Unit 8

Introduction to Chromatographic Methods

 Introduction
 Chromatography, firstly introduced by the Russian botanist Michael
Tswett in 1900.
 Greek words: chroma-color and graphein-to write i.e.
chromatography is writing with colors.
 Chromatography separates the components of a mixture by
differential distribution of the components of the mixture between a
stationary phase and a mobile (moving) phase
 Components of Chromatography
• Stationary Phase: is composed of solid material (including resins,
monolith, membrane, polymers, cellulose and other materials) or
liquid adsorbed on the surface of a solid support (supercritical
fluids).
• Mobile Phase: is composed of liquid or a gaseous component which
pass through the stationary phase to separate the analyte.
• Analytes : is a mixture of components that need to be separated by
adding them to mobile phase and passing them through the stationary
phase where speed of passing the analyte will separate the analyte.
 Principle of Chromatography

The principle of chromatography involves the distribution

of mixture of analytes between a moving fluid (mobile
phase-a liquid or a gas), and a stationary phase (solid or a
liquid).

During the movement of the sample, components get

separated by repeated desorption and sorption in the
direction of the mobile phase migration.
 Types of chromatography
• Based on the shape or nature of the stationary phases support,
chromatography can be divided into two.
• These are planar chromatography and column
chromatography.
• The main difference between planar and column chromatography
is the shape of the stationary phase: planar chromatography uses a
plane, while column chromatography uses a tube/column:

Planar chromatography
 Column chromatography

Planar chromatography Planar chromatography

 Classification based on physical State of mobile phase

 Gas Chromatography: involves the separation of analytes on the

basis of retention time of analyte which was dissolved in a gaseous
mobile phase depending on the affinity of the molecules to the
stationary phase.
• Different structural compounds act differently with the stationary
phase, thereby having different movement speeds in the stationary
phase.

 Liquid Chromatography: It is a separation technique in which the

mobile phase is liquid, and the separation of molecules takes place
in a column or a plain surface.

• When liquid mobile phase passes through the column, analytes tend
to act differently based on its physiochemical properties and show
different speed and get detected by detector.
 Classification based on separation mechanism
 Partition chromatography/Affinity chromatography: the
components of a mixture are separated based on their affinity
towards the stationary phase of the system.
 Ion exchange chromatography: the separation of molecules
based on their net surface charge.
There are two types of ion exchange chromatography:
• Anion Exchange Chromatography: the negatively charge
containing molecules are separated by their interaction with a
positive charged anion-exchange resin.
• Cation Exchange Chromatography: the positively charged
containing molecules are separated by their interaction with
negatively charged ion-exchange resin.
 Gel filtration chromatography: is also known as gel-exclusion /
gel-permeation / molecular-sieve / size-exclusion chromatography.
It is a form of partition chromatography that is used to separate
molecules on the basis of their molecular sizes.
 Partition chromatography principle
 It is based on differential partitioning of components of a sample
mixture between two phases – stationary phase and mobile phase.

Types of partition chromatography

 Liquid-Liquid chromatography–mobile and stationary both

phases are taken in liquid state.
Examples:
• Paper chromatography
• Column chromatography
• Thin layer chromatography

 Gas–Liquid chromatography–mobile phase is an unreactive gas

and stationary phase is non-volatile liquid held on inert solid
support.
• It is also known as vapor-phase chromatography or Gas-liquid
partition chromatography (GLPC).
 Planar Chromatography
 It is a liquid chromatography-based separation technique in which
the stationary phase is placed in the form of a planar or flatbed
through which mobile phase dissolved with analyte move up by
capillary action for the separation of analyte based on adsorption.
 Planer chromatography can be further classified into two types:

Paper Chromatography: It is a separation technique where the

separation of compounds is performed on a specialized paper.
 This type of chromatography is used for separation of complex
mixtures, such as amino acids.

Thin-layer Chromatography: It is a separation technique which is

used to separate the mixture of components using a thin stationary
phase consisting of adsorbent material layer made up of aluminum
oxide and silica supported by a non-reactive layer at the back usually
of glass, aluminum and plastics.
 Retarding force or retention factor (Rf)

 In PC and TLC, the results are represented by Rf value which

represents the movement or migration of solute relative to the
solvent front.
 Method development in paper chromatography

𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒂𝒎𝒑𝒍𝒆

Retention factor (Rf)=
𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕
 TLC method development
• Plate preparation
• Spotting the plate
• Location of spots
• Development solvents
• Developing a Plate
• Visualization
TLC experiment

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 Column Chromatography
 The stationary phase is packed into a glass tube to form a cylinder
or column of granules.
 The sample is applied with care as a layer on top of the stationary
phase.
 Then mobile/solvent is added and flows through the column
carrying the samples.
 Fluid entering the column for separation purpose is called eluent
while fluid emerging from the end of the column is called eluate
 It separates the components in a mixture based on their difference
in adsorption on a stationary phase, which then results in their
different movement speeds when passed through a stationary
column.
 Most common type of stationary phases are silica and alumina
along with organic solvents which are considered as mobile phase.
 This type of chromatography is one the most preferred methods for
protein purification.
 Column Chromatography
 It is a laboratory technique that separates chemical compounds
from a mixture by passing a mobile phase through a column
containing a stationary phase
 The stationary phase is packed into a tube, such as a pipette or
burette.
 The tube can be packed with a solid or coated with a liquid
Column Chromatography
Column Chromatography Experiment

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https://youtu.be/UmWMlKJAdSk?si=lQXfv-gXMge_ZQui
 High performance liquid chromatography (HPLC)
HPLC is an analytical technique used for the separation of
compounds soluble in a particular solvent.
High pressure is applied to the liquid solvent (mobile phase) to
force it through the column.
 HPLC is an analytical technique used to separate, identify or
quantify each component in a mixture.
 The mixture is separated using the basic principle of column
chromatography and then identified and quantified by
spectroscopy.
Retention Times
 The dead time tM is the time it takes for an unretained species to
pass through a column.
 The retention time tR is the time between injection of a sample and
the appearance of a solute peak at the detector of a chromatographic
column.
HPLC Separation
 HPLC can separate and detect each compound by the difference of
each compound's speed through the column.
 The mobile phase is the liquid that dissolves the target compound.
The stationary phase is the part of a column that interacts with the
target compound.
 How to Read a Chromatogram
The word "chromatogram" means a plot obtained via
chromatography.
Retention time (tR) is the time interval between sample injection
point and the apex of the peak.
The required time for non-retained compounds (compounds with no
interaction for the stationary phase) to go from the injector to the
detector is called the dead time (t0).
 Types of HPLC Separation

A) Normal-phase Chromatography
 The columns are packed with polar inorganic particles
stationary phase and a nonpolar mobile phase is used to run
through the stationary phase.
 It is mainly used for purification of crude samples, separation of
very polar samples, or analytical separations by thin layer
chromatography.

B) Reverse-phase Chromatography
 The stationary phase is non-polar (hydrophobic), while the mobile
phase is polar.
 The interactions in RP-HPLC are considered to be the hydrophobic
forces, and these forces are caused by the energies resulting from
the disturbance of the dipolar structure of the solvent.
Mobile phase and stationary phase used for normal phase and
reverse-phase chromatography

Type Stationary Phase Mobile Phase

Normal phase Polar Non polar
Reverse Phase Non polar Polar
 Ion Exchange Chromatography
 The ion exchange mechanism is based on electrostatic interactions
between hydrated ions from a sample and oppositely charged
functional groups on the stationary phase.
 Two types of mechanisms are used for the separation: in one
mechanism, the elution uses a mobile phase that contains
competing ions that would replace the analyte ions and push them
off the column; another mechanism is to add a complexing reagent
in the mobile phase and to change the sample species from their
initial form.

Effluent from the analytical column when analyzing a sample

containing sodium. H+Cl– is the eluent ion.
 Mixture of HCl mixed with the NaCl as it elutes from the column.
 This HCl will interfere with the measurement of conductivity.
 The Cl– ion of the Na+Cl– would exchange with the hydroxide,
converting this into sodium hydroxide (Na+OH–), a conducting
electrolyte because it stays ionized.
 The Cl– ion of the HCl would exchange with the hydroxide,
converting this into H+ and OH–, which is non-conducting water.

Effect of the suppressor column on the eluent from the column

 With any chromatographic method, it helps to know some basic
rules for predicting retention order.
 An interesting example to consider in ion-exchange
chromatography is the retention order for the ions Li+, Na+, and K+
on a cation exchange resin.

Retention order of Li+, Na+ and K+ on a cation-exchange column

based on stationary phase effects.
 Size Exclusion Chromatography
 It is a chromatographic method that separate the molecules in the
solutions based on the size (hydrodynamic volume).
 Separation of constituents of a mixture based on molecular size
and, in some cases, molecular weight
 After the analyte is injected into the column, molecules smaller
than he pore size of the stationary phase enter the porous particles
during the separation and flow through he intricate channels of the
stationary phase.
 Thus smaller components have a longer path to traverse and elute
from the column later than the larger ones.
Principle for Size-exclusion chromatography
 Separation of proteins by size exclusion chromatography
Where will a protein elute in a gel filtration experiment?
 The larger molecular mass (large mass) are completely
excluded from the gel filtration beads.
 The smaller molecular mass (small mass) are completely
included within the pores of the gel filtration beads.
 Solutes between these two ranges of molecular mass will elute
between the excluded and included volumes
 Gas Chromatography(GC)
 GC is always performed in a column, typically “packed” or
“capillary.”
 Made from non-adsorbent and chemically materials to prevent
reactions, these columns are usually made of stainless steel, glass,
and quartz.
 A sample is first vaporized, injected into the column, then
transported by an inert gas such as helium.

 The retention time is given by the time it takes for a compound to

travel through the column, and this value is then compared to
others.
 Generally, the lower the temperature, the better the separation and
the longer it takes.
 Gas chromatography (GC)
 This technique uses a gas as the mobile phase, and the stationary
phase can either be a solid or a non-volatile liquid.

 If a solid stationary phase is used the technique is described as

gas-solid adsorption chromatography (GSC), and
 If the stationary phase is liquid it is called gas-liquid partition
chromatography (GLC).

 The latter is more commonly used, but in both cases the stationary
phase is held in a narrow column in an oven and the stationary
phase particles are coated onto the inside of the column.
Gas-Liquid Chromatography (GLC)/(Gas-Liquid Partition
Chromatography- GLPC)

• It involves the separation of volatile compounds based on their

partitioning between a gaseous mobile phase and a liquid
stationary phase.
• Mostly used for analyzing organic compounds, such as
hydrocarbons, pesticides, and volatile organic compounds (VOCs).

Gas-Solid Chromatography (GSC)

• GSC employs a solid adsorbent as the stationary phase.

• This type of GC is used to separate small, non-polar compounds
like noble gases, light hydrocarbons, and certain inorganic gases.
• Used in analyzing trace impurities in gases, such as air pollution
monitoring and testing semiconductor manufacturing gases.
 Toxicological analysis using HPLC/DAD
Toxicological analysis of human blood or plasma samples
 After single-step liquid/liquid extraction at pH 9.5 using
chloroform/2-propanol/n-heptane (60:14:26, v/v/v),

 The drugs elute isocratically from a NovaPak C18 (Waters) 4-

micrometers column (300 mm x 3.9 mm, i.d.) at 30 degrees C,
with methanol/tetrahydrofuran/pH 2.6 phosphate buffer (65:5:30,
v/v/v) as the mobile phase (flow rate 0.8 mL/min).

 Full UV spectra from 200 to 400 nm (resolution 1.3 nm) are

recorded on-line during the 20 min chromatographic run.

 Solute identification may be automatically performed by

comparison of analytical data (retention times and UV spectra)
with references of 311 pharmaceuticals, toxicants and drugs of
abuse stored in a computerized library.
Toxicology Screening (Urine)
Toxicology screening chromatogram of a urine sample with
HPLC/UV analysis.
Toxicology screening with HPLC-UV analysis
Toxicological analysis using GC
GC-FID chromatograms: (1) methanol, (2) ethanol, (3) acetone, (4) 2-
propanol, (5) t-butanol (internal standard), (6) 1-propanol, (7) methyl
ethyl ketone, (8) 2-butanol, (9) isobutanol, (10) 1-butanol, (11) 2-
methylbutanol, (12) 3-methylbutanol
GC-FID chromatograms: extracted ion chromatograms and production
spectra of tetrahydrocannabinol (THC) (a), 11-OH-THC (b) and THC-
COOH (c)
***Generally, both HPLC and GC play a great role in toxicological
analysis