Practical session no:06

FIS 201G2 Laboratory techniques

Study the following
1) Gas chromatography
• Gas chromatography (GC) is a widely used analytical technique in chemistry and
biochemistry for separating and analyzing compounds in a mixture.
• A small amount of the sample is injected into the system. The sample is often in the
form of a vapour or a gas.
• An inert gas, such as helium or nitrogen, serves as the mobile phase or carrier gas.
• It carries the vaporized sample through a long, coiled column.
• The column is typically a long, narrow tube coated with a stationary phase.
• The stationary phase can be a liquid or a solid material that interacts with the sample
compounds.

▪ Principle: The principle of gas chromatography (GC) is based on the differential

partitioning of components in a sample between a mobile phase (usually an inert
gas) and a stationary phase inside a chromatographic column.
▪ As the sample travels through the column, different components interact with
the stationary phase to varying degrees.
▪ Some compounds spend more time in the gas phase (mobile phase), while
others are more attracted to the stationary phase.
▪ This differential interaction leads to the separation of components based on
their chemical properties.
Advantages:
▪ High separation efficiency in gas chromatography (GC) is a crucial aspect that
contributes to the accuracy and reliability of analyses.
▪ small sample consumption and high detection sensitivity: 1ml of gas sample
consumption, 0.1 micro liter of liquid sample consumption. A few mg of solid sample
consumption.
Disadvantages
▪ GC is most effective for volatile and semi-volatile compounds that can be vaporized
without decomposition. It may not be suitable for high-molecular-weight or non-volatile
compounds.
▪ Some compounds may degrade or decompose at the high temperatures used in gas
chromatography, leading to inaccurate results.
▪ Samples need to be in a gaseous or vaporized state for injection, limiting the types of
samples that can be directly analyzed.
2) Paper chromatography
Paper chromatography is a widely used technique for separating and analyzing mixtures of
compounds. It is a type of chromatography that relies on the differential movement of substances
through a stationary phase (in this case, paper) due to differences in their affinity for the mobile
phase (usually a liquid solvent).
1. Principle:
• Paper chromatography is based on the principle of partition chromatography,
where the components of a mixture distribute themselves between the stationary
phase (paper) and the mobile phase (solvent).
2. Setup:
• A strip of filter paper is used as the stationary phase. A small spot containing the
mixture to be analyzed is applied near one end of the paper.
3. Mobile Phase:
• The mobile phase is a liquid solvent that moves up the paper by capillary action.
Common solvents include water, alcohol, or a mixture of the two.
4. Separation:
• As the solvent moves up the paper, it carries the components of the mixture with
it. Different compounds within the mixture will have varying degrees of affinity
for the paper and the solvent, causing them to move at different rates.
5. Visualization:
• Once the solvent front reaches the top of the paper, the paper is removed, and the
separated components are visualized. This can be done by exposing the paper to a
developing agent or by using various detection methods such as UV light,
staining, or chemical reactions.
6. Rf Value:
• The relative mobility (Rf value) of a compound is a ratio of the distance traveled
by the compound to the distance traveled by the solvent. It is a characteristic
property of a compound under specific chromatographic conditions and is used
for identification.
7. Applications:
• Paper chromatography is commonly used in biology and chemistry laboratories
for separating and identifying amino acids, sugars, dyes, and other organic
compounds. It is a simple and cost-effective method for qualitative analysis.
Despite its simplicity, paper chromatography has some limitations, such as limited resolution and
difficulty in separating complex mixtures. Other more advanced chromatographic techniques like
high-performance liquid chromatography (HPLC) or gas chromatography (GC) are often used
when higher precision and sensitivity are required.

3) Thin layer chromatography

Thin-layer chromatography (TLC) is another chromatographic technique used for separating and
analyzing mixtures. It shares some similarities with paper chromatography but uses a different
stationary phase and allows for greater control and precision. Here's an overview of thin-layer
chromatography:
1. Principle:
• Similar to paper chromatography, TLC is based on the principle of partition
chromatography. It involves the separation of compounds based on their differing
affinities for a stationary phase and a mobile phase.
2. Setup:
• In TLC, the stationary phase is a thin layer of an adsorbent material (usually silica
gel or alumina) coated on a glass, plastic, or aluminum plate. This thin layer
serves as the medium through which the compounds will migrate. A small spot
containing the sample to be analyzed is applied near the bottom of the plate.
3. Mobile Phase:
• The mobile phase is a liquid solvent that moves up the plate by capillary action.
Common solvents include a mixture of organic solvents like ethyl acetate and
hexane.
4. Separation:
• As the solvent moves up the plate, it carries the sample with it. The components
of the mixture interact differently with the stationary phase, leading to varying
rates of movement. This differential movement results in the separation of the
compounds.
5. Visualization:
• After the solvent front reaches the top of the plate, the plate is removed, and the
separated components are visualized. Visualization methods can include exposing
the plate to UV light, using a developing agent, or applying a staining solution.

6. Rf Value:
 • Similar to paper chromatography, the Rf value in TLC is calculated as the ratio of
the distance traveled by the compound to the distance traveled by the solvent. It is
a characteristic value for a particular compound under specific chromatographic
conditions.
7. Applications:
• TLC is widely used in chemistry and biochemistry for the separation and
identification of compounds. It is commonly employed for the analysis of organic
reactions, checking the purity of compounds, and isolating individual components
from mixtures.
TLC is appreciated for its speed, simplicity, and cost-effectiveness. It is often used as a
preliminary technique for assessing the success of a reaction or the purity of a compound.
However, for more quantitative and precise analyses, other chromatographic techniques like
high-performance liquid chromatography (HPLC) may be preferred.

4) GCMS
Gas Chromatography-Mass Spectrometry (GC-MS) is a powerful analytical technique used
for the identification and quantification of compounds in a mixture. It combines the
separation capabilities of gas chromatography with the detection and characterization
abilities of mass spectrometry.
1. Gas Chromatography (GC):
• Sample Introduction: A sample is introduced into the system. It is usually a volatile
liquid or a sample that can be converted into a vapor.
• Vaporization: The sample is vaporized and injected into the chromatograph.
• Separation: The vaporized sample is then injected into a chromatographic column. The
column is typically coated with a stationary phase, and as the sample travels through the
column, different compounds separate based on their chemical properties (such as boiling
points and affinities for the stationary phase).
• Retention Time: Each compound has a characteristic retention time, which is the time it
takes for the compound to travel through the column and reach the detector.
2. Mass Spectrometry (MS):
• Ionization: As the compounds exit the gas chromatograph, they enter the mass
spectrometer. Here, the compounds are ionized, typically by electron impact or chemical
ionization.
• Ion Separation: The resulting ions are then accelerated and passed through a magnetic
field, which causes them to separate based on their mass-to-charge ratio (m/z).
 • Detection: The separated ions are detected by a detector, and the resulting mass spectrum
is a plot of ion abundance against m/z. Each compound produces a unique mass
spectrum, allowing for identification.
3. Data Analysis:
• The data obtained from the GC-MS analysis can be compared to a database of known
mass spectra to identify the compounds present in the sample.
• The abundance of each ion in the mass spectrum can also be used for quantification
purposes.
Applications of GC-MS:
• Environmental Analysis: Detection of pollutants and contaminants.
• Forensic Analysis: Identification of drugs, toxins, and other substances.
• Food and Flavor Analysis: Identification of flavor compounds.
• Pharmaceutical Analysis: Quality control and identification of pharmaceutical
compounds.
• Petroleum Industry: Analysis of hydrocarbons in crude oil.

5) HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC stands for High-performance liquid chromatography. It is a technique in analytical

chemistry used to separate, identify, and quantify specific components in mixtures 1.
HPLC uses high-pressure pumps, solvents, and solid particles to separate the sample
components based on their interactions with the adsorbent material.
How HPLC is working?
Sample Injection:
A small amount of the sample is injected into the HPLC system using an injector.

Mobile Phase:
The sample is carried through the system by a liquid mobile phase. The mobile phase can be
polar or non-polar, depending on the type of chromatography being performed.

Column:
The heart of the HPLC system is the column, which is filled with a stationary phase. The
stationary phase can be a solid (particles) or a liquid (coated surface)

Separation:
As the sample travels through the column, different components interact with the stationary
phase to varying degrees.
In normal phase HPLC, separation is based on polarity differences. In reverse phase HPLC,
separation occurs due to hydrophobic interactions.
Components with stronger affinity for the stationary phase take longer to travel through the
column, leading to separation.

Detection:
The eluent (mobile phase carrying the sample) leaving the column is monitored by a detector.
The detector produces a signal proportional to the concentration of the analyte (component being
analyzed).

Data Analysis:
The data system collects and analyzes the signals from the detector.
Chromatograms are generated, showing peaks that correspond to separated components.
Metabolism and Growth:

Adequate levels of dissolved oxygen support the metabolic processes of aquatic organisms,
promoting their growth, development, and overall well-being

Advantage:
High resolution and sensitivity.
Quantitative analysis

6) Column chromatography
• Principle: It's a separation technique based on the differential partitioning of compounds
between a stationary phase (solid or immobilized) and a mobile phase (liquid or gas).
 • A vertical glass column is packed with a stationary phase (such as silica gel or alumina),
and the sample mixture is loaded on top. The mobile phase (solvent or solvent mixture) is
passed through the column.
• Compounds in the sample interact differently with the stationary phase. Components with
stronger interactions take longer to elute, leading to separation.
• Sample Loading: The sample is loaded onto the column either by dry-packing (placing
the sample on top of the stationary phase) or by dissolving it in a small volume of solvent
and applying it to the top of the column.
• Mobile phase moves through column, compounds separate based on interactions,
collected as fractions with distinct elution times.
• Monitoring: Fraction collection is often done by collecting drops or measuring the eluent
at different time intervals. Monitoring can be done using UV-visible spectroscopy or
other detection methods.
• Applications: Column chromatography is widely used in organic chemistry labs for the
purification and separation of natural products, synthetic compounds, and biomolecules.
• There are various types of column chromatography, including normal phase (polar
stationary phase), reverse phase (non-polar stationary phase), and size exclusion
chromatography (based on molecular size).

7) Liquid chromatography
• Principle: It separates compounds based on their differential partitioning between a
mobile liquid phase and a stationary phase within a column.
• Involves a liquid mobile phase passing through a column packed with a stationary phase
(like silica or a polymer) where separation occurs.
• Samples are injected using an autosampler, allowing precise and automated introduction
into the system.
• Compounds interact differently with the stationary phase, leading to differential retention
and elution times.
• Analytes are detected post-separation by various detectors like UV-Vis, fluorescence,
mass spectrometry, or refractive index detectors.
• Widely used in pharmaceutical, environmental, biochemical, and food industries for
compound analysis, purification, and quantification.
• Offers high resolution, sensitivity, and rapid analysis compared to other chromatographic
methods.
Study the practical ethics we follow in the laboratory.
▪ Follow safety rules, wear your safety gear, and be aware of any dangers in the lab.
▪ Don't eat or drink in the lab. It keeps things clean and avoids any mix-ups with
chemicals.
▪ Pay attention to safety signs. They guide you on what to do and help prevent accidents.
▪ Keep your work area tidy. It helps you find things easily and reduces the chance of spills.
▪ Always put on a lab coat before entering the laboratory.
▪ If you're working with open flames, like a Bunsen burner, be very careful. Follow safety
guidelines, control the flame responsibly, and always turn it off when you're done.