1

06T: Chromatograph Methods of Analysis

Mohammad Ali
Chemist, ACESD
Introduction
The two main chromatographic techniques used in modern analytical chemistry are Gas
Chromatography (GC) and High-Performance Liquid Chromatography (HPLC).
A gas chromatograph (GC) is an analytical instrument that measures the content of various
components in a sample. The technique of analysis by a GC is called gas chromatography. The
various components of sample are separated inside the column when it enters the carrier gas
stream & transports into the column.
HPLC uses a liquid mobile phase to transport the sample components (analytes) through the
column, which is packed with a solid stationary phase material.
Mobil Stationary phase Mobil
Injectio Detect
e (Separation of sample e
n port or
phase mixture) phase

Record
Sample
er
mixture

Fig. 1: Block diagram of chromatographic

method
Principle of chromatography:
Chromatography is a separation technique based on the differential distribution of two phases,
one stationary and other is mobile phase. In this process, the mobile phase carries the sample
components through the stationary phase. The speed of the components is retarded by the
stationary phase on the basis of different affinity for stationary phase. As a result of selective
retardation brought about. The components of the mixture will travel through the column at
different speed causing separation of components. Generally, four different types of principles
are involved in all kinds of chromatography, such as:
1. Adsorption Chromatography
2. Partition Chromatography
3. Ionic-exchange Chromatography and
4. Molecular Exclusion Chromatography.
Classification of Chromatographic Method
Chromatographic methods are of two types:
Column chromatography: the stationary phase is held in a narrow tube and the mobile phase is
forced through the tube under pressure or by gravity. e.g., LC, GC, HPLC
Planar chromatography: the stationary phase is supported on a flat plate or in the pores of a
paper. Here the mobile phase moves through the stationary phase by capillary action or under
the influence of gravity. e.g., PC, TLC
Instrumentation
The essential parts of gas chromatograph mentioned as below:
1) Source and a control device for carrier gas- Helium, Nitrogen, Argon and Hydrogen
2) Sample injection device - Gas sampler / Liquid injection port
3) Columns - Packed, Capillary, Wide bore capillary etc.
4) Detector - Thermal conductivity, Flame ionization, Mass spectrometric
5) Read out device - Recorder / Computer system data integration.
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include Nitrogen, Helium, and
Hydrogen. Probably more than 90% of the present GC instruments run with Helium as a carrier
gas. The choice of carrier gas is often dependent upon the type of detector which is used. The
carrier gas system also contains a molecular sieve to remove water and other impurities.
Analytical Chemistry & Environmental Science Department, TICI, Palash, Narsingdi-1611
Columns
There are two general types of column, packed and capillary (also known as open tubular).
Packed columns contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in
length and have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of
two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-
coated columns consist of a capillary tube whose walls are coated with liquid stationary phase.
Sample Injection
Gas sample injection port: There are six-way ports and eight-way ports valve for introduction
of gaseous samples. 0.5, 1.0, 2.0- & 5.0-ml size sample loops are chosen for charging to
column.
Liquid sample injection port: An aliquot amount (1~5 l) of liquid samples is introduced by
micro syringe through glass insert. Split/Splitless injections are practiced depending on analyte
content in sample. Normally auto-sampler is batch sample analysis.
Column temperature
The performance of a chromatographic separation requires the use of a temperature-controlled
compartment where a column installed which is known as oven. Oven is the fundamental parts
of GC and largest in volume. Separation occurs in column 20.0 o ~ 25.0oC above ambient
temperature to max.350.0oC and accurate to + 5.0oC. for any modern GC. Minimal
temperatures give good resolution, but increase elution times. If a sample has a wide boiling
range, then temperature programming can be useful. The column temperature is increased
(either continuously or in steps) as separation proceeds.
GC Detector
Detector is device that produces electrical signal in presence of eluted components from the
column. In most cases GC detector produces mV as signal. Types of detectors: TCD, FID,
MSD, ECD, NPD, FPD etc.

Retention Time
The overall time that the component spends in column is known as Retention Time.
Death Time
Retention of a compound with no interaction with the stationary phase.

Thermal Conductivity Detector (TCD):

TCD also known as a Katharometer is a universal/bulk property detector and a chemical
specific detector commonly used in gas chromatography. Detector sense changes with the
thermal conductivity of the column effluent and compares it to a reference flow of carrier gas.
Since most compounds have a thermal conductivity much less than that of the common carrier
gases of helium or hydrogen, when an analyte elutes from the column the effluent thermal
conductivity is reduced, and a detectable signal is produced.
Characteristics of TCD
 Non-destructive
 Response depends on the thermal conductivity difference between the carrier gas and the
eluted components
 Wide dynamic range (% to ppm levels)
 Responds also to inorganic gases eg. CO, CO2, NH3, CS2, N2, etc.
Flame Ionization Detector (FID)
FID typically uses a Hydrogen/Air flame into which the sample is passed to oxidize organic
molecules and produces electrically charged particles (ions)/free radical. The ions are collected
and produce an electrical signal which is then measured.

Analytical Chemistry & Environmental Science Department, TICI, Palash, Narsingdi-1611

 3

Characteristics:
 Mass sensitive detector
 Response depends on conducting power of ions/free radical produced on burning of
organic compounds in the flame
 Selective detector but sample detected must be combustible
 No response to inorganic gases CO, CO2, NH3, CS2, N2, etc.
 It is the most widely used detector in Gas Chromatography

Schematic of a gas chromatograph

Most modern commercial GC systems operate in the following way:

 An inert carrier gas, such as helium, is supplied from gas cylinders to the GC where the
pressure is regulated using manual or electronic (pneumatic) pressure controls
 The regulated carrier gas is supplied to the inlet and subsequently flows through the
column and into the detector
 The sample is injected into the (usually) heated injection port where it is volatilized and
carried into the column by the carrier gas
 The sample is separated inside the column - usually a long silica-based column with
small internal diameter. The sample separates by differential partition of the analytes
between the mobile and stationary phases, based on relative vapor pressure and
solubility in the immobilized liquid stationary phase
 On elution from the column, the carrier gas and analytes pass into a detector, which
responds to some physicochemical property of the analyte and generates an electronic
signal measuring the amount of analyte present
 The data system then produces an integrated chromatogram
 Gas chromatography uses ovens that are temperature programmable. The temperature
of the GC oven typically ranges from 5 °C to 400 °C but can go as low as -25 °C with
cryogenic cooling

High Performance Liquid Chromatograph (HPLC):

Chromatographic separation in HPLC is the result of specific interactions of the sample molecules
with both the stationary and mobile phases. With an interactive liquid mobile phase another
parameter is available for selectivity in addition to an active stationary phase. HPLC can be used
whenever the sample can be dissolved in a liquid.

Classification of HPLC:
On the basis of relative polarity of mobile & stationary phases the following two types of
chromatography are seen.
Normal-phase chromatography: Stationary phase is highly polar such as silica or alumina
surfaces or polar liquids such as triethylene glycol supported by silica particles. A relatively
nonpolar solvent such as hexane or i-propylether serves as the mobile phase.

Analytical Chemistry & Environmental Science Department, TICI, Palash, Narsingdi-1611

Reverse-phase chromatography: Stationary phase is nonpolar often a hydrocarbon, and the
mobile phase is relatively polar such as water, methanol, or acetonitrile.
In normal phase chromatography the least polar component is eluted first, increasing the
polarity of the mobile phase decreases the elution time. On the other hand, reversed phase
method, the most polar component appears first & increasing the mobile phase polarity
increases the elution time.
Basic configuration of an instrument (HPLC)

Column: HPLC column consists of 316-type stainless steel for high pressure and glass for
comparatively low pressure. Length of analytical column is 5~25 cm & it's dia 4.6 mm. Column
contains stationary phase which may be either a totally porous layer beds or macro-porous
polymer or thin film covering a solid core.

Detectors:
 UV detector  Fluorescence  Electro-chemical
 Refractometer  Evaporative light scattering

Chromatographic elution:
Isocratic elution: Separation that employs a single solvent of constant composition is termed an
isocratic elution e.g. 50:50 methanol: water for separation of chlorobenzene is used.
Gradient elution: It employs two solvent systems that differs significantly in polarity. 40:60 mixture
of two solvents (methanol & water). Methanol is increased at rate of 8%/min. It shortens the
analysis time.

Advantages
 Rapid analytical procedure
 Very small amount of sample can be used
 Sample can be subjected to both qualitative and quantitative analysis
 The process can be readily automated
 In most cases the test is non destructive
 Members of homologous series can be separated & analyzed easily
 Suitable for analysis of isomeric compounds

Analytical Chemistry & Environmental Science Department, TICI, Palash, Narsingdi-1611