Chromatography

• Chromatography is the collective term for a set of laboratory techniques for the
separation of mixtures (separation technique).

• Received its name from the work of Mikhail Tswett

(Russian Botanist/Plant Scientist) in 1903

• Tswett adsorbed a mixture of plant pigments on to finely divided chalk (CaCO3)and

separated them into colored bands by washing the column with solvents (used
chromatography to separate plant pigments)

• Chromatographien : Chroma: “color” and Graphein: to “write” (Color writing or to

write with color)
Technique is more often used to separate colorless substances, the original name
remains and applied to any process whereby substances are separated by a
continuous redistribution between two phases, one stationary phase and the
other mobile phase
Chromatography
• IUPAC (1993)
“Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary while the
other moves in a definite direction.” (mobile phase)

• USP 2009 
Chromatography is defined as a procedure by which solute are separated by a
dynamic differential migration process in a system consisting of two or more phases,
one of which moves continuously in a given direction and in which the individual
substances exhibit different mobilities by reason of differences in adsorption,
partition, solubility, vapor pressure, molecular size, or ionic charge density. The
individual substances thus separated can be identified or determined by analytical
procedures
 History

The first scientist was Russian botanist Tswett (1906), who used a simple form of
liquid-solid chromatography .

•1931 Lederer & Kuhn - LC of carotenoids

•1938 TLC and ion exchange


In 1940s Martin and Synge- liquid-liquid chromatography- separate acetyl amino
acids.


Martin and A.T. James in 1950 introduced GLC as partition chromatography.


HPLC was invented in 1967 and practical application started in 1970s.

1980s Super Critical chromatography (SFC) Hyphenated techniques
Importance
 Chromatography has application in every branch of the physical
and biological sciences.

 Nobel prizes have been awarded for work in which

chromatography played a vital role
Uses of Chromatography

• 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
 Chromatographic system is composed :
Stationary phase and Mobile phase
a. Stationary phase : column packing material
• may be a solid, liquid supported on a solid or a gel.

b. Mobile Phase: Solvent

 • may be a liquid or a gas

Separation results from differences in the distribution constants of the individual

sample components between the two phases

Purpose of Chromatography
 • Analytical - determine chemical composition of a sample
• Preparative - purify and collect one or more components of a sample
Classification of chromatography

Physical form of stationary phase may be laminar (Planar) or columnar

Planar Chromatography: Stationary phase is in the form of a thin layer or sheet,
through and over which mobile phase flows
Paper chromatography ( Ascending, Descending or circular)
Thin layer chromatography

Column Chromatography: Stationary phase particles are packed into a column

through which mobile phase passes ( mobile phase is forced either by pressure or
by gravity )
Depending upon stationary phase or packing material further subdivided into
Adsorption Chromatography, Ion Exchange Chromatography
Affinity Chromatography, Gel filtration, HPLC, GC
 Classification of Chromatography

By mobile phase:
1. Liquid chromatography.
2. Gas chromatography.
 Important Terms

• Analyte: Substance to be separated during chromatography

• Eluate: MP leaving the column. Also called effluent.

• Eluent :The solvent that carries the analyte.

• Chromatogram: visual output of chromatography.

•  Retention time: Characteristic time it takes for a particular

analyte to pass through the system 

Paper
https://www.youtube.com/watch?v=ZCzgQXGz9Tg
https://www.youtube.com/watch?v=jiPd5CkCkkU
https://www.youtube.com/watch?v=7q5HDMXSdtU
https://www.youtube.com/watch?v=mz_xcNrTK_U
 Paper Chromatography (PC):

The speed depends on the solubility of the

substance (dye) in mobile phase (water) and its
interaction with the paper

Different molecules are with different characteristics

Rf is relative flow

Rf = Distance from the starting point to the middle of a spot

Distance from start to finish point of the water 4/10 = 0.4
6/10 = 0.6
8/10 = 0.8
 Paper chromatography is a
Ascending Chromatography
technique that involves placing a small
dot or line of sample solution onto a
strip of chromatography paper.

The paper is placed in a jar containing

a shallow layer of solvent and sealed.

As the solvent rises through the paper,

it meets the sample mixture, which
starts to travel up the paper with the
solvent. 

Colorless compounds can be visualized by visualizing agents (e.g. amino acid are
treated with Ninhydrin)
Circular paper chromatography
Two-dimensional chromatography

Sometimes, it is rather difficult to

separate a complex mixture of
substances by a single run with one
solvent system.

In such a case, a second run is carried

out by a different solvent system, in a
direction perpendicular to the first run.

This is referred to as two dimensional

chromatography.
Applications of Paper Chromatography
1. Separation of dyes
- To compare ink dyes use in any company.

2. Food coloring
- To differentiate coloring agent used in food
product.

3. Botanist/herbalist
- To isolate plant pigment from root and leaves.
•TLC
•https://www.youtube.com/watch?v=rMGQavOMAmc
•Column
•https://www.youtube.com/watch?v=2R2iq_XR1IY
 Thin Layer Chromatography
TLC is a widely employed laboratory technique and is similar to PC

Separations in TLC involve distributing a mixture of two or more substances between

a stationary phase and a mobile phase.

Stationary phase: It is a thin layer of adsorbent (usually silica gel, or alumina or

cellulose ) coated on a plate (Glass or plastic).

Mobile phase: It is a developing liquid which travels up the stationary phase,

carrying the samples with it.

Compared to paper, it has the advantage of faster runs, better separations, and the
choice between different adsorbents.
 Thin Layer Chromatography (TLC):

Thin-layer chromatography. Molecules separate as they move through the silica gel.
Thin-layer chromatography is used to separate small molecules, such as amino acids
Colorless compounds can be visualized by visualizing agents (e.g. amino acid are
treated with Ninhydrin)
 Column Chromatography

Stationary phase is held in a

narrow tube through which the
mobile phase is forced under
pressure or under the effect of
gravity
Classification of chromatography
According to separation modes and stationary phases chromatography can
be classified into following classes:

1. ADSORPTION CHROMATOGRAPHY

2. MOLECULAR EXCLUSION CHROMATOGRAPHY

3. AFFINITY CHROMATOGRAPHY

4. PARTITION CHROMATOGRAPHY

5. ION EXCHANGE CHROMATOGRAPHY

•Ion exchange
•https://www.youtube.com/watch?v=VOSkyj1dtbc
•https://www.youtube.com/watch?v=lp40a7mtc4E
•https://www.youtube.com/watch?v=rPsJo0jHJ7g
•Affinity
•https://www.youtube.com/watch?v=8_7cdfNO7OY
•Gel
•https://www.youtube.com/watch?v=L-xIHihOGKs
•Adsorption
•https://www.youtube.com/watch?v=YRFSN9q523M
 :Adsorption Chromatography.1
Oldest and most common type

Stationary phase is solid with

adsorption power.

Mixture components adsorbed on

the surface of the stationary
phase with different powers
and that account for separation.

Silica gel is the most common stationary phase.

Natural Products and applied for screening

 :Adsorption Chromatography.1

Some commonly used

adsorbents are 
• Silica gel H, silica gel G, silica
gel N, silica gel S, hydrated gel
silica, modified silica gel

• Cellulose microcrystalline
• Alumina

Adsorbent can be layered on

plate(TLC) or packed in Column
 2:Molecular Exclusion Chromatography

Gel permeation or gel filtration,

Lacks an attractive interaction between the stationary phase and solute
Mobile phase pass through porous gel-
which separates the molecules according to its size.
The pores are normally small and exclude the larger
solute molecules, but allows smaller molecules to
enter the gel, causing them to flow through a
larger volume.
This causes the larger molecules to pass through
the column at a faster rate than the smaller ones.
Commonly used gel
• Dextran Operation of Gel Column
• Polyacrylamide
• Agarose • Column packing
• Polyacrylamide-dextran
• Column size
Selection on the basis of size
• Eluting buffer

Applications
• Sample size
• Desalting
• Partial separation
• Column flow rate
• Estimation of molecular mass
• https://www.youtube.com/watch?v=qrUaZWUM9uw
• https://www.youtube.com/watch?v=S89mAyh6yHU
3:Affinity chromatography
 Most selective type of chromatography employed
• It utilizes the specific interaction between one kind of solute molecule and a
second molecule that is immobilized on a stationary phase (uses the affinity of
proteins to specific ligands) .
• The ligand is attached to suitable matrix

• The immobilized molecule may be an antibody to some specific protein.

When solute containing a mixture of proteins are passed by this molecule, only
the specific protein is reacted to this antibody, binding it to the stationary phase.
This protein is later extracted by changing the ionic strength or pH.

Single step purification: Enzymes, Defense Proteins, Hormones

https://www.youtube.com/watch?v=8_7cdfNO7OY
Ion Exchange Chromatography 
• Resin (the stationary solid phase) is used to which anions or
cations are covalently attach onto it.

• Solute ions of the opposite charge in the mobile liquid phase

are attracted to the resin by electrostatic forces.

https://www.youtube.com/watch?v=i4U4ndf2ayg
Ion Exchange Chromatography
Principle : Process by which ions of an electrolyte solution are brought
into contact with an ion exchange resin.

The ion exchange resin is an insoluble polymer consisting of a "matrix"

(Lattice or framework) that carries fixed charges (not exchangeable) and
mobile active ions "counter ions" which are loosely attached to the
matrix.

In water, the counter-ions move more or less freely in the framework &
can be replaced by ions of the same sign present in the surrounding
solution.
The "matrix" (framework) of a "cation exchanger" is considered as a
crystalline non-ionized "polyanion" & the matrix of an "anion exchanger"
as a non-ionized "polycation".
High Performance Liquid Chromatography
High-performance liquid chromatography (HPLC; formerly referred to as high-
pressure liquid chromatography), is a technique in analytic chemistry used to
separate the components in a mixture, to identify each component, and to quantify
each component.

It relies on pumps to pass a pressurized liquid solvent containing the sample

mixture through a column filled with a solid adsorbent material.

Each component in the sample interacts slightly differently with the adsorbent
material, causing different flow rates for the different components and leading to
the separation of the components as they flow out the column.
 Gas chromatography (GC)
• It is a common type of chromatography used in analytical chemistry for
separating and analyzing compounds that can be vaporized without
decomposition.
• Typical uses of GC include testing the purity of a particular substance, or
separating the different components of a mixture (the relative amounts of such
components can also be determined).
• In some situations, GC may help in identifying a compound.

• In preparative chromatography, GC can be used to prepare pure compounds

from a mixture.

• Two types of gas chromatography are encountered a. Gas-Solid

Chromatography (GSC) b. Gas-Liquid Chromatography (GLC)
Supercritical Fluid Chromatography  Supercritical Fluid Chromatography (SFC) is a
form of normal phase chromatography, that is used for the analysis and purification of
low to moderate molecular weight, thermally labile molecules.  It can also be used for
the separation of chiral compounds.  Principles are similar to those of high
performance liquid chromatography (HPLC), however SFC typically utilizes carbon
dioxide as the mobile phase; therefore the entire chromatographic flow path must be
pressurized.  The supercritical phase represents a state in which liquid and gas
properties converge, supercritical fluid chromatography is sometimes called
"convergence chromatography."
 Partition (liquid-liquid) Chromatography:
Substance having similar chemical type. Both the phases must be liquid.

one having greater partition co-efficient in m.p. than s.p.,

it will move fast and vice versa. Chromatography in which separation is based
mainly on differences between the solubility of the sample components in the
stationary phase (gas chromatography), or on differences between the solubilities
of the components in the mobile and stationary phases (liquid chromatography)
GC
https://www.youtube.com/watch?v=UycPljfrnWo
GCMS
https://www.youtube.com/watch?v=cBXgSPO3pzw
HPLC
https://www.youtube.com/watch?v=eCj0cRtJvJg
https://www.youtube.com/watch?v=p0lRB_ojt_0 agilent
Miscellaneous Chromatography:
a) Ion pair Chromatography:
• Control of hydrophobicity and hydrophilicity counter ion is added.
• Production of ion pair comprising a sample ion and oppositely charged ion component of
mobile phase.
• Non polar interaction of ion-pair with stationary phase.
• Ion pair agent first absorbs onto stationary phase via non polar interaction.
• Sample ion interacts with the agent.
b) Chiral Chromatography:
• S.P.: Optically active
• Thus, entiomer can be separated.
c) Affinity Chromatography:
• Specific interactions between stationary phase and solute molecule.
• Antigen - antibody
• enzyme - substrate or inhibitor
• Hormone - binding protein
• S.P.: Enzyme (highly selective in interaction with substrate) • OR • Substrate
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