A spectrophotometer measures the transmittance or absorbance of light through a sample as a function of wavelength. It consists of five main components: a radiant energy source, a monochromator to select wavelengths, a sample compartment, a detector, and a recorder. The monochromator separates light into individual wavelengths using filters, prisms, or diffraction gratings. The detector converts the transmitted light intensity into an electrical signal proportional to absorbance. Spectrophotometers can operate in single or double beam mode for measuring samples.

1. Spectrophotometry-Instrumentation
A spectrophotometer is an instrument used for:
 measuring the transmittance or absorbance of a sample as a function of
wavelength.
Measuring the absorbance of a series of samples at a single wavelength.
The principle of working of
spectrophotometer depends on
photoelectric phenomenon, where,
the intensity of EMR is measured
through the intensity of electric
current produced by electrons
liberated from a photosensitive metal
under the influence of incident EMR.

2. Instrumentation-Spectrophotometer
1- Radiant energy source.
2- Dispersing system (or monochromator) “ wave length selector”.
3- Sample compartment (cuvette).
4- Detector.
5- Recorder (meter).
Instrument Components
All instruments for absorption measurements with UV, VIS and IR radiation are
made up of 5 components shown in this figure.
The 5 basic components of Spectrophotometer are:

3. Spectrophotometer- Instrument Components
1. Source of Light:
a. Deuterium (hydrogen) Lamp 190~420nm UV region
b. Tungsten Lamp350 ~ 2,500nm Vis and near IR region
 Purpose of monochromator is separation of multi-wavelength light into individual
wavelengths.
Monochromators Components are:
entrance slit; dispersing element; exit slit.
2. Monochromator (Dispersing System)

4. 2. Monochromator (Dispersing System)
Dispersing Element (Device):
Monochromatic light may be obtained by the use of Three types of dispersing elements
Filter: acts by selective absorption of unwanted  and transmit the complementary color
Prism: acts by refraction of light
grating: acts by diffraction and interference.
Dispersing Element (Device):
a. Filters
Act by selective absorption of unwanted  and
transmit the complementary color, which is needed
to be absorbed by the sample to be analyzed.
Wavelength
absorbed (nm)
Absorbed
color
Transmitted
color
400 Violet Yellow-green
450 Blue Yellow
500 Green red
550 Yellow Blue
600 Orange Green-blue
700 red Green
Color substances appears colored because they
selectively absorbed some of wavelengths of
visible light and transmitted other wavelengths
or colors.
For example red substances absorb green
wavelengths from the visible region, so the
transmitted light appears red, table 1.

5. Spectrophotometer –Instrument Components
2. Monochromator
c. Grating
It Acts by diffraction and interference
It consists of a large number of parallel grooves, very close to
each other, on a highly polished surface e.g aluminum or
aluminized glass (600 line/mm).
Each groove functions as a scattering center for light rays falling
on its edge. Through diffraction and interference, the grating make
the light beam into almost single .
b. Prism
 Act by refraction of light.
 In visible range we use glass prism.
 In U.V range we use prism made of quartz (fused silica).

6. a. entrance slit
b. collimating mirror or lens
c. a prism or grating
d. focal plane
e. exit slit
Spectrophotometer –Instrument Components
2. Monochromator

7. Spectrophotometer –Instrument Components
3. Sample Container (Absorption Cell)
The sample compartment contains a cell holder and sample container (cuvette-cell).
Sample Container should be
Transparent to excitation light.
Compatible with samples.
Sample container can made of
1. Quartz, used for UV-Vis region
2. Glass, used for Vis
There is a wide variety of cuvette of different shape and size, choice depends on the
type of analysis and sample volume.
The most common cuvettes for VIS and UV-spectra have a 1.00cm path length.

8. Spectrophotometer Components
4. Detector (Transducer)
 There are many Types of Detectors:
 Phototubes
 Photomultiplier Tubes
 Photodiodes
 Photodiode arrays
 Detector converts electromagnetic ( radiant , photons) energy to an electrical signal.
Ideal detector :
high sensitivity,
high signal/noise,
constant response for λs,
and fast response time.

9. Spectrophotometer Components
4. Detector (Transducer)
 Photons  photoelectrons
the “photocurrent” is proportional to intensity
Application a potential between the electrodes, cause the emitted photoelectrons to
migrate to the wire anode, producing a current.
This current is proportional to the number of photons. This current is readily amplified
and measured.
 Phototube Detector

10. PMT is a very sensitive detector
Spectrophotometer Components
4. Detector (Transducer)
 Photomultiplier tube Detector

11. The Spectrophotometer
1) Spectrophotometer
a) Single-beam
b) Double-beam
 Spectrophotometer may be classified as:
 single or double beam (design of instrument), as shown in figure.
Double Beam Spectrophotometer: The advantages :
(1) Stability of the readings over time
(2) The ability to simultaneous correct for any solvent or
peripheral substance effects.
The disadvantages :
Expense and less sensitivity to measure
highly absorbing samples.

12. 12
wave Length A
430 0.08
450 0.22
470 0.29
490 0.44
510 0.62
520 0.85
540 0.87
570 0.42
600 0.16
630 0.06

13. • Draw the calibration curve then determine the conc. of sample
1 and 2 Concentration
Of standards
Absorbance A
ug/L *
0 0.00
2 0.13
4 0.25
6 0.38
8 0.52
10 0.64
12 0.77
Sample 1 0.34
Sample 2 0.68

14. • Draw the standard addition curve then determine the conc. of
the unknown sample.
Concentration
Added to sample
Absorbance A
ug/L *
0 0.35
2 0.48
4 0.60
6 0.73
8 0.87
10 0.99