CHAPTER 7
BASIC
CHORMATOGRAPHY
PREPARED BY
SITI NUR AZMU’I ABDULLAH
HISTORY
▪ In 1906, Mikhail Tswett used chromatography to
separate plant pigments (chlorophyll).
▪ He called the new technique Chromatography
because the result of the analysis was 'written in
color' along the length of the adsorbent column
▪ Chroma means “color” and graphein means to
Mikhail Tswett (1872-1919)
Russian Botanist
“write”
OUTLINES
Types
Techniques
Term used - Planar &
column
Chromato
Principles Application
graphy
CHROMATOGRAPHY
Technique in which the components of a mixture are separated based on
differences in the rates at which they are carried through a fixed or stationary phase
by a gaseous or liquid mobile phase.
Widely used for the separation, identification and determination of the chemical
components in a complex mixtures.
PRINCIPLES OF CHROMATOGRAPHY
Samples usually is a mixture of many components.
Samples containing unknown compounds,
✓ Each of the components must be separated prior to identification
using other analytical methods.
The intermolecular attraction of each components is different.
✓ Some components in the mixture will be more attracted or
absorbed towards stationary phase,
✓ Some will attract towards mobile phase.
Then, the component will distribute themselves.
How Separation Process Occur?
Components that are strongly retained or absorbed by the stationary
phase, will move slowly with the flow of mobile phase.
Components that are weakly held by the stationary phase will travel
fast (rapid) with the flow of mobile phase.
The differences in mobility for each components in a sample will
separate out into discrete bands.
Then it can be analyzed qualitatively and quantitatively.
The Process
The red molecules are more soluble and strongly retain towards
stationary phase, move slowly with mobile phase. The red
molecules elute later.
The green molecules are less soluble and weakly held towards
stationary phase, travel faster with mobile phase. The green
molecules elute first.
CHROMATOGRAPHY
TERMS
Elution
Process in which
solutes are
washed through
a stationary
phase by the
movement of a
mobile phase
Eluent
Solvent used to Eluate
carry the
components of
a mixtures Mobile phase
through a that exits the
stationary column.
phase.
• Static part
• Porous solid or coated with liquid stationary phase that fixed in a
place (column or planar)
Stationary • To absorb the components that are being separated where the
phase solvents and analyte or components travels through it.
• Moving part (Eluting fluid)
• A phase that moves over or through the stationary phase, carrying
with it the analyte mixture.
Mobile Phase • The mobile phase may be a gas, a liquid or supercritical fluid
Retention Time
Time required for the sample to travel from the injection port
through the column to the detector.
Retention Time
Retention Factor (Rƒ)
The ratio of the distance travelled by the substance to the
distance travelled by the solvent.
Significant of Rf value
Identification the compound
✓ by comparing the sample with standard; similar
compound will have same Rf value.
Detect the difference of the compound in the sample
mixture. (due to differences in solubility in their attraction).
Measure the polarity of the compound
Rf value
If = 0 - solute remains in the stationary phase (immobile)
If = 1 - solute has no affinity towards stationary phase
Travels with the solvent front
TYPES OF SEPARATION
1. PARTITION CHROMATOGRAPHY
Partitioning is a distribution by dissolving of the components
between two immiscible liquid phases.
Based on the surface of a solid support by a liquid stationary phase.
Solute equilibrates between the liquid mobile phase and the liquid
stationary phase.
STATIONARY
MOBILE PHASE:
PHASE:
LIQUID
LIQUID
2. ADSORPTION CHROMATOGRAPHY
Stationary phase is a solid on which sample component are
adsorbed.
Component of the mixture selectively adsorb on the
surface of a finely divided solid stationary phase.
As the mobile phase (gas / liquid) carries the mixture
through the stationary phase, the components of the
mixture stick to its surface with varying degrees of
strength and thus separate.
MOBILE PHASE: STATIONARY PHASE:
LIQUID SOLID
CLASSIFICATION OF CHROMATOGRAPHY
METHODS
1. PLANAR CHROMATOGRAPHY
- Stationary phase is supported on a flat
plate or in the pores of a paper
- Mobile phase moves through capillary
action
- Example : Paper Chromatography & TLC
2. COLUMN CHROMATOGRAPHY
- Stationary phase is held in a narrow tube
- Mobile phase is forced through the tube
under pressure or by gravity
- Example : Gas Chromatography &
Supercritical-fluid chromatography
1. Planar Chromatography
A separation technique in which the stationary phase is
supporting/coated on a flat plane or in the pores of paper
The plane :
A. paper (PAPER chromatography)
B. glass plate (TLC chromatography)
Different compounds in the sample mixture travel different distances
according to how strongly they interact with the stationary phase as
compared to the mobile phase.
Rf of each chemical can be used to:
✓ Identify an unknown substance
A. Paper Chromatography
Techniques:
Use paper chromatography to separate a sample mixture
into its individual components.
The paper is hung and end dips into a solvent at the
bottom of the paper strip.
SP : Water molecules absorbed on cellulose
fibres or on a paper (liquid phase)
MP : solvent (liquid phase)
Type of separation : Partition Chromatography
(liquid-liquid)
Uses:
Identification and separation of coloured mixtures like ▪ Paper: cellulose fibres (contain
trapped water molecules.
pigments. ▪ The trapped water acts as SP
Separation of mixture for polar and nonpolar compounds. ▪ Filter paper as a support.
Concept in Paper Chromatography
Paper is made of cellulose, a polar substance.
As solvent rises up through the paper, it meets the sample
mixture which starts to travel up the paper with the solvent to
separate the component.
Non polar compound is weakly held or retained towards
stationary phase (water molecules on cellulose fibers), travel
further with mobile phase, the compound will elute first.
More polar compound is strongly held or retained towards
stationary phase, travel slower with mobile phase. The
compound will elute later.
Each sample with
one (1)
component only
Sample with
more than one
components
Technique in Paper
Chromatography
After finished
spotting, let all
Dry the first spot spots dry first
before spot before dipped
The spot is again for the into
about 1 cm second time. development
above the Make sure the solvent (MP).
The sample &
surface of the spot is < 5mm The paper is
standard is
solvent, to diameter. placed vertically
Sample & spotted using
prevent the and not touch
standard are the capillary
components the sides of the
dissolved in a tube on the line
from dissolving beaker.
volatile solvent. drawn on the
PC. in solvent.
Identity of
sample can be
Remove the identified by
chromatogram comparing the
The from beaker and Rf sample to the
development immediately standard.
Saturation of solvent will mark the
development carry the solvent front
solvent will various spots with pencil.
Development ensure the spot upward at
chamber should be move upward different rate
close to ensure it is smoothly by until the
saturated with the capillary solvent front is
solvent vapour. action. ¾ of the PC.
Retention Factor (Rƒ) Value Calculation
Rf calculation
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
Rf =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
𝑎
=
𝑏
Rf value
If = 0 - solute remains in the stationary phase (immobile)
If = 1 - solute has no affinity towards stationary phase
Travels with the solvent front
Examples
Advantages vs. Disadvantages of Paper
Chromatography
Cheap, fast, sensitive
and wide application
i) The reproducibility
of the result is low
ii) Cannot withstand
corrosive chemical
B. Thin Layer Chromatography (TLC)
Technique:
Separation of mixture occurs on a thin layer of SP: Thin layer of finely
adsorbent (silica/alumina) which is attached/coated to divided adsorbent
an inert support usually glass plate. supported on a glass/
aluminium plate/plastic
strip
SP : Thin layer plate or strip coated with a
silica or alumina (solid phase).
MP : Organic solvent/inorganic solvent
(liquid phase).
Type of separation : Adsorption Chromatography
(solid-liquid)
Uses:
Separation organic compound, identification purity of
product.
TLC Plate Kieselguhr
In TLC, the stationary phase is a
layer of silica gel fixed onto a glass Alumina
plate. TYPE OF
ADSORBENT
Mobile phase is a solvent which (TLC)
travels up the plate, carrying up the
substance in sample. Cellulose
Silica Gel
GLASS PLATE
SILICA GEL
Concept in Thin Layer Chromatography
As solvent rises up through the silica gel on a plate, it dissolve
the sample mixture, and travel up the plate to separate the
component.
Non polar compound is weakly held or bonded to stationary
phase (silica on TLC plate), moves faster with mobile phase,
travel further. Thus will eluted first.
More polar compound is strongly held or bonded to
stationary phase (silica on TLC plate), moves slowly with
mobile phase, so will eluted later.
Example
Silica gel is a polar stationary phase.
Given twocompounds which differ in polarity,
✓ Compound A (more polar)
✓ Compound B (less polar)
Compound A has a stronger interaction with
the silica.
Therefore, Compound A compound moves
slowly with the mobile phase and difficult to
exit from the binding places, elute later.
Compound B moves higher up the plate, elute
first. Resulting in a higher Rf value.
Technique in Thin Layer Chromatography
1. Preparing and
2. Spotting the TLC 3. Developing the
activating the TLC
plates with sample TLC plate
plate
6. Measuring the Rf 5. Visualizing the
4. Drying the plate
value substance spots
1. Preparing and Activating TLC Plate
The glass plate are cleaned with soaps and any trace of
grease / oil on the plates is removed by using acetone.
An aqueous slurry of powder (SiO2 or Al2O3) is prepared by
mixing with binder such as plaster of Paris, gypsum or
poly(vinyl alcohol) to help it adhere to the baking material.
The slurry is spread on the plate to form a thin film (0.1 –
0.3 mm thick) by using a spreader to assure uniform
thickness.
To activate the plate, the plate is air dried for ½ hour and
then are heated in the oven at 100 °C for ½ hour. Then
cooled in desiccator before use.
2. Spotting the TLC Plate with Sample
Draw a straight line on the bottom of TLC plate with a pencil.
Load the glass capillary tube with the sample mixture solution.
Hold vertically the capillary tube containing the sample mixture to
the drawn line on the TLC plate and slowly let the tube touches the
plate
The sample will deposited on the plate as a small spot.
Allow the solvent to evaporate from the developed spot.
3. Developing the TLC Plate
Chromatogram is
developed by placing
the bottom of the plate
in a suitable solvent.
The development of
components depend on The solvent is drawn up
solubility, retention by
the SP, complexity.
the plate by capillary
action
The developing TIME :10 min – 1hr
4. Drying the TLC Plate
The TLC plates are allow to dry before visualize the spots
5. Visualizing the Substance Spots
If the components are colourless, the spots on the TLC plates can
be visualize using these methods:
Using Ultraviolet light (uv) as fluorescence indicator on TLC
plates (organic compound)
Spraying the TLC plate with ninhydrin (to detect amino acid)
Spraying the plate with sulphuric acid solution H2So4
(develop black spot)
Exposing the TLC plate to iodine vapour (to produce a colour
from interactions)
Example
5. Measuring the Rf Value
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑝𝑜𝑡 (𝑏)
Rf =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑎)
It is about how the compound is interacting with both the solvent
(liquid phase) and the solid support (solid phase)
Can use to compare molecular weight comparison, between known
and unknown molecules
Examples
O
O O
O O
O O
A B C D P Q
standard sample
Sample P – mixture of 2 components, A & B
Sample Q – single component or pure substance, C
Differences between TLC and PC
Substance of
Types of
Type Stationary phase stationary phase
separation
(eg.)
Water
molecules on Partition
PC Liquid cellulose (liquid - sp :
fibres or on a liquid - mp)
paper
Thin layer plate
Adsorption
made up form
TLC Solid solid – sp :
silica or
liquid - mp
alumina
Advantages of TLC over PC
Applications of TLC and PC
Separation of
components in Separation of
a mixture such color pigments
as components in plants
of ink and dyes
Analyse the
Detection of
dye
pesticides or
composition of
insecticides in
fibres in
food
forensics
2. Column Chromatography
Techniques:
Separation of mixture occurs on stationary phase that held in a
narrow tube where mobile phase is forced through the tube under
pressure or by gravity.
SP : Adsorbent in column chromatography (solid phase)
Most common: Silica gel, Alumina
MP : Pure solvents or a mixture of different solvent
(liquid phase)
Type of separation : Column chromatography (solid-liquid)
Uses:
Separation, identification, purity.
Concept in Thin Layer Chromatography
The individual components are retained by the stationary phase differently and
separate from each other while they are running at different speeds through the column
with the mobile phase.
At the end of the column they elute one at a time.
Types of Column Chromatography
Normal Phase Reverse Phase
Stationary Phase – Polar Stationary Phase –
Mobile Phase – Nonpolar
Nonpolar Mobile Phase – Polar
The Stationary Phase
Phase : solid.
The most common stationary phase are silica gel, alumina and
cellulose powder.
The stationary phases are usually finely ground powders or gels.
Microporous for an increased surface.
The Mobile Phase
Phase : liquid
Pure solvent or a mixture of different solvents
There is an optimum flow rate for each particular separation.
✓ an optimum flow rate of eluent → better separation
✓ too slow flow rate → broadening separation (not favour)
A faster flow rate of the eluent minimizes the time required to
run a column
Faster flow rates can be achieved by using a pump or by using
compressed gas (example: air, nitrogen, or argon)
✓ function topush the solvent through the column.
The
Process
The sample pour into a column chromatography, where stationary phase is silica (solid),
and mobile phase is organic solvents (liquid).
After a sample was pass through stationary phase, carried out by the flow of mobile phase,
the green component was elute first and collected . Then, followed by the orange
component.
The green component is weakly absorbed towards stationary phase, travel faster with
mobile phase. The green component is eluted first.
While, the orange component is strongly absorbed towards stationary phase, travel
slower with mobile phase. The orange component is eluted later.
Examples
Column chromatography
sequence
Differences between Planar and Column
Chromatography
Type of Position of Movement of
Types
chromatography stationary phase mobile phase
Stationary phase
supported on a By capillary
Planar flat surface / action or move PC & TLC
planar or in the upward
pores of paper
Stationary By gravity or
Column phase held in a move Column
narrow tube downward
Applications of Column
Chromatography
Pharmaceutical Food industry Forensic Industrial Biochemical
chemistry chemicals • Amino acids,
• Antibiotics, • Antioxidants, • Surfactants, proteins,
sedatives, additives, • Drugs, condensed carbohydrate
steroids artificial poisons, aromatics , lipids
sweeteners blood
alcohol
Summary
Type (mechanism) Stationary phase Mobile phase
Solid Liquid
Adsorption
Eg: Alumina, Eg: Alcohol, NaCl
Chromatography
cellulose, silica Gas
Liquid Liquid
Partition
Adsorbed liquid on Gas
Chromatography
solid surface Eg: Helium
Types Stationary phase Mobile phase
Solid – alumina or
Column silica is placed in a Liquid
Chromatography narrow tube or Eg: Benzene
column
PC
Liquid – water PC
Planar adsorbed on paper Liquid Solvent
Chromatography
TLC TLC
PC & TLC
Solid – adsorbent Liquid Solvent
such as alumina or
silica gel
Uses of Chromatography in Real
Life
Law
Pharmaceutical enforcement Environmental
Manufacturing Hospital
Compare Agency
Determine the Plant Detect blood or
sample found Determine the
amount Purify chemical alcohol levels
at a crime level of
chemical in need to make in a patient’s
scene & pollutants in
new sample. product. blood stream.
samples from water supply.
suspects.
Quiz
1. State the meaning for stationary phase and mobile phase.
2. In paper chromatography the spots A and B are 5.4 cm and 3.8
cm respectively from the starting point. The solvent front is 8.2
cm from the starting point. Calculate the Rf for spots A and B.
THANK YOU