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1
Gas
Chromatography
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
2
 Chromatography-Russian Botanist Mickel Tswett
(1903)
 German Ph D student Fritz Prior (student of Erika
Cremer, University of Innsbruck, Austrelia)developed
Solid state gas chromatography in 1947.
 Archer P.J. Martin(1910-2002) won noble prize in
developing Liquid-liquid (1914) and Paper
(1944)chromatography.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
3
 GC is the common type of chromatography used in
analytical chemistry for separating and analyzing
compounds that can be vapourized without
decomposition.
 In GC mobile phase is carrier gas usually inert gas and
stationery phase is microscopic layer of liquid or
polymer on an inert solid support, inside a piece of
glass or metal tubing called a column.
 The instrument used for performing the gas
chromatography is called gas chromatogram.
 Similar to column chromatography
 Fractional distillation
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Classification based on types of
the stationary and mobile phase
 Gas chromatography
Mobile phase - gas
 Liquid chromatography
Mobile phase - liquid
 Supercritical fluid chromatography
Mobile phase – super critical fluid
LC- performed in Column & planar surfaces
GC&SFC- performed only in Column
4
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Classification of Chromatographic methods
S.No. General
Classificatio
n
Specific
method
Stationary
Phase
Type of
Equilibrium
1
Liquid
Chromatogra
phy (LC)
(Mobile
phase:Liquid)
Liquid- liquid,
or partition
Liquid adsorbed
on a solid
Partition
between
immiscible
liquids
Liquid –
bonded phase
Oraganic species
bonded to a
solid surface
Partition
between liquid
and bonded
surface
Liquid-solid,
or adsorption
Solid Adsorption
Ion exchange Ion –exchange
resin
Ion exchange
size exclusion Liquid in
interstices of a
polymer solid
Partition/sieving
5
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
S.N
o.
General
Classification
Specific
method
Stationary
Phase
Type of
Equilibrium
2
Gas chromatography
Gas- liquid Liquid
adsorbed on a
solid
Partition
between gas
and liquids
Gas- bonded
phase
Organic
species
bonded to a
solid surface
Partition
between liquid
and bonded
surface
Gas- solid solid Adsorption
3 Supercritical – fluid
chromatography
(SFC)
Mobile phase:
supercritical fluid
Organic
species
bonded to a
solid surface
Partition
between
super critical
fluid and
bonded
surface
6
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
S.No. Technique Stationary phase Mobile phase
1
Column Chromatography
or Adsorption
chromatography
Solid liquid
2 Partition chromatography liquid liquid
3 Paperchromatography liquid liquid
4
Thin Layer
chromatography
Liquid orsolid liquid
5
Gas- liquid
chromatography
liquid Gas
6
Gas –solid
chromatography
Solid gas
7
Ion
exchangechromatography
solid liquid
Types of Chromatographic techniques
7
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
S.No. Technique Stationary phase Mobile phase
1 Paper chromatography
Solid liquid
2 Plain chromatography
3 Thin Layer chromatography
4 Adsorption column
chromatography
5 High Performance Liquid
6 Ion exchange chromatography Solid (Ion exchange
resin)
Liquid
7 Gas- solid chromatography Solid Gas
8 Gel permeation chromatography
(Exclusion chromatography)
Solid matrix Liquid
9 Gas-Liquid chromatography Liquid Gas
10 Liquid-Liquid chromatography liquid liquid
Classification of chromatographic methods
8
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
Chromatography is based on a physical equilibrium
that results when a solute is transferred between the
mobile and a stationary phase.
A
A
A
A
A
A
A
A
A
A
A
A
K = distribution
coefficient or
partition ratio
K =
CS
CM
Where CS is the molar
concentration of the solute in
the stationary phase and CM is
the molar concentration in the
mobile phase.
Cross Section of Equilibrium in a
column.“A” are adsorbed to the
stationary phase.“A” are traveling in
the mobile phase.
9
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Flow
As a material travels through the column, it assumes a Gaussian
concentration profile as it distributes between the stationary
packing phase and the flowing mobile gas or liquid carrier phase.
In a chromatography column, flowing gas or liquid continuously
replaces saturated mobile phase and results in movement of A
through the column.
Column is packed
with particulate
stationary phase.
10
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Flow
Flow
Flow
Flow
In a mixture, each component has a different distribution coefficient, and thus
spends a different amount of time absorbed on the solid packing phase vs being
carried along with the flowing gas
More volatile materials are carried through the column more rapidly than less
volatile materials, which results in a separation.
11
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Time spended of analyte in the stationary phase relating to
the mobile phase
The retention factor: k’
k’: relative number of
moles of analytes in the
stationary and mobile
phase
k’ = nS/nM
12
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
13
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Column efficiency and band broadening
The plate theory of chromatography
One theoretical plate (N): the part of the column, where
quasi-equilibrium takes place between stationary and mobile
phase
2
L
2
2
2
t
R
σ
L
σ
t
N =
=
54
,
5
16
2
=






=
w
t
N R
2
2
/
1






w
tR
Where:  standard deviation and 2
Variance
w = 4 
14
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Gauss equation:
HETP: Height equivivalent to the theoretical plate
(H)
N
L
H =
15
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
16
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
The rate theory of chromatography (van Deemter)
A

p
ed
C
Porous silica particle
particle size (diameter): dP
Theory of band broadening
1. Eddy diffusion term (A)
multiple path effects
17
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
u
B

u
D
C M
d
2.The longitudal diffusion term (B/u)
18
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
u
CM

M
2
p
M
D
u
d
C
u
CS

M
2
p
M
S
D
u
d
C
3. Mobile phase mass transfer term (CM/u)
4. Stationary phase mass transfer term (CS/u)
19
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
u
C
u
u
A S



= M
C
B
H
H
u
The van Deemter equation of chromatography
The equation has an optimum (Hopt) where the column efficiency is
highest.
This optimum has been found at a linear velocity:
for gas chromatography at.0.1 – 0.5 cm/s
for liquid chromatography at: 1.0 – 5.0 cm/s 20
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
'
k
1
'
k
α
1
α
N
4
1
R
2
2
2
S


=
Effect of increase of retention factor on resolution
How to increase retention factor:
•By decreasing eluent strength
21
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
 Good for volatile samples (up to about 250 oC)
 0.1-1.0 microliter of liquid or 1-10 ml vapor
 Can detect <1 ppm with certain detectors
 Can be easily automated for injection and data
analysis
Gas Chromatography
22
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
23
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
Types of Gas Chromatography
 Gas solid chromatography
 Gas liquid chromatography
 Gas bonded phase chromatography
24
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
25
Which factors influence the
separation of the components?
 Vapor pressure
 The polarity of components versus the
polarity of stationary phase on column
 Column temperature
 Carrier gas flow rate
 Column length
 Amount of material injected
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
26
Vapor pressure
 Pressure exterted by a vapor in thermodynamic equilibrium with its
condensed phase(solid or liquid) at a given temperature in a closed
system.
 The boiling point of a compound is often related to its polarity.
 The lower the boiling point is, the higher the vapor pressure of the
compound and the shorter retention time usually.
 That is one of the main reasons why low boiling solvents (i.e., diethyl
ether, dichloromethane) are used as solvents to dissolve the sample.
 The temperature of the column does not have to be above the boiling
point because every compound has a non-zero vapor pressure at any
given temperature, even solids.
 That is the reason why we can smell compounds like camphor (0.065
mmHg/25 oC), isoborneol (0.0035 mmHg/25 oC), naphthalene (0.084
mmHg/25 oC), etc. However, their vapor pressures are low compared
to liquids (i.e., water (24 mmHg/25 oC), ethyl acetate (95
mmHg/25 oC).
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
27
The polarity of components versus the polarity
of stationary phase on column
 If the polarity of the stationary phase and compound are similar, the
retention time increases because the compound interacts stronger with the
stationary phase. As a result, polar compounds have long retention times
on polar stationary phases and shorter retention times on non-polar
columns using the same temperature.
 Chiral stationary phases that are based on amino acid derivatives,
cyclodextrins and chiral silanes are capable of separating enantiomers
because one enantiomer interacts slightly stronger than the other one with
the stationary phase, often due to steric effects or other very specific
interactions.
 For instance, a modified -cyclodextrin column is used in the
determination of the enantiomeric excess in the chiral epoxidation
experiment .
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
28
Column temperature
 A excessively high column temperature results in very short
retention time but also in a very poor separation because all
components mainly stay in the gas phase.
 However, in order for the separation to occur the components
need to be able to interact with the stationary phase. If the
compound does not interact with the stationary phase, the
retention time will decrease.
 At the same time, the quality of the separation deteriorates,
because the differences in retention times are not as
pronounced anymore.
 The best separations are usually observed for temperature
gradients, because the differences in polarity and in boiling
points are used here.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
29
Carrier gas flow rate
 A high flow rate reduces retention times, but a poor separation
would be observed as well. Like above, the components have
very little time to interact with the stationary phase and are
just being pushed through the column.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Column length
 A longer column generally improves the separation.
 The trade-off is that the retention time increases
proportionally to the column length and a significant
peak broadening will be observed as well because of
increased longitudinal diffusion inside the column.
 One has to keep in mind that the gas molecules are not
only traveling in one direction but also sideways and
backwards.
 This broadening is inversely proportional to the flow
rate. Broadening is also observed because of the finite
rate of mass transfer between the phases and because
the molecules are taking different paths through the
column.
30
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
31
 Ideally, the peaks in the chromatogram display a symmetric
shape (Gaussian curve).
 If too much of the sample is injected, the peaks show a
significant tailing, which causes a poorer separation.
 Most detectors are relatively sensitive and do not need a lot
of material in order to produce a detectable signal. Strictly
speaking, under standard conditions only 1-2 % of the
compound injected into the injection port passes through the
column because most GC instruments are operated in split-
mode to prevent overloading of the column and the detector.
 The split less mode will only be used if the sample is
extremely low in concentration in terms of the analyte.
Amount of material injected
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Instrumentation
 Carrier gas
 Sample injection system
 The separation column
 Detectors
 Thermo stated chambers for the temperature regulation
of column and detectors
 Amplifier
 recorder
32
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
33
Carrier gas
 The carrier gas (mobile phase) for gas chromatography
should be an inert gas that does not react with the sample
components.
 The GC carrier gas should contribute minimally to the
partitioning process. This differs from the mobile phase
in liquid chromatography.
 In GC the carrier gas is simply stated as just a carrier to
transport the vaporized solute molecules through the
column during the partitioning process.
 Carrier gases are compressible gases that expand with
increasing temperature. This results in a change in the
gas viscosity.
 The selection and linear velocity of the carrier gas will
affect resolution and retention times.
 Carrier gases should be inert to the stationary phase and
free of detectable contaminants.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Efficiency Effects of Carrier Gases
 Carrier gas linear velocity plays a significant role in
the resulting efficiency of a chromatographic system.
 The optimal carrier gas linear velocity is
characteristic for each gas.
 Van Deemter curves and Golay Plots are used to
demonstrate optimal carrier gas linear velocities.
 Van Deemter plots are used for packed columns
since the A term for eddy diffusion is present.
 Golay plots are used for capillary columns (open
tubular) and the A term is dropped.
34
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Retention Time Effects
Analysis can be either isothermal or temperature
programmed runs in GC. The carrier gas linear
veloctiy can influence the overall time of analysis and
the efficiency of the separations. The following
equation demonstrates the affect of linear velocity on
retention time.
t r = L (k+1) µ
where
t r = retention time
L = column length
k = retention factor
µ = carrier gas linear velocity
35
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
LinearVelocity/Flow Control
 Linear velocity or flow can either be run in a
constant pressure or constant flow mode.
 This is important in temperature programmed
analyses. In the constant pressure mode, the
column head pressure is set at a temperature and
the pressure remains constant throughout the
analysis. This can result in linear velocity changes
especially over a wide temperature programming
range.
 In the constant flow mode, the column head
pressure will change throughout a temperature
programmed analysis in order to keep the carrier
gas flow at a constant flow rate.
36
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Carrier GasViscosity
 Carrier gas viscosity is a temperature
dependent parameter.
 As temperature increases, the viscosity of
the gas increases.
 When using a constant pressure mode for
carrier gas and temperature programming,
the viscosity of the gas will increase and
the average linear velocity will decrease.
37
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Detectors
 Flame Ionization Detector (FID)
 Atomic Emission Detector(AED)
 Nitrogen Phosphorus Detector (NPD)
 Electron Capture Detector (ECD)
 Sulfur Chemiluminescence Detectors(SCD)
 Thermal Conductivity Detector (TCD)
 Thermoionic Detector(TID)
 Flame Photometric Detector (FPD)
 Photoionization Detector (PID)
 Mass Spectrometer (MS)
38
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
FLAME IONIZATION DETECTOR
39
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
40
Construction:
The effluent from the column is mixed with hydrogen and air and
then ignited electrically. Most organic compounds, when pyrolyzed
at the temperature of a hydrogen/air flame, produce ions and
electrons that can conduct electricity through the flame.
 The number of ions produced is propositional to number of
reduced carbon atoms in the flame.
 Mass- sensitive rather than concentration sensitive.
 Advantages:
 High sensitive (~10-13g/s), large linear response range, (~107) and
low noise.
 Analysis of most organic samples including those that are
contaminated with water and the oxides of nitrogen and sulfur.
Disadvantages:
 Carbonyl, amine, alcohol, halogen yields few ions or none in the
flame.
 Insensitive toward noncombustible gases such as H2O,CO,SO2 and
NO .
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
THERMAL CONDUCTIVITY
DETECTOR
Thermal conductivity detector cell and
arrangement of two sample detector cells and
two reference detector cells
41
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
 Also known as Katherometer.
Construction:
 An electrically heated element whose temperature at
constant electrical power depends upon the thermal
conductivity of the surrounding gas.
 The heating element –fine platinum, gold or tungsten wire or
alternatively, a semiconducting thermistor.
 The resistance of the wire or thermistor gives a measure of
the thermal conductivity of the gas, in contrast to the wire
detector, the thermistor has a negative temperature
coefficient.
 Two pairs of elements are employed, one pair being located
in the flow of the effluent from the column and the other in
the gas stream ahead of the sample injection chamber.
 The effect of thermal conductivity of carrier gas is canceled,
flow rate, pressure and electrical power are minimized.
42
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
 The resistances of the twin detector pairs are compared
by incorporating them into two arms of a simple
Wheatstone bridge.
 The thermal conductivities of the hydrogen and helium are
roughly six to ten times greater than those of most
amounts of organic materials, a relatively large decrease in
the thermal conductivity of the column effluent takes
place.
Advantages:
 Simple, large linear dynamic range (~105),non destructive,
general response to organic and inorganic species.
 Analysis of permanent gases such as argon, oxygen,
nitrogen and carbon dioxide
Disadvantages:
 Low sensitivity (~10-8solute/mL carrier gas).
43
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
PHOTOIONIZATION DETECTOR
 The column eluent is
irradiated with intense
beam of ultraviolet
radiation varying in
energy from 8.3 to
11.7ev, which causes
ionization of the
molecules.
 Application of a
potential across a cell
containing the ions
leads to an ion current,
which is amplified and
recorded.
44
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
45
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
FLAME PHOTOMETRIC DETECTOR
FLAME PHOTOMETRIC DETECTOR
 To analysis of air and water pollutants, pesticides
and coal hydrogenation products.
 Compounds containing sulfur and phosphorus.
 The eluent is passed into a low- temperature
hydrogen/air flame, which converts part of the
phosphorus to an HPO species that emits bands
of radiation centered about 510 to 526pm.
 Sulfur in the sample is simultaneously converted
to S2, which emits a band centered at 394nm.
Advantages:
 Halogens, nitrogens and several metals such as in
tin, chromium, selenium and germanium.
46
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
47
ELECTRON CAPTURE DETECTOR
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
48
 Detects halogen containing compounds such as
pesticides and polychlorinated biphenyls.
Construction:
 The effluent from the column is passed over a β
emitter, usually nickel-63.
 An electron from the emitter causes ionization of the
carrier gas (often nitrogen) and the production of a
burst of electrons.
 In absence of organic species, a constant standing
current between a pair of electrodes results from this
ionization process.
 The current decreases markedly, however, in the
presence of those organic molecules that tend to
capture electrons.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
49
 Highly sensitive to molecules containing electronegative
funtional groups such as halogens, peroxides, quinones,
organometallic, nitrile and nitro compounds.
 Detection and determination of chlorinated insecticides.
Advantages:
 Highly sensitive, not alters the samples
Disadvantages:
 Linear response range is limited to about two orders of
magnitude.
 Insensitive to functional groups such as amines, alcohols
and hydrocarbons.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
50
SULFUR CHEMILUMINESCENCE
DETECTORS
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
SULFUR CHEMILUMINESCENCE
DETECTORS
 The reaction between sulfur containing
compounds and ozone.
 The luminescence intensity is proportional to
the concentration of sulfur.
 The eluent is mixed with hydrogen and air and
is burned as in FID.
 The resulting gases are then mixed with ozone
and the intensity of the resulting emission is
measured.
 Also used for Super critical fluid
chromatography.
51
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
52
Atomic Emission Detectors
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
53
 The eluent is introduced into a microwave-energized
helium plasma that is coupled to a diode array optical
emission spectrometer.
 The plasma is suffienciently energetic to atomize all of
the elements in a sample and to excite their
characteristic atomic emission spectra.
 These spectra are then observed with a spectrometer
that employs a movable, flat diode array capable of
detecting emitted radiation from about 170 to 780nm.
 The position able diode array is capable of monitoring
simultaneously two to four elements at any given setting.
 At the present time, the software supplied with the
detector allows measurement of the concentration of 15
elements.
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
54
Nitrogen-phosphorus detectors
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
55
 It is selective toward organic compouds containing
phosphorus and nitrogen.
 Its response to phosphorus atom is approximately ten times
greater than to a nitrogen atom and 104 to 106 larger than a
carbon atom.
 Compared to FID it is 500times more sensitive for
phosphorus containing compounds, 50times more sensitive
Construction:
 The column effluent is mixed with hydrogen, passes through
the flame tip assembly, and is ignited.
 The hot gas then flows around an electrically heated
rubidium silicate bead, which is maintained at about 180V
with respect to the collector.
 The heated bead forms a plasma having a temperature of
600°C to 800°C.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Sensitivity of Gas Chromatography
S.
No
.
detector Sensiti
vity
Line
ar
rang
e
comments
1 Thermal conductivity 10-8 104 Universal sensitivity, non-
destructive
2 Flame ionization 10-11 106 Detects all organic compounds
,destructive
3 Electron capture 10-13 102 Detect halo-,nitro- & phosphorus
compounds, non destructive
4 Flame Emission 10-6
103
Sulphur & phosphorus
compounds, destructive
5 Argon ionization 10-12 105 Universal, argon carrier gas
necessary, non-destructive
56
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Support materials and stationary
phases
 The solid support should ideally have large surface area
(atleast 1m2/g) has a good mechanical stability, thermally
stable, inert surface in order to simplify retention
behavior and prevent solute adsorption, has a particle
size in the range of 100-400mm.
 Example: diatomaceous earth, glass beads with suitable
mesh,(80-100mesh,100-120mesh).
 A liquid stationary phase should be inert to analyte, less
volatile and thermally stable.
 In general, the polarity of the stationery phase should
match that of the sample constituents (like dissolves
like).Most stationery phases are based on
polydimethylsiloxane or polythylene glycol (PEG) back
bones.
57
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
58
 The polarity of the stationary phase can be
changed by derivatization with different functional
groups such as a phenyl group. Bleeding of the
column is cured by bonding the stationary phase
to the column; or cross linking the stationery
phase.
 The liquid stationary phases should have the
following characteristics.
 Low volatility, high decomposition temperature
(thermally stable),chemically inert (reversible
interactions with solvents), chemically attached to
support ( to prevent bleeding),appropriate k’ and
a for good resolution
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Stationary phase
 Low volatility
 Thermal stability
 Chemical inertness
 Solvent characteristics
 Polar stationary phases --- –CN,-CO and –OH. non
polar ---Hydrocarbon- type stationary phases and
dialkyl siloxanes
 highly polar -- polyeterphases
 Solutes-polar- alcohol,acids,amines
Medium polarity-ethers, ketones, aldehydes
Non polar-saturated hydrocarbons
59
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
60
Columns
 The column in chromatography is the heart of the
technique.
 A column can either be packed or open tubular.
 Open-WCOT-Wall-coated open tubular tubes
SCOT-Support-coated open tubular tubes
FSOT-Fused – silica open tubular tubes
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
61
Packed columns
 These columns are fabricated from glass,
metal(copper, stainless steel, aluminum) or Teflon
tubes.
 Stainless steel is the most widely used because it is
most inert and easy to work with.
 Length 2 to 3m,inside diameter is 2 to 4mm.
 These tubes are densely packed with a uniform,
finely divided packing material, or solid support,
that is coated with a thin layer (0.05 to1µm) of the
stationary liquid phase.
 In order to fit in a thermostatic oven, the tubes are
formed as coils having diameters of roughly 15cm.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
62
Open tubular / capillary columns
These are mainly two types.
 Wall- coated open tubular (WCOT) < 1mm capillary tubes
coated with a thin layer of the stationary phase.
 Olden days-stainless steel, aluminum, copper or plastic.Then glass.
Often glass etched with gaseous HCl, strong aqueous HCl, or
Potassium hydrogen fluoride to give rough surface, which bonded
the stationary phase more tightly.
 Support-coated open tubular (SCOT) ~30µm thick coated
support on inside of silica tube.
 The most frequently used capillary column, nowadays, is the fused
silica open tubular column (FSOT), which is aWCOT column.
 The external surface of the fused silica columns is coated with a
polyimide film to increase their strength.(internal diameter 320
and 260µm)
 For, high resolution 200 and 150µm.
 Megabore columns-530µm
 Solid Support material-diatomaceous earth.
 Particle size of support- effeciency inversely proportional to
particle diameter.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Properties and characteristics of typical gas-
chromatographic columns
63
Type of column
FSOT WCOT SCOT Packed
Length,m 10-100 10-100 10-100 1-6
Inside
diameter,mm
0.1-0.53 0.25-0.75 0.5 2-4
Efficiency,
plates/m
2000-4000 1000-4000 600-1200 500-1000
total plates (20-400)*103 (10-400)*103 (6-120)*103 (1-10)*103
Sample size,ng 10-75 10-1000 10-1000 10-106
Relative back
pressure
Low Low Low high
Relative speed Fast Fast fast slow
Chemical
inertness
Best Good poor Poorest
Flexible yes No No no
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Hyphenated techniques
 Gas chromatography/Mass Spectrometry
(GC/MS)
 Gas chromatography/Fourier Transform
Infrared Spectroscopy(GC/FTIR)
64
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
65
Gas chromatography/Mass
spectrometry
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
66
Gas chromatography/Fourier Transform
Infrared Spectroscopy(GC/FTIR)
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
67
Derivatization
 Derivatization is the process of chemically modifying a
compound to produce a new compound which has
properties that are suitable for analysis by using GC or
HPLC.
 The chemical structure of the compound remains the same
and just modifies the specific functional groups of reacting
compound to derivative of derivative chemical or physical
properties in order to make them detectable and analyzable.
 Derivatization is needed in GC,HPLC, UV-Visible
spectroscopy.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
68
WHY derivatization
 To permit analysis of compounds which are not directly
amenable to analysis due to its stability and volatility.
 To improve chromatographic behaviour detect ability.
Many compounds do not produce a useable
chromatography or the sample of interest goes undetected.
As a result it may be necessary to derivatize the compound
before GC analysis is done.
 The main reason for derivatizing is to impart volatility to
non volatile compounds.
Derivatization is useful to allowing the
use of GC &GC/MS to be done on sample that would
otherwise not possible in various areas of chemistry such
as medicinal, forensic and environmental.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
69
Ideal characters and disadvantages of
derivatization
 A derivatization reaction should be rapid, quantitative and
produce minimal by product. Excess reagent should not
interfere with the analysis and should be easily removed.
 Derivatization often is a last resort when developing a
method. Introduction of a reaction pre or post column
increases complexity, chance of error, and total analysis
time.
 Care should be taken that the reaction is quantitative and no
additional impurities are introduced into analysis.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
70
What derivatization accomplish ?
 Increases volatility (i.e. sugars)
Eliminates the presence of polar OH,NH & SH
groups.
Derivatization targets O,S,N and P functional
groups ( with hydrogen availability)
 Enhance sensitivity to ECD. The introduction of ECD
detectable groups, such as halogenated acyl groups,
allows detection of previously undetectable groups.
 Increases detectability i.e. steroids
 Increases stability (thermo stability)
 To reduce adsorption of polar samples on active
surfaces of column walls and solid support.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
71
Conditions for choosing a
derivatizing agent
 The derivatizing agent must be stable.
 The derivatizing agent and its products formed during
derivatization should not be detectable or must be
separable from analyte.
 The analyte should be reactive with derivatizing agent
under convenient conditions.
 If possible, it should non-toxic.
 The procedure should be adaptable to automation.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
72
Types of derivatization
 Silylation
 Alkylation
 Acylation
 Chiral derivatization
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
73
Silylation
 Most prevalent method, readily volatizes the sample.
 Mechanism:
 The process produces silyl derivatives which are
more volatile, more thermally stable.
 Replaces active hydrogens with TMS (Trimethyl
silyl groups)
 Silylation occurs nucleophilic attack (SN2). The
better the leaving group, the better the silylation.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
74
Solvents and precautions :
 Silylation reagents will react with water & alcohols. First
care must be taken to ensure that both solvents and sample
are dry.
 Solvents should be pure as possible. This will eliminate
excessive peaks. Try to use little solvent as possible this
will prevent a large solvent peak.
 Pyridine is most commonly used solvent. Although
pyridine may produce peak tailing it is an acid scavenger &
will drive the reaction forward.
 In many cases, the need for a solvent is eliminated with
silylating reagents (if a sample readily dissolves in the
reagent, it usually is a sign the derivatization is complete.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
75
 Ease of reactivity of functional group toward silylation
follows the order:
 Alcohol> Phenol> Carboxyl> Amine> Amide>Hydroxyl
The order of alcohol is 1>2>3
Care needs to be taken not to inject silylating reagent onto
column which have active hydrogen in the stationary
phase, because they will be derivatized.
Eg: column not compatible with silylating reagents are
carbowax &free fatty acid phase
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
Silylation- advantages and
disadvantages
Advantages :
 Ability to silylate a wide variety of compounds
 Large number of silylating reagents are available.
 Easily preapred
Disadvantages:
 Silylating reagents are moisture sensitive.
 Must be aprotic organic solvents
76
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
77
Silylating agents and their mechanisms
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
78
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
79
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
80
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
81
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
82
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
83
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
84
Alkylation
 Alkylation reduces molecular polarity by replacing
active hydrogens with an alkyl group. These
reagents are used to modify compounds with active
hydrogens, such as carboxylic acids and phenols.
These reagents ,make esters, ethers, alkyl amines and
alkyl amides.
 The principal reaction employed for preparation of
these derivatives is nucleophilic displacement.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
85
Alkylation
advantages and disadvantages
Advantages
 Wide range of alkylating reagents are available.
 Reaction can vary from strong acidic to strong basic.
 Alkylation derivatives are generally stable.
Disadvantages
 Limited to amines and acidic hydroxyls
 Reaction conditions are frequently severe
 Reagents are often toxic.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
86
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
87
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
88
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
89
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
90
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
91
Acylation
 Acylation reduces the polarity of amino, hydroxyl, thiol
groups and adds halogenated functionalities for ECD. In
comparision to silylating reagents the acylating reagents
target highly polar, multifunctional compounds such as
carbohydrates and amino acids.
 Acylation converts these compounds with active hydrogens
into esters, thio esters, and amides. They are formed with
acyl anhydride, acid halide and activated amide reagents.
 The anhydrides and acyl halide regents from acid by
products, which must be removed before GC analysis.
 Acylations are normally carried out in pyridine,
tetrahydrofuran or another solvents capable of acid product.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
92
 Fluorinated acyl groups, going from
trifluiroacetyl to heptafluorobutyryl can be used
to increase retention times.
 Acyl derivatives tend to direct the fragmentation
patterns of compounds in MS applications and so
provide helpful information on the structure of
these materials.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
93
Acylation
Advantages and disadvantages
Advantages:
 Addition of halogenated carbons increased detectability
by ECD
 Derivatives are hydrolytically stable
 Increased sensitivity by adding molecular weight
 Acylation can be used as a first step to active
carboxylic acids prior to esterification.
Disadvantages:
 Acylation derivatives can be difficult to prepare.
 Reaction products (acid by products often need to be
removed before analysis).
 Acylation reagents are moisture sensitive.
 Reagents are hazardous and odorous.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
94
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
95
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
96
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
97
Chiral derivatization
 These reagents target one specific functional group and produce
individual distereomers of each of the enantiomers.
 There are two ways of separating enantiomers by
chromatography.
 Separation on an optically active stationary phase
 Prepration of diastereomeric derivatives that can be separated on a
non chiral stationary phase.
Reagents:
 1.TPC(N-Trifluoroacetyl-L-propyl chloride)
Used for optically active amines, most notably amphetamines
2.MCF(Methyl chloroformate)
Used for optically active alcohols.
If an optically pure reagent is used to prepare diastereomeric
derivatives, then only two derivatives are formed. These
enantiomeric ratio is reflected in the relative peak size.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
98
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
99
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
100
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
101
Why derivatization in HPLC?
 To improve detectability
 To prepare soluble derivatives of insoluble
compounds for HPLC analysis
 To change the molecular structure or polarity of
the analyte for better chromatography
 To change to enhance separation
 To reduce tailing, poor peak resolution and/ or
asymmetrical peaks.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
102
Types of HPLC derivatization:
 For UV-Vis spectrophotometric detection
 For Flourimetric detection
 For chiral analysis
According to when and where the derivatization is done:
Pre column derivatization
Post column derivatization
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
103
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
104
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
105
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
106
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
107
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.
108
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
109
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
110
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Applications
 Industrial
 Environmental
 pharmaceutical
 biotechnology.
111
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
Pharmaceuticals
 Analysis of both raw material (drug goods) and finished
goods (drug substances).
 In biopharmaceutical applications, urine drug screens for
Barbiturates and underivatized drugs and for ethylene oxide
in sterilized products such as sutures.
112
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
113
Food/ Flavours/Fragnances
 In quality testing & solvent testing.
 The flavours and fragnances industries use GC for quality
testing and fingerprinting of fragnances for characterization.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
114
Petrochemicals
 Natural gas analysis or refineries, gasoline characterization
and fraction quantification, aromatics in benzene etc.
 Geochemical applications include mapping of oil reserves and
tracking of reservoirs etc.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
115
Chemical Industries
 Determination of product content, determination of purity,
monitoring production processes etc.
 To detect organic acids, alcohols, amines, esters and
solvents.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya
University,Warangal.
116
Environmental
 Detection of pollutants such as pestisides, fungicides,
herbicides, purgeable aromatics etc.
 Industrial environmental protection applications include stack
and waste emissions as well as water discharges.
Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
117
Blessi Priyanka Kolanpaka,University
College of Pharmaceutical
Sciences,Kakatiya University,Warangal.

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Chromatography-Gas chromatography-Principle

  • 1. 1 Gas Chromatography Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 2. 2  Chromatography-Russian Botanist Mickel Tswett (1903)  German Ph D student Fritz Prior (student of Erika Cremer, University of Innsbruck, Austrelia)developed Solid state gas chromatography in 1947.  Archer P.J. Martin(1910-2002) won noble prize in developing Liquid-liquid (1914) and Paper (1944)chromatography. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 3. 3  GC is the common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vapourized without decomposition.  In GC mobile phase is carrier gas usually inert gas and stationery phase is microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column.  The instrument used for performing the gas chromatography is called gas chromatogram.  Similar to column chromatography  Fractional distillation Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 4. Classification based on types of the stationary and mobile phase  Gas chromatography Mobile phase - gas  Liquid chromatography Mobile phase - liquid  Supercritical fluid chromatography Mobile phase – super critical fluid LC- performed in Column & planar surfaces GC&SFC- performed only in Column 4 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 5. Classification of Chromatographic methods S.No. General Classificatio n Specific method Stationary Phase Type of Equilibrium 1 Liquid Chromatogra phy (LC) (Mobile phase:Liquid) Liquid- liquid, or partition Liquid adsorbed on a solid Partition between immiscible liquids Liquid – bonded phase Oraganic species bonded to a solid surface Partition between liquid and bonded surface Liquid-solid, or adsorption Solid Adsorption Ion exchange Ion –exchange resin Ion exchange size exclusion Liquid in interstices of a polymer solid Partition/sieving 5 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 6. S.N o. General Classification Specific method Stationary Phase Type of Equilibrium 2 Gas chromatography Gas- liquid Liquid adsorbed on a solid Partition between gas and liquids Gas- bonded phase Organic species bonded to a solid surface Partition between liquid and bonded surface Gas- solid solid Adsorption 3 Supercritical – fluid chromatography (SFC) Mobile phase: supercritical fluid Organic species bonded to a solid surface Partition between super critical fluid and bonded surface 6 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 7. S.No. Technique Stationary phase Mobile phase 1 Column Chromatography or Adsorption chromatography Solid liquid 2 Partition chromatography liquid liquid 3 Paperchromatography liquid liquid 4 Thin Layer chromatography Liquid orsolid liquid 5 Gas- liquid chromatography liquid Gas 6 Gas –solid chromatography Solid gas 7 Ion exchangechromatography solid liquid Types of Chromatographic techniques 7 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 8. S.No. Technique Stationary phase Mobile phase 1 Paper chromatography Solid liquid 2 Plain chromatography 3 Thin Layer chromatography 4 Adsorption column chromatography 5 High Performance Liquid 6 Ion exchange chromatography Solid (Ion exchange resin) Liquid 7 Gas- solid chromatography Solid Gas 8 Gel permeation chromatography (Exclusion chromatography) Solid matrix Liquid 9 Gas-Liquid chromatography Liquid Gas 10 Liquid-Liquid chromatography liquid liquid Classification of chromatographic methods 8 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 9. Chromatography is based on a physical equilibrium that results when a solute is transferred between the mobile and a stationary phase. A A A A A A A A A A A A K = distribution coefficient or partition ratio K = CS CM Where CS is the molar concentration of the solute in the stationary phase and CM is the molar concentration in the mobile phase. Cross Section of Equilibrium in a column.“A” are adsorbed to the stationary phase.“A” are traveling in the mobile phase. 9 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 10. Flow As a material travels through the column, it assumes a Gaussian concentration profile as it distributes between the stationary packing phase and the flowing mobile gas or liquid carrier phase. In a chromatography column, flowing gas or liquid continuously replaces saturated mobile phase and results in movement of A through the column. Column is packed with particulate stationary phase. 10 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 11. Flow Flow Flow Flow In a mixture, each component has a different distribution coefficient, and thus spends a different amount of time absorbed on the solid packing phase vs being carried along with the flowing gas More volatile materials are carried through the column more rapidly than less volatile materials, which results in a separation. 11 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 12. Time spended of analyte in the stationary phase relating to the mobile phase The retention factor: k’ k’: relative number of moles of analytes in the stationary and mobile phase k’ = nS/nM 12 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 13. 13 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 14. Column efficiency and band broadening The plate theory of chromatography One theoretical plate (N): the part of the column, where quasi-equilibrium takes place between stationary and mobile phase 2 L 2 2 2 t R σ L σ t N = = 54 , 5 16 2 =       = w t N R 2 2 / 1       w tR Where:  standard deviation and 2 Variance w = 4  14 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 15. Gauss equation: HETP: Height equivivalent to the theoretical plate (H) N L H = 15 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 16. 16 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 17. The rate theory of chromatography (van Deemter) A  p ed C Porous silica particle particle size (diameter): dP Theory of band broadening 1. Eddy diffusion term (A) multiple path effects 17 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 18. u B  u D C M d 2.The longitudal diffusion term (B/u) 18 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 19. u CM  M 2 p M D u d C u CS  M 2 p M S D u d C 3. Mobile phase mass transfer term (CM/u) 4. Stationary phase mass transfer term (CS/u) 19 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 20. u C u u A S    = M C B H H u The van Deemter equation of chromatography The equation has an optimum (Hopt) where the column efficiency is highest. This optimum has been found at a linear velocity: for gas chromatography at.0.1 – 0.5 cm/s for liquid chromatography at: 1.0 – 5.0 cm/s 20 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 21. ' k 1 ' k α 1 α N 4 1 R 2 2 2 S   = Effect of increase of retention factor on resolution How to increase retention factor: •By decreasing eluent strength 21 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 22.  Good for volatile samples (up to about 250 oC)  0.1-1.0 microliter of liquid or 1-10 ml vapor  Can detect <1 ppm with certain detectors  Can be easily automated for injection and data analysis Gas Chromatography 22 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 23. 23 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 24. Types of Gas Chromatography  Gas solid chromatography  Gas liquid chromatography  Gas bonded phase chromatography 24 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 25. 25 Which factors influence the separation of the components?  Vapor pressure  The polarity of components versus the polarity of stationary phase on column  Column temperature  Carrier gas flow rate  Column length  Amount of material injected Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 26. 26 Vapor pressure  Pressure exterted by a vapor in thermodynamic equilibrium with its condensed phase(solid or liquid) at a given temperature in a closed system.  The boiling point of a compound is often related to its polarity.  The lower the boiling point is, the higher the vapor pressure of the compound and the shorter retention time usually.  That is one of the main reasons why low boiling solvents (i.e., diethyl ether, dichloromethane) are used as solvents to dissolve the sample.  The temperature of the column does not have to be above the boiling point because every compound has a non-zero vapor pressure at any given temperature, even solids.  That is the reason why we can smell compounds like camphor (0.065 mmHg/25 oC), isoborneol (0.0035 mmHg/25 oC), naphthalene (0.084 mmHg/25 oC), etc. However, their vapor pressures are low compared to liquids (i.e., water (24 mmHg/25 oC), ethyl acetate (95 mmHg/25 oC). Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 27. 27 The polarity of components versus the polarity of stationary phase on column  If the polarity of the stationary phase and compound are similar, the retention time increases because the compound interacts stronger with the stationary phase. As a result, polar compounds have long retention times on polar stationary phases and shorter retention times on non-polar columns using the same temperature.  Chiral stationary phases that are based on amino acid derivatives, cyclodextrins and chiral silanes are capable of separating enantiomers because one enantiomer interacts slightly stronger than the other one with the stationary phase, often due to steric effects or other very specific interactions.  For instance, a modified -cyclodextrin column is used in the determination of the enantiomeric excess in the chiral epoxidation experiment . Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 28. 28 Column temperature  A excessively high column temperature results in very short retention time but also in a very poor separation because all components mainly stay in the gas phase.  However, in order for the separation to occur the components need to be able to interact with the stationary phase. If the compound does not interact with the stationary phase, the retention time will decrease.  At the same time, the quality of the separation deteriorates, because the differences in retention times are not as pronounced anymore.  The best separations are usually observed for temperature gradients, because the differences in polarity and in boiling points are used here. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 29. 29 Carrier gas flow rate  A high flow rate reduces retention times, but a poor separation would be observed as well. Like above, the components have very little time to interact with the stationary phase and are just being pushed through the column. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 30. Column length  A longer column generally improves the separation.  The trade-off is that the retention time increases proportionally to the column length and a significant peak broadening will be observed as well because of increased longitudinal diffusion inside the column.  One has to keep in mind that the gas molecules are not only traveling in one direction but also sideways and backwards.  This broadening is inversely proportional to the flow rate. Broadening is also observed because of the finite rate of mass transfer between the phases and because the molecules are taking different paths through the column. 30 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 31. 31  Ideally, the peaks in the chromatogram display a symmetric shape (Gaussian curve).  If too much of the sample is injected, the peaks show a significant tailing, which causes a poorer separation.  Most detectors are relatively sensitive and do not need a lot of material in order to produce a detectable signal. Strictly speaking, under standard conditions only 1-2 % of the compound injected into the injection port passes through the column because most GC instruments are operated in split- mode to prevent overloading of the column and the detector.  The split less mode will only be used if the sample is extremely low in concentration in terms of the analyte. Amount of material injected Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 32. Instrumentation  Carrier gas  Sample injection system  The separation column  Detectors  Thermo stated chambers for the temperature regulation of column and detectors  Amplifier  recorder 32 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 33. 33 Carrier gas  The carrier gas (mobile phase) for gas chromatography should be an inert gas that does not react with the sample components.  The GC carrier gas should contribute minimally to the partitioning process. This differs from the mobile phase in liquid chromatography.  In GC the carrier gas is simply stated as just a carrier to transport the vaporized solute molecules through the column during the partitioning process.  Carrier gases are compressible gases that expand with increasing temperature. This results in a change in the gas viscosity.  The selection and linear velocity of the carrier gas will affect resolution and retention times.  Carrier gases should be inert to the stationary phase and free of detectable contaminants. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 34. Efficiency Effects of Carrier Gases  Carrier gas linear velocity plays a significant role in the resulting efficiency of a chromatographic system.  The optimal carrier gas linear velocity is characteristic for each gas.  Van Deemter curves and Golay Plots are used to demonstrate optimal carrier gas linear velocities.  Van Deemter plots are used for packed columns since the A term for eddy diffusion is present.  Golay plots are used for capillary columns (open tubular) and the A term is dropped. 34 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 35. Retention Time Effects Analysis can be either isothermal or temperature programmed runs in GC. The carrier gas linear veloctiy can influence the overall time of analysis and the efficiency of the separations. The following equation demonstrates the affect of linear velocity on retention time. t r = L (k+1) µ where t r = retention time L = column length k = retention factor µ = carrier gas linear velocity 35 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 36. LinearVelocity/Flow Control  Linear velocity or flow can either be run in a constant pressure or constant flow mode.  This is important in temperature programmed analyses. In the constant pressure mode, the column head pressure is set at a temperature and the pressure remains constant throughout the analysis. This can result in linear velocity changes especially over a wide temperature programming range.  In the constant flow mode, the column head pressure will change throughout a temperature programmed analysis in order to keep the carrier gas flow at a constant flow rate. 36 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 37. Carrier GasViscosity  Carrier gas viscosity is a temperature dependent parameter.  As temperature increases, the viscosity of the gas increases.  When using a constant pressure mode for carrier gas and temperature programming, the viscosity of the gas will increase and the average linear velocity will decrease. 37 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 38. Detectors  Flame Ionization Detector (FID)  Atomic Emission Detector(AED)  Nitrogen Phosphorus Detector (NPD)  Electron Capture Detector (ECD)  Sulfur Chemiluminescence Detectors(SCD)  Thermal Conductivity Detector (TCD)  Thermoionic Detector(TID)  Flame Photometric Detector (FPD)  Photoionization Detector (PID)  Mass Spectrometer (MS) 38 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 39. FLAME IONIZATION DETECTOR 39 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 40. 40 Construction: The effluent from the column is mixed with hydrogen and air and then ignited electrically. Most organic compounds, when pyrolyzed at the temperature of a hydrogen/air flame, produce ions and electrons that can conduct electricity through the flame.  The number of ions produced is propositional to number of reduced carbon atoms in the flame.  Mass- sensitive rather than concentration sensitive.  Advantages:  High sensitive (~10-13g/s), large linear response range, (~107) and low noise.  Analysis of most organic samples including those that are contaminated with water and the oxides of nitrogen and sulfur. Disadvantages:  Carbonyl, amine, alcohol, halogen yields few ions or none in the flame.  Insensitive toward noncombustible gases such as H2O,CO,SO2 and NO . Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 41. THERMAL CONDUCTIVITY DETECTOR Thermal conductivity detector cell and arrangement of two sample detector cells and two reference detector cells 41 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 42.  Also known as Katherometer. Construction:  An electrically heated element whose temperature at constant electrical power depends upon the thermal conductivity of the surrounding gas.  The heating element –fine platinum, gold or tungsten wire or alternatively, a semiconducting thermistor.  The resistance of the wire or thermistor gives a measure of the thermal conductivity of the gas, in contrast to the wire detector, the thermistor has a negative temperature coefficient.  Two pairs of elements are employed, one pair being located in the flow of the effluent from the column and the other in the gas stream ahead of the sample injection chamber.  The effect of thermal conductivity of carrier gas is canceled, flow rate, pressure and electrical power are minimized. 42 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 43.  The resistances of the twin detector pairs are compared by incorporating them into two arms of a simple Wheatstone bridge.  The thermal conductivities of the hydrogen and helium are roughly six to ten times greater than those of most amounts of organic materials, a relatively large decrease in the thermal conductivity of the column effluent takes place. Advantages:  Simple, large linear dynamic range (~105),non destructive, general response to organic and inorganic species.  Analysis of permanent gases such as argon, oxygen, nitrogen and carbon dioxide Disadvantages:  Low sensitivity (~10-8solute/mL carrier gas). 43 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 44. PHOTOIONIZATION DETECTOR  The column eluent is irradiated with intense beam of ultraviolet radiation varying in energy from 8.3 to 11.7ev, which causes ionization of the molecules.  Application of a potential across a cell containing the ions leads to an ion current, which is amplified and recorded. 44 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 45. 45 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal. FLAME PHOTOMETRIC DETECTOR
  • 46. FLAME PHOTOMETRIC DETECTOR  To analysis of air and water pollutants, pesticides and coal hydrogenation products.  Compounds containing sulfur and phosphorus.  The eluent is passed into a low- temperature hydrogen/air flame, which converts part of the phosphorus to an HPO species that emits bands of radiation centered about 510 to 526pm.  Sulfur in the sample is simultaneously converted to S2, which emits a band centered at 394nm. Advantages:  Halogens, nitrogens and several metals such as in tin, chromium, selenium and germanium. 46 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 47. 47 ELECTRON CAPTURE DETECTOR Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 48. 48  Detects halogen containing compounds such as pesticides and polychlorinated biphenyls. Construction:  The effluent from the column is passed over a β emitter, usually nickel-63.  An electron from the emitter causes ionization of the carrier gas (often nitrogen) and the production of a burst of electrons.  In absence of organic species, a constant standing current between a pair of electrodes results from this ionization process.  The current decreases markedly, however, in the presence of those organic molecules that tend to capture electrons. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 49. 49  Highly sensitive to molecules containing electronegative funtional groups such as halogens, peroxides, quinones, organometallic, nitrile and nitro compounds.  Detection and determination of chlorinated insecticides. Advantages:  Highly sensitive, not alters the samples Disadvantages:  Linear response range is limited to about two orders of magnitude.  Insensitive to functional groups such as amines, alcohols and hydrocarbons. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 50. 50 SULFUR CHEMILUMINESCENCE DETECTORS Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 51. SULFUR CHEMILUMINESCENCE DETECTORS  The reaction between sulfur containing compounds and ozone.  The luminescence intensity is proportional to the concentration of sulfur.  The eluent is mixed with hydrogen and air and is burned as in FID.  The resulting gases are then mixed with ozone and the intensity of the resulting emission is measured.  Also used for Super critical fluid chromatography. 51 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 52. 52 Atomic Emission Detectors Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 53. 53  The eluent is introduced into a microwave-energized helium plasma that is coupled to a diode array optical emission spectrometer.  The plasma is suffienciently energetic to atomize all of the elements in a sample and to excite their characteristic atomic emission spectra.  These spectra are then observed with a spectrometer that employs a movable, flat diode array capable of detecting emitted radiation from about 170 to 780nm.  The position able diode array is capable of monitoring simultaneously two to four elements at any given setting.  At the present time, the software supplied with the detector allows measurement of the concentration of 15 elements. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 54. 54 Nitrogen-phosphorus detectors Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 55. 55  It is selective toward organic compouds containing phosphorus and nitrogen.  Its response to phosphorus atom is approximately ten times greater than to a nitrogen atom and 104 to 106 larger than a carbon atom.  Compared to FID it is 500times more sensitive for phosphorus containing compounds, 50times more sensitive Construction:  The column effluent is mixed with hydrogen, passes through the flame tip assembly, and is ignited.  The hot gas then flows around an electrically heated rubidium silicate bead, which is maintained at about 180V with respect to the collector.  The heated bead forms a plasma having a temperature of 600°C to 800°C. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 56. Sensitivity of Gas Chromatography S. No . detector Sensiti vity Line ar rang e comments 1 Thermal conductivity 10-8 104 Universal sensitivity, non- destructive 2 Flame ionization 10-11 106 Detects all organic compounds ,destructive 3 Electron capture 10-13 102 Detect halo-,nitro- & phosphorus compounds, non destructive 4 Flame Emission 10-6 103 Sulphur & phosphorus compounds, destructive 5 Argon ionization 10-12 105 Universal, argon carrier gas necessary, non-destructive 56 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 57. Support materials and stationary phases  The solid support should ideally have large surface area (atleast 1m2/g) has a good mechanical stability, thermally stable, inert surface in order to simplify retention behavior and prevent solute adsorption, has a particle size in the range of 100-400mm.  Example: diatomaceous earth, glass beads with suitable mesh,(80-100mesh,100-120mesh).  A liquid stationary phase should be inert to analyte, less volatile and thermally stable.  In general, the polarity of the stationery phase should match that of the sample constituents (like dissolves like).Most stationery phases are based on polydimethylsiloxane or polythylene glycol (PEG) back bones. 57 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 58. 58  The polarity of the stationary phase can be changed by derivatization with different functional groups such as a phenyl group. Bleeding of the column is cured by bonding the stationary phase to the column; or cross linking the stationery phase.  The liquid stationary phases should have the following characteristics.  Low volatility, high decomposition temperature (thermally stable),chemically inert (reversible interactions with solvents), chemically attached to support ( to prevent bleeding),appropriate k’ and a for good resolution Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 59. Stationary phase  Low volatility  Thermal stability  Chemical inertness  Solvent characteristics  Polar stationary phases --- –CN,-CO and –OH. non polar ---Hydrocarbon- type stationary phases and dialkyl siloxanes  highly polar -- polyeterphases  Solutes-polar- alcohol,acids,amines Medium polarity-ethers, ketones, aldehydes Non polar-saturated hydrocarbons 59 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 60. 60 Columns  The column in chromatography is the heart of the technique.  A column can either be packed or open tubular.  Open-WCOT-Wall-coated open tubular tubes SCOT-Support-coated open tubular tubes FSOT-Fused – silica open tubular tubes Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 61. 61 Packed columns  These columns are fabricated from glass, metal(copper, stainless steel, aluminum) or Teflon tubes.  Stainless steel is the most widely used because it is most inert and easy to work with.  Length 2 to 3m,inside diameter is 2 to 4mm.  These tubes are densely packed with a uniform, finely divided packing material, or solid support, that is coated with a thin layer (0.05 to1µm) of the stationary liquid phase.  In order to fit in a thermostatic oven, the tubes are formed as coils having diameters of roughly 15cm. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 62. 62 Open tubular / capillary columns These are mainly two types.  Wall- coated open tubular (WCOT) < 1mm capillary tubes coated with a thin layer of the stationary phase.  Olden days-stainless steel, aluminum, copper or plastic.Then glass. Often glass etched with gaseous HCl, strong aqueous HCl, or Potassium hydrogen fluoride to give rough surface, which bonded the stationary phase more tightly.  Support-coated open tubular (SCOT) ~30µm thick coated support on inside of silica tube.  The most frequently used capillary column, nowadays, is the fused silica open tubular column (FSOT), which is aWCOT column.  The external surface of the fused silica columns is coated with a polyimide film to increase their strength.(internal diameter 320 and 260µm)  For, high resolution 200 and 150µm.  Megabore columns-530µm  Solid Support material-diatomaceous earth.  Particle size of support- effeciency inversely proportional to particle diameter. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 63. Properties and characteristics of typical gas- chromatographic columns 63 Type of column FSOT WCOT SCOT Packed Length,m 10-100 10-100 10-100 1-6 Inside diameter,mm 0.1-0.53 0.25-0.75 0.5 2-4 Efficiency, plates/m 2000-4000 1000-4000 600-1200 500-1000 total plates (20-400)*103 (10-400)*103 (6-120)*103 (1-10)*103 Sample size,ng 10-75 10-1000 10-1000 10-106 Relative back pressure Low Low Low high Relative speed Fast Fast fast slow Chemical inertness Best Good poor Poorest Flexible yes No No no Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 64. Hyphenated techniques  Gas chromatography/Mass Spectrometry (GC/MS)  Gas chromatography/Fourier Transform Infrared Spectroscopy(GC/FTIR) 64 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 65. 65 Gas chromatography/Mass spectrometry Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 66. 66 Gas chromatography/Fourier Transform Infrared Spectroscopy(GC/FTIR) Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 67. 67 Derivatization  Derivatization is the process of chemically modifying a compound to produce a new compound which has properties that are suitable for analysis by using GC or HPLC.  The chemical structure of the compound remains the same and just modifies the specific functional groups of reacting compound to derivative of derivative chemical or physical properties in order to make them detectable and analyzable.  Derivatization is needed in GC,HPLC, UV-Visible spectroscopy. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 68. 68 WHY derivatization  To permit analysis of compounds which are not directly amenable to analysis due to its stability and volatility.  To improve chromatographic behaviour detect ability. Many compounds do not produce a useable chromatography or the sample of interest goes undetected. As a result it may be necessary to derivatize the compound before GC analysis is done.  The main reason for derivatizing is to impart volatility to non volatile compounds. Derivatization is useful to allowing the use of GC &GC/MS to be done on sample that would otherwise not possible in various areas of chemistry such as medicinal, forensic and environmental. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 69. 69 Ideal characters and disadvantages of derivatization  A derivatization reaction should be rapid, quantitative and produce minimal by product. Excess reagent should not interfere with the analysis and should be easily removed.  Derivatization often is a last resort when developing a method. Introduction of a reaction pre or post column increases complexity, chance of error, and total analysis time.  Care should be taken that the reaction is quantitative and no additional impurities are introduced into analysis. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 70. 70 What derivatization accomplish ?  Increases volatility (i.e. sugars) Eliminates the presence of polar OH,NH & SH groups. Derivatization targets O,S,N and P functional groups ( with hydrogen availability)  Enhance sensitivity to ECD. The introduction of ECD detectable groups, such as halogenated acyl groups, allows detection of previously undetectable groups.  Increases detectability i.e. steroids  Increases stability (thermo stability)  To reduce adsorption of polar samples on active surfaces of column walls and solid support. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 71. 71 Conditions for choosing a derivatizing agent  The derivatizing agent must be stable.  The derivatizing agent and its products formed during derivatization should not be detectable or must be separable from analyte.  The analyte should be reactive with derivatizing agent under convenient conditions.  If possible, it should non-toxic.  The procedure should be adaptable to automation. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 72. 72 Types of derivatization  Silylation  Alkylation  Acylation  Chiral derivatization Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 73. 73 Silylation  Most prevalent method, readily volatizes the sample.  Mechanism:  The process produces silyl derivatives which are more volatile, more thermally stable.  Replaces active hydrogens with TMS (Trimethyl silyl groups)  Silylation occurs nucleophilic attack (SN2). The better the leaving group, the better the silylation. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 74. 74 Solvents and precautions :  Silylation reagents will react with water & alcohols. First care must be taken to ensure that both solvents and sample are dry.  Solvents should be pure as possible. This will eliminate excessive peaks. Try to use little solvent as possible this will prevent a large solvent peak.  Pyridine is most commonly used solvent. Although pyridine may produce peak tailing it is an acid scavenger & will drive the reaction forward.  In many cases, the need for a solvent is eliminated with silylating reagents (if a sample readily dissolves in the reagent, it usually is a sign the derivatization is complete. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 75. 75  Ease of reactivity of functional group toward silylation follows the order:  Alcohol> Phenol> Carboxyl> Amine> Amide>Hydroxyl The order of alcohol is 1>2>3 Care needs to be taken not to inject silylating reagent onto column which have active hydrogen in the stationary phase, because they will be derivatized. Eg: column not compatible with silylating reagents are carbowax &free fatty acid phase Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 76. Silylation- advantages and disadvantages Advantages :  Ability to silylate a wide variety of compounds  Large number of silylating reagents are available.  Easily preapred Disadvantages:  Silylating reagents are moisture sensitive.  Must be aprotic organic solvents 76 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 77. 77 Silylating agents and their mechanisms Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 78. 78 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 79. 79 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 80. 80 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 81. 81 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 82. 82 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 83. 83 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 84. 84 Alkylation  Alkylation reduces molecular polarity by replacing active hydrogens with an alkyl group. These reagents are used to modify compounds with active hydrogens, such as carboxylic acids and phenols. These reagents ,make esters, ethers, alkyl amines and alkyl amides.  The principal reaction employed for preparation of these derivatives is nucleophilic displacement. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 85. 85 Alkylation advantages and disadvantages Advantages  Wide range of alkylating reagents are available.  Reaction can vary from strong acidic to strong basic.  Alkylation derivatives are generally stable. Disadvantages  Limited to amines and acidic hydroxyls  Reaction conditions are frequently severe  Reagents are often toxic. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 86. 86 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 87. 87 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 88. 88 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 89. 89 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 90. 90 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 91. 91 Acylation  Acylation reduces the polarity of amino, hydroxyl, thiol groups and adds halogenated functionalities for ECD. In comparision to silylating reagents the acylating reagents target highly polar, multifunctional compounds such as carbohydrates and amino acids.  Acylation converts these compounds with active hydrogens into esters, thio esters, and amides. They are formed with acyl anhydride, acid halide and activated amide reagents.  The anhydrides and acyl halide regents from acid by products, which must be removed before GC analysis.  Acylations are normally carried out in pyridine, tetrahydrofuran or another solvents capable of acid product. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 92. 92  Fluorinated acyl groups, going from trifluiroacetyl to heptafluorobutyryl can be used to increase retention times.  Acyl derivatives tend to direct the fragmentation patterns of compounds in MS applications and so provide helpful information on the structure of these materials. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 93. 93 Acylation Advantages and disadvantages Advantages:  Addition of halogenated carbons increased detectability by ECD  Derivatives are hydrolytically stable  Increased sensitivity by adding molecular weight  Acylation can be used as a first step to active carboxylic acids prior to esterification. Disadvantages:  Acylation derivatives can be difficult to prepare.  Reaction products (acid by products often need to be removed before analysis).  Acylation reagents are moisture sensitive.  Reagents are hazardous and odorous. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 94. 94 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 95. 95 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 96. 96 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 97. 97 Chiral derivatization  These reagents target one specific functional group and produce individual distereomers of each of the enantiomers.  There are two ways of separating enantiomers by chromatography.  Separation on an optically active stationary phase  Prepration of diastereomeric derivatives that can be separated on a non chiral stationary phase. Reagents:  1.TPC(N-Trifluoroacetyl-L-propyl chloride) Used for optically active amines, most notably amphetamines 2.MCF(Methyl chloroformate) Used for optically active alcohols. If an optically pure reagent is used to prepare diastereomeric derivatives, then only two derivatives are formed. These enantiomeric ratio is reflected in the relative peak size. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 98. 98 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 99. 99 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 100. 100 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 101. 101 Why derivatization in HPLC?  To improve detectability  To prepare soluble derivatives of insoluble compounds for HPLC analysis  To change the molecular structure or polarity of the analyte for better chromatography  To change to enhance separation  To reduce tailing, poor peak resolution and/ or asymmetrical peaks. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 102. 102 Types of HPLC derivatization:  For UV-Vis spectrophotometric detection  For Flourimetric detection  For chiral analysis According to when and where the derivatization is done: Pre column derivatization Post column derivatization Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 103. 103 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 104. 104 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 105. 105 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 106. 106 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 107. 107 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 108. 108 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 109. 109 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 110. 110 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 111. Applications  Industrial  Environmental  pharmaceutical  biotechnology. 111 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 112. Pharmaceuticals  Analysis of both raw material (drug goods) and finished goods (drug substances).  In biopharmaceutical applications, urine drug screens for Barbiturates and underivatized drugs and for ethylene oxide in sterilized products such as sutures. 112 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 113. 113 Food/ Flavours/Fragnances  In quality testing & solvent testing.  The flavours and fragnances industries use GC for quality testing and fingerprinting of fragnances for characterization. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 114. 114 Petrochemicals  Natural gas analysis or refineries, gasoline characterization and fraction quantification, aromatics in benzene etc.  Geochemical applications include mapping of oil reserves and tracking of reservoirs etc. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 115. 115 Chemical Industries  Determination of product content, determination of purity, monitoring production processes etc.  To detect organic acids, alcohols, amines, esters and solvents. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 116. 116 Environmental  Detection of pollutants such as pestisides, fungicides, herbicides, purgeable aromatics etc.  Industrial environmental protection applications include stack and waste emissions as well as water discharges. Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.
  • 117. 117 Blessi Priyanka Kolanpaka,University College of Pharmaceutical Sciences,Kakatiya University,Warangal.