2. INTRODUCTION
Technique used to separate and identify the components of a mixture
PRINCIPLE: Works by allowing the molecules present in the mixture to
distribute themselves between a stationary and a mobile medium.
Molecules that spend most of their time in the mobile phase are carried
along faster.
The factors effective on this separation process include molecular
characteristics related to adsorption (liquid-solid), partition (liquid-solid),
and affinity or differences among their molecular weights.
3. Gas Chromatography
Also called as Gas liquid chromatography (GLC)
Based on a partition equilibrium of analyte between a solid stationary phase (often a
liquid silicone based material) and a mobile gas (most often Helium).
The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary
column) or a solid matrix inside a larger metal tube (a packed column).
Well suited for use in the petrochemical, environmental monitoring, and industrial
chemical fields. It is also used extensively in chemistry research.
4. GC involves vaporizing a sample and injecting it onto the head of the column.
Under a controlled temperature gradient, the sample is transported through the
column by the flow of an inert, gaseous mobile phase.
Volatiles are then separated based on several properties, including boiling
point, molecular size, and polarity.
Working Principle:
5. Instrumentation:
Mobile Phase: He, Ar, N2, H2
Flow regulators: deliver the gas with uniform pressure/flow rate.
Injection Port: Rubber septum barrier (usually maintained at a higher temperature
than the boiling point of the least volatile component in the sample mixture)
Column: fused silica with a thin coating of stationary phase on the inner surface
Oven: Thermostat controlled forced air oven
Detector:
Data System: recorders & integrators
6. Columns:
Columns in GC are two types
1) Packed column- Glass or metals. Densely packed with packing materials or solid
support coated with thin layer of stationary liquid phase
2) Capillary column-Better resolution – efficient mass transfer between gas and SP
Tubing – fused silica, glass, copper, stainless steel
Packed column Capillary column (Open
tubular column)
7. GC detectors:
The eluted solute particles enter the detector
Then produces electrical signals proportional to the concentration of the
components of solute. This signals are amplified and recorded as peaks at
intervals on the chromatograph.
Different types of detectors are:
• Thermal conductivity detector
• Flame ionization Detector
• Electron Capture Detector
8. Gas chromatography-Mass spectroscopy (GC-MS)
Gas chromatography-Mass spectroscopy (GC-MS) is one of the so-called
hyphenated analytical technique.
It is actually two techniques that are combined to form a single method of
analyzing mixtures of chemicals.
Gas chromatography is a technique capable of separating, detecting and partially
characterizing the organic compounds particularly when present in small quantity.
Mass spectroscopy provides some definite structural information from in small
quantity.
9. Principle of GC-MS:
The sample solution is injected into the GC inlet where it is vaporized and swept
onto a chromatographic column by the carrier gas (usually helium).
The sample flows through the column and the compounds comprising the
mixture of interest are separated by virtue of their relative interaction with the
coating of the column (stationary phase) and the carrier gas (mobile phase).
The latter part of the column passes through a heated transfer line and ends at the
entrance to ion source where compounds eluting from the column are converted
to ions.
10. Gas chromatography:
1. Carrier gas- Served as mobile phase supplied in the steel tank under high pressure. Eg:-
nitrogen, helium; hydrogen and argon can also be used
2. Pneumatic Control- Gas supply is regulated to the correct pressure and then fed to the
required part of instrument.
3. Oven- Temperature programmable, typically range from 5°C - 400°C but can go as low as
- 25°C with cryogenic cooling
4. Sample Injection Port- Sample is made to vaporized rapidly before entering to column
5. Column- Two kinds of column used :Packed column & Capillary column. Gas
chromatograph GC-MS utilizes capillary column
6. Detectors- Thermal Conductivity Detector (TCD)
Flame Ionization Detector (FID)
Electron Capture Detector (ECD)
11. Interface:
Pressure incompatibility problem between GC and MS was solved by inserting
an interface.
Mainly three types of interfaces are there commercially available :
• Jet/Orifice separator
• Direct capillary infusion interface
• Watson-Biemann effusion separator
12. Mass Spectrometer:
1. Ion source- it converts the components of a sample into ions by bombardment with
electrons, ions and molecules. The gas molecules exiting the GC are bombarded by high
energy electron beam. Different types are:
Electron Impact Ionization (EI)- Electrons produced by tungsten filament move to
ion source chamber to produce magnetic flux in parallel to the electron beam.
Chemical Ionization (CI)- Involves ionization of reagent gas like methane at high
pressure & this collides with the analyte molecules producing ions.
Fast Atom Bombardment (FAB)- Technique in which the analyte is bombareded by a
high energy beam of inert gas such as Argon or Xenon.
13. 2. Mass Selective Analyzer
They deflects ions down a curved tubes in a magnetic fields based on their kinetic energy
determined by the mass, charge and velocity.
The magnetic field is scanned to measure different ions.
a) Quadrupole Analyzer- consists of four parallel metal rods with different charges. For
given DC and AC voltage, only ions of certain mass-to-charge ration pass through the
analyzer.
b) Ion Trap Mass Analyzer- The ion trap mass analyzer operates by similar principles
where it consists of circular ring electrode and two end caps that form a chamber. The
ions entering into the chamber are trapped by electromagnetic fields & they oscillates in
concentric trajectories.
c) Time-of-flight Analyzer- analyser uses an electric field to accelerate the ions through
the same potential. Then it measures the time take to reach the detector. If the particles
all have the same charge, the kinetic energies will be identical and their velocities will
depend upon only on their masses.
14. Mass spectrometers employ computer control of same functions and also
use a computerized display and output.
It is used to identify and measure the concentration of one or more analytes
in a complex mixture
3. Data Handling:
15. High Performance Liquid Chromatography (HPLC)
HPLC has the ability to separate, identify, and quantitate the compounds that are
present in any sample that can be dissolved in a liquid
Principle: The stationary phase is usually an inert solid or a liquid held on the inert
solid
Mobile phase travels through the column forcibly with the aid of the high pressure
pump
Solutes of the sample separated on column and eluted with mobile phase
The technique is applicable to thermally fragile samples, e.g. amino acids, proteins,
nucleic acids, hydrocarbons, antibiotics, steroids, drugs, inorganic and may organic
substances.
16.
17. I. NORMAL-PHASE HPLC:
Adsorption of analytes on the polar, weakly acidic surface of silica gel
Stationary Phase.: Silica (pH 2-8), Alumina (pH 2 - 12), Bonded Diol and NH2
Mobile Phase: Non-polar solvents (Hexane, CHCl3)
Polar solutes elute later than non-polar lyophilic ones
II. REVERSED-PHASE HPLC:
Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary
phase.
Stationary Phase: Hydrophobic surfaces of moieties bonded on silica
Mobile phase: Methanol or Acetonitrile and Water.
18. Instrumentation:
1. Solvent/ mobile phase reservoirs- Glass or stainless-steel containers capable of holding
up to 1 liter mobile phase (pure organic solvents or aqueous solutions of salts and
buffers)
2. Degassing & filtration of mobile phase- Degassing is done to prevent formation of gas
bubbles in the pump or detector ( Mobile phases are degassed by stirring of the mobile
phase under vacuum, sonication or sparing with helium gas)
The mobile phase are filtered to remove particulate matter that may clog the system
3. Pump- The solvents or mobile phase must be passed through a column at high pressures
at up to 6000 psi.. Pulse free output & all materials in the pump should be chemically
resistant to solvents.
Flow rates ranging from 0.1 to 10 mL/min
19. 4. Sample injector system- Several injector devices are available either for manual or
auto injection of the sample.
a) Septum Injector-used for injecting the sample through a rubber septum
b) Stop flow- the flow of mobile phase is stopped for a while & the sample is
injected through a valve
c) Rheodyne injector- This has a fixed volume of loop, for holding sample until
its injected into the Column
Rheodyne injector
20. 5. Column-
a) Precolumn-
It contains a packing chemically identical to that in analytical column.
Mainly used to remove the impurities from the solvent and thus prevents
contamination of the analytical column, it can protect analytical column.
It is also called as guard column or protective column.
It is having large particle size.
It is having short length of 2 to 10 cm, so does not affect separation
21. b) Analytical column-
Actual separation is carried out here.
Column is filled with small particles 5
– 10 micron. The solid support can be
silica gel, alumina.
The separation is result of different
components adhering to or diffusion
into the packing particles when the
mobile phase is forced through column.
HPLC Column
22. 6. Detectors:
Used upon the property of the compounds to be separated. Different detectors
available are:
1) Refractive index detectors- detects the change in turbidity
2) U.V detector- based on electronic transitions within molecules
3) Fluorescence detectors- based on emission of excited state molecules
4) Photo diode array detector- allows for the recording of the entire spectrum of
each solute as it passed trough the diode array detector.
23. High Performance Thin Layer Chromatography (HPTLC)
HPTLC- sophisticated form of thin layer chromatography it involves the same
theoretical principle of thin layer chromatography
Key feature of HPTLC:
Simultaneous processing of sample and Standard
Several analysts work simultaneously.
Lower analysis time and less cost per analysis.
Low maintenance cost.
Simple sample preparation
No prior treatment for solvents like filtration and degassing.
Low mobile phase consumption per sample
Visual chromatogram
24. Principle:
The mobile phase flows through the plate because of capillary action. the
components move according to their affinities toward the adsorbent .
The component with more affinity towards stationary phase travels slower. the
component lesser affinity towards stationary phase travel faster
25. Steps Involving in HPTLC
Selection Of Chromatography Plates
Sample And Standard Preparation
Pre Washing Of Pre Coated Plates
Activation Of Pre-coated Plates
Application Of Standard And Sample
Selection Of Mobile Phase
Chromatographic Development And
Drying
Detection and visualization
26. 1. Selection Of Chromatography Plates:
Precoated plates- different supporting materials – different solvents available
80% of analysis takes place by silica gel
Aluminium oxide: Basic substances ,alkaloid and steroids.
2. Sample And Standard Preparation:
Sample and standard should dissolved in the same solvent to ensure comparable
distribution at stationary zones.
For normal phase chromatography solvent for dissolving the sample should be non
polar.
For reverse phase chromatography polar solvents are used.
27. 3. Pre Washing Of Pre Coated Plates: To avoid any possible interference due to
impurities it is recommended to wash the plates is called pre washing
Solvents used for washing are:
Chloroform in methanol(1:1)
Methylene chloride – methanol(1:1)
1%ammonia or 1% acetic acid
4. Activation Of Pre-coated Plates: Freshly open box of plates do not require
activation.
Plates exposed to high humidity or kept on hand for long time to be activated by
placing in oven at 110-120ºc for 30 minutes prior to spotting.
Aluminium sheets should be kept in between two glass plates and placing in
oven at 110-115ºc for 15 minutes
28. 5. Application Of Standard And Sample:
Selection of sample application and devices used
depends on:-
Sample volume
Number of samples to be applied
Samples is applied by use of automatic devices and
graduated capillaries.
Volume recommended for HPTLC 0.5-5μl
a) Capillary tubes- samples applied in form of
spots
b) Micro syringes- sample can apply either as spot
or bands
Capillary tubes
Micro syringes
29. 6. Selection Of Mobile Phase:
Normal phase -
Stationary phase is polar
Mobile phase is non polar
Non polar compounds eluted first because of lower affinity with
stationary phase .
Polar compound retained because of higher affinity with the
stationary phase
Reversed phase-
Stationary phase is non polar
Mobile phase is polar
Polar compound eluted first because of lower affinity with
stationary phase
Non polar compounds retained because of higher affinity with the
stationary phase
30. 7. Chromatographic Development And Drying:
After development remove the plate and mobile phase is removed
from the plate –to avoid contamination of lab atmosphere.
Dry in vacuum desiccator
8. Detection and visualization:
Detection under UV light is first choice –non destructive spots of
fluorescent compounds can be seen at 254nm(near UV range)
31. The Liquid Chromatography-Mass Spectrometry (LC-MS) is combination of Liquid
Chromatography and Mass Spectrometry which is used with separation power of
HPLC with detection power of Mass Spectrometry
Basic Principle of LC/MS:
1. High Performance Liquid Chromatography (HPLC):
Liquid chromatography involves two main phases:
a) Mobile phase: Liquid (solvents i.e. ethanol, Acetonitrile).
b) Stationary phase: Column packed with very small particles
2. Mass Spectrometry:
Analytical technique that measures the mass to charge ratio of charged particles
Liquid Chromatography-Mass Spectrometry (LC-MS)
34. Instrumentation of MS:
1. Ion source- which can convert gas phase sample
molecules into ions.
Different methods of ionization are:
a) Electrospray Ionization:- the eluate
stream leaving the chromatographic
column is introduced into the ionization
source through the capillary.
At the outlet of the capillary, the sample
dissolved in the solvent is exposed to a
strong nebulizing gas (typically, nitrogen)
and a very strong electric field, which
results in the atomization of the sample
into charged microdroplets. Electrospray Ionization
35. b) Atmospheric Pressure Chemical Ionization:- the eluate leaving the
chromatographic column is heated and sprayed from the capillary and then
captured, in gaseous form, by a stream of gas and carried to the electrode where
ions are formed.
c) Atmospheric Pressure Photoionization:- uses photons to excite and ionize
molecules after nebulisation.
2. Mass Analyzer-
Its task is to separate ions in terms of their mass-to charge ratio and to direct
the beam of focused ions to the detector.
Different types of mass analyser are:
36. a) Quadrupole Analyzer:- consists of four
metal electrodes in the form of
symmetrically arranged rods.
It works like a mass filter with the
specific parameters of the
electromagnetic field, allows the
passage of ions with the selected
mass-to-charge ratio values.
b) Time-of-flight mass analyzer:- The
analyzer separates ions accelerated by
an electric field according to their
velocity which depends on their mass
and charge.
Quadrupole Analyzer
Time-of-flight mass analyzer
37. 3. Detectors-
The detector is used to count the ions emergent from the mass analyzer, and may
also amplify the signal generated from each ion. Following are three different
kinds of detectors are used in Mass Spectrometry:-
a) Electron Multipliers:- used to convert either –ve, +ve ions into electrons, that will
be amplified and detected.
b) Dynolyte Photomultiplier:- converts the charged ions into electrons. These
electrons stick to a phosphor and emit photons, and that photons are made to
strike the photomultiplier to achieve multiplied signals for recording.
38. CONCLUSION
Initially chromatographic techniques were used to separate substances based on
their color as was the case with herbal pigments
With time its application area was extended considerably.
Nowadays, chromatography is accepted as an extremely sensitive, and effective
separation method
These modern chromatographic techniques got attention in the field of
pharmaceutical, chemical, food, agrochemical industries, environmental ,forensic
applications, drug research and quality control
