Gas Chromatography

&
Liquid Chromatography

Dr. Jenny Jacob

School of
Bioscience
MACFAST

June 30, 2018 MACFAST

Gas Chromatography

June 30, 2018 MACFAST

 Principle of GC
Components of a vaporized
sample are separated
Based on partitioning between a
mobile gaseous phase and a
liquid/solid stationary phase held
in the column.

June 30, 2018 MACFAST

 Methodology for GC
 Sample (gas/liquid) is injected at the
inlet/injector and vaporized into the
chromatographic column.
 Sample is transported through the
column by the flow of inert gaseous
mobile phase.
 As sample passes through the
column, the components are
separated
 Detected electronically by detector.
June 30, 2018 MACFAST
 Methodology for GC
There are two types.
Gas-liquid chromatography
(GLC)
mobile phase – gas
stationary phase - liquid

Gas-solid chromatography
(GSC)
June 30, 2018
mobile phase – gas MACFAST
Instrument
ation
A. Carrier gas
B. Flow regulator
C. Injector
D. Column
E. Detector
F. Integrator
G.Display system -
printer/monitor
June 30, 2018 MACFAST
 A. Carrier gas

 Must be chemically inert.

 Most common carrier gas is Helium(He)
 Some specific detectors are using Nitrogen
gas(N2), Hydrogen gas(H2), Carbon
dioxide gas(CO2) and Argon.
 The carrier gas should not contain traces
of water or oxygen. Both are harmful to
the stationary phase.

June 30, 2018 MACFAST

 B. Flow regulator

 The function of flow regulator is to control

the flow rate of the carrier gas using the
pressure regulators, gauges and flow meters.
 The pressure at the head of the column is
stabilized
 mechanically
OR
 through the use of an electronic device.

June 30, 2018 MACFAST

 C. Injectors
 Functions
1. An inlet for the sample.
2. To vaporize and mix the sample with the
carrier gas before the sample enters the
head of the column.
 Temperature is set about 50°C higher than
boiling point of the least volatile component of
the sample.
 Modes of injection and characteristics of injectors
vary depending on type of column used whether
split/splitless.
June 30, 2018 MACFAST
 D. Sample Injection System

Examples
 Direct injection using microsyringe
 Loop injectors
 Auto samplers
 Headspace

June 30, 2018 MACFAST

 D. Columns

Two types of columns

1. Packed column 2.
Capillary column

Capillary Column:
Packed column:
10-100m in length, very small i.d
1-5m in length, 2-4mm i.d

June 30, 2018 MACFAST

 1. Packed Column

Less commonly used

Made of glass or steel
Length: 1 to 5 m Cross-sectional view
of packed column

 Internal diameter: 2 to 4 mm
 These column is densely packed with
uniform, finely divided solid support,
coated with thin layer (0.05 to1μm) of
stationary liquid phase.
Accommodate
June 30, 2018
larger samples.
MACFAST
 2. Capillary Column
 Widely used in GC analysis
 Also known as open tubular column
 Length: 10 – 100 m
 Coiled around a light weight of
metallic support.
 Types of capillary column
I. FSOT (Fused Silica Wall Coated) -
i.d. 0.1 - 0.3 mm
II.WCOT (Wall Coated) - i.d. 0.25 –
June 30,0.75
2018 mm MACFAST
 E. Oven
 Must have sufficient space to hold the
column.
 Can be heated to the desired
temperature for analysis.
 Atmosphere inside the oven is
constantly agitated by forced
ventilation which has small thermal
inertia.
 Reproducible of retention time,tR which
require control of the column
temperature
June 30, 2018 within a few tenths of a
MACFAST
 G. Stationary Phase
Desirable properties for the immobilized liquid
stationary phase:
 Low volatility (ideally the boiling point of
the liquid at least 1000C higher than the
maximum operating temperature for the
column)
 Thermal stability.
 Chemical inertness.
 Solvent characteristics such as k and α
values
June 30, 2018 for the solutes to be resolved
MACFASTfall
 Applications of GC
• Analysis of oils and mixtures in
food sample
• Detect drugs and steroids in foods
• Detect fruit esters

June 30, 2018 MACFAST

 Advantages of GC
• High sensitivity
• High resolution
• High speed
• High accuracy
• Better quantitation

June 30, 2018 MACFAST

 HPLC v/s LC TECHNIQUE
 Columns : Small diameter (4.6 mm), stainless steel,
glass or titanium.
 Relatively high inlet pressures and controlled flow of the
mobile phase.
 Detecting very small amounts
 High resolution
 Rapid analysis
 Speed, efficiency, sensitivity and ease of operation
 High degree of versatility
 400 atmospheres.
PUMP PRESSURE
 "Ultra High Performance Liquid Chromatography"
systems
June 30, 2018 1000 atmospheres. MACFAST
 HP Liquid
Chromatography
• Mobile phase - Liquid.
• Column or a plane.
• Very small packing particles and a
relatively high pressure -High
Performance Liquid
Chromatography
June 30, 2018
(HPLC). MACFAST
 HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)

June 30, 2018 MACFAST

 TYPES OF HPLC
I. BASED ON MODE OF SEPARATION
1.Normal phase chromatography - stationary
phase is polar (hydrophilic) and mobile face is
non-polar (hydrophobic).

2.Reverse phase chromatography- stationary

face is non-polar (hydrophobic) and mobile
face is polar (hydrophilic).

June 30, 2018 MACFAST

II. BASED ON PRINCIPLE OF SEPARATION

1. Adsorption Chromatography
2. Ion-exchange chromatography
3. Ion-pair chromatography
4. Gel permeation chromatography
5. Affinity Chromatography
6. Chiral chromatography

June 30, 2018 MACFAST

III. BASED ON ELUTION TECHNIQUE

1.Isocratic elution
•A separation in which the mobile phase
composition remains constant throughout the
procedure is termed isocratic elution

2. Gradient elution

•A separation in which the mobile phase

composition is changed during the
separation process is described as a
gradient
June 30, 2018 elution MACFAST
 IV. BASED ON SCALE OF
OPERATION
1. Analytical HPLC
• No recovery of individual components
of substance

2.Preparative HPLC
• Individual components of substance
can be recovered
June 30, 2018 MACFAST
 V. BASED ON TYPE OF
ANALYSIS
1. Qualitative analysis
• Analysis of a substance in order to
ascertain the nature of its chemical
constituents : No quantitation

2.Quantitaive analysis
• Determining the amounts and proportions
of its chemical constituents
• Quantity of the impurity and individual
Junecomponents
30, 2018 can be assessed MACFAST
 Typical Applications
of HPLC
Field of Separation
Application
Pharmaceuticals Antibiotics, Sedatives, Steroids,
Analgesics

Biochemical Amino acids, Proteins, Carbohydrates,

Lipids
Food Products Artificial Sweeteners, Antioxidants,
Preservatives
Industrical Condensed Aromatics, Surfactants,
Chemicals Propellants, Dyes
Forensic Drugs, Poisons, Blood Alcohol, narcotics
June 30, 2018
Chemistry MACFAST
 Detectors in GC and LC
 Some detectors are universal : Sensitive to all
compounds eluted from column.
 Most detectors are selective : Sensitive to particular
type of compound.
 Characteristics of ideal detector
1. High reliability & ease to use
2. Predictable & selective response
3. Nondestructive.
4. Adequate sensitivity.
5. Good stability and reproducibility.
6. Linear response to solutes
7. Temperature range (from RT to at least
400 0C)
June 30, 2018 MACFAST
 Detectors

Several types of detectors.

1. Flame Ionization Detector (FID)
2. Thermal Conductivity Detector
(TCD)
3. Electron Captured Detector
(ECD)

June 30, 2018 MACFAST

 Detectors

Other detectors
 Nitrogen-Phosphorous Detector
(NPD)
 Flame Photometry Detector (FPD)
 Mass spectrometer (GC-MS)

June 30, 2018 MACFAST

 SUPERCRITICAL FLUID
CHROMATOGRAPHY
 Used for the analysis and purification of low to
moderate molecular weight, thermally labile
molecules.
 Principles are similar to those of HPLC
 Mobile phase - High pressure liquid or Super critical
Carbon Dioxide.
 Modifiers – Methanol, Ethanol, isopropyl alcohol,
acetonitrile and Chloroform.
APPLICATION
 Use
June in industry primarily for separation MACFAST
30, 2018 of Chiral
June 30, 2018