Application of latest techniques likespectroscopy in identification of crude

drugs.

(UV-Vis) spectrophotometry

Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure

light absorbance across the ultraviolet and visible ranges of the electromagnetic
spectrum.

Principle
The absorbance of radiation in the UV-Vis range causes atomic excitation, which
refers to the transition of molecules from a low-energy ground state to an
excited state. Before an atom can change excitation states, it must absorb
sufficient levels of radiation for electrons to move into higher molecular orbits.
A UV-Vis spectrophotometer can use this principle to quantify the analytes in a
sample based on their absorption characteristics. As per the Beer-Lambert law,
the greater the amount of absorbing molecule (that have the ability to absorb
light of a specific wavelength), greater the extent of absorption of the
radiation. So the absorption of the radiation is proportional to the
concentration.

Instrumentation

Monochromator

Light source
Deuterium, Tungsten- halogen and Xenon lamps are used as a high intensity
light source for both UV and visible ranges.
Monochromators
A monochromator separates light into a narrow band of wavelengths. It is used
to select the desired wavelength of light.

Sample holder
Quartz sample holders are required for UV examination because quartz is
transparent to the majority of UV light.

Detection
After the light has passed through the sample, a detector is used to convert the
light into a readable electronic signal called phototubes. Generally, detectors
are based on photoelectric coatings or semiconductors.

Advantages
• The technique is non-destructive, allowing the sample to be reused or
proceed to further processing or analyses.
• Measurements can be made quickly and accurately.
• Easy to use, requiring little user training prior to use.
• Data analysis generally requires minimal processing.
• The instrument is generally inexpensive to acquire and operate, making it
accessible for many laboratories.
• Can be used for qualitative and quantitative.
• Cover the entire of ultraviolet and visible.
Disadvantages
• Only those molecules are analyzed which have chromophores and that
absorb UV wavelength.
• The results of the absorption can be affected by pH, temperature,
contaminants, and impurities.
• Only liquid samples are possible to analyze
• It takes time to get ready to use the instrument - System initiation
• Cuvette handling can affect the reading of the sample

Applications: Wide application of (UV-Vis) spectrophotometry is seen in different

fields. Such as -
• Life Science- Determination of the configurations of geometric isomers. Cis
isomers absorb at shorter wavelength than trans isomers. Analysis of clinical
samples (serum protein, serum cholesterol, uric acid etc)
• Food & Agriculture – Testing of food quality. Analysis of water and waste
waters.
• Pharmaceutical Research - Structure elucidation of organic compounds by
identifying the presence or absence of unsaturation, presence of hetero
atoms (S, O, N) or hlaogens.
• Cosmetic Industry – Analysis of dyes (synthetic or natural) used in cosmetic
formulations.
• Quality Control- Identification, quantification, purity check, stability studies.
Detection of additional peaks for impure drugs can be identified.
• Forensic study – Routinely used in analysis of narcotics and drug for testing.
 IR spectroscopy

Principle:

IR spectroscopy detects the absorption of light by a compound, in the IR region of

the electromagnetic spectrum. To absorb light a molecule must have a bond within
its structure that can exhibit ‘dipole moment’ which means electrons within a bond
are not shared equally. Different bonds within functional groups which possess a
dipole moment will absorb at different areas of the IR spectrum, referred to as
wave numbers. So the major use of infrared spectroscopy is to determine
the functional groups of molecules, relevant to both organic and inorganic
chemistry.

Most of the functional groups present in organic molecules exhibits absorption

bands in the region 4000-1300 cm-1, hence this is known as functional group
region. The region from 1300 cm-1 to 600 cm-1 usually fingerprint region.
Spectrum:

Instrumentation

An IR spectrometer is a relatively simple device consisting of a lamp or heated

rod that will emit light in the IR region. A detector then collects all wavelengths
of IR radiation that have passed through the sample and converts these to wave
numbers. Each chemically distinct molecule will have a different absorption
pattern made up from the number and different types of bond present and the
presence of differing functional groups. The samples used in IR spectroscopy can
be either in the solid, liquid, or gaseous state. Normally thermal detectors and
photoconducting detectors are used.
Advantages:

• Powerful tool for wide range of samples.

• Requires small sample for analysis
• Method is fast.
• Give us lots of structural information of the analytes, such as the type of
compound, the functional group of compound.
• Purity of samples can be checked.

Disadvantages:
• Substance's molecular weight cannot be determined.
• Sample should be moisture free.
• Spectrum complication.
• Maximum for qualitative.

Applications:
• Identification of functional group and structure elucidation. IR spectrum
gives the information about the functional group (region 4000-1300 cm-1)
and characteristic peaks (region 1300 cm-1 to 600 cm-1). Thus helps for
structure elucidation.
• Studying the progress of the reaction. The rate of disappearance of a
characteristic absorption band of the reactant group and/or the rate of
appearance of the characteristic absorption band of the product group due
to formation of product is observed.
• Detection of impurities. Additional peaks due to impurities can be observed
in the spectrum.
• IR can provide a molecular fingerprint that can be used for comparing
samples. If two compounds have identical IR spectra then both of them
must be samples of the same substances.
• Ratio of cis and trans isomers in a mixture can be identified.
 Nuclear Magnetic Resonance (NMR)
Definition:

Nuclear magnetic resonance (NMR) spectroscopy is the study of molecules by

recording the interaction of radiofrequency electromagnetic radiations with the
nuclei of molecules placed in a strong magnetic field. It is based on the
absorption of electromagnetic radiation in the radio frequency region 4 to 900
MHz by nuclei of the atoms.

Principle:

Nuclei (protons) have magnetic properties, called nuclear spin. They behave like
tiny rotating magnets. Within a large external magnetic field, nuclear spins align
with the external field. Some of the spins align with the field (parallel) and some
align against the field (anti-parallel). Transfer of energy is possible from base
energy to higher energy levels when an external magnetic field is applied. It
takes place at a wavelength that matches with radio frequencies and when the
spin returns to ground state, it emits the radio wave of same frequency. This
emitted radio frequency gives NMR spectrum.
There will be variation of nuclear magnetic resonance frequencies of the same
kind of nucleus due to variations in the electron distribution. This is called the
chemical shift. Chemical shift δ is usually expressed in parts per million (ppm).

Spectrum

The signal due to tetramethylsilane, TMS, is adjusted to read zero on the

spectrum.

Instrumentation:

Intrumentation may be for either proton NMR or Carbon NMR ( 1H or 13C)

This instrument consists of the following parts:
⮚ Sample holder – It is a glass tube which is 8.5 cm long and 0.3 cm in diameter.
⮚ Magnetic coils – Magnetic coil generates magnetic field whenever current
flows through it.
⮚ Permanent magnet – It helps in providing a homogenous magnetic field at
60 – 100 MHZ.
⮚ Magnet controller – Modifies the strength of the magnetic field which is
already applied.
⮚ Radiofrequency transmitter (RF transmitter) – It produces a powerful but
short pulse of the radio waves.
⮚ RF detector – It helps in determining unabsorbed radio frequencies.
⮚ Recorder – It records the NMR signals which are received by the RF detector.
⮚ Readout system – A computer that records the data.

Advantages
• Structural details can be obtained
• Non-destruction method
• Lower analysis time
• Automated technique

Disadvantages
• Expensive, need a skilled technician and it is not a portable system
• Limited quantitative results
• Skill required to interpret the spectrum
Applications
• It is used to determine the molecular structure of compounds. Indicates
the number and the position of Hydrogen attached to Carbon or Oxygen
(1H NMR and 13C NMR).
• It is used to check the purity of samples. Identified by comparing the
peaks of impure sample with the peaks of standard sample.
• Environmental monitoring - To detect and characterise contaminants in
air, soil and water samples as well as monitor the metabolic responses of
organisms exposed to these contaminants.
• It is used in food science. Used to map protein structures, profile amino
acids, identify carotenoids and quantify metabolites.
• Identifying human disorders- NMR Spectroscopy allows researchers to
identify these tell-tale biomarkers and treat patients accordingly. It is also
used in the study of biofluids, cancer cells and nucleic acids.
• In MRI (magnetic resonance imaging) scans to reveal detailed images of the
internal organs.
 Mass spectrometry
Definition:

Mass spectrometry, also called mass spectroscopy is an analytic technique by

which chemical substances are identified by the sorting of gaseous ions in
electric and magnetic fields according to their mass-to-charge ratios.

Principle:

In mass spectroscopy, molecules are bombarded with a beam of energetic

electrons. The molecules are ionised and broken up into many fragments, some
of which are positive ions. Each kind of ion has a particular ration of mass to
charge, (m/e). For most ions, the charge is one so m/e is simply the molecular
mass of the ion.

The ions are analysed by the detectors and thus provides data of the
abundances of each ion present in the sample in the form of spectrum.
Instrumentation: The sequence in mass spectroscopy:

⮚ Stage 1: Ionization: Gas phase particles of the sample are ionized through
a collision with a high energy electron yielding a positive ion.

⮚ Stage 2: Acceleration: The ions are accelerated so that they all have the same
kinetic energy and directed into a mass analyzer.

⮚ Stage 3: Deflection: Separation of the ions according to the mass-to charge-

ratio (m/ze). The ions are sorted according to their mass-to charge-ratio
(m/ze).

⮚ Stage 4: Detection: The beam of ions passing through the mass analyzer is
detected as a current.

Advantages
• It is a very precise, rapid method.
• Very sensitive method as ppm level samples can be detected.
• An exceptional technique to identify unknown components in a sample
solution.
• It can work combining with other techniques, (LC-MS) and (GC-MS).
• Gives molecular weight of a molecule.
 Disadvantages
• It is costly, need a skilled technician, and it is not a portable system.
• Unable to differentiate among isomers of the molecule with the same charge-
to-mass ratio.
• Need skill to read (interpret) the spectrum.

Applications
• Proteomics - Characterization of proteins and protein complexes.

• Metabolomics - Cancer screening and diagnosis, metabolic fingerprinting

analysis, lipidomics studies, and metabolic disorder profiling.

• Environmental analysis - Drinking water testing, pesticide screening and

quantitation, soil contamination assessment, carbon dioxide and pollution
monitoring, and trace elemental analysis of heavy metals leaching.

• Pharmaceutical analysis - Drug discovery and absorption, distribution,

metabolism, and elimination (ADME) studies, pharmacokinetic and
pharmacodynamic analyses, metabolite screening, and preclinical
development.

• Forensic analysis - Analysis of trace evidence, confirmation of drug abuse,

and identification of explosive residues (bombing investigation).

• Clinical applications - Clinical drug development, Phase 0 studies, clinical

tests, disease screening, drug therapy monitoring, analysis of peptides used
for diagnostic testing, and identification of infectious agents for targeted
therapies.
 Electrophoresis

Electrophoresis is an electrokinetic process which separates charged particles in

a fluid using a field of electrical charge. It is most often used in life sciences to
separate protein molecules or DNA and can be achieved through several
different procedures depending on the type and size of the molecules.

Principle

Electrophoresis is based on the principle that charged particles in a liquid media

under the influence of an electric field will migrate to the electrode of the
opposite charge. Positive ions (cations) will migrate to the cathode and negative
ions will migrate to anode. Thus in electrophoresis the molecules feel the force
of the applied electric field pulling them in one direction. Molecules with a
larger mass will move slowly. It clears the fact that the electrophoretic mobility
of a molecule depends on its charge to mass ratio.
Gel electrophoresis:

• Gel electrophoresis is a method for separation and analysis of

macromolecules (DNA, RNA and proteins) and their fragments, based on
their size and charge.

Principle and working

In this technique the sorting of molecules is based on size and charge. Using an
electric field, molecules (such as DNA) can be made to move through a gel made
of agarose or polyacrylamide. The gel consists of a permeable matrix, a bit like
a sieve, through which molecules can travel when an electric current is passed
across it. The electric field consists of a negative charge at one end which pushes
the molecules through the gel, and a positive charge at the other end that pulls
the molecules through the gel. The molecules being sorted are dispensed into a
well in the gel material. The gel is placed in an electrophoresis chamber, which
is then connected to a power source. When the electric field is applied, the
larger molecules move more slowly through the gel while the smaller molecules
move faster. The different sized molecules form distinct bands on the gel.
Chromatogram

Advantages

• Useful for qualitative and quantitative analysis.

• Samples can be recovered by gel.
• Useful for separation of large molecules.
• Easy process to handle.
• Gel will be inert and do not denature the samples.

Disadvantages
• Gel can melt due to electric current.
• Chromatogram cannot be preserved as such for documentation.

Applications:
• To analyse results of DNA polymerization.
• DNA fingerprinting- profiling – to distinguish different species.
• Forensic- For investigation by DNA fringerprinting.
• Clinical- To analyse the genes associated with particular illness
• Protein studies- To study structure and function of proteins.
• Genetic engineering- To analyse the results of polymerase chain reaction in
the development of transgenic plants and animals.
• Blotting techniques- Analysis of macromolecules such as DNA, RNA, Proteins
etc.