2. CHROMATOGRAPHY
A laboratory technique in which the
components of a sample are separated
based on how they distribute between two
chemical or physical phases, one of which
is stationary and other of which is allowed
to travel through the separation system.
3. CHROMATOGRAPHY
Introduced first by the Russian botanist
Mikhail Semenovich Tswett.
Mixtures of solutes dissolved in a common
solvent are separated from one another by
a differential distribution of the solutes
between two phases.
4. PRINCIPLE
Fractionalism of mixtures of
substances
In the operation of the
chromatogram, a mobile gaseous or
liquid phase is use to wash the
substances to be separated through a
column of a porous material.
5. PRINCIPLE
The rate of migration of the solute
depends upon the rate of interaction
of the solute with the two phases, one
being the mobile phases and the
other stationary phase as the
compounds travel through the
supporting medium.
6. DEFINITION OF TERMS:
Capillary Action – the movement of
liquid within the spaces of a material
due to the forces of
adhesion, cohesion, and surface
tension.
Retention time
Peak
Viscosity
8. COMPONENTS OF A CHROMATOGRAPH
MOBILE PHASE – A phase that is flowing
through the column and causes sample
components to move toward the column’s end.
STATIONARY PHASE- A fixed phase that is
coated or bonded within the column; it always
remain in the system.
It is responsible for delaying the movement of
compounds as they travel through the column.
SUPPORT- onto which the SP is coated or
attached.
9. COMPONENTS OF A CHROMATOGRAPH
ORIGINAL SAMPLE
AND MOBILE PHASE
COLUMN
SUPPORT AND
STATIONARY
PHASE
Receiving
vessel
11. GAS CHROMATOGRAPHY
A type of Chromatography in which the
mobile phase is a GAS.
First GC system was developed by Erika
Cremer
The presence of a gas mobile phase makes
GC valuable for separating substances like
VOCs that occur naturally as gases that can
easily be placed into gaseous phase.
12. GAS CHROMATOGRAPHY
• It can separate nanograms or
pictograms of volatile substances.
• It is principally a method for the
separation and quantitative
determination of gases and volatile
liquids and substances.
13. GAS CHROMATOGRAPHY
HOW IS IT PERFORMED?
A System called Gas Chromatograph is used
to perform GC.
COMPONENTS:
GAS SOURCE
INJECTION SYSTEM
COLUMN
DETECTOR
14.
15. GAS CHROMATOGRAPHY
∞ GAS SOURCE- supplies the mobile phase. It is
typically a gas cylinder equipped with
pressure regulators to deliver the mobile
phase at a controlled state.
∞ INJECTION SYSTEM- consists of a heated
loop or port into which the sample is placed
and converted into a gaseous form.
16. GAS CHROMATOGRAPHY
∞ COLUMN- contains the stationary phase and
support material for the separation of
components in the sample. This column is
held in an enclosed area known as the column
oven.
∞ COLUMN OVEN- maintains the temperature
at a well-defined value.
∞ DETECTOR- monitors sample components as
they leave the column.
17. GAS CHROMATOGRAPHY
FACTORS THAT AFFECT GC:
Requirements for the Analyte
Volatility and Thermal Stability
Chemical Derivatization
19. GAS CHROMATOGRAPHY
GC SUPPORT MATERIALS
Packed Column
filled with small support particles that acts as an
adsorbent or that are coated with the desired stationary
phase.
Open- Tubular Columns
stationary phase coated on or attached to its interior
surface.
21. GAS CHROMATOGRAPHY
◊ Gas-Solid Chromatography
o Solid adsorbent is used as a stationary phase.
o Uses the same material as both the support and
stationary phase, with retention occurring through the
adsorption of analytes to the support’s surface.
o Example of support is a MOLECULAR SIEVE.
o Other supports include:
o ORGANIC POLYMERS - porous polystyrene
o INORGANIC SUBSTANCES – Silica or Alumina
22. GAS CHROMATOGRAPHY
o Increasing the surface area of the GSC support will
increase the phase ratio and result in higher retention for
analytes
o Pore size is important because only compounds smaller
than the pores are able to contact the surface are within
the space.
o Polarity of Support and its functional groups will also
affect how analytes will bind to them.
23. GAS CHROMATOGRAPHY
◊ Gas-Liquid Chromatography
o A chemical coating or layer is placed onto the support
and used as the stationary phase.
o Most Common types of GC.
o 100% dimethylpolysiloxane, 5%phenyl-95% methyl
polysiloxane – Examples of liquids that are used as
Stationary phase.
24. GAS CHROMATOGRAPHY
◊ Gas-Liquid Chromatography
o All of these liquids have High boiling points and low
volatilities, which allows them to stay within the
column at relatively high temperatures that are often
used in GC for sample injection and elution.
o Liquids are also wettable- easy to place onto a support
in a thin, uniform layer.
25. GAS CHROMATOGRAPHY
◊ Gas-Liquid Chromatography
o All of these liquids have High boiling points and low
volatilities, which allows them to stay within the
column at relatively high temperatures that are often
used in GC for sample injection and elution.
o Liquids are also wettable- easy to place onto a support
in a thin, uniform layer.
26. GAS CHROMATOGRAPHY
◊ Bonded Phases
o Column Bleed- most nonvolatile liquid will slowly
vaporize or break apart and leave the column over
time.
o Column bleed changes the retention characteristics of
the column.
o It can also cause some GC detectors to have a high
background and noisy signal as the stationary phase
leaves the column and enters the detector.
o Use of bonded phase minimize column bleed.
27. GAS CHROMATOGRAPHY
◊ Bonded Phases
o Produced by reacting groups on a polysiloxane
stationary phase with silanol groups that are
located on the surface of a silica support.
28. GAS CHROMATOGRAPHY
Types of Gas Chromatography Detectors
General Detectors
x Thermal Conductivity Detector
-used for both organic and inorganic compounds
-measures the ability of the eluting carrier gas and
analyte mixture to conduct heat away from hot-wire
filament-”thermal conductivity”.
-example: Wheatstone bridge
29. GAS CHROMATOGRAPHY
Flame Ionization Detector
- detects organic compounds by measuring
their ability to produce ions when they are burned
in flame.
30. GAS CHROMATOGRAPHY
Selective Detectors
x Nitrogen-Phosphorous Detector
- selective for the determination of nitrogen- or
phosphorous containing compounds.
- Similar to FID, but does not use a flame for ion
production.
31. GAS CHROMATOGRAPHY
Electron capture detector
- detects compounds that have electronegative
atoms or groups in their structure, such as halogen atoms
( I,Br,Cl and F) and Nitro Groups.
-can also be used to detect polynuclear aromatic
compounds, anhydrides and conjugated carbonyl
compounds.
32. GAS CHROMATOGRAPHY
Applications:
Most effectively used for analyses of organic
compounds, space related, complex mixtures of
volatile substances at column temperature of
less than -40 °C to greater than 550° C.
Geochemical research projects such as
determination of various environmental
pollutants at extremely low concentrations.
33. GAS CHROMATOGRAPHY
ADVANTAGES: DISADVANTAGES:
o Ability to provide qualitative o LIMITED to volatile
information and quantitative samples
information o Not suitable for
o FAST ANALYSIS thermally labile samples
o Efficient, providing high o Fairly difficult for large
resolution preparative samples
o Sensitive o Requires spectroscopy
usually mass
o Nondestructive
spectroscopy for
o Requires small samples confirmation of peak
o Inexpensive identity
34. LIQUID CHROMATOGRAPHY
√ A Chromatographic technique in which the
mobile phase is a liquid.
√ Originally developed by Russian botanist Mikhail
Tswett in 1903.
√ Its popularity is due to the ability of this method
to work directly with liquid samples, which
makes it valuable in such areas as food testing,
environmental testing and biotechnology.
35. LIQUID CHROMATOGRAPHY
HOW IS LIQUID CHROMATOGRAPHY
PERFORMED?
A System known as a Liquid Chromatograph is
used to perform LC.
36. LIQUID CHROMATOGRAPHY
√ Components of the LC System:
Support – enclosed in a Column
Stationary phase- enclosed in a Column
Liquid mobile phase-delivered to Column by means of
Pump
Injection device- on analytical applications it is being
used,to apply samples to the column.
Collection Device (optional)- placed after the column
to capture analytes as they elute.
37.
38. LIQUID CHROMATOGRAPHY
FACTORS THAT AFFECT LIQUID CHROMATOGRAPHY:
*requires both a difference in retention and good efficiency
for it to separate two given chemicals
*Sample
*Analyte Requirements
*Formats
*Role played by the Mobile phase
39. LIQUID CHROMATOGRAPHY
Requirements for the Analyte:
Must be possible to place this chemical
into a liquid that can be injected onto the
column.
There must be a difference in retention
between the analytes to be prepared.
41. LIQUID CHROMATOGRAPHY
1. ADSORPTION CHROMATOGRAPHY
A chromatographic technique that separates solutes
based on their adsorption to the surface of a support.
Also known as Liquid-Solid Chromatography
Equivalent method in GC is Gas-Solid Chromatography
Uses the same material as both stationary phase and
support.
Retention process can be explained on the ff below:
A+ n M-Surface A-Surface + n M
42. LIQUID CHROMATOGRAPHY
Elutropic strength- strength of a mobile
phase in adsorption chromatography
It is a measure of how strongly a particular
solvent or liquid mixture will absorb to the
surface of a given support.
Examples: silica and Alumina supports
A liquid with large elutropic strength will
strongly adsorb to the given support, which
will prevent the analyte from binding to the
support.
45. LIQUID CHROMATOGRAPHY
2. PARTITION CHROMATOGRAPHY
o It is a Liquid Chromatographic technique in
which solutes are separated based on their
partitioning between a liquid mobile phase and a
stationary phase coated on a solid support.
o Support used is usually Silica
o Originally, it involves coating of support with a
liquid stationary phase that was immiscible with
the mobile phase
46. LIQUID CHROMATOGRAPHY
Two Main Categories of Partition Chromatography:
• Normal-phase- uses polar stationary phase
• Reversed phase-uses nonpolar stationary phase
47. LIQUID CHROMATOGRAPHY
Applications:
Used in analytical laboratories
Use of NPLC for separating analytes in organic
solvents and chemicals that contain polar
functional groups.
48. LIQUID CHROMATOGRAPHY
3. ION- EXCHANGE CHROMATOGRAPHY
Solutes are separated by their adsorption
onto a support containing fixed charges on its
surface.
Routinely used in Industry for the removal or
replacement of Ions in products.
Used for the separation of charged compounds
( inorg. Ions, org. ions, AA, Proteins and Nucleic
Acids)
