ANALYTICAL

INSTRUMENTATION

Lecture
on
Chromatography
 Chromatography
• Chromatography is physical process or method of isolating,
separating, quantifying or identifying components of a mixture.
• Mixture is passed through a stationary phase having a large
surface area via a mobile fluid phase.
• Components are classified through this process because the flow
or migration rate of the components in this movement is
different.
• In chromatographic separation process there are basically two
phases: Stationary and Mobile phase.
• Chromatography is generally of two types:
a) Gas chromatography (GC).
b) Liquid chromatography (LC).
 Chromatography
• The mixture can be of liquid or gaseous.
• The stationary phase can either be solid absorbent or liquid
separator.
• The mobile phase is generally of liquid or gaseous state that
transports the mixture through the stationary phase.
• While transportation through the stationary phase the
components of the mixture undergoes selective retardation or
deceleration.
• The deceleration is due to absorption, filtration, solubility or
chemical bonding.
• Hence the molecules or components of the mixture moves at
different rates and tends to be separated.
• Thereafter entering the detector they generate series of
successive peaks in a recorded curve, termed as Chromatogram.
Chromatogram
 Chromatography – Definitions
a) Retention time (tr): It is the total
time spend by a compound both in
mobile and stationary phase. It is
also evident that each analyte in a
sample will have different
retention time. Expressed in
minute.
b) Dead time (tm): It is the time
elapsed by the non-retained
component of a mixture in the
mobile phase. Expressed in
minutes.
c) Capacity factor (C): Ratio of mass
of compound- tr  tm
C
tm
 Chromatography – Definitions
d) Selectivity or Separation factor (S): It is ratio of the capacity
factors of two components in a sample. Also the ratio of the
partition coefficients of two components. The higher the
selectivity, the better separation is there between two
components or peaks.
CA
S
CB
Where, CA and CB are the capacity factors of A and B components
respectively.
e) Efficiency: It is the count number of a column which indicates
the number of component that can be separated through it.
 Gas Chromatography
• Gas chromatography (GC) is an analytical technique which is
used for separating the components of a gas mixture based on
their volatilities.
• Through this process we can get the qualitative and quantitative
information of an individual component in the sample.
• Mobile phase: Inert gases – Helium, Nitrogen.
• Stationary phase: Liquid/Solid.
• The components are partitioned between the mobile and
stationary phases.
• Sample injector: Syringe/Septum.
• Detector: Flame ionization detector (FID)/ Thermal conductivity
(TC).
 Gas Chromatography - Components
The basic parts that are associated with gas chromatography are:
I. Carrier gas supply with pressure regulator and flow monitor.
II. Sample injection process through syringe.
III. Chromatographic column.
IV. Thermostat/Oven.
V. Detector.
VI. Recording system.
 Gas Chromatography - Process
• The inert carrier gases like He or N2 are compressed inside a
cylinder which is attached with a pressure regulator.
• The gas through a valve and flow meter reaches at the sample
injection port.
• A fixed temperature is maintained that enables rapid
vaporization of the solute.
• Liquid or gas samples are injected through silicon rubber
diaphragm.
• Thereafter the solute mixes with the carrier gas and mixed gas is
blown to the chromatographic column.
• The components of the mixed gas are differentiated at this
column through the interaction with the chromatographic
column which is maintained at a different temperature.
 Gas Chromatography - Process
• At the end of the chromatographic column, the solute of
different components enter the detector separately.
• Depending on the volume of the solute leaving the column, a
corresponding electrical signal is generated.
• Then, this signal is taken to the recording system after proper
signal amplification, signal processing and chromatogram is
obtained.
• These records are required to discriminate different components
of the mixture and for obtaining knowledge of their
corresponding concentrations.
 Flame Ionization Detector
• Widely used as detector in gas
chromatography process.
• Here, effluent from the column
is fed to the air-H2 flame.
• An electrical potential is applied
across the electrodes placed
inside a closed housing.
• The H2 flame burns just at the
tip of the jet capillary which
also acts as the cathode.
• The collector electrode is
having a platinum loop and
placed above the flame tip.
 Flame Ionization Detector
• The current across the electrodes remain constant whenever the
inert carrier gas only passes through the flame.
• Now, when the vapor of the mixture from the chromatographic
column passes through the flame, the molecules of the said
vapor become ions by the hydrogen flame.
• Eventually these ions result in an ionization current.
• As the concentration of the solutes in the vapor varies, there will
be a consequent change in the flow of current between the
electrodes.
• The magnitude of the current flow is directly proportional to the
ions formed in the flame gases.
• Through this changing magnitude of the flowing current
between the electrodes, one can discriminate the components
of a mixture and to know the respective concentrations of the
components.
 Liquid Chromatography
• Liquid chromatography is an analytical process of separating ions
or molecules which are dissolved in a solvent.
• If the sample is kept in contact with another liquid or solid
phase, they will interact due to the differences in absorption,
ion-exchange or volume of the solutes.
• Liquid chromatography is basically divided into three broad
categories:
i. Paper chromatography.
ii. Column chromatography.
iii. Thin-layer chromatography.
 High Pressure Liquid Chromatography
• HPLC chromatograph is consisting of the following sections:
a) A high pressure pump programming system to fed the liquid to
the column.
b) The sample injection system.
c) The column.
d) Detector system and future data processing, conditioning
system.
High Pressure Liquid Chromatography
• HPLC is a typical type of liquid chromatography which is
having prolonged column surface area.
• Similarly it is having an injection system through which a small
volume of sample is injected into the mobile phase.
• The mobile phase flows through the chromatographic column
where the separation of the solutes in the solvent takes place.
• Thereafter through the detection system, a corresponding
signal is generated based on the analyte concentration.
Thank You.