The document provides a detailed overview of gas chromatography (GC), a laboratory technique used to separate mixtures into their components using a mobile phase and a stationary phase. It explains the principles, working procedures, types of GC, and the use of detectors such as flame ionization and thermal conductivity detectors to analyze compounds. Additionally, it covers concepts like retention time, the interpretation of chromatograms, and practical examples of analyzing compounds like paracetamol and banana oil.

1. ADVANCED
PHARMACEUTICAL
ANALYSIS

2. Mehedi Hasan Al Amin
MEET OUR TEAM
Sharmin Akter Mukty
AKM Shoaib Mahmud
2019100300061 2019100300062 2019100300063 2019100300064
Abir Hasan Pranto

3. GAS CHROMATOGRAPHY
GROUP
05

4. Chromatography
Planar Column
Paper (PC) Thin layer (TLC) Liquid (LC)
Gas (GC)
CHROMATOGRAPHY
TYPES OF CHROMATOGRAPHY
• In chemical analysis, chromatography is a laboratory technique for
the separation of a mixture into its components.
• The mixture is dissolved in a fluid solvent (gas or liquid) called
the mobile phase, which carries it through a system (a column, a
capillary tube, a plate, or a sheet) on which a material called
the stationary phase.

5. • Eleuent: Fluid entering the column.
• Eluate: Fluid exiting the column.
• Elution: Pass liquid or gas through a column.
• Retention time: The time needed after injection for an
individual solute to reach the detector.
• Chromatogram: Detector response as a function of time.
• Mobile phase: Solvent moving through the column.
• Stationary phase: Substance stays fixed within the column.
C
O
L
U
M
N
Eleuent in
Eleuent out
SOME GENERAL CONCEPTS

6. Gas Chromatography
• It is the process of separating compounds in a mixture by injecting
a gaseous or liquid sample into a mobile phase.
Basic Principle
• Sample vaporized by injection into a heated system, eluted
through a column by inert gaseous mobile phase and detected.
• Helium or nitrogen is used as the so-called carrier gas.
• The various components are separated inside the column.
• To measure a sample with an unknown concentration, a standard
sample with a known concentration is injected into the
instrument.
• The standard sample peak retention time and area are compared
to the test sample to calculate the concentration.
BASIC PRINCIPLE OF GC

7. Two types of Gas Chromatography are encountered:
1) Gas-solid chromatography (GSC)
2) Gas-liquid chromatography (GLC)
1) Gas-solid chromatography (GSC)
Here, the mobile phase is a gas while the stationary phase is a solid. Used
for separation of low molecular gases, e.g., air components, H2S,CS2,CO2,
rare gases, CO and oxides of nitrogen.
2) Gas-liquid chromatography (GLC)
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.
TYPES OF (GC)

8. Carrier Gas
Mobile phase that
runs in Column
Detector
Measures the quantity or
ingredients of sample
Sample Injector
Sample to be
analyzed is injected
Column
Consists of
Stationary Phase
COMPONENTS
COMPONENTS OF GC

9. COMPONENTS OF GC
01
02
03
05
04

10. WORKING PROCEDURE OF GC
• At first, Sample is dissolved into
a suitable volatile solvent
which can evaporate easily.
• Examples of volatile solvents
include Alcohol, Alkanes and
Hydrocarbons.
SAMPLE
PREPARATRION
01

11. WORKING PROCEDURE OF GC
• Carrier Gas is the mobile phase.
• Hydrogen, Helium, Argon or Nitrogen
is mostly used as carrier gas.
• The Helium gas is mostly preferred
because of its efficiency and safety.
• The carrier gas is filled in reservoir
tank and runs in the column.
MOBILE PHASE
02

12. WORKING PROCEDURE OF GC
SAMPLE INJECTION
03
• The prepared sample is now introduced
into the gas chromatograph with a
syringe.
• Here, the solution of the sample &
carrier gas (Mobile Phase) mix with each
other.
• The injected sample is travels with
carrier gas (mobile phase) in column
filled with stationary phase.

13. WORKING PROCEDURE OF GC
SAMPLE
SEPARATION
04
• The column consists of Stationary Phase.
• The temperature of oven increases gradually to
vaporize the injected components.
• The injected sample interacts with stationary phase,
separated here and then carried to the detector.
• Component that adsorbs most strongly to the
stationary phase, has the longest retention time and
adsorbs least strongly to the stationary phase, has
the shortest retention time.

14. WORKING PROCEDURE OF GC
SAMPLE
DETECTION
05
• Once the separated gasses elute from the
column, they pass through a detector which
responds with an output signal.
• This signal is what generates the characteristic
GC peaks in a chromatogram.
• The most commonly used detectors are Flame
Ionization Detector (FID) and Thermal
Conductivity Detector (TCD)

15. Stationary Phase
Polar
Stationary Phase
Non-Polar
Stationary Phase
TYPES OF COLUMNS

16. STATIONARY PHASE RETENTION
MECHANISMS
• Retention mechanisms of NON-POLAR COLUMNS is primarily
dispersive, meaning that they are governed by Van der Waals
forces.
• These are intermolecular attractions that increase with the size of
the compound.
• Thus, larger compounds with higher boiling points have longer
retention. Elution order generally follows the boiling points of the
compounds.
• Retention mechanisms of POLAR COLUMNS is strongly dispersive,
very strongly dipole-dipole, and very strongly dipole-induced
dipole.
• Moderately basic interactions are also possible. Separations are
determined by differences in the overall effects of these
mechanisms.

17. SEPARATION MECHANISMS
Different compounds have different retention times. For a
particular compound, the retention time will vary depending on:
• The boiling point of the compound. A compound which boils at a
temperature higher than the column temperature is going to
spend nearly all of its time condensed as a liquid at the beginning
of the column. So high boiling point means a long retention time.
• The solubility in the liquid phase. The more soluble a compound is
in the liquid phase, the less time it will spend being carried along
by the gas. High solubility in the liquid phase means a high
retention time.
• The temperature of the column. A higher temperature will tend to
excite molecules into the gas phase - because they evaporate
more readily.

18. REAL SEPARATION

19. FLAME IONIZATION DETECTOR (FID)
• The flame ionization detector is the widely used and most
applicable detector for Gas Chromatography. The effluent from
the column is mixed with hydrogen and air and then ignited
electrically. Most organic compounds, when pyrolyzed at the
temperature of a hydrogen/air flame, produce ions and electrons
that can conduct electricity through the flame.
• In FID, the components are burned and ionized; it is used for
almost all organic compounds, which have C-H or C-N structure.
There are several exceptions such as CO, CO2 and CS2 which are
non-organic compounds that cannot be detected. Carbonyl group
and C=O carbon atom of carboxyl group are also not detectable.
FID can detect most hydrocarbons which have a C-H bond in its
structure.

20. FLAME IONIZATION DETECTOR (FID)

21. THERMAL CONDUCTIVITY DETECTOR (TCD)
Thermal Conductivity Detector (TCD) is a detector used in Gas
Chromatography (GC) to analyze inorganic gases (such as argon,
nitrogen, hydrogen, carbon dioxide) and small hydrocarbon
molecules. The TCD compares the thermal conductivity of two
gas flows, the pure carrier (reference) gas and the sample.

22. INTERPRETION OF GAS
CHROMATOGRAM
• The GC produces a graph called a chromatogram, which shows
peaks. The Size of a peak indicates the amount of each
component reaching the detector.
• The Number of peaks shows different compounds present in the
sample. The position of each peak shows the retention time for
each compound.

23. INTERPRETION OF GAS
CHROMATOGRAM
The X-Axis: Retention Time
• The x-axis of the gas chromatogram shows the amount of time
taken for the analytes to pass through the column and reach the
mass spectrometer detector. The peaks that are shown
correspond to the time at which each of the components reached
the detector.
The Y-Axis: Concentration or Intensity Counts
• The y-axis or the area of the peak, is a reflection of the amount of
a specific analyte that’s present. Unknown compounds are
identified based on their retention times of known standards with
other detectors.

24. EXAMPLE OF PARACETAMOL
• Suppose, there are total 4 compounds in Paracetamol.
1. Paracetamol (API)
2. Propylene Glycol
3. Ethanol
4. Glucose
• From GC chromatogram we can identify four different types of
molecule through their retention time. Quantity depends on AUC.
Concentration High = Intensity High.
How to calculate that exact amount?
• Suppose total solution = 100%
• We have to identify individual AUC for each compound.
• Suppose AUC of A = 10, B = 20, C = 15, D = 5
• For A = 10, A (%) = (10/50) × 100 = 20 %

25. EXAMPLE OF BANANA OIL
Peak Retention Time (Min) Type Area Under Curve Area (%)
1. 1.845 BB (s) 9810.8194 99.34327
2. 2.665 BB 64.85681 0.65673

26. EXAMPLE OF BANANA OIL
Total AUC is 9875.67621
• For Peak 1
9810.8194 / 9875.67621 × 100%
= 99.34 %
• For Peak 2
64.85681 / 9875.67621 × 100%
=0.65 %
• Banana Oil has the high concentration than the solvent.