gas chromatography
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About This Presentation
gas chromatography in detail
Slide Content
Gas Chromatography 2016
1
INDEX
Contents Page No.
1. Introduction 02
2. Principle 06
3. Split-Split less Injector 09
4. Head Space Sampling 13
5. Columns For GC 14
6. Detectors 18
7. Derivatization Techniques 27
8. Applications 30
9. Conclusion 33
10. Bibliography 34
1
INDEX
Contents Page No.
1. Introduction 02
2. Principle 06
3. Split-Split less Injector 09
4. Head Space Sampling 13
5. Columns For GC 14
6. Detectors 18
7. Derivatization Techniques 27
8. Applications 30
9. Conclusion 33
10. Bibliography 34
Gas Chromatography 2016
2
CHROMATOGRAPHY
INTRODUCTION:
(Greek = chroma “color” and graphein “writing”) Tswett named this new
technique chromatography based on the fact that it separated the components of a
solution by color.
Mikhail Tswett invented chromatography in 1901 during his research on plant
pigments. He used the technique to separate various plant pigments such as
chlorophylls, xanthophylls and carotenoids.
Chromatography is usually introduced as a technique for separating and/or
identifying the components in a mixture. The basic principle is that components in
a mixture have different tendencies to adsorb onto a surface or dissolve in a
solvent. It is a powerful method in industry, where it is used on a large scale to
separate and purify the intermediates and products in various syntheses.
Theory:
There are several different types of chromatography currently in use – i.e. paper
chromatography; thin layer chromatography (TLC); gas chromatography (GC);
liquid chromatography (LC); high performance liquid chromatography (HPLC);
ion exchange chromatography; and gel permeation or gel filtration
chromatography.
Basic Principles:
All chromatographic methods require one static part (the stationary phase) and one
moving part (the mobile phase). The techniques rely on one of the following
phenomena: adsorption; partition; ion exchange; or molecular exclusion.
1. Adsorption:
Adsorption chromatography was developed first. It has a solid stationary phase and
a liquid or gaseous mobile phase. (Plant pigments were separated at the turn of the
20th century by using calcium carbonate stationary phase and a liquid hydrocarbon
mobile phase. The different solutes travelled different distances through the solid,
carried along by the solvent.) Each solute has its own equilibrium between
adsorption onto the surface of the solid and solubility in the solvent, the least
soluble or best adsorbed ones travel more slowly. The result is a separation into
bands containing different solutes. Liquid chromatography using a column
containing silica gel or alumina is an example of adsorption chromatography.
2
CHROMATOGRAPHY
INTRODUCTION:
(Greek = chroma “color” and graphein “writing”) Tswett named this new
technique chromatography based on the fact that it separated the components of a
solution by color.
Mikhail Tswett invented chromatography in 1901 during his research on plant
pigments. He used the technique to separate various plant pigments such as
chlorophylls, xanthophylls and carotenoids.
Chromatography is usually introduced as a technique for separating and/or
identifying the components in a mixture. The basic principle is that components in
a mixture have different tendencies to adsorb onto a surface or dissolve in a
solvent. It is a powerful method in industry, where it is used on a large scale to
separate and purify the intermediates and products in various syntheses.
Theory:
There are several different types of chromatography currently in use – i.e. paper
chromatography; thin layer chromatography (TLC); gas chromatography (GC);
liquid chromatography (LC); high performance liquid chromatography (HPLC);
ion exchange chromatography; and gel permeation or gel filtration
chromatography.
Basic Principles:
All chromatographic methods require one static part (the stationary phase) and one
moving part (the mobile phase). The techniques rely on one of the following
phenomena: adsorption; partition; ion exchange; or molecular exclusion.
1. Adsorption:
Adsorption chromatography was developed first. It has a solid stationary phase and
a liquid or gaseous mobile phase. (Plant pigments were separated at the turn of the
20th century by using calcium carbonate stationary phase and a liquid hydrocarbon
mobile phase. The different solutes travelled different distances through the solid,
carried along by the solvent.) Each solute has its own equilibrium between
adsorption onto the surface of the solid and solubility in the solvent, the least
soluble or best adsorbed ones travel more slowly. The result is a separation into
bands containing different solutes. Liquid chromatography using a column
containing silica gel or alumina is an example of adsorption chromatography.
Gas Chromatography 2016
3
The solvent that is put into a column is called the eluent, and the liquid that flows
out of the end of the column is called the eluate.
Figure: Adsorption Chromatography using a column
2. Partition:
In partition chromatography the stationary phase is a non-volatile liquid which is
held as a thin layer (or film) on the surface of an inert solid. The mixture to be
separated is carried by a gas or a liquid as the mobile phase. The solutes distribute
themselves between the moving and the stationary phases, with the more soluble
component in the mobile phase reaching the end of the chromatography column
first. Paper chromatography is an example of partition chromatography.
3
The solvent that is put into a column is called the eluent, and the liquid that flows
out of the end of the column is called the eluate.
Figure: Adsorption Chromatography using a column
2. Partition:
In partition chromatography the stationary phase is a non-volatile liquid which is
held as a thin layer (or film) on the surface of an inert solid. The mixture to be
separated is carried by a gas or a liquid as the mobile phase. The solutes distribute
themselves between the moving and the stationary phases, with the more soluble
component in the mobile phase reaching the end of the chromatography column
first. Paper chromatography is an example of partition chromatography.
Gas Chromatography 2016
4
Figure: Partition Chromatography
3. Ion exchange
Ion exchange chromatography is similar to partition chromatography in that it has
a coated solid as the stationary phase. The coating is referred to as a resin, and has
ions (either cations or anions, depending on the resin) covalently bonded to it and
ions of the opposite charge are electrostatically bound to the surface. When the
mobile phase (always a liquid) is eluted through the resin the electrostatically
bound ions are released as other ions are bonded preferentially. Domestic water
softeners work on this principle.
Figure: Ion Exchange
4
Figure: Partition Chromatography
3. Ion exchange
Ion exchange chromatography is similar to partition chromatography in that it has
a coated solid as the stationary phase. The coating is referred to as a resin, and has
ions (either cations or anions, depending on the resin) covalently bonded to it and
ions of the opposite charge are electrostatically bound to the surface. When the
mobile phase (always a liquid) is eluted through the resin the electrostatically
bound ions are released as other ions are bonded preferentially. Domestic water
softeners work on this principle.
Figure: Ion Exchange
Gas Chromatography 2016
5
4. Molecular exclusion:
Molecular exclusion differs from other types of chromatography in that no
equilibrium state is established between the solute and the stationary phase.
Instead, the mixture passes as a gas or a liquid through a porous gel. The pore size
is designed to allow the large solute particles to pass through uninhibited. The
small particles, however, permeate the gel and are slowed down so the smaller the
particles, the longer it takes for them to get through the column. Thus separation is
according to particle size.
Figure: Gel permeation chromatography
5
4. Molecular exclusion:
Molecular exclusion differs from other types of chromatography in that no
equilibrium state is established between the solute and the stationary phase.
Instead, the mixture passes as a gas or a liquid through a porous gel. The pore size
is designed to allow the large solute particles to pass through uninhibited. The
small particles, however, permeate the gel and are slowed down so the smaller the
particles, the longer it takes for them to get through the column. Thus separation is
according to particle size.
Figure: Gel permeation chromatography
Gas Chromatography 2016
6
GAS CHROMATOGRAPHY
Modern gas chromatography (GC) was invented by Martin and James in 1952, and
has become one of the most important and widely applied analytical techniques in
modern chemistry. Major milestones in the development of GC, especially in
column technology, detection and sample introduction are described in this
historical review. Many trends in current progress can be seen to originate in the
first two decades of the history of GC, but the invention of fused-silica capillary
columns greatly increased the application of high-resolution GC across the field of
organic analysis; the development of low-cost, bench-top mass spectrometers led
to further advances. Progress continues to be rapid in comprehensive 2D GC, fast
analysis, detection by atomic emission and time-of-flight mass spectrometry, and
in applications to process analysis.
GC is analytical technique that helps to separate and analyze a mixture of organic
vaporizable or volatile compounds without their decomposition. It is carried out at
suitable temperature in a glass or metal tubing known as a column, which contains
the liquid or solid stationary phase. Inert gases like helium or unreactive gases like
nitrogen are used as mobile phase which is passed over stationary phase. The
instrument used to perform gas chromatography is known as gas chromatogram or
gas separator or aerograph.
PRINCIPLE:
The basic principle of GC is partition.
In GC, the separation of mixture of components occurs between a gaseous mobile
phase and a liquid stationary phase.
The mixture of components to be separated is converted to vapor and mixed with
gaseous mobile phase.
The component which is more soluble in stationary phase travels slower and eluted
later. The component which is less soluble in stationary phase travels faster and
eluted out first.
No two components has same partition coefficient for fixed combination of
stationary phase, mobile phase and other conditions.
6
GAS CHROMATOGRAPHY
Modern gas chromatography (GC) was invented by Martin and James in 1952, and
has become one of the most important and widely applied analytical techniques in
modern chemistry. Major milestones in the development of GC, especially in
column technology, detection and sample introduction are described in this
historical review. Many trends in current progress can be seen to originate in the
first two decades of the history of GC, but the invention of fused-silica capillary
columns greatly increased the application of high-resolution GC across the field of
organic analysis; the development of low-cost, bench-top mass spectrometers led
to further advances. Progress continues to be rapid in comprehensive 2D GC, fast
analysis, detection by atomic emission and time-of-flight mass spectrometry, and
in applications to process analysis.
GC is analytical technique that helps to separate and analyze a mixture of organic
vaporizable or volatile compounds without their decomposition. It is carried out at
suitable temperature in a glass or metal tubing known as a column, which contains
the liquid or solid stationary phase. Inert gases like helium or unreactive gases like
nitrogen are used as mobile phase which is passed over stationary phase. The
instrument used to perform gas chromatography is known as gas chromatogram or
gas separator or aerograph.
PRINCIPLE:
The basic principle of GC is partition.
In GC, the separation of mixture of components occurs between a gaseous mobile
phase and a liquid stationary phase.
The mixture of components to be separated is converted to vapor and mixed with
gaseous mobile phase.
The component which is more soluble in stationary phase travels slower and eluted
later. The component which is less soluble in stationary phase travels faster and
eluted out first.
No two components has same partition coefficient for fixed combination of
stationary phase, mobile phase and other conditions.
Gas Chromatography 2016
7
So the components are separated according to their partition coefficient.
[Partition Coefficient is the ratio of solubility of a substance distributed between
two immiscible liquids at a constant temperature.]
INSTRUMENTATION :
1. Gas Inlets:
Gas is fed from cylinders through supply piping to the instrument. It is usual
to filter gases to ensure high gas purity and the gas supply may be regulated
at the bench to ensure an appropriate supply pressure.
Required gases might include:
Carrier - (H2, He, N2)
Make-up gas - (H2, He, N2)
Detector Fuel Gas -(H2& Air,Ar or Ar & CH4,N2) depending on the
detector type
2. Pneumatic controls:
The gas supply is regulated to the correct pressure (or flow) and then fed to
the required part of the instrument. Control is usually required to regulate the
gas coming into the instrument and then to supply the various parts of the
instrument. A GC fitted with a Split/Splitless inlet, capillary GC column and
7
So the components are separated according to their partition coefficient.
[Partition Coefficient is the ratio of solubility of a substance distributed between
two immiscible liquids at a constant temperature.]
INSTRUMENTATION :
1. Gas Inlets:
Gas is fed from cylinders through supply piping to the instrument. It is usual
to filter gases to ensure high gas purity and the gas supply may be regulated
at the bench to ensure an appropriate supply pressure.
Required gases might include:
Carrier - (H2, He, N2)
Make-up gas - (H2, He, N2)
Detector Fuel Gas -(H2& Air,Ar or Ar & CH4,N2) depending on the
detector type
2. Pneumatic controls:
The gas supply is regulated to the correct pressure (or flow) and then fed to
the required part of the instrument. Control is usually required to regulate the
gas coming into the instrument and then to supply the various parts of the
instrument. A GC fitted with a Split/Splitless inlet, capillary GC column and
Gas Chromatography 2016
8
Flame Ionization detector may have the following different gas
specifications:
Carrier gas supply pressure, column inlet pressure (column carrier gas flow),
inlet split flow, inlet septum purge flow, detector air flow, detector hydrogen
flow, detector make-up gas flow.
Modern GC instruments have Electronic Pneumatic pressure controllers –
older instruments may have manual pressure control via regulators
3. Injector:
Here the sample is volatilized and the resulting gas entrained into the carrier
stream entering the GC column.
Many inlet types exist including:
-on-column (COC) etc.
The COC injector introduces the sample into the column as a liquid to avoid
thermal decomposition or improve quantitative accuracy.
4. Column:
In GC, retention of analyte molecules occurs due to stronger interactions
with the stationary phase than the mobile phase. This is unique in GC and,
therefore, interactions between the stationary phase and analyte are of great
importance. The interaction types can be divided into three broad categories:
The sample is separated into its constituent components in the column.
Columns vary in length and internal diameter depending on the application
type and can be either packed or capillary. Packed columns (typical
dimension 1.5 m x 4 mm) are packed with a solid support coated with
immobilized liquid stationary phase material (GLC). Capillary columns
(typical dimension 30 m x 0.32 mm x 0.1 mm film thickness) are long
hollow silica tubes with the inside wall of the column coated with
immobilized liquid stationary phase material of various film thickness.
Many different stationary phase chemistries are available to suit a host of
8
Flame Ionization detector may have the following different gas
specifications:
Carrier gas supply pressure, column inlet pressure (column carrier gas flow),
inlet split flow, inlet septum purge flow, detector air flow, detector hydrogen
flow, detector make-up gas flow.
Modern GC instruments have Electronic Pneumatic pressure controllers –
older instruments may have manual pressure control via regulators
3. Injector:
Here the sample is volatilized and the resulting gas entrained into the carrier
stream entering the GC column.
Many inlet types exist including:
-on-column (COC) etc.
The COC injector introduces the sample into the column as a liquid to avoid
thermal decomposition or improve quantitative accuracy.
4. Column:
In GC, retention of analyte molecules occurs due to stronger interactions
with the stationary phase than the mobile phase. This is unique in GC and,
therefore, interactions between the stationary phase and analyte are of great
importance. The interaction types can be divided into three broad categories:
The sample is separated into its constituent components in the column.
Columns vary in length and internal diameter depending on the application
type and can be either packed or capillary. Packed columns (typical
dimension 1.5 m x 4 mm) are packed with a solid support coated with
immobilized liquid stationary phase material (GLC). Capillary columns
(typical dimension 30 m x 0.32 mm x 0.1 mm film thickness) are long
hollow silica tubes with the inside wall of the column coated with
immobilized liquid stationary phase material of various film thickness.
Many different stationary phase chemistries are available to suit a host of
Gas Chromatography 2016
9
applications. Columns may also contain solid stationary phase particles
(GSC) for particular application types.
5. Column Oven:
Temperature in GC is controlled via a heated oven. The oven heats rapidly
to give excellent thermal control. The oven is cooled using a fan and vent
arrangement usually at the rear of the oven.
A hanger or cage is usually included to support the GC column and to
prevent it touching the oven walls as this can damage the column.
The injector and detector connections are also contained in the GC oven. For
Isothermal operation, the GC is held at a steady temperature during the
analysis. In temperature programmed GC (pTGC) the oven temperature is
increased according to the temperature program during the analysis.
6. Detector:
The detector responds to a physicochemical property of the analyte,
amplifies this response and generates an electronic signal for the data system
to produce a chromatogram.
Many different detector types exist and the choice is based mainly on
application, analyte chemistry and required sensitivity – also on whether
quantitative or qualitative data is required.
Detector choices include:
a) Flame Ionization (FID)
b) Electron Capture (ECD)
c) Flame Photometric (FPD)
d) Nitrogen Phosphorous (NPD)
e) Thermal Conductivity (TCD)
f) Mass Spectrometer (MS)
SPLIT/SPLITLESS INJECTOR:
1. Split Injection:
The sample, in most cases a liquid, is introduced into a heated space, the
liner, where fast evaporation takes place. As a result of the fast evaporation
and the (required) turbulent flow, the sample vapor is mixed with the carrier
gas in the liner. This diluted gas mixture flows with a high velocity past the
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applications. Columns may also contain solid stationary phase particles
(GSC) for particular application types.
5. Column Oven:
Temperature in GC is controlled via a heated oven. The oven heats rapidly
to give excellent thermal control. The oven is cooled using a fan and vent
arrangement usually at the rear of the oven.
A hanger or cage is usually included to support the GC column and to
prevent it touching the oven walls as this can damage the column.
The injector and detector connections are also contained in the GC oven. For
Isothermal operation, the GC is held at a steady temperature during the
analysis. In temperature programmed GC (pTGC) the oven temperature is
increased according to the temperature program during the analysis.
6. Detector:
The detector responds to a physicochemical property of the analyte,
amplifies this response and generates an electronic signal for the data system
to produce a chromatogram.
Many different detector types exist and the choice is based mainly on
application, analyte chemistry and required sensitivity – also on whether
quantitative or qualitative data is required.
Detector choices include:
a) Flame Ionization (FID)
b) Electron Capture (ECD)
c) Flame Photometric (FPD)
d) Nitrogen Phosphorous (NPD)
e) Thermal Conductivity (TCD)
f) Mass Spectrometer (MS)
SPLIT/SPLITLESS INJECTOR:
1. Split Injection:
The sample, in most cases a liquid, is introduced into a heated space, the
liner, where fast evaporation takes place. As a result of the fast evaporation
and the (required) turbulent flow, the sample vapor is mixed with the carrier
gas in the liner. This diluted gas mixture flows with a high velocity past the
Gas Chromatography 2016
10
column entrance where a small portion is introduced into the column, but
most is carried away along the split outlet.
The splitting of the sample serves two purposes. Fast evaporation and a short
residence time in the liner results in a small injection plug. Secondly,
splitting reduces the size of the sample to an amount compatible with the
sample capacity of the capillary column. To improve mixing between the
vaporized sample and the carrier gas, packed liners containing a plug of
glass wool are sometimes used. With such liners better reproducibility is
normally obtained. Due to catalytic activity, however, even properly
deactivated glass wool can result in serious degradation of unstable solutes.
The ratio of the amount of material entering the column to the amount lost
via the split can be calculated from the ratio of the column flow and the split
flow. The split ratio is:
Split ratio = Fcolumn
Fsplit + Fcolumn
F column= column flow [ml/min]
F split= split flow [ml/min]
Because generally the column flow is much lower than the split flow, this equation
can be rewritten as:
Split ratio= F column
Fsplit
10
column entrance where a small portion is introduced into the column, but
most is carried away along the split outlet.
The splitting of the sample serves two purposes. Fast evaporation and a short
residence time in the liner results in a small injection plug. Secondly,
splitting reduces the size of the sample to an amount compatible with the
sample capacity of the capillary column. To improve mixing between the
vaporized sample and the carrier gas, packed liners containing a plug of
glass wool are sometimes used. With such liners better reproducibility is
normally obtained. Due to catalytic activity, however, even properly
deactivated glass wool can result in serious degradation of unstable solutes.
The ratio of the amount of material entering the column to the amount lost
via the split can be calculated from the ratio of the column flow and the split
flow. The split ratio is:
Split ratio = Fcolumn
Fsplit + Fcolumn
F column= column flow [ml/min]
F split= split flow [ml/min]
Because generally the column flow is much lower than the split flow, this equation
can be rewritten as:
Split ratio= F column
Fsplit
Gas Chromatography 2016
11
2. Splitless Injection:
The hardware required for splitless injection is very similar to that used for
split injections. As in the case of split injection the sample is evaporated in a
heated liner.
The split line however, is now closed (closed split/splitless valve). Transport
of sample vapors onto the column can only take place by means of the
column flow. After the largest part of the sample has been introduced into
the column, usually 10-40 secs.
After the injection (i.e. the so-called splitless time), the split line is opened
and the liner is quickly flushed. Sample is introduced onto the column
during the entire splitless time.
A very serious broadening of the peaks would result without re-
concentration of the sample in the column. The use of a suitable initial
column temperature ensures condensation and re-concentration of the
sample takes place in the column. Two re-concentration mechanisms can be
distinguished:
1. Cold trapping:
Re-concentration of high boiling components takes place by a cold trapping
mechanism. In the first centimeters of the column there is a negative
11
2. Splitless Injection:
The hardware required for splitless injection is very similar to that used for
split injections. As in the case of split injection the sample is evaporated in a
heated liner.
The split line however, is now closed (closed split/splitless valve). Transport
of sample vapors onto the column can only take place by means of the
column flow. After the largest part of the sample has been introduced into
the column, usually 10-40 secs.
After the injection (i.e. the so-called splitless time), the split line is opened
and the liner is quickly flushed. Sample is introduced onto the column
during the entire splitless time.
A very serious broadening of the peaks would result without re-
concentration of the sample in the column. The use of a suitable initial
column temperature ensures condensation and re-concentration of the
sample takes place in the column. Two re-concentration mechanisms can be
distinguished:
1. Cold trapping:
Re-concentration of high boiling components takes place by a cold trapping
mechanism. In the first centimeters of the column there is a negative
Gas Chromatography 2016
12
temperature gradient, where the temperature drops from the injection
temperature (± 250°C) to the oven temperature (e.g.40°C). Due to this
temperature drop the mobility of the heavy components reduces to virtually
zero. The components remain in a small band and will only start to migrate
when the oven temperature has risen sufficiently during a temperature
programme. Optimal re-concentration takes place if the initial oven
temperature is about 150°C or more, below the boiling point of the
components.
2. Solvent effect:
Re-concentration of low boiling components (B.P. less than roughly 50 to
100 degrees above the boiling point of the solvent), takes place by the so-
called solvent effect. When the starting temperature of the column is about
20°C below the boiling point of the chosen solvent, then the lighter
components will condense in the column together with the solvent. The
liquid film formed will start to evaporate from the back and the sample
components will concentrate in a continuously shortening liquid film. This
results in a very small band of re-concentrated sample components.
For a proper re-concentration of the sample in the column, mixing of
the sample vapor and carrier gas in the liner should be suppressed. This can
be achieved by a combination of a small non-curved liner and a slow
injection. In general, long and narrow inserts are preferred to obtain minimal
sample dilution. This in contrast to split injection where mixing of the
sample with carrier gas in the liner is a prerequisite. Therefore wide, fritted
or baffled liners are used in split injection.
The initial temperature of the column is of the utmost importance. A
fast condensation of the vapor can be achieved by selecting an oven
temperature of about 20°C below the boiling point of the solvent. In contrast
to what the name suggests, the split exit is open during most of the GC run.
It is only a few seconds before and a short time after injection that the split
flow is switched off. The time the split is closed is called the splitless time.
Besides the correct choice of temperature and liner geometry the
optimization of splitless time is of crucial importance. If the split line is
opened too soon losses of sample will occur, furthermore, reproducibility is
often poor. If the splitless time is too long sample components and solvent
will show severe tailing.
12
temperature gradient, where the temperature drops from the injection
temperature (± 250°C) to the oven temperature (e.g.40°C). Due to this
temperature drop the mobility of the heavy components reduces to virtually
zero. The components remain in a small band and will only start to migrate
when the oven temperature has risen sufficiently during a temperature
programme. Optimal re-concentration takes place if the initial oven
temperature is about 150°C or more, below the boiling point of the
components.
2. Solvent effect:
Re-concentration of low boiling components (B.P. less than roughly 50 to
100 degrees above the boiling point of the solvent), takes place by the so-
called solvent effect. When the starting temperature of the column is about
20°C below the boiling point of the chosen solvent, then the lighter
components will condense in the column together with the solvent. The
liquid film formed will start to evaporate from the back and the sample
components will concentrate in a continuously shortening liquid film. This
results in a very small band of re-concentrated sample components.
For a proper re-concentration of the sample in the column, mixing of
the sample vapor and carrier gas in the liner should be suppressed. This can
be achieved by a combination of a small non-curved liner and a slow
injection. In general, long and narrow inserts are preferred to obtain minimal
sample dilution. This in contrast to split injection where mixing of the
sample with carrier gas in the liner is a prerequisite. Therefore wide, fritted
or baffled liners are used in split injection.
The initial temperature of the column is of the utmost importance. A
fast condensation of the vapor can be achieved by selecting an oven
temperature of about 20°C below the boiling point of the solvent. In contrast
to what the name suggests, the split exit is open during most of the GC run.
It is only a few seconds before and a short time after injection that the split
flow is switched off. The time the split is closed is called the splitless time.
Besides the correct choice of temperature and liner geometry the
optimization of splitless time is of crucial importance. If the split line is
opened too soon losses of sample will occur, furthermore, reproducibility is
often poor. If the splitless time is too long sample components and solvent
will show severe tailing.
Gas Chromatography 2016
13
HEAD SPACE SAMPLING:
Headspace analysis is generally defined as a vapor-phase extraction, involving the
partitioning of analytes between a non-volatile liquid or solid phase and the vapor
phase above the liquid or solid. It is expected that the vapor-phase mixture contains
fewer components than the usually complex liquid or solid sample and that this
mixture is transferred to a GC or other instrument for analysis. There are a number
of techniques for sampling headspace vapors and introducing them to a GC.
Most consumer products and biological samples are composed of a
wide variety of compounds that differ in molecular weight, polarity, and volatility.
For complex samples like these, headspace sampling is the fastest and cleanest
method for analyzing volatile organic compounds. A headspace sample is normally
prepared in a vial containing the sample, the dilution solvent, a matrix modifier,
and the headspace. Volatile components from complex sample mixtures can be
extracted from non-volatile sample components and isolated in the headspace or
vapor portion of a sample vial. An aliquot of the vapor in the headspace is
delivered to a GC system for separation of all of the volatile components.
In order to achieve the best performance when using headspace/GC, careful
attention should be used in sample preparation and instrument setup. Key issues to
address when setting up headspace/GC systems include minimizing system dead
volume, maintaining inert sample flow paths, and achieving efficient sample
transfer. These issues, as well as other instrument setup-related topics, are
addressed later in the System Optimization section of this guide.
13
HEAD SPACE SAMPLING:
Headspace analysis is generally defined as a vapor-phase extraction, involving the
partitioning of analytes between a non-volatile liquid or solid phase and the vapor
phase above the liquid or solid. It is expected that the vapor-phase mixture contains
fewer components than the usually complex liquid or solid sample and that this
mixture is transferred to a GC or other instrument for analysis. There are a number
of techniques for sampling headspace vapors and introducing them to a GC.
Most consumer products and biological samples are composed of a
wide variety of compounds that differ in molecular weight, polarity, and volatility.
For complex samples like these, headspace sampling is the fastest and cleanest
method for analyzing volatile organic compounds. A headspace sample is normally
prepared in a vial containing the sample, the dilution solvent, a matrix modifier,
and the headspace. Volatile components from complex sample mixtures can be
extracted from non-volatile sample components and isolated in the headspace or
vapor portion of a sample vial. An aliquot of the vapor in the headspace is
delivered to a GC system for separation of all of the volatile components.
In order to achieve the best performance when using headspace/GC, careful
attention should be used in sample preparation and instrument setup. Key issues to
address when setting up headspace/GC systems include minimizing system dead
volume, maintaining inert sample flow paths, and achieving efficient sample
transfer. These issues, as well as other instrument setup-related topics, are
addressed later in the System Optimization section of this guide.
Gas Chromatography 2016
14
Figure: Head space sampling
COLUMNS FOR GAS CHROMATOGRAPHY:
In gas chromatography, the column is the heart of the system where the separation
of sample components takes place. They are classified in terms of tubing
dimensions and type of packing material. Packed columns are generally 1.5 – 10m
in length and 2 – 4mm id. These are generally made of stainless steel or glass. On
the other hand capillary columns are 0.1 – 0.5 mm id and can be 10 – 100m long.
Types of Columns:
a) Packed Columns:
Packed columns are prepared from glass or metals. They are 2-3 m long with
an internal diameter of 2-4 mm. In GLC, these columns are densely packed
with finely divided solid support which is in turn coated with a thin layer of
14
Figure: Head space sampling
COLUMNS FOR GAS CHROMATOGRAPHY:
In gas chromatography, the column is the heart of the system where the separation
of sample components takes place. They are classified in terms of tubing
dimensions and type of packing material. Packed columns are generally 1.5 – 10m
in length and 2 – 4mm id. These are generally made of stainless steel or glass. On
the other hand capillary columns are 0.1 – 0.5 mm id and can be 10 – 100m long.
Types of Columns:
a) Packed Columns:
Packed columns are prepared from glass or metals. They are 2-3 m long with
an internal diameter of 2-4 mm. In GLC, these columns are densely packed
with finely divided solid support which is in turn coated with a thin layer of
Gas Chromatography 2016
15
liquid stationary phase. In GSC, the columns are packed with adsorbents or
porous polymers. Packed columns are shaped as coils.
Packing of coiled columns is relatively difficult. However, it may be
achieved by applying vaccum from one of its ends and filling the coated
support from the other end.
Figure: Packed column for GC
b) Capillary Column/Open Tubular Columns:
Capillary columns are gas chromatography (GC) columns that have the
stationary phase coating their inner surfaces rather than being packed into
the cavity.
Capillary GC columns are used to analyze samples for the individual
chemical compounds that they contain. The capillary column is used in the
petroleum and pharmaceutical industries to test for impurities and in clinical
laboratories to help determine the chemical makeup of a sample.
A capillary CG column has a more efficient separation of the sample than a
packed column, but it is more easily overloaded by introducing too much of
the sample.
Three types of capillary columns are commonly used in gas
chromatography:
15
liquid stationary phase. In GSC, the columns are packed with adsorbents or
porous polymers. Packed columns are shaped as coils.
Packing of coiled columns is relatively difficult. However, it may be
achieved by applying vaccum from one of its ends and filling the coated
support from the other end.
Figure: Packed column for GC
b) Capillary Column/Open Tubular Columns:
Capillary columns are gas chromatography (GC) columns that have the
stationary phase coating their inner surfaces rather than being packed into
the cavity.
Capillary GC columns are used to analyze samples for the individual
chemical compounds that they contain. The capillary column is used in the
petroleum and pharmaceutical industries to test for impurities and in clinical
laboratories to help determine the chemical makeup of a sample.
A capillary CG column has a more efficient separation of the sample than a
packed column, but it is more easily overloaded by introducing too much of
the sample.
Three types of capillary columns are commonly used in gas
chromatography:
Gas Chromatography 2016
16
1) Wall Coated Open Tubular (WCOT):
A wall coated open tubular column, as the name suggests, consists of a tube
in which the wall is coated with a material acting as a stationary phase.
In general the tube itself is a capillary tube with a narrow inner diameter,
less than 1 mm, but of very long length measuring up to tens of meters. The
tubes are so narrow that they are easily coiled up and suspended in an oven
for temperature control.
The coating is usually a film of a polymer that uniformly wets the inside of
the column. A variety of functional groups may be present in such a polymer
so that specific polarity and selectivity is provided. The film is thermostable,
within reasonable temperatures, so that a WCOT can work over a range of
temperatures. The polymer is also non extractable meaning that the column
can be flushed with pure solvents to remove contaminants.
The thickness of the coating allows one to optimize columns for separation
of very volatile (thick films, 3- 5 mm) or high molecular weight compounds
(thin films, < 1 mm) and achieve separations within a reasonable analysis
time. The usual thickness of the film is 1-2 mm.
Advantages:
The advantages of open tubular columns over packed columns are:
1. Faster Analysis (High Flow Of Carrier Possible)
2. Shorter Retention Times
16
1) Wall Coated Open Tubular (WCOT):
A wall coated open tubular column, as the name suggests, consists of a tube
in which the wall is coated with a material acting as a stationary phase.
In general the tube itself is a capillary tube with a narrow inner diameter,
less than 1 mm, but of very long length measuring up to tens of meters. The
tubes are so narrow that they are easily coiled up and suspended in an oven
for temperature control.
The coating is usually a film of a polymer that uniformly wets the inside of
the column. A variety of functional groups may be present in such a polymer
so that specific polarity and selectivity is provided. The film is thermostable,
within reasonable temperatures, so that a WCOT can work over a range of
temperatures. The polymer is also non extractable meaning that the column
can be flushed with pure solvents to remove contaminants.
The thickness of the coating allows one to optimize columns for separation
of very volatile (thick films, 3- 5 mm) or high molecular weight compounds
(thin films, < 1 mm) and achieve separations within a reasonable analysis
time. The usual thickness of the film is 1-2 mm.
Advantages:
The advantages of open tubular columns over packed columns are:
1. Faster Analysis (High Flow Of Carrier Possible)
2. Shorter Retention Times
Gas Chromatography 2016
17
3. More Inert
4. Longer Life
5. Less Bleed Of Coating Material
6. Higher Efficiencies
7. Greater Reproducibility.
2) Support Coated Open Tubular (SCOT):
Capillary tube wall is lined with a thin layer of solid support on to which
liquid phase is adsorbed. The separation efficiency of SCOT columns is
more than WCOT columns because of increased surface area of the
stationary phase coating.
3) Fused Silica Open Tubular (FSOT):
Walls of capillary fused silica tubes are strengthened by a polyimide coating.
These are flexible and can be wound into coils.
Column Characteristics:
i. Column Materials:
Fused silica and stainless steel columns offer high degree of inertness
and flexibility. When breakage is not of much concern fused silica is
the best choice.
ii. Internal Diameter:
Sample concentration is the deciding factor for the internal diameter
of the column. Loss of resolution, poor reproducibility and peak
distortion result if sample concentration exceeds column capacity.
17
3. More Inert
4. Longer Life
5. Less Bleed Of Coating Material
6. Higher Efficiencies
7. Greater Reproducibility.
2) Support Coated Open Tubular (SCOT):
Capillary tube wall is lined with a thin layer of solid support on to which
liquid phase is adsorbed. The separation efficiency of SCOT columns is
more than WCOT columns because of increased surface area of the
stationary phase coating.
3) Fused Silica Open Tubular (FSOT):
Walls of capillary fused silica tubes are strengthened by a polyimide coating.
These are flexible and can be wound into coils.
Column Characteristics:
i. Column Materials:
Fused silica and stainless steel columns offer high degree of inertness
and flexibility. When breakage is not of much concern fused silica is
the best choice.
ii. Internal Diameter:
Sample concentration is the deciding factor for the internal diameter
of the column. Loss of resolution, poor reproducibility and peak
distortion result if sample concentration exceeds column capacity.
Gas Chromatography 2016
18
iii. Length:
Longer columns provide greater resolution of sample components.
However, increasing column length increases analysis time.
iv. Film Thickness:
Film thickness determines the retention and elution temperature of
each sample component. Thick films increase the time a compound
stays on the stationary phase and thinner films reduce retention time.
Compounds having high volatility require more residence time for
better separation and should be analyzed on thicker films. The
commonly used film thickness in gas chromatography columns ranges
from 0.1 to 5.0µm.
Columns are selected for use in a particular application based on
column length and type of packing. Guidelines on selection of
columns are provided in more detail in the certificate programme
which will be launched in due course.
The most important criteria in selection of column are the stationary
phase packing which will be discussed in greater detail in the next
module.
DETECTORS:
1) Flame ionization detector:
Most common detector for GC.
In an FID, effluent from the column is directed into a small air-
hydrogen flame. Most carbon atoms (except C=O) produce radicals
(CHO
+
) in the flame:
CH + O→ CH
+
+ e
-
Electrons are used to neutralize the CHO
+
atoms and the ions are
collected at an electrode to create a current to be measured. This
current is proportional to the number of molecules present.
The ionization of carbon compounds in the FID is not fully
understood, although the number of ions produced is roughly
proportional to the number of reduced carbon atoms in the flame.
18
iii. Length:
Longer columns provide greater resolution of sample components.
However, increasing column length increases analysis time.
iv. Film Thickness:
Film thickness determines the retention and elution temperature of
each sample component. Thick films increase the time a compound
stays on the stationary phase and thinner films reduce retention time.
Compounds having high volatility require more residence time for
better separation and should be analyzed on thicker films. The
commonly used film thickness in gas chromatography columns ranges
from 0.1 to 5.0µm.
Columns are selected for use in a particular application based on
column length and type of packing. Guidelines on selection of
columns are provided in more detail in the certificate programme
which will be launched in due course.
The most important criteria in selection of column are the stationary
phase packing which will be discussed in greater detail in the next
module.
DETECTORS:
1) Flame ionization detector:
Most common detector for GC.
In an FID, effluent from the column is directed into a small air-
hydrogen flame. Most carbon atoms (except C=O) produce radicals
(CHO
+
) in the flame:
CH + O→ CH
+
+ e
-
Electrons are used to neutralize the CHO
+
atoms and the ions are
collected at an electrode to create a current to be measured. This
current is proportional to the number of molecules present.
The ionization of carbon compounds in the FID is not fully
understood, although the number of ions produced is roughly
proportional to the number of reduced carbon atoms in the flame.
Gas Chromatography 2016
19
Figure: Flame ionization detector
Advantages:
1. Universal detector for organics
2. Does not respond to common inorganic compounds
3. Mobile phase impurities not detected
4. Carrier gases not detected
5. Limit of detection: fid is 1000x better than TCD
6. Linear and dynamic range better than TCD
Disadvantage:
1. Destructive detector
2) Thermal conductivity detector:
Thermal conductivity detectors (TCD) were one the earliest
detectors developed for use with gas chromatography. The TCD
works by measuring the change in carrier gas thermal conductivity
caused by the presence of the sample, which has a different thermal
conductivity from that of the carrier gas. Their design is relatively
19
Figure: Flame ionization detector
Advantages:
1. Universal detector for organics
2. Does not respond to common inorganic compounds
3. Mobile phase impurities not detected
4. Carrier gases not detected
5. Limit of detection: fid is 1000x better than TCD
6. Linear and dynamic range better than TCD
Disadvantage:
1. Destructive detector
2) Thermal conductivity detector:
Thermal conductivity detectors (TCD) were one the earliest
detectors developed for use with gas chromatography. The TCD
works by measuring the change in carrier gas thermal conductivity
caused by the presence of the sample, which has a different thermal
conductivity from that of the carrier gas. Their design is relatively
Gas Chromatography 2016
20
simple, and consists of an electrically heated source that is
maintained at constant power. The temperature of the source
depends upon the thermal conductivities of the surrounding gases.
The source is usually a thin wire made of platinum or gold. The
resistance within the wire depends upon temperature, which is
dependent upon the thermal conductivity of the gas.
TCDs usually employ two detectors, one of which is used as the
reference for the carrier gas and the other which monitors the
thermal conductivity of the carrier gas and sample mixture. Carrier
gases such as helium and hydrogen has very high thermal
conductivities so the addition of even a small amount of sample is
readily detected.
The advantages of TCDs are the ease and simplicity of use, the
devices' broad application to inorganic and organic compounds, and
the ability of the analyte to be collected after separation and
detection. The greatest drawback of the TCD is the low sensitivity
of the instrument in relation to other detection methods, in addition
to flow rate and concentration dependency.
Figure: Thermal Conductivity Detector
20
simple, and consists of an electrically heated source that is
maintained at constant power. The temperature of the source
depends upon the thermal conductivities of the surrounding gases.
The source is usually a thin wire made of platinum or gold. The
resistance within the wire depends upon temperature, which is
dependent upon the thermal conductivity of the gas.
TCDs usually employ two detectors, one of which is used as the
reference for the carrier gas and the other which monitors the
thermal conductivity of the carrier gas and sample mixture. Carrier
gases such as helium and hydrogen has very high thermal
conductivities so the addition of even a small amount of sample is
readily detected.
The advantages of TCDs are the ease and simplicity of use, the
devices' broad application to inorganic and organic compounds, and
the ability of the analyte to be collected after separation and
detection. The greatest drawback of the TCD is the low sensitivity
of the instrument in relation to other detection methods, in addition
to flow rate and concentration dependency.
Figure: Thermal Conductivity Detector
Gas Chromatography 2016
21
Advantages:
1. Simplicity
2. Large linear dynamic range
3. Non-destructive
Disadvantages:
1. Low sensitivity (precludes their use with WCOT columns with small
amounts of sample)
3) Electron Capture Detector (ECD):
Radioactive decay-based detector.
Selective for compounds containing electronegative atoms, such as
halogens, peroxides, quinones, and nitro groups
The sample effluent from a column is passed over a radioactive β
emitter, usually
63
Ni. An electron from the emitter causes ionization of
the carrier gas (often N2) and the production of a burst of electrons.
In the absence of organic species, a constant standing current between
a pair of electrode results from this ionization process. The current
decreases significantly in the presence of organic molecules
containing electron negative functional groups that tend to capture
electrons.
21
Advantages:
1. Simplicity
2. Large linear dynamic range
3. Non-destructive
Disadvantages:
1. Low sensitivity (precludes their use with WCOT columns with small
amounts of sample)
3) Electron Capture Detector (ECD):
Radioactive decay-based detector.
Selective for compounds containing electronegative atoms, such as
halogens, peroxides, quinones, and nitro groups
The sample effluent from a column is passed over a radioactive β
emitter, usually
63
Ni. An electron from the emitter causes ionization of
the carrier gas (often N2) and the production of a burst of electrons.
In the absence of organic species, a constant standing current between
a pair of electrode results from this ionization process. The current
decreases significantly in the presence of organic molecules
containing electron negative functional groups that tend to capture
electrons.
Gas Chromatography 2016
22
Figure: Electron capture Detector
Advantages:
1. Useful for environmental testing detection of chlorinated pesticides or
herbicides; polynuclear aromatic carcinogens, organ metallic compounds.
2. Selective for halogen-(I, Br, Cl, F), nitro-, and sulfur-containing compounds.
3. Detects polynuclear aromatic compounds, anhydrides and conjugated
carbonyl compounds.
Disadvantages:
1. Could be affected by the flow rate.
4) Thermionic Detector/Nitrogen-Phosphorous Detector (NPD):
A NPD is based on the same basic principles as an FID.
However, small amounts of alkali metal vapor in the flame, which
greatly enhances the formation of ions from nitrogen and phosphorus-
containing compounds.
The NPD is about 500-fold more sensitive that an FID in detecting
phosphorous-containing compounds, and 50-fold more sensitive to
nitrogen-containing compounds.
22
Figure: Electron capture Detector
Advantages:
1. Useful for environmental testing detection of chlorinated pesticides or
herbicides; polynuclear aromatic carcinogens, organ metallic compounds.
2. Selective for halogen-(I, Br, Cl, F), nitro-, and sulfur-containing compounds.
3. Detects polynuclear aromatic compounds, anhydrides and conjugated
carbonyl compounds.
Disadvantages:
1. Could be affected by the flow rate.
4) Thermionic Detector/Nitrogen-Phosphorous Detector (NPD):
A NPD is based on the same basic principles as an FID.
However, small amounts of alkali metal vapor in the flame, which
greatly enhances the formation of ions from nitrogen and phosphorus-
containing compounds.
The NPD is about 500-fold more sensitive that an FID in detecting
phosphorous-containing compounds, and 50-fold more sensitive to
nitrogen-containing compounds.
Gas Chromatography 2016
23
Applications:
Organophosphate in pesticides and in drug analysis for determination of amine-
containing or basic drug.
Figure: Thermionic Detector
5) Electrolytic Conductivity Detector:
Element-selective detector for halogen-, sulfur-and nitrogen-
containing compounds.
Compounds containing halogens, sulfur, or nitrogen are mixed with a
reaction gas in a small reactor tube, usually made of Ni. The products
from the reaction tube are then dissolved in a liquid, which produces a
conductive solution. The change in conductivity as a result of the
ionic species is then measured.
23
Applications:
Organophosphate in pesticides and in drug analysis for determination of amine-
containing or basic drug.
Figure: Thermionic Detector
5) Electrolytic Conductivity Detector:
Element-selective detector for halogen-, sulfur-and nitrogen-
containing compounds.
Compounds containing halogens, sulfur, or nitrogen are mixed with a
reaction gas in a small reactor tube, usually made of Ni. The products
from the reaction tube are then dissolved in a liquid, which produces a
conductive solution. The change in conductivity as a result of the
ionic species is then measured.
Gas Chromatography 2016
24
Figure: Electrolytic Conductivity Detector
6) Photo Ionization Detector:
Another different kind of detector for GC is the photo ionization
detector which utilizes the properties of chemiluminescence
spectroscopy.
Photo ionization detector (PID) is a portable vapor and gas detector
that has selective determination of aromatic hydrocarbons, organo-
heteroatom, inorganic species and other organic compounds.
PID comprise of an ultraviolet lamp to emit photons that are
absorbed by the compounds in an ionization chamber exiting from a
GC column.
Small fractions of the analyte molecules are actually ionized,
nondestructive, allowing confirmation analytical results through
other detectors.
In addition, PIDs are available in portable hand-held models and in a
number of lamp configurations. Results are almost immediate.
PID is used commonly to detect VOCs in soil, sediment, air and
water, which is often used to detect contaminants in ambient air and
soil.
The disadvantage of PID is unable to detect certain hydrocarbon that
has low molecular weight, such as methane and ethane.
24
Figure: Electrolytic Conductivity Detector
6) Photo Ionization Detector:
Another different kind of detector for GC is the photo ionization
detector which utilizes the properties of chemiluminescence
spectroscopy.
Photo ionization detector (PID) is a portable vapor and gas detector
that has selective determination of aromatic hydrocarbons, organo-
heteroatom, inorganic species and other organic compounds.
PID comprise of an ultraviolet lamp to emit photons that are
absorbed by the compounds in an ionization chamber exiting from a
GC column.
Small fractions of the analyte molecules are actually ionized,
nondestructive, allowing confirmation analytical results through
other detectors.
In addition, PIDs are available in portable hand-held models and in a
number of lamp configurations. Results are almost immediate.
PID is used commonly to detect VOCs in soil, sediment, air and
water, which is often used to detect contaminants in ambient air and
soil.
The disadvantage of PID is unable to detect certain hydrocarbon that
has low molecular weight, such as methane and ethane.
Gas Chromatography 2016
25
Figure: Photo Ionization Detector
Limitations:
1. Not suitable for detecting semi-volatile compounds
2. Only indicates if volatile organic compounds are presents.
3. High concentration so methane is required for higher performance.
4. Frequent calibrations are required.
5. Units of parts per million range
6. Environmental distraction, especially water vapor.
7. Strong electrical field’s Rapid variation in temperature at the detector and
naturally occurring compounds may affect instrumental signal.
7) Atomic Emission Detector:
Atomic emission detectors (AED), one of the newest additions to the
gas chromatographer's arsenal, are element-selective detectors that
utilize plasma, which is a partially ionized gas, to atomize all of the
elements of a sample and excite their characteristic atomic emission
spectra.
25
Figure: Photo Ionization Detector
Limitations:
1. Not suitable for detecting semi-volatile compounds
2. Only indicates if volatile organic compounds are presents.
3. High concentration so methane is required for higher performance.
4. Frequent calibrations are required.
5. Units of parts per million range
6. Environmental distraction, especially water vapor.
7. Strong electrical field’s Rapid variation in temperature at the detector and
naturally occurring compounds may affect instrumental signal.
7) Atomic Emission Detector:
Atomic emission detectors (AED), one of the newest additions to the
gas chromatographer's arsenal, are element-selective detectors that
utilize plasma, which is a partially ionized gas, to atomize all of the
elements of a sample and excite their characteristic atomic emission
spectra.
Gas Chromatography 2016
26
AED is an extremely powerful alternative that has a wider
applicability due to its based on the detection of atomic emissions.
There are three ways of generating plasma: microwave-induced
plasma (MIP), inductively coupled plasma (ICP) or direct current
plasma (DCP).
MIP is the most commonly employed form and is used with a
position able diode array to simultaneously monitor the atomic
emission spectra of several elements.
Figure: Atomic Emission Detector
8) Flame Photometric Detector:
The flame photometric detector (FPD) allows sensitive and selective
measurements of volatile sulphur and phosphorus compounds.
The detection principle is the formation of excited sulphur (S2*) and
excited hydrogen phosphorous oxide species (HPO*) in a reducing
flame.
A photomultiplier tube measures the characteristic
chemiluminescent emission from these species.
The optical filter can be changed to allow the photomultiplier to
view light of 394 nm for sulphur measurement or 526 nm for
phosphorus.
26
AED is an extremely powerful alternative that has a wider
applicability due to its based on the detection of atomic emissions.
There are three ways of generating plasma: microwave-induced
plasma (MIP), inductively coupled plasma (ICP) or direct current
plasma (DCP).
MIP is the most commonly employed form and is used with a
position able diode array to simultaneously monitor the atomic
emission spectra of several elements.
Figure: Atomic Emission Detector
8) Flame Photometric Detector:
The flame photometric detector (FPD) allows sensitive and selective
measurements of volatile sulphur and phosphorus compounds.
The detection principle is the formation of excited sulphur (S2*) and
excited hydrogen phosphorous oxide species (HPO*) in a reducing
flame.
A photomultiplier tube measures the characteristic
chemiluminescent emission from these species.
The optical filter can be changed to allow the photomultiplier to
view light of 394 nm for sulphur measurement or 526 nm for
phosphorus.
Gas Chromatography 2016
27
The detector response to phosphor is linear, whereas the response to
sulphur depends on the square of the concentration.
Figure: Flame photometric detector
GC DERIVATIZATION:
Derivatization is the process of chemically modifying a compound to produce a
new compound which has properties that are suitable for analysis using a GC.
Derivatisation is done:
To permit analysis of compounds not directly amenable to analysis
due to, for example, inadequate volatility or stability
Improve chromatographic behavior or detectability
Many compounds do not produce a useable chromatograph (i.e.
multiple peaks, or one big blob), or the sample of interest goes
undetected. As a result it may be necessary to derivatize the
compound before GC analysis is done.
Derivatization is a useful tool allowing the use of GC and GC/MS to
be done on samples that would otherwise not be possible in various
areas of chemistry such as medical, forensic, and environmental.
27
The detector response to phosphor is linear, whereas the response to
sulphur depends on the square of the concentration.
Figure: Flame photometric detector
GC DERIVATIZATION:
Derivatization is the process of chemically modifying a compound to produce a
new compound which has properties that are suitable for analysis using a GC.
Derivatisation is done:
To permit analysis of compounds not directly amenable to analysis
due to, for example, inadequate volatility or stability
Improve chromatographic behavior or detectability
Many compounds do not produce a useable chromatograph (i.e.
multiple peaks, or one big blob), or the sample of interest goes
undetected. As a result it may be necessary to derivatize the
compound before GC analysis is done.
Derivatization is a useful tool allowing the use of GC and GC/MS to
be done on samples that would otherwise not be possible in various
areas of chemistry such as medical, forensic, and environmental.
Gas Chromatography 2016
28
Types of derivatization:
i. Silylation:
It is the most commonly practiced derivatization technique in GC and
widely used group for this purpose is trimethylsilyl (TMS) or
[Si(CH3)3].
Derivatization by silylation involves the conversion of polar
NH2, NH, OH, SH and COOH groups into non-polar, more volatile
and thermally stable groups. The silyl groups upon reaction with
amines, amides, alcohols, acids and thiols undergo replacement with
their active hydrogen resulting in the formation of silyl derivatives. A
large number of silylation reagents are available for this purpose.
Most of these reagents and their derivatives have good thermal
stability and are compatible with vast range of injection port and
column conditions. Both silylation reagents as well as their derivatives
undergo decomposition in the presence of moisture. Hence, they
should be protected from moist environment.
Table: Silylation Reagents
Silylation reagent Good silylation Moderate
silylation
No
silyalation
N, O – Bis
(trimethylsilyl)acetamide
Acids, alcohols,
amines and phenols
Thiols -
N, O – Bis (trimethylsilyl)
trifluoroacetamide
Acids, alcohols,
amines and phenols
Thiols -
Chlorotrimethylsilane Acids, alcohols and
phenols
Amines and
thiols
-
1-(trimethylsilane)
imidazole
Acids, alcohols and
phenols
Thiols Amines
Hexamethyldisilazane Alcohols and
phenols
Acids,
amines and
thiols
-
ii. Alkylation:
Derivatization in GC by alkylation reaction involves the replacement of
active hydrogen in R-OH, R-SH, R-NH2 or R-COOH with an alkyl group.
This results in conversion of organic acids into esters, especially methyl
esters. The technique is commonly employed in GC because the column
chromatography produced by esters is better than that by organic acids.
28
Types of derivatization:
i. Silylation:
It is the most commonly practiced derivatization technique in GC and
widely used group for this purpose is trimethylsilyl (TMS) or
[Si(CH3)3].
Derivatization by silylation involves the conversion of polar
NH2, NH, OH, SH and COOH groups into non-polar, more volatile
and thermally stable groups. The silyl groups upon reaction with
amines, amides, alcohols, acids and thiols undergo replacement with
their active hydrogen resulting in the formation of silyl derivatives. A
large number of silylation reagents are available for this purpose.
Most of these reagents and their derivatives have good thermal
stability and are compatible with vast range of injection port and
column conditions. Both silylation reagents as well as their derivatives
undergo decomposition in the presence of moisture. Hence, they
should be protected from moist environment.
Table: Silylation Reagents
Silylation reagent Good silylation Moderate
silylation
No
silyalation
N, O – Bis
(trimethylsilyl)acetamide
Acids, alcohols,
amines and phenols
Thiols -
N, O – Bis (trimethylsilyl)
trifluoroacetamide
Acids, alcohols,
amines and phenols
Thiols -
Chlorotrimethylsilane Acids, alcohols and
phenols
Amines and
thiols
-
1-(trimethylsilane)
imidazole
Acids, alcohols and
phenols
Thiols Amines
Hexamethyldisilazane Alcohols and
phenols
Acids,
amines and
thiols
-
ii. Alkylation:
Derivatization in GC by alkylation reaction involves the replacement of
active hydrogen in R-OH, R-SH, R-NH2 or R-COOH with an alkyl group.
This results in conversion of organic acids into esters, especially methyl
esters. The technique is commonly employed in GC because the column
chromatography produced by esters is better than that by organic acids.
Gas Chromatography 2016
29
Moreover, alkylation derivatives are less polar, highly stable and can be
separated and stored for longer durations than the parent compound.
Table: Alkylation Reagents
Alkylation Reagent Applicable to
Triethyloxonium tetrafluoroborate Sterically hindered carboxylic acids
N,N-dimethylformamide di-tert-
butylacetal
Sterically hindered amines
N,N-dimethylformamide
dimethylacetal
Sterically hindered amines
iii. Acylation:
This derivatization technique includes the conversion of compounds
with active hydrogen by the action of a carboxylic acid or its
derivatives.
E.g.: OH to esters, NH to amides, SH to thioesters.
The derivatives so formed are better chromatographed and detected
than a parent compound. Acylation may be carried out by the
following two types of acylation reagents, fluoro acid anhydride and
fluoroacylimidazoles.
Table: Acylation Reagents
Acylation reagent Examples Undergo acylation
by reacting with
Fluoro acid anhydride Trifluoroacetic anhydride,
Heptafluorobutyric anhydride
Amino, hydroxyl
and thiol groups
Fluoro acylimidazoles 1-(trifluoroacetyl) imidazole
1-(heptafluorobutyryl)
imidazole
1
o
and 2
o
amines
2
o
and 3
o
amines
ADVANTAGES OF GC:
Fast analysis
High efficiency leading to high resolution
Sensitive detectors (ppb)
29
Moreover, alkylation derivatives are less polar, highly stable and can be
separated and stored for longer durations than the parent compound.
Table: Alkylation Reagents
Alkylation Reagent Applicable to
Triethyloxonium tetrafluoroborate Sterically hindered carboxylic acids
N,N-dimethylformamide di-tert-
butylacetal
Sterically hindered amines
N,N-dimethylformamide
dimethylacetal
Sterically hindered amines
iii. Acylation:
This derivatization technique includes the conversion of compounds
with active hydrogen by the action of a carboxylic acid or its
derivatives.
E.g.: OH to esters, NH to amides, SH to thioesters.
The derivatives so formed are better chromatographed and detected
than a parent compound. Acylation may be carried out by the
following two types of acylation reagents, fluoro acid anhydride and
fluoroacylimidazoles.
Table: Acylation Reagents
Acylation reagent Examples Undergo acylation
by reacting with
Fluoro acid anhydride Trifluoroacetic anhydride,
Heptafluorobutyric anhydride
Amino, hydroxyl
and thiol groups
Fluoro acylimidazoles 1-(trifluoroacetyl) imidazole
1-(heptafluorobutyryl)
imidazole
1
o
and 2
o
amines
2
o
and 3
o
amines
ADVANTAGES OF GC:
Fast analysis
High efficiency leading to high resolution
Sensitive detectors (ppb)
Gas Chromatography 2016
30
Non-destructive –enabling coupling to Mass Spectrometers (MS) –an
instrument that measures the masses of individual molecules that have been
converted into ions, i.e. molecules that have been electrically charged
High quantitative accuracy (<1% RSD typical)
Requires small samples (<1 mL)
Rugged and reliable techniques
Well established with extensive literature and applications
DISADVANTAGES OF GC:
Limited to volatile samples
Not suitable for samples that degrade at elevated temperatures (thermally
labile)
Not suited to preparative chromatography
Requires MS detector for analyte structural elucidation (characterization)
Most Non-MS Detectors are destructive.
APPLICATIONS OF GC:
There are various applications of gas chromatography (GC) which are discussed
here so let us check it out one by one which are as follows:
1. Gas chromatography used in determination and identification of fatty acids.
2. Determination of drugs in body fluids such as urine, serum, plasma etc.
3. Analysis of alcoholic beverages.
4. GC is also used in determination of steroids.
5. Analysis of various solvents.
6. Analysis of different organic functional groups.
7. Gas chromatography is also useful in isolation and identification of volatile
oils, proteins, lipids, carbohydrates, colorants, plant extracts etc.
8. Gas chromatography is also useful in analysis of cosmetics, fertilizers,
perfumes, petroleum products and food products.
9. GC is also used in analysis of dairy products such as cheese, butter, milk etc.
Pharmaceutical Applications:
Gas Chromatography is useful in analysis of pharmaceutical products, so let us
check it out some of the applications in pharmaceutical industry. Uses of GC are as
follows:
30
Non-destructive –enabling coupling to Mass Spectrometers (MS) –an
instrument that measures the masses of individual molecules that have been
converted into ions, i.e. molecules that have been electrically charged
High quantitative accuracy (<1% RSD typical)
Requires small samples (<1 mL)
Rugged and reliable techniques
Well established with extensive literature and applications
DISADVANTAGES OF GC:
Limited to volatile samples
Not suitable for samples that degrade at elevated temperatures (thermally
labile)
Not suited to preparative chromatography
Requires MS detector for analyte structural elucidation (characterization)
Most Non-MS Detectors are destructive.
APPLICATIONS OF GC:
There are various applications of gas chromatography (GC) which are discussed
here so let us check it out one by one which are as follows:
1. Gas chromatography used in determination and identification of fatty acids.
2. Determination of drugs in body fluids such as urine, serum, plasma etc.
3. Analysis of alcoholic beverages.
4. GC is also used in determination of steroids.
5. Analysis of various solvents.
6. Analysis of different organic functional groups.
7. Gas chromatography is also useful in isolation and identification of volatile
oils, proteins, lipids, carbohydrates, colorants, plant extracts etc.
8. Gas chromatography is also useful in analysis of cosmetics, fertilizers,
perfumes, petroleum products and food products.
9. GC is also used in analysis of dairy products such as cheese, butter, milk etc.
Pharmaceutical Applications:
Gas Chromatography is useful in analysis of pharmaceutical products, so let us
check it out some of the applications in pharmaceutical industry. Uses of GC are as
follows:
Gas Chromatography 2016
31
1. Gas Chromatography is used to determine the identity of natural products
which contains complex mixture of the similar compounds.
2. Gas chromatography is used in separation of volatile mixture.
3. Amount of chemicals in the drugs can be identified by gas chromatography.
4. Gas chromatography is widely used in cosmetic manufacturing companies to
measure amount of volatile chemicals used in cosmetics.
5. GC is used in Analysis of various solvents used in manufacturing of
pharmaceutical products.
6. GC is also used in analysis of various organic functional groups.
7. Qualitative and quantitative estimation of solid, liquid and gaseous organic
compounds.
Environmental Applications:
Gas Chromatography is useful in Environmental Analysis, so let us check it out
some of the applications in environmental analysis. Uses of GC are as follows:
1. Gas Chromatography (GC) is used in quantification of pollutants in drinking
and waste water.
2. Gas chromatography is used in identification of unknown organic compound
in hazardous waste.
3. Gas chromatography is also used in analysis of industrial products as well
identification of reaction products.
4. Environmental toxins can be identified with gas chromatography.
5. Air samples can be analyzed by gas chromatography for quality control.
6. Pestisides, fertilizers can by analyzed by GC.
7. Pollution studies, environmental analysis by GC.
Clinical Applications:
Gas Chromatography is useful in Clinical analysis, so let us check it out some of
the applications in clinical analysis. Uses of GC are as follows:
1. Blood, Saliva and other secretions which contains large amount of organic
volatiles can be easily analyzed by gas chromatography.
2. Isolation of volatile proteins, lipids, carbohydrates by gas chromatography.
3. Gas chromatography is used to analyze the chemicals and drugs present in
the body.
4. Various biological volatile organic compounds can be analyzed by gas
chromatography.
31
1. Gas Chromatography is used to determine the identity of natural products
which contains complex mixture of the similar compounds.
2. Gas chromatography is used in separation of volatile mixture.
3. Amount of chemicals in the drugs can be identified by gas chromatography.
4. Gas chromatography is widely used in cosmetic manufacturing companies to
measure amount of volatile chemicals used in cosmetics.
5. GC is used in Analysis of various solvents used in manufacturing of
pharmaceutical products.
6. GC is also used in analysis of various organic functional groups.
7. Qualitative and quantitative estimation of solid, liquid and gaseous organic
compounds.
Environmental Applications:
Gas Chromatography is useful in Environmental Analysis, so let us check it out
some of the applications in environmental analysis. Uses of GC are as follows:
1. Gas Chromatography (GC) is used in quantification of pollutants in drinking
and waste water.
2. Gas chromatography is used in identification of unknown organic compound
in hazardous waste.
3. Gas chromatography is also used in analysis of industrial products as well
identification of reaction products.
4. Environmental toxins can be identified with gas chromatography.
5. Air samples can be analyzed by gas chromatography for quality control.
6. Pestisides, fertilizers can by analyzed by GC.
7. Pollution studies, environmental analysis by GC.
Clinical Applications:
Gas Chromatography is useful in Clinical analysis, so let us check it out some of
the applications in clinical analysis. Uses of GC are as follows:
1. Blood, Saliva and other secretions which contains large amount of organic
volatiles can be easily analyzed by gas chromatography.
2. Isolation of volatile proteins, lipids, carbohydrates by gas chromatography.
3. Gas chromatography is used to analyze the chemicals and drugs present in
the body.
4. Various biological volatile organic compounds can be analyzed by gas
chromatography.
Gas Chromatography 2016
32
5. Urine sample is also analyzed by gas chromatography.
Applications in food analysis:
Gas Chromatography is useful in Food Analysis, so let us check it out some of the
applications in food analysis. Uses of GC are as follows:
1. Food products can be analyzed by gas chromatography.
2. Gas chromatography is used in analysis of alcohol beverages.
3. Analysis of various solvents in food preparation.
4. GC is used in analysis of dairy products such as milk, butter, cheese etc.
5. Analysis of food and beverages for nutrition person.
6. Quantification of volatile organic food products.
Forensic Applications:
Gas Chromatography is useful in Forensics, so let us check it out some of the
applications in forensics. Uses of GC are as follows:
1. Gas Chromatography is used in determination of blood alcohol content in
the body.
2. Blood alcohol analysis test is used by law enforcement to determine if a
driver was unlawfully driving or operating a vehicle.
3. Certain drugs are prohibited in certain states, so in order to check whether
person has taken drugs in the body Gas chromatography is used.
4. Gas chromatography is used to test samples found at crime scene.
5. GC is used to identify which fluids and chemicals are present in the body.
6. Testing of blood and fiber at the crime scene by GC.
32
5. Urine sample is also analyzed by gas chromatography.
Applications in food analysis:
Gas Chromatography is useful in Food Analysis, so let us check it out some of the
applications in food analysis. Uses of GC are as follows:
1. Food products can be analyzed by gas chromatography.
2. Gas chromatography is used in analysis of alcohol beverages.
3. Analysis of various solvents in food preparation.
4. GC is used in analysis of dairy products such as milk, butter, cheese etc.
5. Analysis of food and beverages for nutrition person.
6. Quantification of volatile organic food products.
Forensic Applications:
Gas Chromatography is useful in Forensics, so let us check it out some of the
applications in forensics. Uses of GC are as follows:
1. Gas Chromatography is used in determination of blood alcohol content in
the body.
2. Blood alcohol analysis test is used by law enforcement to determine if a
driver was unlawfully driving or operating a vehicle.
3. Certain drugs are prohibited in certain states, so in order to check whether
person has taken drugs in the body Gas chromatography is used.
4. Gas chromatography is used to test samples found at crime scene.
5. GC is used to identify which fluids and chemicals are present in the body.
6. Testing of blood and fiber at the crime scene by GC.
Gas Chromatography 2016
33
CONCLUSION:
This technique has a high resolution power compared to others. Complex
mixture can be resolved into its components by this GC method. The
separation, determination and identification of many compounds
with negligible differences in boiling points are possible by this technique.
Sensitivity in detection is very high with thermal conductivity detectors. One
can detect up to 100 ppm, while flame detectors, electron capture and
phosphorus detectors can detect ppm, parts per billion or picograms
respectively.
It gives relatively good precision and accuracy.
It is a micro method hence very small size is required hence micro litre of
sample is sufficient for complete analysis.
The speed of analysis is very fast.
The use of a gas as the moving phase has the advantage of rapid equilibrium
between the moving and stationary phases and allows use of high carrier gas
velocities, leading to fast analysis in seconds, minutes or in hours.
It involves relatively simple instrumentation operation of gas
chromatography and related calculations don not require highly skilled
personnel and thus the technique is suitable for routine analysis.
Qualitative as well as quantitative analysis at a time is possible.
The area produced for each peak is proportional to that concentration.
The cost of gas chromatography is very low as compared to the data
obtained.
33
CONCLUSION:
This technique has a high resolution power compared to others. Complex
mixture can be resolved into its components by this GC method. The
separation, determination and identification of many compounds
with negligible differences in boiling points are possible by this technique.
Sensitivity in detection is very high with thermal conductivity detectors. One
can detect up to 100 ppm, while flame detectors, electron capture and
phosphorus detectors can detect ppm, parts per billion or picograms
respectively.
It gives relatively good precision and accuracy.
It is a micro method hence very small size is required hence micro litre of
sample is sufficient for complete analysis.
The speed of analysis is very fast.
The use of a gas as the moving phase has the advantage of rapid equilibrium
between the moving and stationary phases and allows use of high carrier gas
velocities, leading to fast analysis in seconds, minutes or in hours.
It involves relatively simple instrumentation operation of gas
chromatography and related calculations don not require highly skilled
personnel and thus the technique is suitable for routine analysis.
Qualitative as well as quantitative analysis at a time is possible.
The area produced for each peak is proportional to that concentration.
The cost of gas chromatography is very low as compared to the data
obtained.
Gas Chromatography 2016
34
BIBLIOGRAPHY:
K. Grob, ‘Classical Split and Split less Injection’, in Capillary GC, A.
Huethig, Heidelberg, 1987
R. Buffington, M.K. Wilson, Detectors for Gas Chromatography – a
Practical Primer, Hewlett-Packard Corporation, Part No. 5958-9433, 1987.
D.J. David, “Gas Chromatographic Detectors”, John Wiley & Sons, 1974.
J.P. Bantley, “Principles of Measurement Systems”, Longman, Singapore,
1998.
Instrumental Methods of Chemical Analysis” by B.K.Sharma.
“Pharmaceutical Analysis by G.Vidya Sagar”.
“Instrumental Methods Of Chemical Analysis” by Gurdeep R.Chatwal,
Sham K.Anand.
“Pharmaceutical Analysis” by Dr.S.Ravi Sankar.
“Quantitative Analysis Of Pharmaceutical Formulations”
“Pharmaceutical Analysis” by Dr.A.V.Kasture, Dr.K.R.Mahadik,
Dr.H.N.More.
“Pharmaceutical Analysis”, 2nd Edition by David G Watson.
P.G. Jeffery and P.J. Kipping, “Gas Analysis by Gas Chromatography”,
Pergamon, Oxford, 1972.
34
BIBLIOGRAPHY:
K. Grob, ‘Classical Split and Split less Injection’, in Capillary GC, A.
Huethig, Heidelberg, 1987
R. Buffington, M.K. Wilson, Detectors for Gas Chromatography – a
Practical Primer, Hewlett-Packard Corporation, Part No. 5958-9433, 1987.
D.J. David, “Gas Chromatographic Detectors”, John Wiley & Sons, 1974.
J.P. Bantley, “Principles of Measurement Systems”, Longman, Singapore,
1998.
Instrumental Methods of Chemical Analysis” by B.K.Sharma.
“Pharmaceutical Analysis by G.Vidya Sagar”.
“Instrumental Methods Of Chemical Analysis” by Gurdeep R.Chatwal,
Sham K.Anand.
“Pharmaceutical Analysis” by Dr.S.Ravi Sankar.
“Quantitative Analysis Of Pharmaceutical Formulations”
“Pharmaceutical Analysis” by Dr.A.V.Kasture, Dr.K.R.Mahadik,
Dr.H.N.More.
“Pharmaceutical Analysis”, 2nd Edition by David G Watson.
P.G. Jeffery and P.J. Kipping, “Gas Analysis by Gas Chromatography”,
Pergamon, Oxford, 1972.
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