Chromatography

The document provides an overview of chromatography, including its definition, types, and principles of separation. It covers various chromatographic methods such as TLC, HPLC, and GC, and highlights the importance of chromatography in pharmaceutical quality control and environmental testing. Additionally, it discusses the historical context of chromatography's invention and its applications in research and industry.

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Chromatography

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Fundamentals of Analytical Chemistry

4FHH2001-0905-2020

Chromatography
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography

By the end of this lecture, you should be aware of the following:

• An introduction to chromatography
• Understand the difference between normal
and reverse phase chromatography
• Learn chromatographic terms:
Chromatographic methods, Retention time,
Column efficiency, Plate theory, Selectivity
and Resolution.
• TLC, HPLC, GC
Types of chromatography separation :
Adsorption
Partition
Ion exchange
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography

Instrumental Analytical Methods

Spectroscopic methods

Chromatographic methods
Thin layer Chromatography Column Chromatography
Liquid Chromatography (LC)
Using liquid mobile phase
Gas Chromatography (GC)
Using gas mobile phase
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Chromatographic terms
Chromatography is a separation method, where the separation is based on
differences in rates of migration of sample components in a mobile phase
transported through a stationary phase.
-Sample components are the analyte(s) or the compound(s) of interest in a
mixture. Sometimes called solutes in chromatography.
-Phase that moves (mobile phase )
-Phase that is stationary (stationary phase)
The stationary phase is a layer or coating on the supporting medium that interacts
with the analyte. The stationary phase may be a solid, a liquid or a gel. If the
stationary phase is packed in a tube, the tube is called a column.

The mobile phase or eluent is a solvent or gas that flows through the supporting
medium. This can be a gas, a liquid or a supercritical fluid.

The supporting medium is a solid surface on which the stationary phase is bound
or coated.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Chromatography uses
To check the identity, purity and amount of impurities in a sample.
Can be used to identify and give approximation of:
Accelerants used in a fire
Contamination in rivers/ other ecosystems
Counterfeit items – alcohol, art work, medicine etc.
Evidence of doping in sports testing

Chromatography examples - Pharma

Prime reason is to protect public health with regard to pharmaceutical and clinical
products
✓ Quality control: In-process checking, residual solvents and is the purity of your active
pharmaceutical ingredient (API) to specification?
✓ Does your dosage contain enough of the API, is it within specification (generally 100-
110% of stated amount), are impurities below thresholds?
✓ Stability testing – storage temperature, humidity and package material etc.
✓ Identification and quantification of other active ingredients in medicines
✓ To investigate drug dosage properties: absorption, distribution, metabolism and
elimination (ADME)
Read more: http://www.ehow.com/about_6501907_importance-hplc-pharmaceuticals-medicines.html#ixzz2zcqXHd00
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Invention of Chromatography
➢ Chromatography was invented by the Russian botanist
Michael Tswett in 1906.
➢ He separated plant pigments using a glass column
packed with finely divided calcium carbonate.
➢ The separated species appeared as coloured bands on
the column. - chroma = colour - graphy = write
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Invention of Chromatography
The Nobel Prize in Chemistry (1952) was awarded jointly to
Archer John Porter Martin and Richard Laurence Millington
Synge "for their invention of partition chromatography"

Common Types of Chromatography

Tswett’s technique is based on Liquid Chromatography. There are
now several common chromatographic methods. These include:
Porter Martin
❑Paper Chromatography
❑Thin Layer Chromatography (TLC)
❑Liquid Chromatography (LC, HPLC, UPLC)
❑Gas Chromatography (GC, GC-MS)
❑Supercritical Fluid Chromatography (SFC)
Richard Synge
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Introduction
IUPAC definition for Chromatography: “it is a physical method of separation in which the
components to be separated are distributed between 2 phases – one that is stationary (the
stationary phase) and one that moves in a definite direction (the mobile phase), usually but
not necessary in a column.”

The stationary phase is usually in a column but may take a planar form such as in TLC.
How Does Chromatography Work?
In all chromatographic separations, the sample is transported in a mobile phase.
The mobile phase can be a gas, a liquid, a supercritical fluid or a subcritical water.
The mobile phase is then forced through a stationary phase held in a column or
on a solid surface. The stationary phase needs to be something that does not
react with the mobile phase or the sample.
The sample then has the opportunity to interact with the stationary phase as it
moves past it. Samples that interact greatly, then appear to move more slowly.
Samples that interact weakly, then appear to move more quickly. Because of this
difference in rates, the samples can then be separated into their components.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Principles of chromatographic separation
1- A small volume of sample is placed at the top of
the column, which is filled with the
chromatographic particles (stationary phase) and
solvent.

2- Mobile phase solvent is added to the column

and is allowed to slowly emerge from the bottom
of the column.

3- The individual components interact with the

stationary phase to different degrees.
Component A has the same speed as the mobile phase;
Component C is the most retarded by the stationary phase.
When the differences in retention are sufficiently large, the
components elute from the column at different times and are
separated.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Principles of chromatographic separation
• At a molecular level, migration is not smooth.
• A molecule that is sorbed by the stationary phase is temporarily
stopped, while others of the same type pass over.
• Each individual molecule follows a rapid, random alternation
between the mobile and the stationary phases, but on average
the distribution between the two phases is a constant, called the
distribution equilibrium or distribution constant Kc.
Kc is the Equilibrium Constant = the Distribution Coefficient =
Partition Coefficient in partition chromatography.
Cs is the concentration of component in the stationary phase
Cm is the concentration of component in the mobile phase.

Migration speed depends on Cm (no. of molecules in mobile

phase at any instant)
Solutes with a large Kc value will be retained more strongly by the
stationary phase than those with a small value. The result is that
the latter will move along the column (be eluted) more rapidly.
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Principles of chromatographic separation
• The detector detects the compounds that are eluted from
the column.
Chromatogram
the detector response to compounds eluted from the colum
recorded as a function of time.

Detector signal

A, B and C are separated

Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography
Ultimate aim of chromatography is to ensure
• All analytes (of interest) are well
separated
• The peak shapes are symmetrical
and sharp
• The run time being as short as
possible (resource saving).

Detector response
Question
How is chromatography used in
research and industry? Why are
the peak characteristics noted
aimed for if good results are to be
Elution time (10 minutes)
obtained?
Figures adapted from
Dr S. Evans Lecture
Fundamentals of Analytical Chemistry (4FHH2001)
Chromatography

Chromatography

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Chromatography

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Fundamentals of Analytical Chemistry

By the end of this lecture, you should be aware of the following:

Instrumental Analytical Methods

Chromatography examples - Pharma

Common Types of Chromatography

2- Mobile phase solvent is added to the column

3- The individual components interact with the

Migration speed depends on Cm (no. of molecules in mobile

A, B and C are separated

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