CHAPTER 7

INTRODUCTION TO CHROMATOGRAPHIC SEPARATION

Types of chromatography
Paper chromatography
Thin layer chromatography
Column chromatography
Efficiency of separation
Application (Qualitative and
quantitative information)
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 Introduction
 Chromatography is a separation technique
based on the differential distribution of
the components of a mixture between two
phases: a stationary phase and a mobile
phase.
 The basic principle revolves around how
different substances interact with these
two phases.

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 Introduction…
 The mobile phase (also called eluent) penetrates or passes
through a solid or immiscible stationary phase.
 Solutes (eluates) in the sample usually have differential
partitioning or interactions with the mobile and stationary
phases.
 Since the stationary phase is the fixed one then those
solutes which have stronger interactions with the
stationary phase will tend to move slower (have higher
retention times) than others which have lower or no
interactions with the stationary phase will tend to move
faster.
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 Phases in Chromatography
1. Stationary Phase:
 The phase that remains fixed inside the column
or on a plate (solid or a liquid supported on a
solid). It acts as the surface or medium that
retains some components more strongly than
others.
2. Mobile Phase:
 The phase that moves through or across the
stationary phase (liquid or gas). It carries the
sample mixture with it.

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 The analytes
interacting most
strongly with the
stationary phase
will take longer to
pass through the
system than those
with weaker
interactions.

These interactions
are usually
chemical in
nature, but in
some cases
physical
interactions can
also be used.

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 Key Mechanisms of Separation
A. Adsorption
• Based on the adhesion of molecules onto the
surface of a solid stationary phase (e.g., silica
gel).
• More strongly adsorbed compounds move
slower.
B. Partition
• Based on solubility between two immiscible
phases (liquid-liquid system).
• Used in paper chromatography and partition
column chromatography.

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 Key Mechanisms of Separation…
C. Ion-Exchange
• Based on attraction between charged
analytes and oppositely charged
stationary phase.
• Used for separating ions and polar
molecules.

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Key Mechanisms of Separation…
D. Size-Exclusion (Gel Filtration)
• Based on molecular size.
• Larger molecules elute faster as they cannot
enter the pores of the stationary phase.
E. Affinity
• Based on specific biological interactions (e.g.,
antigen-antibody, enzyme-substrate).
• Highly selective.

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 Retention and Movement
 Each component of a mixture spends
different amounts of time in the mobile and
stationary phases.
 The retention time or migration distance is
determined by:
Polarity
Molecular size
Charge
interaction strength with the stationary
phase
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 Types of Chromatography
Chromatography techniques are categorized based
on:
1. Physical State of Mobile Phase
A. Gas Chromatography (GC)
• Mobile Phase: Inert gas (e.g., helium, nitrogen)
• Stationary Phase: Liquid or solid inside a
column
• Used for: Volatile and thermally stable compounds
• Applications: Drug testing, food flavor analysis,
environmental pollutant detection

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 Types of Chromatography…
B. Liquid Chromatography (LC)
• Mobile Phase: Liquid solvent
• Stationary Phase: Solid adsorbent
• Variants Include:
• High-Performance Liquid Chromatography
(HPLC): High-pressure system for fast and
accurate separation
• Normal Phase LC: Polar stationary phase, non-
polar mobile phase
• Reverse Phase LC (RP-HPLC): Non-polar
stationary phase, polar mobile phase

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 Types of Chromatography…
2. Mode of Separation
A. Adsorption Chromatography
• Based on different affinities for surface of
stationary phase (e.g., silica, alumina)
• Used in TLC, column chromatography
B. Partition Chromatography
• Based on differences in solubility between two
immiscible liquids
• Used in paper chromatography and some forms of
HPLC
C. Ion-Exchange Chromatography
• Based on ionic interactions between charged
stationary phase and analytes
• Ideal for separating amino acids, proteins, and
nucleotides
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 Types of Chromatography…
D. Size-Exclusion Chromatography (SEC) /
Gel Filtration
• Based on molecular size
• Larger molecules elute faster as they cannot
enter the pores of the stationary phase
• Used in protein purification
E. Affinity Chromatography
• Based on specific biological interactions (e.g.,
antigen-antibody, enzyme-substrate)
• Highly selective and used in biotechnology and
immunology

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 Types of Chromatography…
3. Planar vs. Column Chromatography
A. Planar Chromatography
 Paper Chromatography: Cellulose paper as
stationary phase
 Thin Layer Chromatography (TLC): Thin
adsorbent layer on glass or plastic
• Simple, used for small sample analysis
B. Column Chromatography
• Stationary phase packed into a tube or column
• Can be gravity-driven or pressure-driven (as in
HPLC)
• Suitable for preparative and analytical purposes

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 Paper Chromatography
• Paper Chromatography (PC) was first
introduced by German scientist Christian
Friedrich Schonbein (1865).

• PC is considered to be the simplest and most

widely used of the chromatographic
techniques because of its applicability to
isolation, identification and quantitative
determination of organic and inorganic
compounds.

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Paper Chromatography…
 A simple and economical technique used especially in
educational labs.

- Principle: Partition chromatography

- Stationary Phase: Water adsorbed on cellulose
fibers of paper
- Mobile Phase: Organic solvent or mixture (e.g.,
water:ethanol)
- Process: A small drop of mixture is spotted on a
pencil line. The end is dipped in solvent. Components
move at different rates.
- Rf Value = Distance travelled by solute / Distance
travelled by solvent front
- Applications: Separation of pigments, amino acids,
inks.
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APPLICATION OF SAMPLE
 The sample to be applied is dissolved in the
mobile phase and applied as a small spot on the
origin line, using capillary tube or micropipette.
very low concentration is used to avoid larger
zone
 The spot is dried on the filter paper and is placed
in developing chamber.

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 APPLICATIONS of PAPER CHROMATOGRAPHY
• Separation of mixtures of drugs
• Separation of carbohydrates, vitamins, antibiotics,
proteins, etc.
• Identification of drugs
• Identification of impurities
• Analysis of metabolites of drugs in blood , urine ….
Advantages of PC
 Simple ,rapid ,inexpensive ,excellent resolving
power
Disadvantage
 Not sensitive
 Not accurate for quantification
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 Thin Layer Chromatography (TLC)
• A fast and more sensitive technique than paper
chromatography.
- Principle: Adsorption chromatography
- Stationary Phase: Silica gel or alumina on glass,
metal, or plastic
- Mobile Phase: Suitable solvent system
- Procedure: Spot the sample, place in chamber,
develop, and visualize under UV or iodine.
- Advantages: Better resolution, reusable plates,
more solvent flexibility
- Applications: Purity checking, identifying
compounds, monitoring reactions
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 Stationary Phase: Silica (SiO2)

OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O O
O O
Si
Si O
O
O O
O

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 Stationary Phase: Alumina

O OH OH OH OH

Al Al Al Al Al
O O O O O O

Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-

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 Thin-Layer Chromatography: Determination of
Rf Values

solvent front
Rf of component A =
dA component B
dS
dS
dB
Rf of component B =
dB component A

dS dA
origin
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
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 Detection of spots

 Spots in TLC chromatograms can be detected in 4

different ways:
1. By their natural color
2. By their fluorescence
3. By their chemical reactions that take place after
the TLC has been sprayed with various reagents for
example: during paper chromatography of amino
acids, the chromatograms are sprayed with
ninhydrin.
4. By radioactivity

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 TLC

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 COULUMN CHROMATOGRAPHY

• Typical response obtained by chromatography (i.e., a

chromatogram): chromatogram - concentration versus elution
time

Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
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Wh = half-height width of the peak in time units
 Column Chromatography...

 Well-separated components appear as distinct peaks

(in column chromatography) or spots (in TLC or
paper chromatography).
 Efficient separation depends on:
 Composition and flow rate of the mobile phase
 Type and surface area of the stationary phase
 Temperature and column dimensions (for column
chromatography)

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 Column Chromatography…
Solute Retention time or volume:
 A solute’s retention time or retention volume in
chromatography is directly related to the strength of the
solute’s interaction with the mobile and stationary phases.
 Retention on a given column pertain to the particulars of
that system:
- size of the column
- flow rate of the mobile phase

Capacity factor (k’): more universal measure of

retention, determined from tR or VR.
k’ = (tR –tM)/tM or k’ = (VR –VM)/VM
capacity factor is useful for comparing results
obtained on different systems since it is independent on
column length and flow-rate.
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 Column Chromatography…
The value of the capacity factor is useful in understanding the
retention mechanisms for a solute, since the fundamental definition
of k’ is:
moles Astationary phase
k’ =
moles Amobile phase

k’ is directly related to the strength of the interaction

between a solute with the stationary and mobile phases.
 Moles Astationary phase and moles Amobile phase represents the amount
of solute present in each phase at equilibrium.
Equilibrium is achieved or approached at the center of a
chromatographic peak.

When k' is less than 1.0, separation is poor

When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum

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 Column Chromatography…
Number of theoretical plates (N): compare efficiencies of a
system for solutes that have different retention times
N = (tR/s)2
or for a Gaussian shaped peak

N = 16 (tR/Wb)2

N = 5.54 (tR/Wh)2
 The larger the value of N is for a column, the better the
column will be able to separate two compounds.
- the better the ability to resolve solutes that have small
differences in retention
- N is independent of solute retention
- N is dependent on the length of the column

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 Column Chromatography…
Plate height or height equivalent of a theoretical
plate (H or HETP): compare efficiencies of columns
with different lengths:
H = L/N
where: L = column length
N = number of theoretical
plates for the colum
Note: H simply gives the length of the column that
corresponds to one theoretical plate. H can be also
used to relate various chromatographic parameters
(e.g., flow rate, particle size, etc.) to the kinetic
processes that give rise to peak broadening:

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 Applications (Qualitative and
Quantitative Information)
 Chromatography has broad applications in both
qualitative and quantitative analysis across
scientific, industrial, and regulatory domains.
 Chromatographic methods are widely accepted in
regulatory standards due to their accuracy,
sensitivity, and reproducibility.
1. Qualitative Applications:
• Identification of Compounds: Based on Rf values in
TLC or paper chromatography, or retention times in
GC and HPLC.
• Purity Testing: Detection of impurities or
byproducts in pharmaceuticals and chemicals.
• Monitoring Reactions: Observation of reactants and
products during the course of chemical synthesis.

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 Applications…
2. Quantitative Applications:
• Concentration Determination: Peak areas in HPLC
or GC are directly proportional to analyte concentration.
• Calibration Curves: Standard curves are prepared
using known concentrations to calculate unknowns.

 Drug Testing and Forensics: Quantitative detection of

drugs, toxins, and other substances in biological samples.
 Environmental Monitoring: Measuring levels of
pollutants, pesticides, or heavy metals in air, water, and
soil.
 Food and Beverage Analysis: Determining additives,
preservatives, or nutrient content (e.g., vitamins, amino
acids).

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