1. Instrumentation of Column and
Gas Chromatography

2. Contents
• Column chromatography
• Principle
• Instrumentation
• Applications
• Procedure
• Gas chromatography
• Instrumentation
• Application
• references

3. Column chromatography
• Column chromatography is basically a type of
adsorption chromatography techniques.
• Here the separation of components depends
upon the extent of adsorption to stationary
phase.
• Here the stationary phase is a solid material
packed in a vertical column made of glass or
metal.

4. Principle
• When a mixture of mobile phase and sample to
be separated are introduced from top of
the column, the individual components of
mixture move with different rates.
• Those with lower affinity and adsorption to
stationary phase move faster and eluted out first
while those with greater adsorption affinity
move or travel slower and get eluted out last.

5. Instrumentation
Column chromatography consists of
• A packed, three-dimensional stationary phase
inside a glass, plastic, or metal column and can
be used for both preparative and analytical
purposes.

6. Procedure:
• The stationary phase material is suitably moistened
with mobile phase and packed sufficiently in the
column with a cotton or asbestos pad at the bottom.
• The extract material or sample to be separated is
placed on the top of packed stationary phase with a
second cotton or asbestos pad in between.
• The mobile phase is poured into the column over the
sample. A collecting beaker is placed at the bottom
of column near the end to collect the elute.

7. Classification
Column chromatography is therefore classified
according to the type of fluid flow system used:
• Gravity chromatography
• Low-pressure chromatography
• Medium-pressure chromatography (including
fast protein liquid chromatography)
• High-pressure/high-performance liquid
chromatography (HPLC)

8. Gravity Chromatography
• Gravity chromatography uses gravity to pass
sample and buffers across the column resin.
• Small-volume columns (~1.5 ml) designed for
quick flowthrough by spinning in a
microcentrifuge provide a convenient method
for rapidly purifying many small samples.

9. Applications:
• small-scale, rapid preparative chromatography
of nucleic acids, peptides, and proteins.

10. Low-Pressure Chromatography
• Low-pressure chromatography systems are
operated at less than 50 psi (0.35 MPa).
• These systems require a sample pump and are
often equipped with fraction collectors, gradient
capabilities, and detectors to monitor column
elution.

11. Applications:
• Medium-scale preparative chromatography of
native or recombinant proteins.

12. Medium-Pressure Chromatography
• Medium-pressure chromatography is conducted
at operating pressures that are actually rather
high, up to 3,500 psi (24 MPa).
• Medium-pressure chromatography systems
often include additional capabilities such as
column switching valves, advanced gradient
capabilities, and multi-wavelength detectors.

13. Applications:
• Preparative and analytical chromatography of a
wide variety of molecules, ranging from
nonvolatile organics to nucleic acids, peptides,
and proteins.

14. High-Pressure Liquid
Chromatography (HPLC)
• Conducted at very high pressures — up to 5,000
psi (34 MPa).
• HPLC is a powerful analytical tool providing
high resolution and sensitivity, with the ability
to detect concentrations down to parts per
trillion while having very small sample
requirements.

15. Instrumentation
• Solvent Reservoir : Solvent must be deaerated prior
to use either by boiling or by applying a partial
vacuum to the solvent reservoir.
• Pump : The pump used in HPLC is pulseless (to
avoid pressure changes) with adjustable flow-rate.
They may pump solvents from more than one
reservoir.
• Pre-column : It contains same stationary phase as
that in separating column. It is used to remove any
impurities present in the mobile phase, which may
contaminate stationary phase in separating column.

16. • Injector : The sample to be separated is
introduced into injector by the help of a syringe.
It contains a valve which in one position allows
mobile phase to directly enter the separating
column. While, in the other position, it passes
mobile phase through a loop containing sample
mixture. Mobile phase flushes sample mixture
into separating column.
• Separating Column : It is made up of stainless
steel with length ranging from 10-100 cm and
diameter ranging from 2-6 mm. Micro bore
columns, often glass-lined, with diameter of
1 mm or less and length of 25 cm are also used.
The column can be placed in a thermo stated
oven.

17. • Detector : After exiting the column, the eluate
enters a flow-through detector, where it is
continuously monitored.
• Amplifier : The electrical signal obtained from the
detector is amplified and routed to recorder by
amplifier.
• Recorder : The recorder records the developed
chromatogram.

18. Applications:
• Preparative and analytical chromatography of a
wide variety of molecules, ranging from
nonvolatile organics to nucleic acids, peptides,
and proteins.
• Bio-Rad does not manufacture HPLC systems,
but we do carry a full selection of HPLC
columns.

19. Precautions:
1. Keep the column in a clean and dust free place.
2. Do not disturb the column till the separation is
complete.
3. Avoid gaps within the stationary phase packing.

20. Applications
• Column chromatography is best suited to
separate active principle from plant materials.
• In separation of compounds after organic
synthesis to obtain desired molecule.
• To separate or purify natural compound
mixtures like alkaloids, glycosides.

21. instrumentation of Gas chromatography

22. Gas Chromatography
• In gas chromatography, the components of a
sample are dissolved in a solvent and vaporized
in order to separate the analytes by distributing
the sample between two phases:
• a stationary phase and
• a mobile phase.

23. • The mobile phase is a chemically inert gas that
serves to carry the molecules of the analyte
through the heated column.
• The stationary phase is either a solid adsorbant,
termed gas-solid chromatography (GSC), or a
liquid on an inert support, termed gas-liquid
chromatography (GLC).

24. Instrumentation
Consist of
• Sample Injection
• Carrier Gas cylinder with pressure regulator
• Column Oven
• Open Tubular Columns and Packed Columns
• Detection Systems

25. Sample Injection
• A sample port is necessary
for introducing the sample
at the head of the column.
• A calibrated microsyringe is
used to deliver a sample
volume in the range of a few
microliters through a rubber
septum and into the
vaporization chamber.

26. Carrier Gas
• The carrier gas plays an important role, and
varies in the GC used.
• Carrier gas must be dry, free of oxygen and
chemically inert mobile-phase employed in gas
chromatography.
• Helium is most commonly used because it is
safer than, but comprable to hydrogen in
efficiency, has a larger range of flow rates and is
compatible with many detectors.

27. Gas cylinder with pressure regulator
• A pressure regulator is used to control the
amount of gas to be passed to column.

28. Column Oven
• The thermostated oven serves to control the temperature of
the column within a few tenths of a degree to conduct precise
work.
• The oven can be operated in two manners:
• isothermal programming
Or
temperature programming.
• In isothermal programming, the temperature of the column is
held constant throughout the entire separation.
• The optimum column temperature for isothermal operation
is about the middle point of the boiling range of the sample.

30. Open Tubular Columns and Packed
Columns
• Open tubular columns, which are also known as
capillary columns, come in two basic forms.
• The first is a wall-coated open tubular (WCOT)
column and
• The second type is a support-coated open
tubular (SCOT) column.

32. Detection Systems
• The detector is the device located at the end of
the column which provides a quantitative
measurement of the components of the mixture
as they elute in combination with the carrier
gas.

33. Types of Gas Chromatography
Detectors
Non-selective
• Responds to all compounds present in carrier
gas stream except the carrier gas itself
Selective
• Responds to range of compounds with a
common physical or chemical characteristic
Specific
• Responds to a single specific compound only

34. • Detectors can also be grouped into
concentration or mass flow detectors
• Concentration Dependent
• The response of such Gas
Chromatography detectors is proportional to the
concentration of the solute in the detector such
as TCD. Dilution of sample with makeup gas will
lower detector response.
• Mass Flow Dependent
• Signal is dependent on the rate at which solute
molecules enter the detector such as FID.
Response of such detectors is not affected by
makeup gas flow rate changes.

35. Desirable characteristics of
detectors
• Reproducible response to changes in eluent
composition in carrier gas stream
• High sensitivity
• Large linear dynamic range
• Low noise
• Small volume to avoid peak broadening and
resultant loss of resolution
• Preferably non – destructive

36. Common Gas
Chromatography detectors

37. Flame Ionization Detector (FID)
• Mass sensitive detector
• Response depends on
conducting power of ions or
electrons produced on burning
of organic compounds in the
flame
• Selective detector but sample
detected must be combustible
• Large linear dynamic range
(107)
• No response to inorganic
and permanent gases such
as CO, CO2, NH3, CS2, N2,
etc.
• It is the most widely used
detector in Gas
Chromatography

38. Thermal Conductivity Detector (TCD)
• Non-destructive universal detector
• Response depends on the thermal conductivity
difference between the carrier gas and the eluted
components
• Wide dynamic range (107 – % to ppm levels)
• Responds also to inorganic gases such as CO,
CO2, NH3, CS2, N2, etc.

39. Advantages of TCD
• Sample is not wasted
• Easy to operate

40. Applications
• Applications
• Gas chromatography is a physical separation method in where
volatile mixtures are separated.
• It can be used in many different fields such as
pharmaceuticals, cosmetics and even environmental toxins.
• Since the samples have to be volatile, human breathe, blood,
saliva and other secretions containing large amounts of
organic volatiles can be easily analyzed using GC.
• Knowing the amount of which compound is in a given sample
gives a huge advantage in studying the effects of human
health and of the environment as well.
• Air samples can be analyzed using GC.
• GC/MS is also another useful method which can determine
the components of a given mixture using the retention times
and the abundance of the samples.

41. REFERENCES
• www.chemwiki.ucdavis.edu.com
• www.lab-training.com
• www.bio-rad.com
• www.bheem.hubpages.com
• Modern Practice of Gas Chromatography
edited by Robert L. Grob, PhD, Eugene F. Barry,
PhD page no 37
• Liquid Column Chromatography: A Survey of
Modern Techniques and Applications
edited by K. Macek, Z. Deyl, J. Janák page no 57