1

CHROMATPGRAPHY
Ms.Heena Goswami
2 Chromatography
 Principle
3

 Separation of a
mixture into individual
component using a
stationary phase and
a mobile phase.
 Types of Chromatography
4

1. Based upon the nature of stationary and mobile phase

I. Gas- Solid chromatography
II. Gas – liquid chromatography
III. Solid – liquid chromatography ( column chromatography, TLC, HPLC)
IV. Liquid – liquid chromatography (Paper partition chromatography, column
partition chromatography)
2. Based on the principle of separation
I. Adsorption chromatography
II. Partition chromatography
3. Based on the modes of chromatography
I. Normal phase chromatography
II. Reverse phase chromatography
4. Other types
I. Ion-exchange chromatography
II. Gel permeation chromatography
 Adsorption chromatography
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Used for anionic compounds

 mobile phase + analytes and
stationary phase
Surface interaction of analytes
with stationary phase
 As a result analytes stick to
the stationary phase and mobile
phase
Passes towards gravity.
 Partition chromatography
6
 Normal and Reverse phase
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chromatography

 Stationary phase is
polar and mobile phase
non-polar normal phase
chromatography.
 Stationary phase is non-
polar and mobile phase
polar reverse phase
chromatography
8 Paper Chromatography
 Introduction
9

 Paper chromatography is defined as technique in

which the analysis of unknown substance is carried
out mainly by the flow of solvents on specially
designed filter paper.
 There are two types of paper chromatography
1. Paper adsorption chromatography
2. Paper partition chromatography
 Instruments
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Stationary phase
Mobile phase
Application sample
Development technique
Detecting agents
Paper Chromatography Experiment
Which pen wrote the note?
Overview of the Experiment

Purpose:
To introduce students to the principles and terminology
of chromatography and demonstrate separation of
the dyes in various pens with paper chromatography.
 Materials List
 1 beaker
 Distilled H2O
 1 strip of filter paper
 Different pens (A, B, & C)
 Pencil
 Skewer
 Preparing the Chromatography Strips

 Cut a strip of filter paper

 Draw a line 1 cm above the bottom
edge of the strip with the pencil
 Use pencil to label each strip for its
corresponding pen
 Place a spot from each pen on your
starting line
 Thread the paper strips onto the
skewer.
 Place the skewer so the strips hang
into the water in the beaker making
sure the ink dots are above the
waterline.
 Let strips process for several
minutes.
Illustration of Chromatography
Stationary Phase
(filter paper)

Separation
Mixture Components

(Ink)

Mobile Phase
(water)

As the water is soaked up through the filter paper and reaches the ink dots, the
component colors of the ink separate and are carried with the water along the
filter paper. The different components travel to different heights on the paper.
1. Examine the chromatogram of each of the three pens (A, B, C)

2. Examine the chromatogram made from the note.

3. Determine if chromatograms A, B, or C match the evidence.

17 Thin Layer Chromatography
 Principle of TLC
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 The principle of separation is adsorption.

 One of more compounds are spotted on thin layer
of adsorbent coated on a chromatographic plate.
 The mobile phase solvent flows through because of
capillary action(against the gravitational force).
 Compounds moves according to their affinities
towards adsorbent.
 Advantages of TLC
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1. Simple method and cost of equipment is low.

2. Rapid technique and not time consuming like
column chromatography.
3. Separation of μg of substance can be achieved.
4. Capacity of thin layer can be altered.
5. Corrosive spray reagents can be used without
damaging the plates.
 Practical Requirements
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1. Stationary phase
2. Glass phase
3. Preparation and activation of TLC plates
4. Application of sample
5. Development tank
6. Mobile phase
7. Development technique
8. Detecting agents
 Stationary phase
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 There are several adsorbents which can be used as

stationary phase.
Name Composition Adsorbent:water ratio
Silicagel H Silicagel without binder 1:1.5
Silicagel G Silicagel + CaSO4 1:2
Silicagel GF Silicagel + binder + 1:2
fluorescent indicator
Alumina Al2O3 without binder 1:1.1
Neutral
Basic
Acidic
Al2O3 G Al2O3 with binder 1:2
Cellulose powder Cellulose with binder 1:5
Polyamide powder Polyamide 1:9
 Glass phase
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20X20 cm 20X10 cm 20X5 cm

 Preparation of TLC plates
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1. Pouring
2. Dipping
3. Spraying
4. Spreading
 Activation of TLC plates
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 Activation of TLC plate is nothing but removing

water/moisture and other adsorbed substances
from the surface of any adsorbent, by heating at
high temperature.
 The activation plates can be stored in
thermostatically controlled oven and can be used
whenever required.
 Application of sample
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 Usually to get good spots the concentration of

sample or standard solution should be minimum.
 Capillary tube or micropipette should be used.
 Spots can be placed random or equidistant.
 Spot should be at least 2cm above the base plate
& spotting area should be immersed in the mobile
phase in the development tank.
 Mobile Phase
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 The solvent or mobile phase used depends upon

various factors which are as follows:
1. Nature of substance to be separated
2. Nature of stationary phase
3. Mode of chromatography
4. Separation to be achieved
 Development technique
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 Different development techniques are used for

efficient separation:
I. One dimensional development

II. Two dimensional development

III. Multiple development

 Detecting or Visualizing agents
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 After the development of TLC plates, the spots should

be visualized.
 Colored spots detection can be done visually, but for
the detection colorless following techniques can be
used:
a. Non specific methods
I. Iodine chamber method
II. Sulphuric acid spray reagent
III. UV chamber for fluorescent compounds
IV. Using fluorescent stationary phase
b. Specific methods
 Types of detecting techniques
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1. Destructive techniques
Specific spray reagents, sulphuric acid spray reagent,
etc.
2. Non-Destructive techniques
Iodine chamber method, UV chamber for fluorescent
compounds
 Qualitative analysis
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 Retardation factor (Rf value) is the ratio of distance

travelled by the solute to the distance travelled by the
solvent front.

Rf = Distance travelled by solute

Distance travelled by solvent front
31 Gas Chromatography
 Introduction of GC
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 Gas chromatography consist of Gas Solid

chromatography(GSC) and Gas Liquid
chromatography(GLC).
 In both types, gas is used as mobile phase and
either solid or liquid is used as stationary phase.
 In GSC principle of separation is adsorption.
 GSC is used only in the case of less solubility of
solutes in stationary phase which is rare so GLC is
used most.
 Principle of GLC
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 The principle of separation in GLC is partition.

 Gas is used as mobile phase.
 Liquid which is coated on to a solid support is used
as stationary phase.
 The mixture of components to be separated is
converted to vapor and mixed with gaseous mobile
phase.
 Criteria for compounds to be analysed
34
by GC
1. Volatility
Unless a compound is volatile it cannot be mixed
with mobile phase.

2. Thermo stability
All the compounds will not be in the form of vapor.
There will be solid or liquid samples too. Hence to
convert them in vapor form they have to be
heated at high temperature. At that temperature
the compounds have to be thermostable.
 Practical Requirements
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1. Carrier gas
2. Flow regulators and flow meters
3. Injection devices
4. Columns
5. Temperature control devices
6. Detectors
7. Recorders
 Gas Chromatographic Apparatus
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Inject point Detector

Flow
Flow
Regulato
Meter
G r
A
S

S
U Amplifier
P
P
L
Y
Column in thermostatically Recorder
Controlled oven
 Carrier Gas
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 The choice of carrier gas determines the efficiency

of chromatographic separation.
 Most widely used are:
1. Hydrogen –better thermal conductivity, low
density. Disadvantage is it react with unsaturated
compounds.
2. Helium-excellent thermal conductivity but
expensive.
3. Nitrogen- inexpensive but has reduced sensitivity.
 Flow regulators and flow meters
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Rotameter Soap bubble meter

Soap bubble

Calibrated tube Calibrated tube

Float
Spring
Rubber buld

Soap solution
Gas inlet
Inert gas
 Flow regulators and flow meters
39

Rotameter Soap bubble meter

Soap bubble

Calibrated tube Calibrated tube

Float
Spring
Rubber buld

Soap solution
Gas inlet
Inert gas
 Injection devices
40

 Sample can be either solid, liquid or gas in nature.

 Gases can be introduced into the column by valve
devices.
 Liquids can be injected through loop or spectrum
devices. Most GC has good quality of silicone
rubber spectrum which can withstand high
temperature.
 Solid samples are dissolved in suitable solvents and
then injected through rubber spectrum.
 Columns
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 Decides the separation efficiency.

 Made up of glass or stainless steel.
 Stainless steel columns has long life and can be
handled easily, but some samples reacted with them
so in that case glass columns are used.
 Glass columns are inert but disadvantage is they
are highly fragile.
 Columns can be classified according to
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nature as well as its use.
1. Depending on its use
I. Analytical column
II. Preparative column
2. Depending on its nature
I. Packed column
II. Open tubular column or capillary column or Golay
column
III. SCOT column(Support Coated Open Tubular
column)
 Temperature Control Devices and
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Detectors
Control devices
1. Preheaters
2. Thermostatically controlled oven
Detectors
1. Flame Ionization detector(FID)
2. Argon Ionization detector(AID)
3. Electron Capture detector(ECD)
44 HPLC
High Performance Liquid Chromatography
 Introduction
45

 The technique of high performance liquid

chromatography is so called because of its
improved performance compared to classical
column chromatography.
 It is called as high performance liquid
chromatography since high pressure is used.
 Types of HPLC techniques
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A. Based on modes of chromatography

B. Based on principle of separation
C. Based on elution technique
D. Based on scale of operation
E. Based on the type of analysis
 Based on modes of chromatography Normal
and Reverse phase chromatography
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 Stationary phase is
polar and mobile phase
non-polar normal
phase chromatography.
 Stationary phase is non-
polar and mobile phase
polar reverse phase
chromatography
 Based on principle of separation
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1. Adsorption chromatography
2. Ion exchange chromatography
3. Ion pair chromatography
4. Size exclusion or Gel permeation chromatography
5. Affinity chromatography
6. Chiral phase chromatography
 Based on elution technique
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1. Isocratic separation

2. Gradient separation
 Based on scale of operation
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1. Analytical HPLC

2. Preparative HPLC
 Based on the types of analysis
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 Qualitative analysis

 Quantitative analysis
 Principle of separation in HPLC
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 The principle of separation in normal phase mode

and reverse phase mode is adsorption.
 When a mixture of components are introduced into
a HPLC column, they travel according of relative
affinity towards stationary phase.
 No two components have the same affinity towards
the stationary phase, the components are
separated.
 Instrument Requirements
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 Pumps- Solvent delivery system

 Mixing unit, gradient controller and solvent
degassing
 Injector-Manual or auto injector
 Guard column
 Analytical columns
 Detector
 Recorders and integrators
 Instrumentation
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 Pumps- Solvent delivery system
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 The solvents or mobile phases used must be passed

through the column at high pressure at about 1000-
3000 psi.
 This is because as the particle size of stationary
phase is few μ (5-10) the resistance to the flow of
solvent is high.
 Hence such high pressure is recommended.
 There are different types of pumps available.
 Types of pumps
56

Mechanical pump Pneumatic pump

 Operates with constant  Operates with constant
flow rate and uses pressure and uses
sapphire piston. highly compressed
gas.
 Used in analytical
 The solvent used must
scale. be high purity grade.
 Preferred filter
through 0.45µ.
 Parts of Pump
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 Check valves-These are present to control the flow

rate of solvent and back pressure.

 Pulse dampners: These are used to reduce the pulse

observed from the wavy baseline caused by the
pumps.
 Mixing unit, gradient controller and
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solvent degassing
 Mixing units is used to mix solvent in different
proportions and pass through the column.
 There are two types of mixing units:
1. Low pressure mixing chamber which uses helium for
degassing solvents.
2. High pressure mixing chamber does not require helium
for degassing solvents.
 Mixing of solvents is done either with static mixer
which is packed with beads or a dynamic mixer which
uses magnetic stirrer and operates under high
pressure.
 Gradient controller
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 In isocratic separation mobile phase is prepared

using pure solvents or mixtures of solvent(solvent of
same eluting power or polarity is used)
 In gradient elution technique, the polarity of
solvents increases gradually so solvents composition
has to be changed.
 Hence a gradient controller is used when two or
more solvent pumps are used for separation.
 Solvent degassing
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Several gases are soluble in organic solvents. When

solvents are pumped under high pressure, gas
bubbles are formed which will interfere with the
separation process, steady baseline and the
shapes of the peak. Hence degassing of solvents is
important.

a. Vacuum filtration
b. Helium purging
c. Ultrasonication
 Injectors
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a. Septum injectors
b. Stop flow(on line)
c. Rheodyne injectors (Loop valve type)
 Guard column
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 Guard column has very small quantity of absorbent

and improves the life of the analytical column.
 It also acts as a prefilter to remove particulate
matter, if any and other materials.
 It is made up of same material that of analytical
column.
 Analytical column is most important part of HPLC
which decides efficiency of separation.
 There are several stationary phases depending
upon the technique or mode of separation used.
 Detectors
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1. Flame Ionization detector(FID)

2. Argon Ionization detector(AID)
3. Electron Capture detector(ECD)
4. UV detectors
5. Flourimetry detectors
6. Mass detectors