CHROMATOGRAPHY

Chromatography is a laboratory technique used to separate and analyze

mixtures of compounds into their individual components. It mechanisms based
on the principle that different compounds in a mixture will interact differently
with the chromatography medium, causing them to move at different rates. It
involves a stationary phase (such as chromatography paper) and a mobile phase
(solvent) that moves through the stationary phase, separating the mixture into its
individual components based on their different affinities for the stationary and
mobile phases.
Each of these chromatographic techniques can be used for different purposes,
such as purification, quantitative analysis, or qualitative analysis, depending on
the nature of the sample and the components to be separated.
There are various chromatography techniques, which are crucial in separating
mixtures into their individual components. Each method uses a different
mechanism for separation, based on the physical and/or chemical properties of
the components involved.
1. Paper Chromatography
2. Thin Layer Chromatography (TLC)
3. Gas Chromatography (GC)
4. Liquid Chromatography (LC)
5. Ion-Exchange Chromatography
6. Gel Filtration Chromatography (also known as Size-Exclusion
Chromatography).

Paper Chromatography
In paper chromatography, a paper serves as the stationary phase, and a solvent
or a mixture of solvents acts as the mobile phase. The mixture to be separated is
applied as a spot near the base of the paper. As the solvent moves up the paper
by capillary action, different components of the mixture travel at different rates
and thus get separated along the paper length. It's often used in teaching
laboratories because it's simple and requires minimal equipment.

Gas Chromatography (GC)

Gas chromatography separates mixtures based on their distribution between a
stationary liquid phase and a mobile gas phase. It's primarily used for the
analysis of volatile compounds. The mixture gets vaporized and carried by an
inert gas through a column containing the stationary phase. Different
components of the mixture are retained for different lengths of time based on
their interaction with the stationary phase and are detected as they exit the
column.
Liquid Chromatography (LC)
This term encompasses several chromatographic techniques that use a liquid
mobile phase to separate the components of mixtures. It including High
Performance Liquid Chromatography (HPLC), uses a liquid as the mobile phase
and a variety of materials as the stationary phase. The stationary phase can be a
solid or a liquid. High-performance liquid chromatography (HPLC) is a
common variant that uses high pressure to push the solvent through the column,
allowing for the separation of compounds that are not volatile or stable at high
temperatures. It's a very versatile technique, capable of analyzing a wide range
of compounds. Separation is based on differences in the solubility of the
compounds in the mobile phase and their adsorption to the stationary phase.

Gel Filtration Chromatography (Size-Exclusion Chromatography)

Gel filtration, also known as size-exclusion chromatography, separates

molecules based on size. The stationary phase consists of microscopic beads
with pores of a specific size. Smaller molecules enter the pores and are thus
retained longer than larger molecules, which are excluded/ bypass from the
pores and travel faster through the column. Each of these techniques serves
specific purposes and offers advantages in different scenarios, from analyzing
the composition of chemical or biological mixtures to purifying individual
components.

During the process, a mixture of molecules of different sizes is loaded onto the
column. As the mixture moves through the column, smaller molecules enter into
the pores of the beads and take a longer path to travel through the column, while
larger molecules bypass the beads and travel a shorter path. This difference in
path length causes the molecules to separate based on size, with larger
molecules eluting (coming off the column) before smaller molecules.

Gel Filtration Chromatography is used in biochemistry and molecular biology

for purifying and analyzing proteins, nucleic acids, and other biomolecules. It is
particularly useful for desalting (removing small molecules and salts from a
solution), buffer exchange, and size-based separation of biomolecules for
further analysis or purification steps.

ION EXCHANGE CHROMATOGRAPHY

This technique separates ions and polar molecules based on their affinity to
ionic groups attached to the stationary phase. The stationary phase is typically a
resin or gel matrix with charged groups, while the mobile phase (column) is
typically a gradient of increasing ionic strength which is used to elute bound
ions from the column. It uses a charged stationary phase to separate charged
compounds including anions, cations, amino acids, peptides, and proteins.
Molecules are separated based on differences in charge, size, and the strength of
the interaction with the ionic groups on the resin.
There are two main types of ion exchange chromatography:
1. Cation Exchange Chromatography: This involves the use of a
negatively charged stationary phase, which attracts and binds positively
charged ions (cations). Molecules are eluted (washed away) by increasing
the salt concentration or changing the pH of the mobile phase, which
affects the charge and/or binding affinity of the molecules.
2. Anion Exchange Chromatography: Conversely, this uses a positively
charged stationary phase, binding negatively charged ions (anions).
Similar to cation exchange, the molecules are eluted by altering the salt
concentration or pH.
The process of ion exchange chromatography typically involves the following
steps:
Equilibration: The column is prepared with a buffer at a certain pH and salt
concentration, ensuring the ions on the stationary phase are ready to interact
with the sample.
Sample Loading: The sample is applied to the column, and the molecules of
interest bind to the stationary phase based on their charge.
Washing: Unbound or weakly bound molecules are washed out of the column
with the same buffer used for equilibration, removing impurities.
Elution: The bound molecules are eluted by increasing salt concentration or
changing the pH. This can be done gradually (gradient elution) or in steps (step
elution), depending on the method.
The separated molecules can then be detected, often by UV absorbance, and
collected for further analysis.
Ion exchange chromatography is widely used in biochemical and molecular
biology research, as well as in the purification of proteins, peptides, nucleic
acids, and other charged biomolecules for industrial and medical applications.

THIN LAYER CHORMATOGRAPHY (TLC)

TLC is quite similar to paper chromatography but uses a thin layer of absorbent
material (like silica gel, alumina, or cellulose) spread on a supporting plate as
the solid stationary phase and coated on a glass, metal, or plastic plate. It's used
for separating non-volatile mixtures. A drop of the mixture to be separated is
placed on the plate, and then the edge of the plate is placed in a solvent. The
components of the mixture move up the plate at different rates. This method
offers better resolution than paper chromatography due to a more uniform
stationary phase since the process is relatively simple, quick, and requires only
small quantities of the sample. TLC is a widely used analytical technique in
chemistry and biochemistry that allows for the separation, identification, and
quantification of the components in a mixture especially small organic
molecules.
Here's a basic overview of how Thin Layer Chromatography works:
Basic Components of TLC
 Stationary Phase: A thin layer of silica gel or alumina is coated onto a
glass, metal, or plastic plate. This acts as the stationary phase.
 Mobile Phase: A solvent or a mixture of solvents (eluent) moves up the
stationary phase, carrying the components of the mixture with it.
 Sample: The mixture to be analyzed.
Steps in TLC
 Preparation of the TLC Plate: The stationary phase is prepared on a
suitable support (plate).
 Application of the Sample: A small amount of the mixture to be tested is
applied as a spot near the bottom of the plate.
 Development of the Chromatogram: The bottom of the plate is placed in
a shallow pool of solvent, without submerging the spot(s) of the sample.
The solvent moves up the plate by capillary action, carrying the sample
compounds with it.
 Visualization: Once the solvent has reached a predetermined height, the
plate is removed from the developing chamber. The separated
components of the sample can be visualized using various methods
depending on the nature of the compounds (e.g., UV light, staining).
Principles of Separation
The separation of components is primarily based on the differences in their
polarity and their interaction with the stationary and mobile phases. Compounds
that are more non-polar will tend to have less interaction with the stationary
phase and more with the mobile phase, thus moving farther up the plate.
Conversely, more polar compounds will interact more with the stationary phase
and less with the mobile phase, moving less far up the plate.
Results and Analysis
Retention Factor (Rf): The distances travelled by the compounds are used to
calculate their Retention Factor (Rf) values, which are characteristic for given
compounds under specific conditions.
Rf = Distance travelled by the compound / Distance travelled by the solvent
front
The Rf values and the appearance of the spots (colour, size) help in identifying
the components of the mixture.
Applications
TLC is used in various fields for:
 Purity testing of substances.
 Monitoring the progress of reactions.
 Identifying compounds in a mixture.
 Preparative TLC (for purification purposes).
  TLC is favored for its simplicity, speed, and the small amount of sample
required, making it an essential tool in analytical chemistry laboratories.

GAS (GAS LIQUID) CHROMATOGRAPHY

Gas-liquid chromatography (GLC), also known as gas chromatography (GC), is
a widely used analytical technique for separating and analyzing volatile
compounds in a mixture. The mobile phase is a gas (carrier gas), and the
stationary phase is a high boiling point liquid adsorbed onto a solid. Samples
vaporize and are carried by the gas through a column with the stationary phase.
Components of the sample separate based on their volatility (i.e., how easily
they can be vaporized) and their interaction with the stationary phase. This
technique is very sensitive and can separate complex mixtures with high
resolution.
Here's a brief overview of how gas-liquid chromatography works:
Basic Components of GLC
 Mobile Phase (Carrier Gas): In GLC, an inert gas (commonly helium,
nitrogen, or hydrogen) serves as the mobile phase. It carries the sample
through the chromatographic column.
 Stationary Phase (Liquid Phase): The stationary phase is a high-boiling
liquid coated or bonded to the inside wall of a capillary column in the GC
apparatus.
 Sample Injection Port: Where the sample is introduced into the carrier
gas before entering the column.
 Separation Column: The heart of the GC system, this is where the
separation of the sample components occurs. It is packed or coated with
the stationary phase.
 Detector: Various detectors (e.g., Flame Ionization Detector, Thermal
Conductivity Detector) are used to measure the concentration of the
analyte as it exits the column.

Steps in Gas-Liquid Chromatography

  Sample Introduction (Injection): The sample is vaporized and injected
into the heated inlet of the gas chromatograph.
 Vaporization: The sample is volatilized into the carrier gas and carried
into the column.
 Separation in the Column: The carrier gas carries the sample vapor
through the column, where the components interact with the stationary
liquid phase. These interactions cause the components to separate based
on their chemical properties.
 Detection: As the separated components exit the column, they pass
through a detector that responds to the compounds and produces a signal
proportional to the concentration of each component.
 Data Analysis: The detector signal is recorded as a chromatogram, which
is a graph showing the detector response against time or volume. This
chromatogram can be analyzed to identify and quantify the components
in the sample.

Principles of Separation
The separation in gas chromatography is based on differences in the distribution
of the sample components between the stationary and mobile phases.
Compounds with stronger interactions with the stationary phase will take longer
to move through the column, leading to separation.

Applications of Gas-Liquid Chromatography:

1. Analysis Qualitative: Identification of components in a mixture by
comparing retention times with known standards.
2. Quantitative Analysis: Determining the concentration of components in a
mixture by comparing peak areas or heights.
3. Purity Testing: Assessing the purity of compounds by analyzing
impurities and degradation products.
4. Environmental Analysis: Monitoring pollutants in air, water, and soil.
5. Forensic Analysis: Identifying substances in criminal investigations.
 6. Pharmaceutical Analysis: Analyzing drugs and their metabolites.
7. Flavour and fragrance analysis in the food industry.
8. Analysis of petroleum products in the petrochemical industry.

Advantages of Gas-Liquid Chromatography:

1. High Separation Efficiency: Capable of resolving complex mixtures.
2. Sensitivity: Can detect compounds at trace levels.
3. Speed: Provides rapid analysis compared to traditional methods.
4. Versatility: Suitableh for a wide range of compounds.
5. Automation: Easily automated for high-throughput analysis.
Gas-Liquid Chromatography is a vital tool in analytical chemistry, providing
precise separation and identification of compounds in various fields, from
research labs to industrial applications.

GEL FILTRATION
Gel filtration is a type of column chromatography that separates molecules
based on their size. In gel filtration chromatography, the stationary phase is
composed of porous beads. Larger molecules move through the column more
quickly as they are excluded from the beads' pores, while smaller molecules
enter the beads and take longer to elute. This technique is commonly used for
purifying proteins, nucleic acids, and other biomolecules based on their size.
Gel filtration is a type of column chromatography used to separate molecules in
a mixture based on their size. In gel filtration chromatography, a porous gel
matrix is used as the stationary phase. The smaller molecules can enter the pores
of the gel matrix, resulting in longer retention times, while larger molecules are
excluded and elute first, resulting in shorter retention times. This method is
commonly used in biochemistry to separate proteins or nucleic acids based on
their molecular size.

Gel filtration chromatography is a type of size exclusion chromatography used

for separating biomolecules based on their size. In this method, a porous gel
matrix is used to separate molecules based on their size. Smaller molecules can
enter the pores and therefore take longer to elute, while larger molecules pass
more quickly through the column because they are excluded from the gel
matrix. This results in the molecules getting separated based on their size.
Gel filtration chromatography is a type of size exclusion chromatography used
to separate biomolecules based on their size. In this method, a porous gel matrix
is used to separate molecules in a sample based on their size. Smaller molecules
can enter the pores and take longer to travel through the column, while larger
molecules are excluded and elute first. This technique is commonly used for
purification and separation of proteins, nucleic acids, and other biomolecules
based on their molecular weight.