as %T or T x 100 • T = I/Io • Where Io is the intensity of the blank

  14. Graphical Relationship • % transmission and % absorption are not linearly related to concentration • For a graph to be useful, a straight line is needed • ABSORBANCE = log(1/T) = -log(T)

  15. Beer’s Law • The intensity of a ray of monochromatic light decreases exponentially as the concentration of the absorbing medium increases. • More dissolved substance = more absorption and less transmittance

  16. Spectral Transmission Curve • Optimum wavelength

  17. Standardization Graph • Standards (solutions of known concentration) of the compound of interest are made, treated, and their absorbances (ABS) and concentration values are used to create a Standardization Graph.

  18. Standardization Graph

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