Spectrophotometer
SPECTROPHOTOMETER
AND
COMPONENTS
SPECTROPHOTOMETER
S
S
Components of Spectrophotometer
Single Beam Spectrophotometer
Operation of single beam spectrophotometer
1 Light Source: 4 Types of Light Sources
UV to Mid Visible Visible & Near Infrared
4
Medium & high pressure Incandescent Tungston or
Mercury lamp, emits continum Tungston-iodide
Low pressure Mercury lamp Deutrium discharge lamp &
gives sharp line spectrum Mercury arc lamp, continuous
UV & Visible Ultraviolet
5 most important factors for light source
Range
Spectral distribution
Source of radiant production
Stability of radiant energy
Temperature
2 Monochromator & its function
Many light wavelengths
One wavelength isolated
Degree of wavelength isolation depends on 3 factors
Bandpass of the type of monochromator
Width of entrance slit
Width of exit slit
4 types of monochromators are;
Colored Filters
Interference Filters
Prism
Diffraction gratings
1. Colored filters
Pass wide band
Colored
Light ray enters Low transmittance
filter
colored filter
Not precise but simple,
Inexpensive and useful
2. Interference filters produce monochromatic
light based on constructive interference of waves
Glass 1 Spacer Glass 2
Multiwavelength Waves with
Light constructive
interference
will go out
Destructive Interference
waves cancel each other
3. Prism: Refraction of light waves
Diffraction grating mechanism of operation
Wavelengths bend as they
move past corners
Degree of bending
depends on wavelength
In phase wave front
Wave fronts form reinforce each other
& come out
Not in phase wave fronts
cancel each other
Results in complete
spectra
Diffraction grating consisting
of many parallel groves
3 Sample cell/ Flowcell
4 Photodetector
1. Photocell: Structure & Function
Silver
Selenium
4 Advantages of Photocell
1 The produced current is proportional to
incident radiation
2 Require no external voltage source
3 Inexpensive
4 Durable
3 Disadvantages of Photocell
1 Output electrical energy not easily amplified
2 Used in filter photometers with high illumination
3 Non-linear at low and high level of illumination
2: Phototube: Structure & Function
Advantage & disadvantage of Phototube
1 Photocurrent is linear
2 However external voltage is required
3: Photomultiplier Tube: Structure & Function
2 Advantages of Photomultiplier tube
1 PM tube is 200 times more sensitive than
the phototube
2 PM tubes are used in extremely sensitive
instruments
4: Photodiode: Structure & Function
3 Advantages of Photocell
1 Excellent linearity
2 Good Speed
3 Small size
QUALITY ASSURANCE
OF
SPECTROPHOTOMETER
QUALITY ASSURANCE
S
5 Checks for Quality Assurance
1 Absorbance Correction
2 Wavelength Accuracy
3 Stray Light
4 Linearity
5 Lipemia
Absorbance must be blanked with a reference solution for single
beam spectrophotometers
4 reference solutions for blanking single beam
spectrophotometers
Water
Air (enzymes)
Reagent (most cases)
Sample (bilirubin)
Double beam spectrophotometers permit automatic
correction of sample and reference absorbance
Wavelength indicated on control dial must match
actual wavelength passed by monochromator
Standard Absorbing Filter with known Mx.Abs
Solution
Bandpass of a monochromator defines the range of
wavelengths transmitted
Wavelength calibration using filter of known
absorbance maxima
Filter Abs Maxima 340 nm
Grating
PM Tube
Max Abs
Display
340 nm
Max Wavelength matched
Control Dial
Stray light refers to wavelengths outside the band
transmitted by monochromator
Grating
Absorbance
PM Tube
Display
Control Dial
3 most common causes of stray light are;
1 Reflection of light from scratches on optics
2 Reflection from dust Particles
3 Higher order spectra of gratings
Use of cutoff filters to detect stray light
Cutoff filter
Grating
Absorbance
PM Tube
Display
Stray light detected
Control Dial
Preparation of calibration Curve (Linear)
1.5
1.4
1.3
1.2
1.1
1
Absorbance
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Concentration g/dL
Use of diluted colored solutions to check linearity
Filter
Grating
PM Tube
Max Abs
Display
340 nm
Control Dial
High centrifugation clears lipemia of a turbid sample
(14000 RPM)
14000 RPM
25 min
