This document discusses the principles of spectroscopy. It describes how spectroscopy works by measuring electromagnetic radiation absorbed or emitted by sample atoms. It also discusses the Beer-Lambert law, which states that absorbance is directly proportional to concentration. Finally, it distinguishes between qualitative spectroscopy, which identifies samples based on their absorption peaks, and quantitative spectroscopy, which determines concentration by measuring absorbance against a standard curve.

1. JAI NARAIN VYAS UNIVERSITY
JODHPUR
Department Of Botany
(COSIST )
Seminar on- PRINCIPLE OF SPECTROSCOPY
Submited to Submited by
Dr. Rachna Dinesh Modi Narpat Singh
(Assistant Prof.) (Msc. 3rd SEM)

2. • Spectroscopy is the measurement and
interpretation of electromagnetic radiation
which is absorbed or emitted by atoms of a
sample.
• This absorption or emission happens when
the atoms of the sample move from one
energy state to another in presence of light.

3. • Light is an electromagnetic radiation which is
made up of discrete particles called photons.
• In short, wave length is the distance between
two crests or troughs while frequency is the
number of wavelength units passing through
an unit time.
• Wave length is represented by ‘λ’ and
frequency is denoted by ‘ν’.

4. • Natural light is a combination of many spectra.
These spectra are the light rays of different
wave lengths and frequency.
• The spectra used in spectroscopy varies from
ultra-violet, visible, infra red ranges. The
wavelength range for the three spectra are 0-
400, 400-700 and above.

5. • When a light rays falls on a compound, it gets
absorbed to certain extent and remaining is
reflected. The wave length of absorbed light is
specific to the material taken.
• Spectroscopy is extended to study the substance
based on their characteristic absorbance of the
above three spectra.
• Based on the two phenomenon, we try to
identify and also measure the quantity of any
given substance.

6. Spectroscopy Principle:
• Every sample has molecules consisting of some
functional groups by which they may incur color
or some nature to absorb light of specific
wavelength. This wavelength at which sample
absorbs to a greater extent is called as λ max.
• When the light beam is passed on to the sample,
the electrons in the molecules absorb energy in
the light, and go for exited state. During this
transition some of the light energy is absorbed
while the remaining light falls on the photo-
electric detector.

7. • There are different types of spectroscopy
based on the technique and use.
• Spectroscopy is suitable for both qualitative
analysis and quantitative analysis.

8. Beer-Lambert Law
• Introduction
• The Beer-Lambert law (also called the Beer-Lambert-Bouguer law or simply Beer's law) is the linear
relationship between absorbance and concentration of an absorber of electromagnetic radiation.
The general Beer-Lambert law is usually written as:A = aλ · b · c
• where A is the measured absorbance, aλ is a wavelength-dependent absorptivity coefficient, b is
the path length, and c is the analyte concentration. When working in concentration units of
molarity, the Beer-Lambert law is written as:
• A = el · b · c
• where el is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1. The λ
subscript is often dropped with the understanding that a value for ε is for a specific wavelength. If
multiple species that absorb light at a given wavelength are present in a sample, the total
absorbance at that wavelength is the sum due to all absorbers:
• A = (ε1 · b · c1) + (e2 · b · c2) + ...
• where the subscripts refer to the molar absorptivity and concentration of the different absorbing
species that are present.
•

9. Theory
• Experimental measurements are usually made in terms of
transmittance (T), which is defined as:
• T = P/Po
• where P is the power of light after it passes through the sample and Po
is the initial light power. The relation between A and T is:
• A = − log(T) = − log(P/Po)
• The figure shows the case of absorption of light through an optical
filter and includes other processes that decreases the transmittance
such as surface reflectance and scattering.
•
•

11. • In analytical applications we often want to measure the concentration of an analyte independent of
the effects of reflection, solvent absorption, or other interferences. The figure to the right shows
the two transmittance measurements that are necessary to use absorption to determine the
concentration of an analyte in solution. The top diagram is for solvent only and the bottom is for an
absorbing sample in the same solvent. In this example, Ps is the source light power that is incident
on a sample, P is the measured light power after passing through the analyte, solvent, and sample
holder, and Po is the measured light power after passing through only the solvent and sample
holder. The measured transmittance in this case is attributed to only the analyte.
• Depending on the type of instrument, the reference measurement (top diagram) might be made
simultaneously with the sample measurement (bottom diagram) or a reference measurement
might be saved on computer to generate the full spectrum.
• Absorption of light by a sample
• Modern absorption instruments can usually display the data as either transmittance, %-
transmittance, or absorbance. An unknown concentration of an analyte can be determined by
measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity
coefficient is not known, the unknown concentration can be determined using a working curve of
absorbance versus concentration derived from standards.

12. Limitations of the Beer-Lambert law
• The linearity of the Beer-Lambert law is limited by chemical
and instrumental factors. Causes of nonlinearity
include:deviations in absorptivity coefficients at high
concentrations (>0.01M) due to electrostatic interactions
between molecules in close proximity
• scattering of light due to particulates in the sample
• fluoresecence or phosphorescence of the sample
• changes in refractive index at high analyte concentration
• shifts in chemical equilibria as a function of concentration
• non-monochromatic radiation, deviations can be minimized
by using a relatively flat part of the absorption spectrum
such as the maximum of an absorption band
• stray light

13. Types of Spectroscopy
• Spectroscopy is mostly 2 types.
• Qualitative spectroscopy:
• This is the technique to know the type of
sample molecule there by one can tell what
the sample is and its chemical nature after
comparing the obtained analysis curve peaks
with that of standard sample from official
books like Pharmacopeias or books on
chemical standards etc..

14. • The point or wavelenght where the sample shows
maximum absorbance is noted as it’s λ max. This λ
max is fixed for every sample and and there by any
unknown sample can be identified by knowing its λ
max after comparing with standard.

15. Quantitative spectroscopy:
• This is a method to determine the exact
concentration of a substance in a given
sample.
• At a specified wave length (λ max) when a
given sample is analyzed by spectroscopy, the
concentration in the sample can be known by
plotting it against a standard substance graph
as shown in the pic.

16. • For this a series of dilution of standard sample
and test sample are taken and absorbance is
measure by spectroscopy.
• The absorbance for difference concentrations
of standard and test are plotted on a graph.
•