Download h - Pharos University in Alexandria

Spectrophotometer
Prof.Dr. Moustafa M. Mohamed
Vice Dean
Faculty of Allied Medical Science
Pharos University in Alexandria,
EGYPT
Absorption Spectroscopy
Sample
h
M
M*
in
reflection
M
M
M
M*
*
M
M*
M
M
h
out
scattering
Absorption Spectroscopy
Generic Instrument
P0
Light
Source
Monochromator
P
Sample
Detector
P0 = 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)
P
T
P0
• 0T1
• T is independent of P0
• %T = T x 100
Absorption Spectroscopy
• sample with T = 50%
P0 = 1.00
P = 0.50
P = 0.25
P = 0.125
Absorption Spectroscopy
T
1
0.5
0
Concentration or Path Length
Absorption Spectroscopy
• Absorbance (A)
P0
1
A  log  log   log T
P
T
%T
T
;
A  2  log %T
100
0A 
A
Absorption Spectroscopy
Concentration or Path Length
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, 200-400 nm - electrical
discharge in D2
– Laser source
Monochromators
•
•
Mono - one; chromatic - color
Prisms and gratings - disperse (spread out) light
according to wavelength
h
Prism
Monochromators
• n = d(sin I + sin r)
• I = constant; therefore   r
r
I
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
eamplifier
-V
dynode
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 * I 0
I0 = Initial light intensity and I1= attenuated light
intensity
• The results are displayed in percent optical
color transmittance or absorption to indicate
hemoglobin concentration.
I1
T 
x100 percent
Io
Single Beam Instrument
Slit
Light
Source
(Tungsten
Lamp)
Filter,
MonoChromator,
Grating
Sample
Detector
Quartz
Cuvette
Photomultiplier
Double Beam Instrument
Beam Chopper
Semi-transparent
Mirror
Sample
Tungsten
Lamp
Grating
Slit
Quartz
Cuvette
Photomultiplier
Reference
Mirror
Mirror
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 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.