UV/VIS

SPECTROPHOTOMETER
 Theory of light
• Light is electromagnetic radiation that is visble to
the human eye, and is responsible for the sense of
light
• Visible light- 380-740 nanometres- between the
invisible infrared with longer wavelengths and the
invisible ultraviolet with shorter wavelengths.
• Light is emitted and absorbed in tiny “packets”
called photons, and exhibits the properties of both
waves and particles. This property is referred to as
the “wave particle duality”
• The study of lights is known as optics
 Electromagnetic Radiation
• Electromagnetic radiation is a type of energy
that is transmitted through space as a traverse
wave at enormous velocity
• It takes numerous forms known as
electromagnetic spectrum.
• The electromagnetic spectrum include gamma
ray, ultraviolet (UV), visible, infrared (IR),
microwave and radio wave radiation
Spectroscopy – Radiation Terminology
• Wavelength (λ)- the length between two
equivalent points on successive waves
• Wavenumber (n)- the number of waves in a
unit length or distance per cycle
• Frequency (μ)- is the number of oscillations of
the field per second (Hz)
• Velocity- independent of wavelength – in
vacuum is 3.00 x 108 m/s
Colors and Wavelengths
 Properties of electromagnetic
radiation(EMR)
1. Wave Properties
The wave is described ether in terms of its
wavelength(λ), or in terms of the frequency(μ), the
number of oscillation of the field per second.
Effect of Medium on a Light wave
2. Particle Properties
• EMR can be viewed as a stream of discrete
wave packets of distinct particles called photons
• The energy E of photon depends upon the
frequency of the radiation
E = hv
h = Planck’s constant
v = frequency of the radiation
• Energy is inversely proportional to wavelength
What are the basic properties of light?

• Light travels in straight lines

• Light can be reflected
• Light can be refracted
• Light is a form of energy
• Light can be dispersed
• Light can be diffracted
 Spectroscopy
• Spectroscopy is the interaction of radiated
energy with matter, resulting in absorption,
reflection or scattering
 Types of spectroscopy
1. Absorption – UV/vis spec
2. Emission – Fluorescent spec
3. Scattering
 Absorption spectroscopy
• Transmittance : the passing of light through a
sample
• Absorbance: amount of light absorbed by a sample
• %Transmittance: The manner in which a
spectrophotometer reports the amount of light in a
beam
• Absorbance spectrum: a graph of a sample’s
absorbance at different wavelengths
• Lambdamax: The wavelength that gives the highest
absorbance value for a sample
 UV/vis spectrophotometry
• It is an analytical machine that measures the
transmission of a beam of light through a
sample (%Transmission)
• Spectrophotometer deals with visible light,
near UV and near IR
• The spectrophotometer either measures the
amount of light transmitted through a solution
(transmittance) or the amount of light absorbed
by the solution (absorbance)
 BEER LAMBERT’S LAW
(Beer-Lambert-Bouguer law)
• Relates the absorption of light to the propertied of the
material through which the light is travelling.
BEER’S LAW
• When monochromatic light passes through a solution there
is usually a quantitative relationship between the solute
concentration and the intensity of the transmitted light.
• The amount of light absorbed by the medium (solution /
sample) is proportional to the absorbing material or solute
present.
• Thus the concentration of a colored solute in a solution may
be determined in the lab by measuring the ABSORBANCE
OF LIGHT AT A GIVEN WAVELENGTH
Lambert’s Law
• Lambert described how intensity changes
with distance in an absorbing medium.
• The amount of light absorbed by the medium
(solution/sample) at a given wavelength is
proportional to the thickness of the absorbing
layer: path length of the light
 Different types of spectrophotometers
Single beam
• All the light passes through the sample
• To measure the intensity of the incident light
the sample must be removed so that all the
light can pass through
• It is usually inexpensive and less complicated
Double beam
• Before reaching the sample the light source is
split into two separate beams
• From these one passed through the sample and
second one is used for reference
• This gives the advantage of reading both
reference and sample at the same time
Visible light
• Vis Spec can use incandescent, halogen, LED
or a combination of these sources and these
specs vary in accuracy
• Plastic and glass cuvettes can be used
UV Light
• UV spec is used for fluids and even solids.
• Cuvettes, only made of quarts, are used
 Applications
• Quantitative and qualitative analysis
• To measure light intensity at different
wavelengths
• To determine the unknown concentration of
solution