Chromatography

Presentation by Hariprasad
Chromatography is a laboratory technique
(analytical technique) for the separation of
a mixture into its components.

Chromatography is a technique for separating

mixtures into their components in order to
identify, purify, and/or quantify the mixture or
components.

The mixture is dissolved in a fluid solvent (gas

or liquid) called the mobile phase, which carries
it through a system (a column, a capillary tube,
a plate, or a sheet) on which a material called
the stationary phase is fixed

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 • Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components

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 Invention of Chromatography

Mikhail Tswett invented chromatography in

1901 during his research on plant pigments.
He used the technique to separate various
plant pigments such as chlorophylls,
xanthophylls and carotenoids.

Mikhail Tswett
Russian Botanist
(1872-1919)
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 Original Chromatography Experiment
Start: A glass column is End: A series of colored
filled with powdered bands is seen to form,
Limestone (CaCO3). corresponding to the different
pigments in the original plant
extract. These bands were
Later
later determined to be
An Ethyl alcohol extract of leaf
pigments is applied to the top of the
chlorophylls, xanthophylls and
column. carotenoids.
Ethyl alcohol is used to flush the
pigments down the column.

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Chromatography: (Greek = chroma “color” and graphein
“writing”) Tswett named this new technique chromatography since it
separated the components of a solution by colour.

Common Types of Chromatography

• Tswett’s technique is based on Liquid Chromatography.
• There are now several common chromatographic methods.
• These include:
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid Chromatography (LC)
High Pressure Liquid Chromatography (HPLC)
Ion Chromatography
Gas Chromatography (GC)

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Terminology:

• Differential – showing a difference, distinctive

• Affinity – natural attraction or force between
things
• Mobile Medium – gas or liquid that carries the
components (mobile phase)
• Stationary Medium – the part of the apparatus
that does not move with the sample (stationary
phase)

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 Uses of Chromatography
• Chromatography is used by scientists to:
• Analyze – examine a mixture, its components, and their relations to one
another
• Identify – determine the identity of a mixture or components based on
known components
• Purify – separate components in order to isolate one of interest for further
study
• Quantify – determine the amount of a mixture and/or the components
present in the sample

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 Uses of Chromatography
Real-life examples of uses for chromatography:
• Pharmaceutical Company – determine amount of each chemical found in
new product
• Hospital – detect blood or alcohol levels in a patient’s blood stream
• Law Enforcement – to compare a sample found at a crime scene to
samples from suspects
• Environmental Agency – determine the level of pollutants in the water
supply
• Manufacturing Plant – to purify a chemical needed to make a product

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Applications of Chromatography

Forensics

Research
Pharmaceutical industry

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Illustration of Chromatography
Stationary Phase

Separation

Mobile
Phase
Mixture Components
Components Affinity to Stationary Phase Affinity to Mobile Phase
Blue ---------------- Insoluble in Mobile Phase

Black  

Red  

Yellow          

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Chromatography terms
Analyte – the substance to be separated during chromatography. It is also normally what is needed from
the mixture.

Analytical chromatography – the use of chromatography to determine the existence and possibly also
the concentration of analyte(s) in a sample.

Bonded phase – a stationary phase that is covalently bonded to the support particles or to the inside wall
of the column tubing.

Chromatogram – the visual output of the chromatograph. In the case of an optimal separation, different
peaks or patterns on the chromatogram correspond to different components of the separated mixture.

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Chromatography terms

Chromatograph – an instrument that enables a sophisticated separation, e.g., gas chromatographic

or liquid chromatographic separation.

Chromatography – a physical method of separation that distributes components to separate between

two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite
direction.

Mobile phase – the phase that moves in a definite direction. It may be a liquid (LC and capillary
electrochromatography (CEC)), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography,
SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves
the sample through the column.
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Eluent (sometimes spelled eluant) – the solvent or solvent fixure used in elution
chromatography and is synonymous with mobile phase.

Eluate – the mixture of solute and solvent exiting the column.

Effluent – the stream flowing out of a chromatographic column. In practise, it is used

synonymously with eluate, but the term more precisely refers to the stream independent of
separation taking place.

Eluite – a more precise term for solute or analyte. It is a sample component leaving the
chromatographic column.

Eluotropic series – a list of solvents ranked according to their eluting power.

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 Liquid Chromatography – separates liquid
samples with a liquid solvent (mobile phase) and a
column composed of solid beads (stationary phase)
Gas Chromatography – separates vaporized
samples with a carrier gas (mobile phase)
and a column composed of a liquid or of solid
Types of beads (stationary phase)

Chromatography Paper Chromatography – separates dried liquid

samples with a liquid solvent (mobile phase) and a
paper strip (stationary phase)
Thin-Layer Chromatography – separates dried
liquid samples with a liquid solvent (mobile phase)
and a glass plate covered with a thin layer of
alumina or silica gel (stationary phase)

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Types of Chromatography…

Thin layer
Paper

HPLC Gas Column

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High-performance liquid chromatography (HPLC), formerly referred to as high-
pressure liquid chromatography, is a technique in analytical chemistry used to
separate, identify, and quantify each component in a mixture.

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 Paper chromatography is a technique that involves
placing a small dot or line of sample solution onto a strip
of chromatography paper.

The paper is placed in a container with a shallow layer

of solvent and sealed.

Paper
Chromatography
As the solvent rises through the paper, it meets the
sample mixture, which starts to travel up the paper with
the solvent.

This paper is made of cellulose, a polar substance, and

the compounds within the mixture travel further if they
are less polar. More polar substances bond with the
cellulose paper more quickly, and therefore do not travel
as far.
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Thin layer chromatography

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Basic Steps of TLC Technique

Preparation of the Plate

Sample Application

Chromatogram Development

Locating of the Spots

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 Preparation of the Plate

• Slurry of the active material is uniformly spread over the plate by

means of a commercially available spreader.
• Air-drying overnight, or oven-drying at 80-90 C for about 30 minutes.
• Ready to use thin layers (pre-coated plates) are commercially
available.

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Sample Application

1-2 cm 1-2 cm
 Base line
2-2.5 cm

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 Locating of the Spots

For Colored Compounds:

Solvent front

Rf = b/a a

b
Base line 

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For Colorless Compounds:
Where is the spots ??
We do not know.

Solvent front

a Rf = b/a

 Base line

•Iodine or sulphuric acid is used for most organic mixtures.

•Ninhydrin is used for amino acids.
•2,4-Dinitrophenylhydrazine is used for aldehydes and ketones
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Applications of TLC Technique
Identification of Unknown Compounds

     

Co-spot
Co-spot

Unknown

Authentic
Unknown

Authentic

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Determination of the Purity of a Product Compound

Product compound

Impurities


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Column chromatography
Column chromatography is a separation technique in which the stationary bed is within a
tube.

The particles of the solid stationary phase or the support coated with a liquid stationary
phase may fill the whole inside volume of the tube (packed column) or be concentrated on
or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the
middle part of the tube (open tubular column).

Differences in rates of movement through the medium are calculated to different retention
times of the sample

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1.Column loaded with silica/column medium
2.Eluting solvent added to compact silica layer and to remove air bubbles
3.Purple mixture as a thin layer is added to top of silica layer
4.Eluting solvent added and eluted (purple layer separates into a red and blue layer)
5.Eluting solvent added and eluted (red and blue layers separate further)
6.Red layer collected (the faster moving layer)
7.Blue layer collected (the slower moving layer)
8.No more compounds are eluted, process ended

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 Experiment
Purpose:
To introduce students to the principles and terminology of
chromatography and demonstrate separation of the dyes in
Sharpie Pens with paper chromatography.

Time Required:
Prep. time: 10 minutes
Experiment time: 45 minutes

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Materials List
• 6 beakers or jars
• 6 covers or lids
• Distilled water (H2O)
• Isopropanol (C3H7OH)
• Graduated cylinder
• 6 strips of filter paper
• Different colors of Sharpie pens
• Pencil
• Ruler
• Scissors
• Tape

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Preparing the Isopropanol Solutions
• Prepare 15 ml of the following isopropanol solutions
in
appropriately labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%

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 Preparing the Chromatography Strips

• Cut 6 strips of filter paper

• Draw a line 1 cm above the bottom
edge of the strip with the pencil
• Label each strip with its corresponding
solution
• Place a spot from each pen on your
starting line

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 Developing the Chromatograms

• Place the strips in the beakers

• Make sure the solution does not come
above your start line
• Keep the beakers covered
• Let strips develop until the ascending
solution front is about 2 cm from the top of
the strip
• Remove the strips and let them dry

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 Developing the Chromatograms
50% Isopropanol

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 Developing the Chromatograms
50% Isopropanol

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 Observing the Chromatograms

0% 20% 50% 70% 100%

Concentration of Isopropanol
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 Black Dye
1. Dyes separated – purple and black
2. Not soluble in low concentrations of isopropanol
3. Partially soluble in concentrations of isopropanol >20%

0% 20% 50% 70% 100%

Concentration of Isopropanol

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 Blue Dye
1. Dye separated – blue
2. Not very soluble in low concentrations of isopropanol
3. Completely soluble in high concentrations of
isopropanol

0% 20% 50% 70% 100%

Concentration of Isopropanol
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 Green Dye
1. Dye separated – blue and yellow
2. Blue – Soluble in concentrations of isopropanol >20%
3. Yellow – Soluble in concentrations of isopropanol >0%

0% 20% 50% 70% 100%

Concentration of Isopropanol
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 Red Dye
1. Dyes separated – red and yellow
2. Yellow –soluble in low concentrations of isopropanol and
less soluble in high concentrations of isopropanol
3. Red – slightly soluble in low
concentrations of isopropanol,
and more soluble in
concentrations of isopropanol
>20%.

0% 20% 50% 70% 100%

Concentration of Isopropanol
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 Case Study 1: Pharmaceutical Analysis
Introduction: Chromatography is widely used in the pharmaceutical industry for the analysis and
purification of compounds.
Details:
• Application: Quality control of drugs.
• Chromatography Type: High-Performance Liquid Chromatography (HPLC).
• Example: Analyzing the purity of a batch of aspirin tablets.
Process:
• A sample of the aspirin tablets is dissolved in a suitable solvent.
• The solution is injected into the HPLC system.
• The chromatogram produced helps in identifying the presence of impurities or degradation
products.
Impact and Applications:
• Ensures that pharmaceuticals meet regulatory standards for safety and efficacy.
• Identifies and quantifies impurities, ensuring the drug's purity and quality.
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 Case Study 2: Environmental Monitoring
Introduction: Chromatography is used in environmental science to detect and quantify pollutants in air, water,
and soil.
Details:
• Application: Monitoring pesticide residues in water.
• Chromatography Type: Gas Chromatography-Mass Spectrometry (GC-MS).
• Example: Detection of organochlorine pesticides in river water.
Process:
• Water samples are collected from various points along the river.
• Samples are extracted using an organic solvent.
• The extract is injected into the GC-MS system.
• The chromatogram and mass spectrum are analyzed to identify and quantify the pesticide residues.
Impact and Applications:
• Helps in assessing the pollution levels in water bodies.
• Provides data for environmental protection agencies to take necessary action.
• Ensures the safety of drinking water by monitoring contamination levels.
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 Case Study 3: Food Safety
Introduction: Chromatography is crucial in the food industry to ensure the safety and quality of food
products.
Details:
• Application: Detecting food additives and contaminants.
• Chromatography Type: Liquid Chromatography-Mass Spectrometry (LC-MS).
• Example: Detection of aflatoxins in peanuts.
Process:
• Peanut samples are ground and extracted with a solvent.
• The extract is injected into the LC-MS system.
• The chromatogram helps in identifying and quantifying the aflatoxins present.
Impact and Applications:
• Ensures that food products comply with safety regulations.
• Protects consumers from harmful substances.
• Helps manufacturers monitor and control the quality of their products.
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 Case Study 4: Forensic Science
Introduction: Chromatography is an essential tool in forensic science for the analysis of crime scene evidence.
Details:
• Application: Analysis of drug residues.
• Chromatography Type: Thin Layer Chromatography (TLC) and GC-MS.
• Example: Detecting narcotics in a suspect’s belongings.
Process:
• Samples (e.g., hair, clothing) are collected from the suspect.
• TLC is used as a preliminary test to identify the presence of drugs.
• Confirmatory analysis is performed using GC-MS.
• The chromatogram helps in identifying the specific drugs and their quantities.
Impact and Applications:
• Provides crucial evidence in criminal investigations.
• Helps in identifying substances and linking suspects to crime scenes.
• Assists in the judicial process by providing reliable analytical results.

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 Case Study 5: Biochemical Research
Introduction: Chromatography is used extensively in biochemical research for separating and analyzing
biomolecules.
Details:
• Application: Protein purification and analysis.
• Chromatography Type: Affinity Chromatography.
• Example: Purification of a recombinant protein expressed in bacterial cells.
Process:
• Bacterial cells expressing the protein are lysed.
• The lysate is loaded onto an affinity chromatography column containing a ligand specific to the protein.
• The protein binds to the ligand, while other components are washed away.
• The protein is eluted using a specific buffer.
• The purity of the protein is analyzed using SDS-PAGE or HPLC.
Impact and Applications:
• Enables the purification of proteins for structural and functional studies.
• Essential for the production of therapeutic proteins and vaccines.
• Facilitates the study of protein interactions and functions.
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 Case Study 6: Metabolomics
Introduction: Chromatography plays a key role in metabolomics, the study of small molecules in biological
systems.
• Details:
• Application: Profiling metabolites in biological samples.
• Chromatography Type: Liquid Chromatography-Mass Spectrometry (LC-MS).
• Example: Analysis of metabolic changes in blood samples from diabetic patients.
Process:
• Blood samples are collected and processed.
• The metabolite extract is injected into the LC-MS system.
• The chromatogram provides a profile of metabolites present in the samples.
• Changes in metabolite levels are analyzed to understand disease mechanisms.
Impact and Applications:
• Helps in identifying biomarkers for diseases.
• Provides insights into metabolic pathways and their alterations in diseases.
• Aids in the development of new diagnostic and therapeutic strategies.

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