Spectrophotometer Prof.Dr. Moustafa M. Mohamed Vice Dean Faculty of Allied Medical Science Pharos...
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Transcript of Spectrophotometer Prof.Dr. Moustafa M. Mohamed Vice Dean Faculty of Allied Medical Science Pharos...
Spectrophotometer
Prof.Dr. Moustafa M. MohamedVice Dean
Faculty of Allied Medical SciencePharos University in Alexandria,
EGYPT
Absorption Spectroscopy
Sample
M
MM
M
M
MM
M
M
M
reflection scattering
h
in out
h
****
Absorption SpectroscopyGeneric Instrument
DetectorLightSource
Mono-chromator
Sample
P0 P
P0 = intensity of light into sampleP = 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
0P
PT
Absorption Spectroscopy
P0 = 1.00 P = 0.50 P = 0.25 P = 0.125
• sample with T = 50%
Absorption Spectroscopy
0
0.5
1
Concentration or Path Length
T
Absorption Spectroscopy
• Absorbance (A)
TlogT
1log
P
PlogA 0
TAT
T %log2 ;100
%
0 A
Absorption Spectroscopy
Concentration or Path Length
A
Beer’s Law
A = bcBeer’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, 200-400 nm - electrical
discharge in D2
– 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
Ir
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 Visibleh
e-
-Vdynode
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:I1= T * I0
I0 = Initial light intensity and I1= attenuated light intensity
• The results are displayed in percent optical color transmittance or absorption to indicate hemoglobin concentration.
percentxI
IT
o
1001
Single Beam Instrument
DetectorLightSource
(TungstenLamp)
Filter, Mono-
Chromator, Grating
Sample
QuartzCuvette
Photo-multiplier
Slit
Double Beam Instrument
Slit
Beam Chopper
Reference
Mirror Mirror
Semi-transparentMirror
TungstenLamp
Grating Photo-multiplier
Quartz Cuvette
Sample
Mirror
Colorimeter- filter photometer
• The concentration of the unknown solution can be found from the following relationship:
Cu= Cs Au/As
where
Cu= unknown concentration
Cs= standard concentration (for calibration)
Au= unknown absorbance
As= 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 (V1) and adjust potentiometer (R1) for 0 volt
2- Remove the ground and place reference concentration in cuvettes 1 an 2
3- Adjust potentiometer R1 for 0 V4- 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.