M.Prasad Naidu MSC Medical Biochemistry, PH.D Research Scholar

M.
PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.
What is Chromatography?
Chromatography is a technique for
separating mixtures into their components
in order to analyze, identify, purify,
and/or quantify the mixture or
components.
• Analyze
Separate • Identify
• Purify
Mixture Components
• Quantify
Uses for 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 the a mixture
and/or the components present in the sample
Uses for 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
Definition of Chromatography
Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile
medium and for a stationary adsorbing medium through
which they pass.
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)
Definition of Chromatography
Simplified Definition:
Chromatography separates the components of a
mixture by their distinctive attraction to the mobile
phase and the stationary phase.
Explanation:
• Compound is placed on stationary phase
• Mobile phase passes through the stationary phase
• Mobile phase solubilizes the components
• Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the
phases
Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture Components
Affinity to Stationary Affinity to Mobile
Components
Phase Phase
Blue ---------------- Insoluble in Mobile Phase
Black  
Red  
Yellow          
Types of Chromatography
• 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 beads (stationary phase)
• 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)
(A) uses charge, (B) uses
pores, and (C) uses
covalent bonds to create
the differential affinities
among the mixture
components for the
stationary phase.
Principles of Paper Chromatography
 Capillary Action – the movement of liquid within the spaces of a

porous material due to the forces of adhesion, cohesion, and surface
tension. The liquid is able to move up the filter paper because its
attraction to itself is stronger than the force of gravity.
 Solubility – the degree to which a material (solute) dissolves into a

solvent. Solutes dissolve into solvents that have similar properties.
(Like dissolves like) This allows different solutes to be separated by
different combinations of solvents.
Separation of components depends on both their solubility in the

mobile phase and their differential affinity to the mobile phase and the
stationary phase.
Paper Chromatography Experiment
What Color is that Sharpie?
Overview of the 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
Costs:
Less than $10
Materials List
 6 beakers or jars
 6 covers or lids
 Distilled H2O
 Isopropanol
 Graduated cylinder
 6 strips of filter paper
 Different colors of Sharpie
pens
 Pencil
 Ruler
 Scissors
 Tape
Preparing the Isopropanol
Solutions
• Prepare 15 ml of the following isopropanol solutions
in
appropriately labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%
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
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
Observing the Chromatograms
0% 20% 50% 70% 100%

Concentration of Isopropanol
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%

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%

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%

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%

Alternative Experiments
• Test different samples:
– Other markers, pens, highlighters
– Flower pigments
– Food Colors
• Test different solvents:

– Other alcohols: methanol, ethanol,
propanol, butanol
• Test different papers:
– Coffee filters
– Paper towels
– Cardstock
– Typing paper
TEKS Standards
• 6.1, 7.1, 8.1 Student conducts laboratory investigations using safe,

environmentally appropriate, and ethical practices.
• 6.2, 7.2, 8,2 Student uses scientific inquiry methods during laboratory
investigations.
• 6.3, 7.3, 8.3 Student uses critical thinking and scientific thinking and
problem solving to make informed decisions.
• 6.4, 7.4, 8.4 Student knows how to use a variety of tools and methods
to conduct science inquiry.
• 6.7 , 7.7, 8.9 Student knows that substances have physical and chemical
properties.
Thank you

M.Prasad Naidu MSC Medical Biochemistry, PH.D Research Scholar

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Chromatography is used by scientists to:

• Analyze – examine a mixture, its components,

• Gas Chromatography – separates vaporized samples

• Paper Chromatography – separates dried liquid

• Thin-Layer Chromatography – separates dried liquid

 Capillary Action – the movement of liquid within the spaces of a

 Solubility – the degree to which a material (solute) dissolves into a

Separation of components depends on both their solubility in the

 Cut 6 strips of filter paper

 Place the strips in the beakers

0% 20% 50% 70% 100%

0% 20% 50% 70% 100%

0% 20% 50% 70% 100%

0% 20% 50% 70% 100%

0% 20% 50% 70% 100%

• Test different solvents:

• 6.1, 7.1, 8.1 Student conducts laboratory investigations using safe,

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