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