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

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

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

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

Sample cuvettes 5

Scale 6

-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.

Light Spectrum(UV- Visible light -IR) 200-400 UV 400-700 Visible light 700-900 IR

Any solution containing a compound that absorb light in range of 400-700 nm will appear colored to the eye Incident light (I0) Transmitted light (I) Eye Absorbed light

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

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

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

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

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

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

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

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

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