Ajay Analysis PDF

SRIKRUPA INSTITUTE OF PHARMACEUTICAL SCIENCES
(approved by AICTE, PCI)

(affiliated to Osmania university)
ASSIGNMENT ON
GAS CHROMATOGRAPHY
SUBMITTED TO
Dr.S.Y. MANJUNATH
ASSOCIATE PROFESSOR
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
SUBMITTED BY
G.AJAY KUMAR
H.NO: 636218886001
M. Pharmacy 1ST semester
DEPARTMENT OF PHARMACEUTICS
CONTENT
INTRODUCTION:
PRINCIPLE:
 Gas chromatography sample vaporized by injection into a heated system,
eluted through a column by inert gaseous mobile phase and detected.
 The principle of separation in GC is “partition.” The mixture of
component to be separated is converted to vapour and mixed with
gaseous mobile phase.
 The component which is more soluble in stationary phase travel slower
and eluted later. The component which is less soluble in stationary phase
travels faster and eluted out first.
 No two components have same partition coefficient conditions. 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.’
DEFINACTION: Gas chromatography It is a process of separating
component from the given crude drug by using a gaseous mobile phase
 It involves a sample being vaporized and injected on to the head of the
chromatographic column. The sample is transported through the column
by the flow of inert, gaseous mobile phase. The column itself contains a
liquid stationary phase which is adsorbed onto the surface of an inert
solid.
Two major types:
1. Gas-solid chromatography:
(stationary phase: solid)
2. Gas-liquid chromatography:
(stationary phase: immobilized liquid)
1. Gas-solid chromatography:
The mobile phase is a gas while the stationary phase is a
solid. Used for separation of low molecular gases,
E.g., air components, H2 S, CS2, CO2, rare gases, CO and oxides of nitrogen.
2. Gas-liquid chromatography:
The mobile phase is a gas while the stationary phase is a liquid retained
on the surface as an inert solid by adsorption or chemical bonding.
INSTRUMENTATION:
A. Carrier gas: He (common), N2, H2, Argon
B. Sample injection port: micro syringe
C. Columns:
D. Detectors:
1) Flame ionization (FID).
2) Thermal conductivity (TCD).
3) Electron capture (ECD).
4) Flame photometric (FPD).
SCHEMATIC DIAGRAM OF A GAS CHROMATOGRAPH
INSTRUMENTATION:
A. CARRIER GAS: (He (common), N2, H2, Argon)
The choice of carrier gas determines the efficiency of chromatographic
separation o. Most widely used carrier gases are hydrogen helium nitrogen and
argon
HYDROGEN: it as better thermal conductivity. Low density if it is useful in
case of thermal conductivity detector and flame ionisation detector the dis
advantages is that it reacts with unsaturated compounds and it is inflammable
HELIUM: it also has excellent thermal conductivity but is expensive it is good
carrier gas in the thermal conductivity detectors>
NITROGEN; it is inexpensive but as reducing sensitivity
B.SAMPLE INJECTION PORT: micro syringe

Sample for introducing into the column can be of any type either gas,
liquid or solid in nature
 Gases can be introduced into the column by walve device
 Liquid can be injected through loop or septum device. Most GC
instruments have a high-quality rubber septum through which sample
solution is injected the rubber is made up of good quality rubber which
can withstand high temperature of preheating device and withstand
repeated injection over a period of time
 Solid sample are dissolved in suitable solvent and then they are injected
through a septum
C. COLUMNS:
Column is one of the important part f GC which decides the separation
efficiency. Columns are made up of glass or stainless steel.
 Stainless steel columns have the advantage of long life and can be easily
handled without the fear of fragility.
 But some sample react with them. Hence in such cases glass columns are
used.
I. Depending on the its use:
a) Analytical column: Analytical columns have a length of 1-1.5metres and
outer diameter of 3-6mm. They are packed columns and are made up of glass
or stainless steel. Only small quantity of sample can be loaded on to the
columns.
b) Preparative columns: preparative columns are larger when compared to
analytical columns since large amount of sample has to be loaded, they have
a length of 3-6 metres and outside diameter of 6-9 mm.
II. Depending on its nature:
Packed columns: columns are available in packed manner commercially and
hence are called as packed columns. Different columns ranging from low polar
nature to high polar nature are available.
Capillary columns: they are made up of long capillary tubing of 30-90metres in
length and have uniform and narrow internal diameter of 0.025-0.075cm.
 These are made up of stainless steel and are in the form of a coil.
 The inner wall of the capillary is coated with the stationary phase liquid in the
form of a thin film.
 These columns offer efficient than packed columns which offer more
resistance to the flow of carrier gas. But the disadvantage is that more sample
cannot be loaded.
Support-coated columns: the inner wall of the capillary is lined with a thin layer
of support material such as diatomaceous earth, onto which the stationary phase
has been adsorbed. It is also known as PLOT (porous layer opens tubular
columns).
SCOT columns are generally less efficient than WCOT columns. Both types of
capillary column are more efficient than packed columns.
Column temperature:
 This can be controlled by jackets equipped with vapours of a boiling liquid,
electrically heated metal blocks or circulating air baths.
 Compounds of low B.P- eluted at lower temperature.
 Compounds of high B.P- boils at higher temperature resulting in broader and
shallower peaks, require temperature programming.
D. Detectors:
1. Flame Ionization Detector (FID): one of most widely used GC detectors -
good sensitivity to almost all organic compounds.
 It operates by the principle that by change in conductivity of the flame as
the compound is burnt.
 The change in conductivity of the flame does not arise by simple
ionization of the compound, it is partial or complete stripping of the
compound to give charged hydrogen- deficient polymers or aggregates of
carbon of low ionization potential.
Flame Ionization Detector
2. Thermal Conductivity Detector (TCD):
 Thermal Conductivity Detector is based upon the fact that the heat lost from
a filament depends upon the thermal conductivity of the stream of
surrounding gas as well as its specific heat.
 When only carrier gas flows heat loss to metal block is constant, filament T
remains constant.
 When an analyte species flows past the filament generally thermal
conductivity changes, thus resistance changes which is sensed by
Wheatstone bridge arrangement.
 The imbalance between control and sample filament temperature is
measured and a signal is recorded.
Advantages:
 Simple and inexpensive
 Durable and posses long life
 Accurate results
 Non-selective, hence known as universal detectors
Disadvantages:
 Low sensitivity
 Affected by fluctuations in temperature and flow rate.
Thermal Conductivity Detector
3. Electron capture Detector (ECD): Molecules of compounds, which

possess affinity for electrons, differ in their electron absorbing capacities. This
difference is utilized in this detector for identification of the compounds.
Working:
A foil made up of a radioactive metal like Ni63 (β- emitter) is placed
inside a Teflon coated cell which also contains a cathode and an anode.
 In the absence of organic species, the produced electrons migrate towards
positive electrode and produce a certain constant standing current.
 When a sample/eluent is present it captures the electrons, elutes from
column, there is a drop in this constant current.
 The potential across two electrodes is adjusted to collect all the ions and a
steady saturation current, is therefore, recorded.
Advantages:
 Highly selective
 Highly sensitive for the detection of compounds like halogens, quinones,
peroxides, nitrites, etc.
 It is non-destructive
 More sensitive than TCD and FID.
Disadvantages:
 Least sensitive to compounds whose molecules have negligible affinity for
electrons.
 Carrier gas used should be of pure form like pure nitrogen
Electron capture Detector
4. Flame photometric Detector (FPD): It is a selective detector that is

responsive to compounds containing sulphur or phosphorous.
 The detection principle is the formation of excited sulphur and excited
hydrogen phosphorous oxide species 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.
Flame photometric Detector

Working:
1. Fill the syringe with sample.
2. Record the setting column temperature, detector temperature and injection
port temperature.
3. Introduce sample into the injection port by completely inserting the needle
into the rubber septum. Note down the injection time.
4. The sample gets vaporized due to higher temperature of injection port and is
swept into column by carrier gas.
5. This sample components now get distributed between the gas and stationary
liquid phase depending upon their solubilizing tendencies.
6. The components with minimal solubility move faster and those with
maximum solubility travel slowly.
7. The components leaving the column activate detector and recorder to give a
plot.
8. The components that slowly leave the column give a bell-shaped curve of
shorter peak while the one which travels faster gives a bluntly pointed curve
of larger peak.
Applications:
1) Qualitative Analysis: By comparing the retention time or volume of the
sample to the standard / by collecting the individual components as they
emerge from the chromatograph and identifying these compounds by other
methods like UV, IR, NMR.
2) Quantitative Analysis: Area under a single component elution peak is
proportional to the quantity of the detected component/response factor of the
detectors. It is done by
a) Direct comparison method:
By injecting a sample and standard separately and comparing
their peak area the quality of the sample can be determined.
A1/A2= αw1/w2
b) Calibration curve:
A graph is plotted by taking peak areas on Y axis and
concentration of standard compound on X axis. Concentration of
unknown sample is then determined by plotting its peak area on same
graph.
c) Internal standard method:

A known concentration of internal standard, which has similar
retention characteristics as that of sample is added to both reference
standard and test sample.

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SRIKRUPA INSTITUTE OF PHARMACEUTICAL SCIENCES

(approved by AICTE, PCI)

M. Pharmacy 1ST semester

SCHEMATIC DIAGRAM OF A GAS CHROMATOGRAPH

B.SAMPLE INJECTION PORT: micro syringe

3. Electron capture Detector (ECD): Molecules of compounds, which

4. Flame photometric Detector (FPD): It is a selective detector that is

Flame photometric Detector

c) Internal standard method:

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