1. Spectrophotometer Prof.Dr. Moustafa M. Mohamed Vice Dean Faculty of Allied Medical Science Pharos University in Alexandria, EGYPT

2. Absorption Spectroscopy Sample M M M M M M M M M M reflection scattering h in out h * * **

3. Absorption Spectroscopy Generic Instrument Detector Light Source Mono- chromator Sample P0P0 P P 0 = intensity of light into sample P = intensity of light out of sample

4. Absorption Spectroscopy The more photons the sample absorbs, the lower the intensity (transmission) at the detector. Transmittance (T) 0  T  1 T is independent of P0 %T = T x 100

5. Absorption Spectroscopy P 0 = 1.00 P = 0.50P = 0.25P = 0.125 sample with T = 50%

6. Absorption Spectroscopy

7. Absorbance (A) 0  A  

8. Absorption Spectroscopy

9. Beer’s Law A =  bc Beer’s Law

10. Light sources Different light sources for different regions of the spectrum UV/Vis –Tungsten Lamp 320-2,500 nm - run an electrical current through a wire in vacuum –Deuterium arc lamp, 200-400 nm - electrical discharge in D 2 –Laser source

11. Monochromators Mono - one; chromatic - color Prisms and gratings - disperse (spread out) light according to wavelength Prism h

12. Monochromators n = d(sin  I + sin  r )  I = constant; therefore   r II rr d

13. Sample Holders Cuvettes –flat surface best - better reproducibility –avoid fingerprints, dust, etc. on surface –must be transparent in region of interest

14. Sample Holders UV - quartz Visible - glass, quartz IR - NaCl

15. Detectors Detector converts incident light to an electrical signal that we can measure and process

16. Detectors Ideal Characteristics –sensitive –linear –flat response v. –stable –fast

17. Detectors - photo tube Good for UV and Visible h e-e- -V dynode amplifier

18. Detectors - photo multiplier Characteristics –sensitive (single photons) –linear –flat response v. within limitations –stable w/ time (sensitive decreases over time, weeks to months) –fast

19. Detectors - Array Channel plate (similar to multiplier) photodiode array (less sensitive than multiplier) charge coupled device (CCD) (more sensitive than multiplier) Do not need a monochromator

20. Colorimeter An optical electronic device that measures the color concentration of a substance in solution. Optical color filters are used to select a narrow wavelength. Basic colorimeter analysis involves the precise measurement of light intensity.

21. Transmittance is defined as: I 1 = T * I 0 I 0 = Initial light intensity and I 1 = attenuated light intensity The results are displayed in percent optical color transmittance or absorption to indicate hemoglobin concentration.

22. Single Beam Instrument Detector Light Source ( Tungsten Lamp) Filter, Mono- Chromator, Grating Sample Quartz Cuvette Photo- multiplier Slit

23. Double Beam Instrument Slit Beam Chopper Reference Mirror Semi-transparent Mirror Tungsten Lamp Grating Photo- multiplier Quartz Cuvette Sample Mirror

24. Colorimeter- filter photometer

25. The concentration of the unknown solution can be found from the following relationship: C u = C s A u /A s where C u = unknown concentration C s = standard concentration (for calibration) A u = unknown absorbance A s = standard absorbance

26. Light passes through an optical color filter, is focused by lenses on the reference and sample cuvettes and falls on the reference and sample photodetectors. The difference in voltage between the two detectors is increased by a dc amplifier and applied to a meter.

27. A calibration procedure is as follows: 1- Ground the amplifier input (V 1 ) and adjust potentiometer (R 1 ) for 0 volt 2- Remove the ground and place reference concentration in cuvettes 1 an 2 3- Adjust potentiometer R 1 for 0 V 4- Leave the reference concentration in cuvette 1 and replace cuvette 2 with a cuvette containing the sample 5-Read the unbalanced voltage on the meter in percent transmittance or absorbance units.