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

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

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

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

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

Absorption Spectroscopy

Absorbance (A) 0  A  

Absorption Spectroscopy

Beer’s Law A =  bc Beer’s Law

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, nm - electrical discharge in D 2 –Laser source

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

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

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

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

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

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

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

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

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

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.

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.

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

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

Colorimeter- filter photometer

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

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.

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.