Spectrophotometer

Measures the light that passes through

a liquid sample

Spectrophotometer gives readings in
Percent Transmittance (%T) and in
Absorbance (A)
The Electromagnetic Spectrum
n = c / l E = hn
Absorbance and
Complementary Colors
I
o

I
Cell with
Pathlength, b,
containing solution
light
source
detector
blank where I
o
= I
concentration 2
concentration 1
b
with sample
I < I
o
The process of light being absorbed by a solution
As concentration
increased, less
light was
transmitted (more
light absorbed).

The law states that the amount of light absorbed by a solution
(colored) is proportional to the concentration of the
absorbing substance and to the thickness of the absorbing
material (path length). Absorbance is also called optical
density
A = abc

where a molar absorptivity, b
pathlength, and c molar concentration

Some terminology
I intensity where I
o
is initial intensity

T transmission or %T = 100 x T
(absorption: Abs = 1 T or %Abs = 100 - %T)
T = I/ I
o

A absorbance
A = - log T = -log I/ I
o

The blank contains all substances
expect the analyte.
Is used to set the absorbance to zero:
A
blank
= 0
This removes any absorption of light
due to these substances and the cell.
All measured absorbance is due to
analyte.

Conventional
Spectrophotometer
1. A stable and cheap energy source.
2. A monochromator to break the polychromatic radiation into component and
wavelength/bands of wave length.
3. Transparent vessels (cuvettes) to hold the sample.
4. A photo sensitive detector and associated amplifier and recorder
Conventional
Spectrophotometer
Optical system of a split-beam spectrophotometer
LIGHT SOURCES
UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
IR Spectrophotometer
1. Carborundum (SIC)

Light Source

Deuterium Arc Lamp
UV Region
Wavelength Range :
190~420nm
Tungsten Lamp
Wavelength Range : Part of the UV and the whole of the
Visible
range (350 ~ 2,500nm)
Xenon Lamp
Wavelength Range : 190~800nm
Monochromator
Accepts polychromatic input light from a lamp and
outputs monochromatic light
Components : Entrance slit, Dispersion device, Exit slit.
The resolving element are of two kinds namely,
prisms and diffraction gratings. Simple glass prisms
are used for visible range. For UV region silica, fused
silica or quartz prism is used. Fluorite is used in vaccum
UV range.
Gratings are often used in the monochromators of
spectrophotometers operating in UV, visible and infra red
regions. Their resolving power is far superior to that of
prisms & they yield a linear resolution of spectrum.

Dispersion Devices
Non-linear dispersion
Temperature sensitive
Linear Dispersion
Different orders
CELLS
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer
NaCl
Open-topped rectangular standard cell (a)
and apertured cell (b) for limited sample volume
Cell Types I
Cell Types II
Micro cell (a) for very small volumes
and flow-through cell (b) for automated applications
Detection Devices
Most detectors depend on the photoelectric
effect, where incident light photons) liberates
electrons from a metal or other material surface.
Important requirements for a detector
(1)high sensitivity to allow the detection of low levels of radiant
energy,
(2)short response time,
(3)long term stability, and
(4)an electronic signal which is easily amplified for typical read out
apparatus, Ultraviolet and visible radiation detectors are photocells,
phototubes and photo multiplier tubes.

Photomultiplier Tube Detector
Anode
High sensitivity at
low light levels
Cathode material
determines spectral
sensitivity
Good signal/noise
Shock sensitive

Amplification and Readout

Radiation detectors generate electronic
signals which are proportional to the
transmitter light.
These signals need to be translated to a
form that is easy to interpret.
This is accomplished by using amplifiers,
ammeters, potentiometers, and
potentiometric recorders.



1. Qualitative Analysis
2. Quantitative Analysis
3. Molecular weight determination
4. Study of cis-trans Isomerism
5. Other Physiochemical studies
6. Control of Purification
7. Difference Spectroscopy
8. Turbidimetry




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