Fundamentals of Analytical Chemistry

4FHH2001-0905-2020

Classification of
Chromatography
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography

Learning Outcomes
After studying this topic, student should be able to :

• Define Normal phase and reversed phase chromatography

• Understand separation modes based on polarity, ion exchange and

size exclusion chromatography

• Understand how analyte separation is achieved in TLC.

• Identify the visualisation techniquest in TLC

• Appreciate the Advantages and disadvantages of TLC.

Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography

Classification of chromatographic techniques

Chromatographic separation principles (modes)
Normal phase chromatography
Reversed phase chromatography

Types of interaction in chromatography:

• Adsorption
• Partition
• Ion exchange
• Pore penetration (size exclusion)
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Modes of Chromatography
Normal phase chromatography
• In normal phase chromatography the stationary phase is more polar than
the mobile phase.

• Organic solvents are used for the mobile phases and silica is the most
common stationary phase.

• Retention of analytes is determined by the properties of the compounds, the

nature of the stationary phase and the nature of the mobile phase.

• Non-polar compounds are eluted quickly, whereas polar compounds are

retained more and elute later. The highest retention is obtained using non-
polar solvents with low solvent strengths. Increasing the polarity of the solvent
(mobile phase) decreases retention time of compounds.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Modes of Chromatography
Reversed phase chromatography
• In reverse phase chromatography the stationary phase is less polar than the
mobile phase. The stationary phase consists of a hydrophobic long chain alkyl
group chemically bonded to a support matrix, usually silica. The mobile phase,
i.e. the solvent, is water based initially, with an organic component such as
methanol or acetonitrile.
• Polar compounds elute rapidly, i.e. are not retained by the stationary phase. Non-
polar compounds interact with the stationary phase and so are retained and
elute later.
• The organic component of the mobile phase can be increased gradually (a gradient
program) to speed up elution of more non-polar compounds in the sample.
• For compounds that are ionisable, the pH of the mobile phase affects retention
time. Lowering the pH of the mobile phase will increase the retention of acidic
(polar) compounds.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Modes of Chromatography
Summary of Normal phase (NP)
• The original mode for chromatography
• Polar stationary phase (e.g. bonded siloxane with a polar functional group)
with non-polar mobile phase (e.g. n-hexane, heptane etc.).
• LEAST polar analyte elutes first.
• If polarity of mobile phase increased the retention times decrease.
Summary of Reversed phase (RP)
• Now the most popular type of chromatography.
• Non-polar stationary phase (e.g. C18) with polar mobile phase (e.g.
methanol, acetonitrile or a mixture of buffer with an organic solvent).
• MOST polar analyte elutes first.
• Increasing the mobile phase polarity increases the retention times.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Types of Interaction in Chromatographic
Adsorption chromatography
• The stationary phase is a solid on which the sample
components are adsorbed. In column chromatography,
the column may be packed with small particles that
act as the stationary phase.

• The mobile phase may be a liquid (liquid-solid

D.C. Harry. Exploring chemical analysis. 3rd
chromatography) or a gas (gas-solid chromatography).
ed. W.H. Freeman & co. 2004

• The components distribute between the 2 phases

Adsorption through a combination of sorption and desorption
processes.

• TLC is an example of adsorption chromatography in

which the stationary phase is a plane, in the form of a
solid supported on an inert plate and the mobile
phase is a liquid.

Normal chromatography is used for polar samples E.g. sugars

Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Types of Interaction in Chromatographic
Partition chromatography • The stationary phase is a liquid supported on an
inert solid. In column chromatography, the
column may be packed with small particles
which are coated with a thin layer of liquid
phase.

• The mobile phase may be a liquid (liquid-liquid

D.C. Harry. Exploring chemical analysis. 3rd
partition chromatography) or a gas (gas-liquid
ed. W.H. Freeman & co. 2004
chromatography (GLC)).
Partition
Reversed chromatography is used for nonpolar
samples (Most of drugs)

E.g. hormones/vitamins/polyphenols/some
amino acids and small chain peptides
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Types of Interaction in Chromatographic
Ion exchange chromatography

• Ion exchange uses an ion exchange resin as the

stationary phase.
• The mechanism of separation is based on ion
exchange equilibria
Removal of ionic compounds (water deionisation)
E.g. Ca2+ SO42-
D.C. Harry. Exploring chemical analysis. 3rd
ed. W.H. Freeman & co. 2004
Separation of amino acids

+ - - -+ - + + + -
- - + +
+- - -+ +
- -+ + +-
- - - ++ +
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Types of Interaction in Chromatographic
Size exclusion chromatography
• In size exclusion chromatography (SEC), solvated
molecules are separated according to their size by
their ability to penetrate a sieve-like structure (the
stationary phase).
Separation of Peptides

D.C. Harry. Exploring chemical analysis. 3rd

ed. W.H. Freeman & co. 2004

Size exclusion
Separation of Proteins

Separation of Polymers
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography
Paper Chromatography
Paper chromatography is a type planar chromatography in which the stationary
phase is the cellulose filter paper and the mobile phase is liquid.
The method is not useful for testing a large number of samples and data is not
as accurate as in the case of the TLC technique.
Advantages
It offers simple, low cost and hassle-free operations.
The setup of paper chromatography occupies less space and it requires less
amount of sample for analysis.
Disadvantage
Volatile or complex substances cannot be separated using paper
chromatography techniques.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography

Steps to carry out TLC

• Stationary phase is dispersed as a thin layer placed on the
surface of a plate (planar chromatography). The plate is
made of glass or alumina foil.
▪ stationary adsorbent phase is either silica gel (SiO2)for
more polar compounds or alumina (Al2O3)for less polar
compounds.
• A dilute solution (1%) of sample prepared in a volatile
solvent, are placed as spots on the plate parallel to one of
its edges
• Solvents are then evaporated and the plate inserted
upright in a tank with a mobile phase at the bottom of
the tank
• Mobile phase migrates up the plate by capillary forces
• the components travel at different rates based on
competing interactions with the mobile (solvent) and
adsorbent phases.
Figure retrieved from: Introduction to Pharmaceutical Chemical Analysis; S Hanson, S Pedersen-Bjergaard, K Ramussen; Chapter 10; Wiley 2012
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography

Reporting Results; Retention factor (R f ) Solvent

front
• A polar solvent will carry a polar compound farther while a non-polar
solvent will carry a non-polar compound farther.
• Rf value is the ratio of the distance the spot travels from the origin to
the distance the solvent travels.

Optimisation of mobile phases for better separation Start line

Assume you have a sample containing 3 components (solutes)
How to optimize mobile phase polarity for better separation
distance travelled by solute (a)
𝑅𝑓 =
distance travelled by solvent front (b)

Heptane / Toluene Toluene Toluene/Methanol

(non-polar) (polar) Rf values will be in the range between 0
Less retained
and 1 and analytes with the strongest
More retained interaction with the stationary phase are
retarded most and have the lowest Rf
values
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography

Reporting Results; Retention factor (R f )

distance travelled by solute

𝑅𝑓 =
distance travelled by solvent front

Advantages and uses

TLC is a fast, simple, and inexpensive analytical technique used to determine or monitor:
- The identity of few substances.
- The effectiveness of a purification.
- The progress of a reaction.
- The appropriate conditions for a column chromatographic separation.
-Column chromatography effectiveness.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography

TLC Visualisation
Physical Chemical
- UV light at 254 nm (mercury lamp) - Iodine; Any organic compound
- fluorescence - Ninhydrin; Selective for amino acids
- sulphuric acid
- pH indicators
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography

High Performance TLC – HPTLC

HPTLC is an automated version of TLC where an instrument is used for the separation to
aid with inconsistencies when done by hand e.g. application of the sample (e.g. may be
done via ink jet printer)

Example:
Paracetamol (PCM) with
lornoxicam (LORN) are non-
steroidal anti-inflammatory drugs
available in tablets containing
500mg PCM and 8mg LORN.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography

Conclusions
• Analytes in the spots migrate up the plate based on their distribution
between mobile and stationary phase
• When the mobile phase has moved a certain distance towards the top of the
plate, the TLC plate is removed from the tank and the mobile phase is
evaporated in a fume hood.
• The retention factor Rf determined after spots are visualised
• Advantages: Inexpensive, simple, easy to detect strongly retained analytes.
• Disadvantage: higher detection limits than HPLC, GC or CE
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Thin layer chromatography
Applications

• TLC is applicable in the field of the medicinal preparations

• Assaying the radiochemical purity of radiopharmaceuticals
• Determination of pigments in plants
• Detection of pesticides and insecticides
• In Forensic science laboratory
• Monitoring organic reaction
• Analysing of biological and metabolic samples to detect disease
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography

Recommended Readings
Essential reading:

Introduction to
Pharmaceutical
Chemical Analysis

S Hanson
-Chapters 10- 11;
Wiley 2012