This document provides information about chromatography techniques. It begins with an overview of chromatography and its inventor, Mikhail Tswett. It then describes several common chromatography methods including thin layer chromatography, paper chromatography, gas chromatography, ion exchange chromatography, and discusses their components and principles. Key terms like mobile phase, stationary phase, retention factor (Rf) are explained. Various chromatography applications and advantages are also mentioned.

1. Dr.A.DINESH KARTHIK
HEAD, P G & RESEARCH DEPT. OF CHEMISTRY
SHANMUGA INDUSTRIES ARTS & SCIENCE
COLLEGE,
TIRUVANNAMALAI-606603.

2. Is a technique used to separate and identify
the components of a mixture.
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.
Chromatography

3. Invention of Chromatography
Mikhail Tswett
Russian Botanist
(1872-1919)
Mikhail Tswett invented
chromatography in 1901
during his research on
plant pigments.
He used the technique to
separate various plant
pigments such as
chlorophylls, xanthophylls
and carotenoids.

4. Chromatography: (Greek = chroma “color” and
graphein “writing” ) Tswett named this new technique
chromatography based on the fact that it separated the
components of a solution by color.
Common Types of Chromatography
Tswett’s technique is based on Liquid Chromatography.
There are now several common chromatographic
methods. These include:
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid Chromatography (LC)
High Pressure Liquid Chromatography (HPLC)
Ion Chromatography
Gas Chromatography (GC)

5. Thin Layer Chromatography
Here the mobile phase is a liquid
Flowing past a thin layer of powder on a solid support.
Substances that are less attracted to the solid or are more
soluble in the liquid move faster.
And so move further up the plate by the time that the
process has been stopped by taking the plate out of the
liqiud. - larger Rf

6. THIN LAYER CHROMATOGRAPHY
Stationary Phase ---------> Silica Gel
Mobile Phase -------------> Solvent
(developing)

7. SOLVENT
SPOT
DEVELOPING
CHAMBER
Origin
Solvent
Front
1.1 cm
5.5 cm
Rf =
Distance from starting origin to center of zone
Distance from starting origin to solvent front
=
5.5
11
= 0.5
THIN LAYER CHROMATOGRAPHY

8. Paper and Thin Layer Chromatography
Later
The solvent moves up paper by capillary action,
carrying mixture components at different rates.
solvent
solvent
front

10. Rf = distance moved by substance
distance moved by solvent front
For substances that are very soluble in the liquid
Rf will be close to ....
For substances that are rather insoluble in the liquid
Rf will be close to ....
1
0

11. Comparing Chromatography to the
Flow of a River...
Base
Water flow
Light leaf
Heavy stone

12. How Does Chromatography Work?
In all chromatographic separations, the sample is transported
in a mobile phase. The mobile phase can be a gas, a liquid,
or a supercritical fluid.
The mobile phase is then forced through a stationary phase
held in a column or on a solid surface. The stationary phase
needs to be something that does not react with the mobile
phase or the sample.
The sample then has the opportunity to interact with the
stationary phase as it moves past it. Samples that interact
greatly, then appear to move more slowly. Samples that
interact weakly, then appear to move more quickly. Because
of this difference in rates, the samples can then be
separated into their components.

13. Mobile Phase / Stationary Phase
● A site in which a moving phase
(mobile phase) and a non-
moving phase (stationary
phase) make contact via an
interface that is set up.
● The affinity with the mobile
phase and stationary phase
varies with the solute. 
Separation occurs due to
differences in the speed of
motion.
Strong Weak
Mobile
phase
Stationary
phase

14. Gas Liquid Chromatography
Here the mobile phase is an unreactive gas ( eg
Nitrogen) flowing through a tube.
And the stationary phase is an involatile liquid
held on particles of a solid support.

15. Column Chromatography and Planar
Chromatography
Separation column
Packing material
Column Chromatography
Paper or a
substrate coated
with particles
Paper Chromatography
Thin Layer Chromatography (TLC)

16. Separation Process and Chromatogram for
Column Chromatography
Output
concentration
Time
Chromatogram

17. Chromatogram
tR
t0
Intensityofdetectorsignal
Time
Peak tR : Retention time
h
A
t0 : Non-retention time
A : Peak area
h : Peak height

18. Ion Exchange Chromatography
N+
R
R
R
SO3
-
+
+
+
++
++
+
+
+
Electrostatic interaction
(Coulomb force)
Anion exchange
Cation exchange

19. Stationary Phase Used in Ion
Exchange Mode
● Base Material
– Resin is often used.
– Silica gel is also used.
● Cation Exchange Column
– Strong cation exchange (SCX) -SO3
-
– Week cation exchange (WCX) -COO-
● Anion Exchange Column
– Strong anion exchange (SAX) -NR3
+
– Week anion exchange (WAX) -NHR2
+

20. Dependence of Exchange Capacity of Ion
Exchanger on pH of Eluent
Exchange
capacity
Exchange
capacity
pH
0 7 14
pH
0 7 14
Weakly acidic
cation
exchanger
Strongly acidic
cation
exchanger
Weakly basic
anion
exchanger
Strongly basic
anion
exchanger
Cation exchange mode Anion exchange mode

21. Ion Exclusion Chromatography
H+
H+
H+
Strong acid ions are repelled by
charge and cannot enter the
pore.
Depending on the level of
dissociation, some weak acid ions can
enter the pore.

22. Relationship between Retention Time and Salt
Concentration of Eluent in Ion Exchange Mode
Resin ResinResin
The exchange
groups are in
equilibrium with
anions in the eluent.
An eluent ion is
driven away
and a solute ion
is adsorbed.
The solute ion is
driven away by an
eluent ion and is
adsorbed by the next
exchange group.
If the salt concentration of the eluent increases, the solutes are eluted sooner.
Solute ions and eluent ions compete for ion exchange groups.

23. Gas Chromatography (GC)
*Gas chromatography is a chromatographic technique that
can be used to separate volatile organic compounds.
*It consists of
a flowing mobile phase
an injection port
a separation column (the stationary phase)
an oven
a detector.

24. In the animation below the red molecules are more soluble
in the liquid (or less volatile) than are the green molecules.

25. The organic compounds are separated due to
differences in their partitioning behavior between
the mobile gas phase and the stationary phase in
the column.
Principle

26. Mobile phases are generally inert gases such as
helium, argon, or nitrogen.
The injection port consists of a rubber septum
through which a syringe needle is inserted to inject
the sample.
The injection port is maintained at a higher
temperature than the boiling point of the least
volatile component in the sample mixture.

27. Since the partitioning behavior is dependent on
temperature, the separation column is usually
contained in a thermostat-controlled oven.
Separating components with a wide range of boiling
points is accomplished by starting at a low oven
temperature and increasing the temperature over time
to elute the high-boiling point components.
The more volatile (Low Boiling Point / Higher
Vapor Pressure) compounds arrive at the end of the
column first and pass into the detector

28. Components of a Gas Chromatograph
Gas Supply: (usually N2 or He)
Sample Injector: (syringe / septum)
Column: 1/8” or 1/4” x 6-50’ tubing packed with
small uniform size, inert support coated with
thin film of nonvolatile liquid
Detector: TC - thermal conductivity
FID - flame ionization detector

30. Oven
Detector
Injection
port
Nitrogen
cylinder
Column
Recorder

32. GC Theory
To
detector
Analyte
Column + packing
Time

33. Separations
To
detector
Time 1 Time 2 Time 3

35. GC Columns
Capillary columnsPacked columns
•Typically a glass or stainless
steel coil.
•1-5 total length and 5 mm
inner diameter.
• Filled with the st. ph. or a
packing coated with the st.ph.
•Thin fused-silica.
•Typically 10-100 m in length
and 250 mm inner diameter.
•St. ph. coated on the inner
surface.
•Provide much higher
separation eff.
•But more easily overloaded
by too much sample.

36. 1. Add about 1 tsp of dry/sifted Tide
to fill pipet within 1/4” of top
3. Use a plug of fiberfill
to hold Tide in place
(Tide has been sifted and dried,
so keep lid closed on container.)
2. Tap column with a pencil
to settle the powder.
Filling the Column
*Do not compact Tide into column.
**Do not leave any dead space at head of column.

37. A gas chromatography oven, open to
show a capillary column

38. GC Detectors
After the components of a mixture are separated using gas
chromatography, they must be detected as they exit the GC
column.
Thermal-conduc. (TCD) and flame ionization (FID) detectors
are the two most common detectors on commercial GCs.
The others are
1. Atomic-emmision detector (AED)
2. Chemiluminescence detector
3. Electron-capture detector (ECD)
4. Flame-photometric detector (FPD)
5. Mass spectrometer (MS)
6. Photoionization detector (PID)

39. GC Detectors Cont’d
The requirements of a GC detector depend on the separation
application.
E.g.
An analysis may require a detector selective for chlorine
containing molecules.
Another analysis might require a detector that is non-
destructive so that the analyte can be recovered for
further spectroscopic analysis. You can not use FID in
that case because it destroys the sample totally. TCD on the
other hand is non-destructive.

40. TCD Detector
A TCD detector consists of an electrically-heated wire.The
temperature of the sensing element depends on the thermal
conductivity of the gas flowing around it.
Changes in thermal conductivity, such as when organic
molecules displace some of the carrier gas, cause a
temperature rise in the element which is sensed as a change in
resistance.
The TCD is not as sensitive as other detectors but it is non-
specific and non-destructive.

41. ECD Detector
Uses a radiactive Beta emitter
(electrons) to ionize some of the
carrier gas and produces a current
between a biased pair of
electrodes.
When an org. mol. that contains
electornegative functional gr.,
such as halojens, phosphorous
and nitro groups, pass by the
detector, they capture some of the
electrons and reduce the current.

42. FID Detector
Consists of a hydrogen/air
flame and a collector plate.
The eff. from the GC
column passes through the
flame, shich breaks down org.
mol. and produces ions.
The ions are collected on a
biased electrode and produce
an elec. sig.
Extremely sensitive, large
dynamic range.

43. MS Detector
Uses the difference in mass-to-charge ratio (m/e) of ionized
atoms or molecules to separate them from each other.
Molecules have distinctive fragmentation patterns that provide
structural information to identify structural components.
The general operation of a mass spectrometer is:
1. create gas-phase ions
2. separate the ions in space or time based on their mass to
charge ratio
3. Measure the quantity of ions of each mass-to-charge ratio.

44. MS Detector Cont’d
The ion separation power of an MS is described by the
resolution:
R = m/Dm
Where m is the ion mass and Dm is the difference in mass
between two resolvable peaks in a mass spectrum.
E.g., an MS with a resolution of 1000 can resolve an ion with
a m/e of 100.0 from an ion with an m/e of 100.1.

45. GAS LIQUID CHROMATOGRAPHY
Principles
Partition of molecules between gas (mobile
phase) and liquid (stationary phase).

46. Gas-liquid chromatography (GLC)
Packed columns are fabricated from glass,
metal, or Teflon with 1 to 3 m length and 2 to
4 mm in internal diameter. The column is
packed with a solid support (100-400 mm
particle diameter made from diatomaceous
earth) that has been coated with a thin layer
(0.1-5 mm) of the stationary liquid phase.
Efficiency increases with decreasing particle
size as predicted from van Deemter equation.
The retention is based on absorption of
analyte (partition into the liquid stationary
phase) where solutes must have differential
solubility in the stationary phase

47. Open tubular capillary columns, either
WCOT, SCOT are routinely used. In WCOT
the capillary is coated with a thin film (0.1-
0.25 mm) of the liquid stationary phase while
in SCOT a thin film of solid support material
is first affixed to the inner surface of the
column then the support is coated with the
stationary phase. WCOT columns are most
widely used. Capillary columns are typically
made from fused silica (FSOT) and are 15 to
100 m long with 0.10 to 0.5 mm i.d.

48. The thickness of the stationary phase affects the
performance of the column as follows:
1. Increasing thickness of stationary phase allows the
separation of larger sample sizes.
2. Increasing thickness of stationary phase reduces
efficiency since HS increases.
3. Increasing thickness of stationary phase is better for
separation of highly volatile compounds due to
increased retention.

49. 49
Much more efficient separations can be achieved
with capillary columns, as compared to packed
columns, due to the following reasons:
1. Very long capillary columns can be used which
increases efficiency
2. Thinner stationary phase films can be used with
capillary columns
3. No eddy diffusion term (multiple paths effect) is
observed in capillary columns

50. Gas-solid chromatography (GSC)
Gas-solid chromatography is based upon adsorption
of gaseous substances on solid surfaces.
Distribution coefficients are generally much larger
than those for gas-liquid chromatography.
Consequently, gas-solid chromatography is useful
for the separation of species that are not retained
by gas-liquid columns, such as the components of
air, hydrogen sulfide, carbon disulfide, nitrogen
oxides, and rare gases.
Gas-solid chromatography is performed with both
packed and open tubular columns.

51.  Separation and analysis of organic compounds
 Testing purity of compounds
 Determine relative amounts of components in mixture
 Compound identification
 Isolation of pure compounds (microscale work)
 Similar to column chromatography, but differs in 3 ways:
 Partitioning process carried out between Moving Gas Phase and
Stationary Liquid Phase
 Temperature of gas can be controlled
 Concentration of compound in gas phase is a function of the vapor
pressure only.
 GC also known as Vapor-Phase Chromatography (VPC) and Gas-
Liquid Partition Chromatography (GLPC)
Gas Chromatography Application

52. SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
C
C
C
Detector Response
Retention Time
14
16
18 Peak Area (cm )
Sample Concentration (mg/ml)
2
4
6
8
10
0.5 1.0 1.5 2.0 2.5 3.0
2
The content % of C fatty acids =
C
C + C + C



= the content % of C fatty acids14
14

53. TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS
Response
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane
Decane
1.6 min = RT
Response
Unknown compound may be Hexane
1.6 min = RT
Retention Time on Carbowax-20 (min)

54. GLC ADVANTAGES
1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - ml
4. Good detection system
5. Quantitatively analyzed

55. Food and Cancer
● Chemicals that can cause cancer have a wide
variety of molecular structures and include
hydrocarbons, amines, certain drugs, some metals
and even some substances occurring naturally in
plants and molds.
● In this way, many nitrosamines have carcinogenic
properties and these are produced in a number of
ways such as cigarette smoke.
● GC can be used to identify these nitro-compounds
in trace quantities.

56. Drugs
● There are still numerous GC applications involving both
quantitative and qualitative identification of the active
components and possible contaminants, adulterants or
characteristic features which may indicate the source of
the particular sample.
● Forensic analysis frequently users GC to characterize
drugs of abuse, in some cases the characteristic
chromatographic fingerprint gives an indication of the
source of manufacture of the sample or worldwide
source of a vegetable material such as cannabis.
● Analytical procedures, chromatographic methods and
retention data are published for over 600 drugs, poisons
and metabolites. These data are extremely useful for
forensic work and in hospital pathology laboratories to
assist the identification of drugs.

57. DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250C without decomposition.
R C OH CH3OH H2SO4
O
R C O CH3
O
CH2 O C R
CH O C R
CH2 O C R
O
O
O
CH3OH
O
R C O CH3
CH3ONa
Fatty Acids Methylester
Reflux
+ 3
Volatile in Gas
Chromatography
Volatile in Gas
Chromatography
+ +

58. Dr.A.DINESH KARTHIK
dineshkarthik2008@gmail.com