1. Spectrophotometry Lecture

3. Interaction of Radiation and Matter

6. Absorption and Fluorescence

7. Terms

8. Interaction of Light with Matter

10. Molecules Absorption Wavelengths

11. Spectrophotometry Although a number of different types of spectrophotometers exist all have one thing in common.  Utilize light energy to detect molecules in a solution  Light energy is reported to the user as wavelengths in nanometers (nm) Different spec’s utilize wavelengths that fall into different ranges.  Visible (VIS) 350-700 nm  Ultraviolet (UV) 200-350 nm

12. Absorption Spectrophotometer

13. Spectrophotometer

14. spectrophotometer measures intensity of a light beam after it is directed through and emerges from a solution  Ex: solution of copper sulfate (CuSO4) absorbs light  The red part of spectrum has been almost complete absorbed by CuSO4 and blue light has been transmitted  Gain greater sensitivity by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum  But to do this, we have to isolate the red wavelengths

15. Spectrum of visible light How do you isolate red wavelengths of light?  In a spectrophotometer, a light source gives off white light which strikes a prism, separating light into its component wavelengths:  Red wavelengths pass through CuSO4 solution and measure amount of red light absorbed Colored compounds absorb light differently depending on the l of incident light l = Wavelength, nm=nanometers

16. Design of Spectrophotometer THE BLANK  In order to effectively use a spectrophotometer we must first zero the machine  Blank contains everything except compound of interest which absorbs light. By zeroing machine using "the blank," any measured absorbance is due to the presence of solute of interest ABSORPTION SPECTRUM  Different compounds having dissimilar atomic and molecular interactions have characteristic absorption phenomena and absorption spectra which differ  The point (wavelength) at which any given solute exhibits maximum absorption of light (the peaks on the curves on the figure below) is defined as that compounds particular lmax

17. Design of Spectrophotometer

18. Electromagnetic Spectrum

19. How is Does a Spectrophotometer Work? Amount of a particular molecule of interest is measured according to amount of light that is absorbed  Absorbance data is compared to a standard of a known concentration to determine the concentration of the unknown.

20. How is Does a Spectrophotometer Work? All spec’s share following common features:  Lamp i.e. tungsten or deuterium  Prism or grating  Sample holder  Display

21. Absorption Spectrophotometer

22. Absorption Curve

23. Background, B

24. Detection Limit DL or LOD

25. Dynamic or linear range

26. Sensitivity

27. Calibration Curve

28. Calibration Curve Procedures

29. Calibration Curve Plot

30. Example Nitrite Analysis

31. Results

32. Plot of Results

33. Best Fit Line

34. How are Concentrations Obtained Using a Spec? More molec…more to absorb light  Note peaks of absorption curves Lambda max  Wavelength at which a molecule absorbs the most light Proteins, like other molecules, interact with certain wavelengths of light  Proteins absorption spectrum can be determined by measuring proteins light absorbance at different wavelengths.  Determine the lambda max for protein

35. How are Concentrations Obtained Using a Spec? Most proteins are colorless  Light in visible range will not work  Light in UV range will work for a colorless solution ~280 nm Does NOT distinguish between different protein types in a solution

36. Using Bradford Reagent Way to colorize proteins and use white light spectroscopy  Solution changes from brown to blue when proteins present. Degree of “blueness” of Bradford-protein mixture can be used to determine concentration of protein in a solution

37. How are Concentrations Obtained Using a Spec? Calculating protein concentration in an unknown sample  Known standards are mixed with Bradford reagent and their absorbance values are determined Standard curve generated.  known absorbance values can be plotted and concentrations determined

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