KANHAIYA KUMAWAT

INTRODUCTION TO
CHROMATOGRAPHY
• Invention:
• By M.Twsett in 1906
• ‘chroma’ means color and ‘graphy’ means writing
• Definition :
It is an instrumental method for separation and
identification of chemical compounds.
• Principle :
This technique is based on the difference in the rate at
which the components of a mixture move through a
porous medium (stationary phase) under the influence of
some solvent or gas (moving phase).
 T=2 min
T=0
Gas flow rate 3 ft. / min.

6 ft. long tube

T=3 min
T=0

Gas flow rate 2 ft. / min.

Sand filled tube has some properties of GC column

Liquid “A” is non volatile
Dynamic Equilibrium
Gas B spends 40 % time in liquid phase
 T=2 min
T=0
Gas flow rate 3 ft. / min.

6 ft. long tube

T=3 min
T=0

Gas flow rate 2 ft. / min.

T=5 min
T=0

(60/100) * 2 ft. / min. = 1.2 ft. / min. will be the flow rate.

• Gas B spend 60 % time in gaseous phase

T=0 T=3.75 min

(80/100) * 2 ft. / min. = 1.6 ft. / min. will be the flow rate.
• Gas C spend 80 % time in gaseous phase
Detector Response 

Gas C Gas B

0 1 2 3 4 5 6 7
Time (minutes)
Rt = 3.75 Rt = 5 min.
min. slower
faster moving
moving more
less retained retained
 Flow of Mobile Phase
Injector Detector
T=0

T=10’

T=20’

Most Interaction with Stationary Phase Least

 CLASSIFICATION OF
CHROMATOGRAPHIC METHODS
Chromatography

Partition Adsorption
(liquid stationary phase) (solid stationary phase)

Mobile Mobile Liquid Gaseous

Gaseous Liquid Mobile Mobile
Phase Phase Phase Phase
- GLC - HPLC -Column GSC
-Ion Exchange -Thin layer
-Paper
 PARTITION TYPE
GLC (Gas-Liquid chromatography):
The separation is achieved due to difference in
solubilities and hence distribution of the solute
between liquid(stationary phase) and gaseous
phase(mobile phase).
HPLC (High pressure liquid chromatography):
In this high pressure is used to push a mobile phase
solution.
Ion Exchange:
It involves the exchange of ions between the
solution phase and inert solid material.
 ADSORPTION TYPE

Column chromatography:
The mixture is dissolved in a suitable solvent and
passed through a tube containing adsorbent.
Paper chromatography:
The components of mixture are migrated at
different rates and appear as spots on paper.
Thin-Layer chromatography:
In this a thin layer of solid adsorbent is coated on
a glass or plastic plate.
 In GAS CHROMATOGRAPHY

• The mobile phase is an inert carrier gas.

• The stationary phase is a solid or a liquid coated

on a solid contained in a coiled column.
 INSTRUMENTATION OF GAS
CHROMATOGRAPHY

• Operating principle of GC:

A sample is,
• Introduced into a injector port.
• Passed through column with inert carrier gas.
• Detected as a series of peaks by detector.
Experimental setup of GC
Carrier gas/ Gas Computer Controls for
Regulator Chromatograph Method and Output
 CARRIER GAS
• It is stored in gas cylinder under pressure.
• Its flow rate is controlled by two stage regulator.
• carrier gas should have following properties :
• Inert
• Suitable for detector and sample.
• Readily available in pure form.
• Cheap
• Best column performance with required
speed of analysis.
• Non explosive
• In GC H2, He, and N2 are widely used as carrier
gas.
• Choice of Carrier Gas depends on sample to be
analyzed.
Thermal Conductivities Of Gases
Gas Thermal conductivity
cal/cm/0C/Sec.
H2 44.5
He 36.0
Ne 11.6
O2 6.35
N2 6.24
CO2 3.96
CH3OH 3.68
CH4 3.18
 SAMPLE INJECTION SYSTEM
• Sample:
• Pure, less quantity.
• It can be solid, liquid or gas.
• Prepared as a dilute solution.
• Sample injection system:
• It is one of the important part of GC.
• Sample is injected using a micro syringe which enters
through a replaceable rubber septum (self sealing).
• As liquid sample is injected into a column it gets.
vaporized instantaneously so it enters in a column at
once.
• This system is temperature controlled.
Dilute
Solution
Pure
Sample
 Column
• Columns are called as the brain of chromatograph.
• Generally used for the analytical purpose.
• Made up of glass or metal tube.
• Columns are usually placed in coiled form in a oven.
• There are two types of columns –
1) Packed column
2) Capillary columns
• Except columns all the components are same for
GLC and GSC.
 Packed Columns
• Made up of glass or a metal tube having
– Diameter 1 to 8 mm.
– Length 2 to 20 meter.
• The number of theoretical plates are 20,000 or
more.
• Metal tube is packed with granular stationary phase.

• For GLC- Packing is prepared by coating a liquid

phase over an inert solid support.
• For GSC- Packed with size graded absorbent or a
porous polymer.
Efficiency of Chromatographic Column

• The efficiency of chromatographic column

is a measure of its ability to separate the
components of a mixture.
• The efficiency of chromatographic column
is expressed in terms of number of
Theoretical plates, a term borrowed from
distillation process.
Efficiency of Column in terms of Theoretical plates.

No. of Theoretical plates, r = 16(x /y) 2

r = 16*(x/y) 2 Theoretical plate height, H = L / r
= 16*((5-1)/0.5) 2 = 6 ft./1024
= 16(4/0.5)2
= 16*64
= 1024
Factors that affects theoretical plate height are discussed in
Van Deemtor equation

• H = A + B/V + CV

• Where,
• H is Theoretical plate height
• A = Depends on Eddy currents in flowing gas.
• B = Depends on diffusion of sample in gas phase and liquid phase. It is affected by
temperature.
• C = Depends on mass transfer of sample between two phases. (How fast the equilibrium
is achieved).
• V = Flow rate of the carrier gas.
Fig. A typical Van Deemter graph
 Stationary phase
In GLC, Support must satisfy the following properties-
• High surface area.
• Chemically inert.
• Material used as a support is crushed fire brick and used even in high
temperature furnaces for extended time periods.
• Surface of support is coated with liquid film, which is also chemically
inert and it’s vapor pressure must be low.
• Column packing is based on the type of the sample to be analyzed.
For Example,
• Carbowax 20 M (Polyethylene Glycol) is preferred for the
separation of alcohols, esters, pesticides and essence of oils.
• DEG adipate (Diethylene Glycol) column is used for the separation
of fatty acids, esters and pesticides.
 Stationary phase
• For GSC, Use of porous polymer beads as a
packing material have certain advantages-
• Stable up to 250 0C and causes no base line drift
and hence allows the use of the highly sensitive
detectors.
• no adsorption of polar components such as
water, alcohols, acids and are eluted rapidly as a
sharp symmetrical peaks.
• Sample overload recovery is rapid and without
trailing.
• Polymer beads are mechanically strong.
• Retention data are highly reproducible.
• Some of the separation provided are unique.
 Column packing
• It is important that the packing is evenly done and
contains no gaps. Following method is used for the
packing of column:
Column is left uncoiled and held vertically on a
suitable plane. The column material is slowly added
to it from the top. In this way every time small
quantity of material is added and column is well
shaken.
At this stage the ultrasonic vibrator is used to vibrate
the column.
After packing, both the ends are sealed by glass wool.
Then the column is bent into a coil so that it will be
more compact for handling.
Now the column is ready to use in the instrument.
 Capillary column
• These are also called as the open tabular columns.
• Made up of stainless steel, copper or a glass etc.
but stainless steel is most popular.
– Diameter is 0.25 to 0.75 mm.
– Length is 30 to 90 meter.
• Inside walls are coated with liquid phase in the
form of thin (0.5 to 1 micron) and uniform film.
• Number of theoretical plates are about several
100 to several 1000.
• Disadvantage – low capacity of resolution.
• It is very delicate.
 Enter from Exit to
Injector Detector

Packed Column
installed in Oven
Compartment.
 Temperature Programming in GC

• In order to get reproducible analytical results, it is

necessary to control the temperature of the column
very carefully. The low-boiling components are
eluted quickly and bunch together on the record
chart, while the less volatile species take much
longer and their peaks are much broader. This can
be overcome by increasing the temperature of the
column at a uniform rate.
Relation between the Retention time (Rt) and some parameters

Retention time (min.) Retention time (min.)

35

30

25

Retention Time (min.)

20

15

10

0
0 50 100 150 200 250
0
temperature of a column ( C)
 Qualitative analysis

• For a given column, if flow rate and temperature is

kept constant then the retention time data can be
highly reproducible and be used to identify the
compound.
• To check the quality of Product one has synthesized.
• To find the trace impurity if present in the sample.
 Quantitative analysis
Quantitative analysis is based on the comparison of the area of the peak.

height h

W
base

Area = (Height * Base) / 2 Area = h * w

 DETECTOR
• Function: To detect and measure different components
of sample as they emerge from column.
• Choice of detector depends upon type of analysis being
performed.
• Characteristics of an ideal detector :
• Rapid and reproducible response though quantity
of sample is less.
• Linear response over large concentration range.
• Good stability over entire temperature range.
• Uniform response to compounds of same
concentration having different chemical nature.
No single detector has all above characteristics.
• First family detectors: • Second family detectors :
• It responds to concentration • It responds to mass flow
(in mole fraction) of solute. rate of solute (in moles per
unit time).
• It do not destroys the • It destroys the sample.
sample.
e.g Thermal conductivity e.g. Flame ionization detector,
detector, Flame emission detector,
Electron capture detector, Microwave plasma detector,
Cross section detector, Argon Ionization detector,
Helium Ionization detector
Gas density balance,etc.
 Thermal Conductivity Detector
• It belongs to first family detectors.
( Nondestructive detection technique)
• It is simple in construction.
• It is the most common.
• It is non selective i.e. Adequate sensitivity for many
compounds.
• It gives good linear range of signal.
• Signal is quite stable, provided carrier gas flow rate,
block temperature, and filament power are controlled.

• Principle : It is based on the difference between

the thermal conductivities of carrier gas and
sample.
Thermal Conductivity Detector
 Thermal Conductivity Basics
The TCD is a nondestructive, When the carrier gas is contaminated by
concentration sensing detector. A sample , the cooling effect of the
heated filament is cooled by the flow gas changes. The difference in cooling
of carrier gas . is used to generate the detector signal.
Flow

Flow
 Unequal loss of heat from filament
• When a compound elutes, the thermal
conductivity of the gaseous mixture of carrier gas
and compound gas is lowered, and the filament in
the sample column becomes hotter than the other
control column.
• Its resistance increased, and this imbalance
between control and sample filament resistances is
measured by a simple gadget and a signal is
recorded.
• For maximum sensitivity, change in resistivity
should be large. Hence carrier gas of high thermal
conductivity is used.
• H2, He has thermal conductivity 6 to 10 times
that of most organic compound. But they are
hazardous and costly.
• N2,Co2 has same order of thermal conductivity
as that of most organic compound. They are
cheap and non hazardous.
• TCD is not most sensitive but satisfactory for a
wide variety of analytical applications.
 ELECTRON CAPTURE
DETECTOR
• It is non destructive in nature.
• It has extreme sensitivity.
• It is selective in nature, i.e. it is insensitive
to amines,alcohols and hydrocarbons and
sensitive to halogen compounds,compounds
containing functional groups like nitro
group, peroxides, quinone.
Principle: Decrease in ion current due to
presence of sample.
ELECTRON CAPTURE DETECTOR

Ni-63
β-Ray electron
 Flame Ionization Detector
• It is destructive in nature.
• It is one of the most sensitive detector.
• It is 1000 times sensitive than TCD.
• It is sensitive to all hydrocarbons and hetero-organic
compounds.
• Insensitive to many inorganic compounds.
• It is superior for quantitative analysis.
Detectors Sensitivity Linear range Comments

Thermal conductivity 10-8 104 Universal sensitivity

nondestructive

Flame ionization 10-11 106 Detects all organic

compounds, the most
widely used GC
detector

Electron capture 10-13 102 Detector Halo, Nitro and

Oxygenated
compound response
varies significantly
non destructive
• The detector information can also sent to electronic
devices where it is amplified and plotted against time.
This is called as chromatograph.
 Analysis of Data
• Each component of the mixture reaches the detector at
a different time and produces a signal at a
characteristic time called a retention time.
• In the printout of the chromatographic analysis:
• the number of peaks correlates with the number
of components in the sample,
• the area under each peak correlates with the
relative amount of that component in the sample,
• and if standard information is available, the
retention time under defined conditions can be
used to identify each component.
CHROMATOGRAPH :
A plot of detectors response VS Time is called as
Chromatograph
Response

X 2

X1

X0

1 4 7
Retention Time, in min.
 Approximation
of peak area by
triangulation
Absorbance 

Peak A Peak B

height

0 1 2 3 4 5 6 7
Time (minutes)
base

Rt = 3.0 min. Rt = 5.6 min.

faster moving slower moving base x height
Area =
less retained more retained 2
 TENTATIVE IDENTIFICATION OF UNKNOWN
Response COMPOUNDS
Sample having three components
3.2 min = RT
1.6 min = RT

GC Retention Time

Response Response
Standard response for X Standard response for Y

1.6 min = RT 3.2 min = RT

GC Retention Time GC Retention Time

 MAIN ADVANTAGES OF GC
• Strong separation power
• High sensitivity
• Good precision and accuracy
• Short time analysis
• Low cost and long life
• Easy to handle: does not require highly
qualified person.
 THANK YOU

for hearing me patiently

 Back

Response
Response

GC Retention Time GC Retention Time

Good Separation Poor Separation

 Van Deemter Equation

• H = A + B/V + CV

Where,
H is Theoretical plate height
A = Depends on Eddy currents in flowing gas.
B = Depends on diffusion of sample in gas phase and liquid
phase. It is affected by temperature.
C = Depends on mass transfer of sample between two phases
V = Flow rate of the carrier gas.
Detector Response 

Gas C Gas B

0 1 2 3 4 5 6 7
Time (minutes)

Rt = 3.75 min. Rt = 5 min.

faster moving slower moving
less retained more retained