Spectrophotometry uses light absorption properties of substances to quantitatively analyze samples. It follows Beer's Law, where absorbance is directly proportional to concentration. A spectrophotometer splits light into wavelengths, passes a sample beam through the sample, and measures the intensities of light transmitted versus a reference beam. This allows measurement of absorbance across wavelengths. Main applications include concentration measurement, detection of impurities, and studying chemical kinetics.

1. Spectrophotometry
Presenter:-
Dr Arun Singh
2nd Year Resident Doctor
Department of Pharmacology
SMS Medical College,Jaipur

2. Outliners
Introduction
Basic concepts and principles
Types of spectrometry
Device and mechanism
Beer-lambert-law
Applications

3. Introduction
Spectrometry is a method in which the
absorption or transmission properties of
a material is quantitatively measured as a
function of wavelength.
Spectrophotometry is a measurement of
the intensity of light at selected
wavelengths.
The method depends on the light
absorbing property of either the
substance or a derivative of the
substance being analyzed.

4. Spectrophotometer is an instrument which measures light absorption as a function of
wavelength in the UV as well as visible regions.
It also follows essentially the laws of light absorption viz the beer-lambert’s law.
Unlike colorimeters in spectrophotometers the compound can be measured at precise
wavelength.
The light absorbed by the sample is directly proportional to the concentration of
sample in the solution.
As concentration increases , absorption increases exponentially.

5. Basic concepts
and principles
The basic principle behind this method is that :
“Each compound absorbs or transmits light over a
certain range of wavelength.
When light passes through a solution, a certain
fraction is being absorbed.
This fraction is detected, measured and used to
relate the light absorbed or transmitted to the
concentration of the substance.
This enables both qualitative and quantitative
analyses of substances.

6. • The spectrophotometric technique measures the light intensity due to :
– Diffracting the light beam into a spectrum of wavelengths
– Direct it to an object
– Receiving the light reflected or returned from the object
– Detecting the intensities with a charge-coupled device
– Displaying the results as a graph on the detector and then the display
device

7. • The light absorption is directly related to the
concentration of the compound in the sample.
• As Concentration increases, light Absorption
increases linearly and light Transmission
decreases, exponentially.

8. Spectrometer
Instrumentation
• A spectrometer consists
of a light source, a prism
that separate the lights
into different
wavelengths, a slit
through which a narrow
beam of a desire
wavelength passes, a
sample holder, a detector
and a recording device.

9. Transmittance and Absorbance
• When a sample is illuminated, it absorbs some of the light and
transmits the rest.
• The transmitted light (Is ) is of lower intensity than the incident light
(Io ), and the transmitted light is defined as: T = Is / Io

10. • To ensure accuracy (by eliminating effects of reflection by surface of the
cell, absorption by the cell wall and by solvent) an identical reference cell
without the compound of interest is also used.
• Thus, the amount of light absorbed (A) as the incident light passes through
the sample is equivalent to:
A = - log Is / IR = - log T
• In practice, the Reference cell is inserted, and the instrument adjusted to
an arbitrary scale corresponding to 100% transmittance, after which the
percentage transmittance reading is made on the sample.

11. Types of
spectrometry Spectrophotometer can be classified
into two different types :
• Single Beam Spectrometer
• Double Beam Spectrometer

12. Single Beam Spectrometer
• To measure the intensity of the incident light the sample must be
removed so that the reference can be placed each time.
• This type of spectrometer is usually less expensive and less
complicated.

13. Double Beam Spectrometer
• In this type, before it reaches the sample, the light source is split into two
separate beams.
• From these one passes through the sample and second one is used for
reference.
• This gives an advantage because the reference reading and sample reading
can take place at the same time.

14. Based on the
wavelength of light
used it can be
classified into:
(A)Visible Spectrometer:-
• Uses visible range (400 – 700nm) of electromagnetic
radiation spectrum.
• Visible spectrophotometers vary in accuracy.
• Plastic and glass cuvettes can be used for visible
light spectroscopy.
(B)UV Spectrometer:-
• Uses light over the UV range (180 - 400 nm).
• UV spectroscopy is used for fluids, and even solids.
• Cuvettes, only made of quartz, are used for placing
the samples.
(C) IR Spectrophotometer:-
• Uses light over infra red range (700 -15000) of
electromagnetic radiation spectra

15. (D)Fluorescence Spectrometer:-
• Its are instruments used to scan the fluorescence
spectrum emitted by liquid fluorescent labels, which
used in scientific research, chemical
industry,medicine,biochemistry,environmental
protection,clinical testing,food testing,teaching
experiments and other fields.
(E)Atomic absorption Spectrometer:-
• This method is mainly applied to detect trace
components in the sample analysis.
• It is powerful tool for material analysis and
elemental analysis of trace metals(semimetals).

16. Device and Mechanism
• The spectrophotometer, in general, consists of two devices. They are the following :
1.Spectrometer : A device that produce, typically disperse and measure the light.
2.Photometer : Indicates the photoelectric detector that measures the light.
• The spectrometer consists of the following parts :
(i) Light source :It produces a desired range of wavelength of light.
(ii) Collimator : It transmits a straight beam of light.
(iii)Monochromator : It split the light into its component wavelength.
(iv)Wavelength selector : transmits only the desired wavelength.

17. (v) Cuvettes : The optically transparent cells (cuvett) are made up of glass,
plastic, silica or quartz, glass and plastic absorb UV light below 310 nm.
(vi) Photocell and photo-multiplier tubes: It a photocell is a photoelectric
device which converts light energy into electrical energy, which is then
amplified, detected and recorded.’’
• The photometer detects the light absorbed by the sample as the light from
the slit is passed through the solution and then it sends signal to the
galvanometer or digital display.

18. Beer – Lambert Law
• It states that the absorbance of light by a material in a solution is directly
proportional to its concentration in that solution.
A = ϵlc
Where,
A -absorbance
ϵ -molar absorptivity
l -length of solution
c -concentration

19. Standardization
Graph
• Standards (solutions of
known concentration) of
the compound of interest
are made, treated, and their
absorbances (ABS) and
concentration values are
used to create a
Standardization Graph.

20. Absorption spectra
• A spectrum of electromagnetic radiation transmitted through a
substance, showing dark lines or bands due to absorption at specific
wavelengths.

21. Applications of Spectrometry
Concentration measurement
Detection of impurities
Chemical kinetics
Detection of functional group
Molecular weight determination