Yazmin Bustami, PhD.

(Modified by Prof. Dr. K. Sudesh Kumar)

School of Biological Sciences, USM
▪ All types of chromatography are based on a very simple principle.
▪ The sample to be examined (called the solute) is allowed to interact with two
physically distinct entities- a mobile phase and a stationary phase.
▪ Material to be purified must be capable of ‘binding’ (retarded) or retain to the
solid phase and re–entering the solution later in order to be eluted.

2
3
▪ There are several different
▪ Paper chromatography
▪ Thin layer chromatography (TLC)
▪ Column chromatography
▪ Gas chromatography (GC)
▪ Liquid chromatography (LC)
▪ High performance liquid chromatography (HPLC)
▪ Ion exchange chromatography
▪ Gel permeation or gel filtration chromatography.

4
→ separation of volatile substances, or
substances that can be made volatile, from one
another in a gaseous mixture at high
temperatures.

→ Partition of molecules between liquid (stationary

phase) and gas (mobile phase)
▪ Mobile phase → is a carrier gas, usually an
inert gas e.g. helium, nitrogen
▪ Stationary phase → solid like silica or alumina
▪ most useful for the separation and analysis of
gases like CH4, CO2, CO, ... etc.
▪ The mobile phase → is a carrier gas,
usually an inert gas e.g. helium,
nitrogen
▪ The stationary phase → microscopic
layer of liquid or polymer on an inert
solid support, inside glass or metal
tubing, called a column.
1. Separation of mixture of polar compounds
▪ (polyethylene glycol)
2. Separation of mixtures of non-polar
compounds
▪ (polymer of methyl silicone)
3. Methyl ester of fatty acids
▪ DEGS (diethyl glycol succinct)
Estimation of price: ≈ RM70,000
 BASIC COMPONENTS TO A GAS CHROMATOGRAPHY
SYSTEM
Filters/Traps Data system
H
H

RESET

RESET

Regulators Syringe/Sampler

Inlets

Detectors
 gas system
 inlet
Gas Carrier
Hydrogen
Air

Column  column
 detector
 data system
Schematic Diagram of Gas Chromatography

A gas
chromatograph is a
chemical analysis
instrument for
separating chemicals
in a complex sample.

11
▪ Column → flow-through narrow tube
– different chemical constituents of a sample pass
in a gas stream (carrier gas, mobile phase) at
different rates depending on:
1.Their various chemical and physical
properties
2.Their interaction with a specific column
filling, called the stationary phase.
▪ As the chemicals exit the end of the column,
they are detected and identified electronically.
▪ stationary phase → to separate different
components at a different time (retention time).
▪ Other parameters that can be used to alter the
order / time of retention:
▪ carrier gas flow rate
▪ temperature
▪ strength of adsorption
Auto samplers/
Autoinjector
▪ The auto sampler provides the means to introduce
automatically a sample into the inlets.
▪ Manual insertion of the sample is possible but is no longer
common.
▪ Automatic insertion provides better reproducibility and
time-optimization.

GC Vial
1. Solvent vial
3. Chloroform
2. Waste liquid vial
2- The column inlet (or
injector) provides the
means to introduce a
sample into a
continuous flow of
carrier gas.
▪ The inlet is a piece of
hardware attached to the
column head.
3. Columns
Two types of columns are used in GC:

a. Packed columns
▪ 1.5 - 10 m in length and have an internal diameter of 2 - 4 mm.
▪ tube → usually stainless steel / glass and contains a packing of solid support material that
is coated with a liquid or solid stationary phase.
▪ The nature of the coating material determines what type of materials will be most strongly
adsorbed.
▪ Thus numerous columns are available that are designed to separate specific types of
compounds.
b. Capillary Columns
▪ They have a very small internal diameter, on the
order of a few tenths of millimeters, and lengths
between 25-60 meters are common.
▪ The inner column walls are coated with the active
materials.
▪ Most capillary columns are made of fused-silica
with a polyimide outer coating. These columns are
flexible, so a very long column can be wound into
a small coil.
 Column
(Fused Silica Capillary
Column)

Price estimation of column: ≈ RM 3,000

4. Detector → to monitor the outlet stream from the column
▪ Determine
– the time at which each component reaches the
outlet
– the amount of that component can be determined.
1. Flame Ionization Detector (sensitivity 10 µg)

▪ High temperature of hydrogen flame (H2 +O2 +

N2) ionizes compounds eluted from column into
flame.
▪ The ions collected on collector or electrode and
were recorded on recorder due to electric
current.
2. Thermal Conductivity
Detector (sensitivity 10 µg)
▪ Measures the changes of
thermal conductivity due to
the sample (mg).
3. Electron Capture
Detector (sensitivity 10
µg)
• uses a radioactive (electron)
source to measure the degree of
electron capture.
• Sensitive to electronegative
groups
• Insensitive to amines, alcohols
4. GC-MS Detector
▪Some gas chromatographs are connected to a
mass spectrometer which acts as the detector.
▪The combination is known as GC-MS.
 1. SEMI-
Detector Response
APPLICATION QUANTITATIVE
C18 Peak Area (cm2 ) ANALYSIS OF
10

C 16 8 FATTY ACIDS
6

C14 4

0.5 1.0 1.5 2.0 2.5 3.0

Sample Concentration (mg/ml)
Retention Time C
The content % of C14 fatty acids =  
C + C+ C 


= the content % of C14 fatty acids

2. TENTATIVE
IDENTIFICATION
OF UNKNOWN
COMPOUNDS
▪ Very good separation
▪ Time (analysis is short)
▪ Small sample is needed - ml
▪ Good detection system
▪ Quantitatively analyzed
28
 Lyophilized cells (≈20 mg)
was created in small screw-
cap test tube

Added with 2 mL chloroform &

2mL methanolysis solution (85%
methanol and 15% sulfuric acid)
This method degrades the
intracellular PHA by methanolysis to
Sample heated for 140 min at 100°C in its constituent P-hydroxycarboxylic
heating block acid methyl esters. Concentrated
sulfuric acid was added in to convert
poly(3HB) quantitatively to crotonic
acid
Sample cooled to room temperature and
added with 1 mL of distilled water. The
suspension was vortexed.

After phase separation, the organic phase

(bottom layer) was removed and transferred to Natrium Sulphate is to
a flat-bottom test tube contains sodium remove any trace amount of
water
sulphate (Na2SO4)

Sample were subjected to gas chromatography (GC)

analysis after 0.5ml of hydroxyacyl methyl esters was
mixed with 0.5ml of 0.2% CME solution small screw-cap The accuracy and
reproducibility of this
gc glass vial.
method can be
increased as usual by
employing an internal
standard
▪ performed on a Shimadzu GC-14B instrument equipped with flame-ionization
detector
▪ Column: SPBTM–1 (Supelco) non polar fused silica capillary column (30 m x 0.25
mm x 0.25 μm
▪ operating in a split mode (split ratio, 1:10)
▪ Nitrogen (14 ml/min) was used as the carrier gas.
▪ The temperatures of the injector and detector were 270°C and 280°C, respectively.
▪ https://www.slideshare.net/gopinathkarnam/gas-chromatography-24600103
▪ https://www.slideshare.net/bhavyamitta/gas-chromatography-44699218
▪ http://bionmr.unl.edu/courses/chem221/lectures/chapter-24.ppt