1. Principles of Spectrophotometer
Spectrophotometer - an instrument which can measure the optical density of a sample at any wavelength.

2. Construction:
Light Source Wave Length Selector (Filter/ Monochromator)  Slit  Sample (Cuvette) Detector (photo cells or tubes) Quantitative Analysis(Meter)

3. Wave Length Selector (Filter/ Monochromator)
  X ?
Each color needs a proper monochromatic light to be read properly by the photometer. This is done by choosing the proper monochromator ( filter). 3

4. DISPERSION OF POLYCHROMATIC LIGHT WITH A PRISM or Diffraction grating
Prism - spray out the spectrum and choose the certain wavelength (l) that you want by slit. 4

5. Sample cuvettes 5

6. Scale 6

7. -Light Spectrum(UV- IR- Visible light). Absorbed &Transmitted Light .
To study the principles
Certain terms should be understood!
-Light Spectrum(UV- IR- Visible light).
Absorbed &Transmitted Light .
Scattered, reflected Light.

8. Light Spectrum(UV- Visible light -IR) UV
Visible light IR

9. Any solution containing a compound that absorb light in range of nm will appear colored to the eye
Incident
light (I0)
Transmitted
light (I)
Eye
Absorbed light

12. Light reflection means that the radiation is not absorbed, but returns back.

13. Light scattering means that radiation is absorbed, then instantaneously remitted uniformly in all direction.

14. Beer’s Law
States that the concentration of a substance in solution is directly proportional to the absorbance (A) (O.D) of the solution.
Concentration
Absorbance
A  C

15. Spectrophotometer Analysis
II-Quantitative(direct or Indirect)= Allow to measure absorbance of a sample in solution and calculate the actual concentration of the sample
I-Qualitative = identifying unknown compounds by their absorption spectra

16. Qualitative Spectrophotometric assays
What is meant by absorption Spectra? An absorption spectrum can be generated by measuring the absorbance of the pure compound in solution at variety of wavelength

17. 1- Heme proteins or Cytochromes show a distinct absorbance in the visible range from 500-600 nm
2- The nitrogenous bases that comprises nucleic acids are known to absorb strongly at 260 nm

18. 3- The aromatic rings on tryptophan and tyrosine are known to absorb strongly at 280 nm.

19. Quantitative Spectrophotometric assays
Reference=Blank
Test
Known concentration=Standard 19

20. Thank You