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INDEX
2. Principle 06
5. Columns For GC 14
6. Detectors 18
7. Derivatization Techniques 27
8. Applications 30
9. Conclusion 33
10. Bibliography 34
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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.
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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.
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.
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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.
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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.
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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.
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.
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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
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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:
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:
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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.
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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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:
Because generally the column flow is much lower than the split flow, this equation
can be rewritten as:
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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
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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.
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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
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liquid stationary phase. In GSC, the columns are packed with adsorbents or
porous polymers. Packed columns are shaped as coils.
Capillary columns are gas chromatography (GC) columns that have the
stationary phase coating their inner surfaces rather than being packed into
the cavity.
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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:
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3. More Inert
4. Longer Life
5. Less Bleed Of Coating Material
6. Higher Efficiencies
7. Greater Reproducibility.
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.
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.
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iii. Length:
DETECTORS:
1) Flame ionization detector:
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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
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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)
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Disadvantages:
1. Could be affected by the flow rate.
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Applications:
Organophosphate in pesticides and in drug analysis for determination of amine-
containing or basic drug.
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Limitations:
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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.
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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.
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.
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Moreover, alkylation derivatives are less polar, highly stable and can be
separated and stored for longer durations than the parent compound.
iii. Acylation:
This derivatization technique includes the conversion of compounds
with active hydrogen by the action of a carboxylic acid or its
derivatives.
ADVANTAGES OF GC:
Fast analysis
High efficiency leading to high resolution
Sensitive detectors (ppb)
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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:
Pharmaceutical Applications:
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Environmental Applications:
Clinical Applications:
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.
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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:
Forensic Applications:
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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.
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BIBLIOGRAPHY:
K. Grob, ‘Classical Split and Split less Injection’, in Capillary GC, A.
Huethig, Heidelberg, 1987
D.J. David, “Gas Chromatographic Detectors”, John Wiley & Sons, 1974.
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