INTRODUCTION TO SPECTROSCOPY

Spectroscopy is the study of the interaction between matter&electromagnetic radiation while

spectrometry deals with the measurements&analysis of a specific spectrum.
Spectrophotometry is a method to measure how many chemical substances absorb light by
measuring the intensity of light as a beam of light passes through sample solution.
Mainly there are two types of spectroscopy, Emission spectroscopy and Absorption
spectroscopy. Different regions of the electromagnetic spectrum provide different kinds of
information as a result of such interactions. This electromagnetic spectrum ranges from
very short wavelengths (including gamma&x-rays) to very long wavelengths (including
microwaves&broadcast radio waves)

UV SPECTROSCOPY
Ultraviolet-Visible Spectroscopy: Absorption of this relatively high-energy light causes
electronic excitation. The easily accessible part of this region (wavelengths of 200 to 800 nm)
shows absorption only if conjugated pi-electron systems are present. A specific range of
energies are sufficient to promote or excite a molecular electron to a higher energy orbital.
The interaction between radiation matter is a fascinating area. Most drug molecules absorb
radiation in the ultraviolet region of the spectrum, although some are colored & thus absorbed
radiation in the visible region, e.g., a substance with a blue color absorbs radiation in the red
region of the spectrum. The absorption of UV/visible radiation occurs through the excitation
of electrons within the molecular structure to a higher energy state.
These transitions occur from the bottom vibrational state in the electronic ground
state of the molecule to any one of a number of vibrational levels in the electronic excited
state. The transition from a single ground state energy to one of a number of excited states
gives width to UV spectra. Vibrational fine structure can be seen, although the bands overlap
extensively; the vibrational bands themselves have width due to rotational transitions that are
intermediate in energy between each vibrational transition. The relative energy of electronic:
vibrational: rotational transitions is 100:1 :0.01. In most molecules the vibrational behavior is
complex & the degree of overlap of the different energies of the vibrational transitions is too
great for vibrational fine structure to be observed.

SPECTROPHOTMETER
It is an instrumental method is comprising of two devices i.e., spectrometer&photometer.
✓ Spectrometer-it produces a desired range of wavelength of light.
✓ Photometer-it detect the amount of light of selected wavelength when pass through the
sample.
Spectrophotometry: it is measurement of intensity of light at selected wavelength.
 Type of instruments:
1. Single Beam Instruments: A single beam is passed through the sample & reference
cell one by one & the difference of transmittance through both solutions to the
photodetector is amplified & shown.
1. Double beam instruments: A single beam is split into two & both resultant beams
passthrough the reference & sample cells simultaneously to strike the photo detector.

Spectrophotometer Consist of Following Components:

➢ Source Of Light: It provides a sufficient polychromatic light which is suitable for making
measurement, sources include
• Deuterium lamp: produces an ultraviolet light. (190-380nm)
• Tungsten lamp: produces a visible light (380- 800nm)
they can be of two types.
a. Continue source: which provides a ‘continued’ source of radiation over a broadspectrum of
wavelength.
b. Line source: which provide only a limited range of radiation, e.g., Mercury arc
lamp(253.7nm), Hollow Cathode Lamps&Laser like He-Ne laser etc.
➢ Collimator: transmits a straight beam of light.

➢ Monochromator: they allow only a specific wavelength to pass on through them so that
only specific wavelengths are measured for absorption&transmittance.
• Prism: It is used to isolate different wavelengths.
 Glass prism---visible range spectrum
 Quartz prism--ultraviolet spectrum
• Filter: It separate different parts of electromagnetic spectrum by either absorbing or
transmitting certain wavelengths.
 Absorption filters
 Interference filters
• Diffraction grating: It diffracts light into several beams travelling in different directions.
It is a combination of lenses, slit & mirrors. It gives a direction to beam of light. (e.g.,
Echellete Grating, Holographic Grating, & Concave Grating)

➢ Wavelength selector: It transmits only the desired wavelength.

➢ Beam splitter: it splits the beam into two so that light of equal wavelength must pass through
both cuvettes simultaneously.

➢ Cuvettes: They are container in which sample or reference is filled (holds the sample).
Sample must be transparent along with cuvettethe latter ones being perpendicular to the light
beam their widthis the path length through which the light interacts with the particles of the
sample&usually, constant i.e., 1 cm. They are available in
• Optical glass or Pyrex: For visible work.
• Quartz: For UV work narrow refractive index).
• Plastic: Inexpensive, uses for some uv work too.
 ➢ Detector: they are also called Transducers as they convert nonelectrical signals into
electrical impulses&signals. They receive the radiation source&display the result
according to the strength of the incident beams. Since the UV rays are highly energetic, these
detectors function by photo electric.
• Charge coupled device detectors: it is used mainly for detection of extremely low
intensity light signals
• Phototube • Photomultiplier
• Array • Photodiode

➢ Display devices: data from the detector is transmitted to a digital display such as a computer
or printer

 PRINCIPLE(LAW)
❖ Beers Law
“When a monochromatic light passes through a medium, the absorbance of a medium is
directly proportional to its concentration.”
aαc
❖ Lambert Law
“When a monochromatic light passes through a medium, the absorbance of a medium is
directly proportional to its path length.”
aαL
❖ Beer Lambert Law These two laws are combined in beer-lambert law according to this law:
“The absorbance of a medium is directly proportional to its length & concentration.”

 Molar Absorptivity Constant (a corrected absorption value):

The Molar Absorptivity Constant is specific for every single solution, & at every wavelength.
When you are taking an absorbance spectrum, & measuring the absorbance at different
wavelengths, this is the only factor that is changing, as the concentration of the solution
remains the same, &so does the pathlength. It is a measure of how well a chemical species
absorbs a given wavelength of light. The standard units for molar absorptivity are liters per
mole centimeter (L mol-1 cm-1)
The absorbance of a solution will change based on the wavelength that is passed through the
solution. Some wavelengths will be absorbed more than others depending upon the makeup
of the solution. Absorbance between readings can vary due to the concentration of the
solution&the shape of the container used to measure intensity. Molar absorptivity
compensates for these variations.

 Specific absorbance: it is defined as the maximum absorption of 1% solution over 1cm of

path length measured with spectrophotometer.
 Relationship Between Absorbance & Transmittance
Absorbance measures how much of an incident light is absorbed when it travels in a material
while transmittance measures how much of the light is transmitted. if all the light passes
through a solution without any absorption,
then absorbance is zero, & percent transmittance is
100%. If all the light is absorbed, then
percent transmittance is zero, & absorption is infinite.
In spectrophotometer we determine both absorbance transmittance

Applications of UV Spectroscopy:
1- Detection of Impurities:
• It is one of the best methods for determination of impurities in organic molecules.
• Additional peaks can be observed due to impurities in the sample & it can be
compared with that of standard raw material.
• By also measuring the absorbance at specific wavelength, the impurities can be
detected.
2- Structure elucidation of organic compounds
It is useful in the structure elucidation of organic molecules, such as in detecting the
presence or absence of unsaturation, the presence of hetero atoms.
3- UV absorption spectroscopy can be used for the quantitative determination of compounds
that absorb UV radiation.
4- UV absorption spectroscopy can characterize those types of compounds which absorbs
UV radiation thus, used in qualitative determination of compounds. Identification is done
by comparing the absorption spectrum with the spectra of known compounds.
5- This technique is used to detect the presence or absence of functional group in the
compound. Absence of a band at particular wavelength regarded as an evidence for
absence of particular group.
6- Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is
passed through the reaction cell & the absorbance changes can be observed.
7- Many drugs are either in the form of raw material or in the form of formulation. They can
be assayed by making a suitable solution of the drug in a solvent & measuring the
absorbance at specific wavelength.
8- UV spectrophotometer may be used as a detector for HPLC

CHROMOPHORES
Chromophore is the part of a molecule or chemical group which is a covalently unsaturated
group responsible for absorption of UV or visible radiation (responsible for its color) &may
or may not impact in color to the compound. A compound which contains chromophore it is
called chromogen. In unsaturated linkage such as -c=c-,-N=N-, the π electron are loosely
bound. These loosely bound electron required less energy for electronic transition&the
absorption band occur in near UV region.
Example Acetylene possessed -C=C- in structure it λmax is 175-180 nm.