CHEMISTRY
INVESTIGATORY PROJECT
Investigation of the properties of different types of
chromatography
Name- Nishant Shukla
Class- XII
School- Narayana Junior College
TABLE OF CONTENTS
➢ INTRODUCTION
➢ THEORY
➢ CLASSIFICATION OF CHROMATOGRAPHY
➢ TYPES OF CHROMATOGRAPHY
➢ APPLICATIONS OF CHROMATOGRAPHY
➢ ADVANTAGES OF CHROMATOGRAPHY
➢ LIMITATIONS OF CHROMATOGRAPHY
➢ CONCLUSION
➢ BIBLIOGRAPHY
INTRODUCTION
Chromatography is a widely used technique in chemistry and biology
for separating and analysing mixtures based on the distribution of
components between two phases – the stationary phase and the mobile
phase.
The word ‘chromatography’ was first
used by the Russian scientist Mikhail Tsvet
in 1906 while separating plant pigments.
Tswett poured a plant pigment solution on
top of a column with an adsorptive material
and added a solvent to wash it through the
column. As the solution moved down, the
pigments separated into bands of different
colours. Over time, chromatography has evolved into various types,
allowing for the separation of small molecules to complex biomolecules.
This theoretical project examines the properties of various
chromatography techniques, including paper, thin-layer, column, gas, and
high-performance liquid chromatography (HPLC). The project explores their
principles, advantages, limitations, and applications in scientific research
and industry.
THEORY
➢ Principle of Chromatography
The main principle of chromatography is that different substances interact
differently with the stationary and mobile phases, and this difference in
interaction separates the compounds. As the mobile phase moves the sample
through the fixed stationary phase, each compound moves at a different
speed based on its properties, such as size, charge, or affinity to the
stationary phase, resulting in separation. The separated components are
represented as peaks on a chromatogram and identified by their retention
times, which are the times it takes for compounds to move through the
system.
➢ Components and Parts of Chromatography
a. Sample or Analyte
The sample or analyte is the mixture that needs to be separated. Different
components of the sample behave differently and are divided based on how
they interact with the mobile and stationary phases.
b. Stationary Phase
This is the fixed material that can be solid or liquid coated on a surface. As
the analyte passes through it, the stationary phase holds or slows down
specific molecules based on their properties. Some of the commonly used
stationary phases are filter paper, silica, and beads.
c. Mobile Phase
This is the moving fluid or solvent that carries the mixture and flows through
the stationary phase. The mobile phase can be a liquid or a gas. Some of the
commonly used mobile phases include water, acetic acid, and gases such as
hydrogen and nitrogen.
d. Chromatography Column
It is the container that holds the stationary phase, where separation occurs.
It is used in column-based methods, such as gas chromatography and HPLC.
Chromatography columns are usually cylindrical in shape. The size and
material can vary depending on the type of chromatography.
➢ Retention Factor
In paper and thin-layer chromatography, the Retention Factor (Rf) value
helps identify compounds.
Distance travelled by the solute (spot)
𝑅𝑓 =
Distance travelled by the solvent front
Each compound has a characteristic Rf value under given experimental
conditions (i.e., the same solvent, temperature, and stationary phase).
CLASSIFICATION
➢ BASED ON THE GEOMETRY OF THE SYSTEM:
a. Planar chromatography
b. Paper chromatography
c. Thin-layer chromatography
d. Column chromatography
➢ BASED ON THE RETENTION MECHANISM:
a. Adsorption chromatography
b. Partition chromatography
c. Size exclusion chromatography
d. Ion exchange chromatography
e. Affinity chromatography
➢ BASED ON THE PHASE INVOLVED:
a. Gas Phase chromatography
b. Liquid Phase chromatography
PAPER CHROMATOGRAPHY
Paper chromatography is a simple and affordable technique used to
separate and identify components in a mixture based on their differing
affinities for a stationary phase (paper) and a mobile phase (solvent). In this
method, a small spot of the sample is placed near the base of a filter paper,
which is placed vertically in a sealed container with solvent just below the
sample spot.
The solvent moves up the paper along
with the sample due to capillary action,
separating the sample components based
on their affinity for the solvent and the
paper. The polarity of the mobile phase,
relative to the stationary phase, determines
how components move. Less polar
Components that are more soluble in compounds tend to dissolve better in a less
the solvent move faster and travel polar mobile phase and travel farther.
farther up the paper, while those
that are more soluble in the
stationary phase (water) move
more slowly. It is quite efficient in
analysing pharmaceutical
compounds or determining rates in
synthetic chemicals. It is also used
in separating and identifying
coloured compounds, such as plant
pigments.
THIN-LAYER CHROMATOGRAPHY
Thin Layer Chromatography (TLC) uses a thin layer of solid particles like
silica gel or alumina as the stationary phase. This thin layer of adsorbent
material is coated on a flat plate where separation occurs. It is characterised
by its speed, sensitivity, and high reproducibility. It works similarly to paper
chromatography but has better separation and faster results. TLC is used
mainly for separating and identifying organic compounds.
TLC plates containing a small
amount of a
fluorescent compound
(usually manganese-
activated zinc silicate) in the
adsorbent layer allow for the
visualisation of some
compounds under UV-C light
The adsorbent layer will fluoresce a light green,
(254 nm).
while spots containing compounds that absorb UV-C
light will not. Placing the plate in a container filled with iodine vapours temporarily
Stains the spots. They typically become a yellow or brown colour.
TLC helps show the purity of a sample. A pure sample should only contain one
spspot by TLC. TLC is also useful for small-scale purification. Because the separated
cocompounds will be in different areas of the plate, a scientist can scrape off the
ststationary phase particles containing the desired compound and dissolve them in
an appropriate solvent.
COLUMN CHROMATOGRAPHY
Column chromatography in chemistry is a chromatography method used to
isolate a single chemical compound from a mixture. Chromatography can
separate substances based on the differential absorption of compounds by the
adsorbent; compounds move through the column at different rates, allowing
them to be divided into distinct fractions. The technique is widely applicable,
as many different adsorbents (normal phase, reversed phase, or otherwise)
can be used with a wide range of solvents.
The mixture is loaded onto the top of a column
packed with a stationary adsorbent, and a
solvent (mobile phase) is passed through
it. The components separate as they travel
down the column at different rates and are
collected in fractions. The chromatographic
technique of extracting an adsorbed substance
from a solid adsorbing medium using a solvent.
The eluent is the solvent or mobile phase that
passes through the column. When the polarity
of the eluent matches the polarity of the
molecules in the sample, the molecules desorb
from the adsorbent and dissolve in the eluent.
Silica gel is used to separate a wide variety of compounds, including
hydrocarbons, alcohols, ketones, esters, acids, azo compounds, and amines.
Alumina is also used extensively, and comes in three forms: acidic, basic and
neutral. It is pretty helpful in isolating active ingredients and removing
impurities. It can also be used for drug estimations and separating compounds.
APPLICATION OF CHROMATOGRAPHY
➢ Chromatography can be used in environmental testing to monitor air and
water quality.
➢ It has applications in forensic science to detect toxic substances in the body
and analyse samples, such as blood, urine, or saliva.
➢ It can be used in bioprocessing or product purification, such as for the
production of monoclonal antibodies and vaccines.
➢ It has applications in the food industry to check the safety and quality of
food products. It helps to detect contaminants and additives in food
products. It can also determine the ingredient composition, which is used for
nutritional assessment and accurate labelling.
➢ It has applications in the chemical industry for checking the purity and
determining the chemical properties of various compounds.
➢ It has applications in the pharmaceutical industry for developing drugs and
checking the quality of products.
➢ Used in biotechnology to analyse the
composition, metabolic pathways,
and fermentation products.
➢ It can be used to test the quality of
things like ink in pens.
ADVANTAGES OF CHROMATOGRAPHY
➢ It can separate all the components of a mixture with little to no
prior information about the sample composition.
➢ It is a versatile method as it is compatible with a wide range of
substances.
➢ It is accurate and can handle complex mixtures.
➢ It is highly sensitive and can
detect substances in extremely
small amounts.
➢ It can be easily combined with
additional detectors like mass
spectrometry.
➢ It can be applied to different
molecules, from the smallest
gases to large biological
macromolecules.
➢ It can be used for both analytical
purposes (small-scale) and
preparative purposes (large-scale
purification).
LIMITATIONS OF CHROMATOGRAPHY
➢ Chromatography requires expensive instruments and consumables. Setting
up chromatographic systems, such as HPLC or GC, requires a significant
initial investment. Operational expenses, including regular maintenance
and consumables, are also substantial.
➢ Preparing samples for chromatography is complex.
➢ It may face difficulties when
separating compounds with
similar properties.
➢ It requires skilled personnel with
specialised training and
expertise to operate
chromatography instruments,
interpret results, and
troubleshoot.
➢ Temperature-sensitive or
unstable analytes may
decompose during
chromatographic separation.
➢ High-molecular-weight
polymers, particulates, or
viscous samples can clog
columns or impede flow.
CONCLUSION
Chromatography is a valuable method for separating and analysing the
components of a mixture. The basic idea of all chromatographic methods is
that different substances move at different speeds through a material,
allowing them to be separated and analysed. From simple paper
chromatography to advanced HPLC, the method chosen depends on the
nature of the mixture, the required accuracy, and the available resources.
It has various applications, including chemical analysis, forensic
investigations, environmental studies, pharmaceutical testing, and food
safety. New advances in stationary phase materials, such as the development
of monolithic columns and nanoparticle-based phases, as well as integration
with other analytical methods like Mass Spectrometry, enhance separation.
Chromatographic methods are becoming more accurate and efficient.
BIBLIOGRAPHY
➢ NCERT Chemistry Class XII Textbook.
➢ NCERT Chemistry Practical Journal Class XII
➢ Vogel’s Textbook of Practical Organic Chemistry.
➢ [Link]
applications/
