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Gas Chromatography

Gas Chromatography
Presented By -
Mr. Shaise Jacob
Faculty
Nirmala College of Pharmacy
Muvattupuzha, Kerala
India
Email – jacobshaise@gmail.com
What is Gas Chromatography?

• It is also known as…


– Gas-Liquid Chromatography (GLC)
GAS CHROMATOGRAPHY

 Separation of gaseous & volatile substances


 Simple & efficient in regard to separation
GC consists of GSC (gas solid chromatography)
GLC (gas liquid chromatography
Gas → M.P
Solid / Liquid → S.P
GSC not used because of limited no. of S.P
GSC principle is ADSORPTION
GLC principle is PARTITION
Sample to be separated is converted into vapour
And mixed with gaseous M.P
Component more soluble in the S.P → travels slower
Component less soluble in the S.P → travels faster
Components are separated according to their
Partition Co-efficient

Criteria for compounds to be analyzed by G.C


1.VOLATILITY:
2.THERMOSTABILITY:
What is Gas Chromatography?
• The father of modern
gas chromatography
is Nobel Prize winner
John Porter Martin,
who also developed
the first liquid-gas
chromatograph.
(1950)
The Next Generation in Gas
Chromatography
How a Gas Chromatography Machine
Works
– First, a vaporized sample is injected onto the
chromatographic column.
– Second, the sample moves through the
column through the flow of inert gas.
– Third, the components are recorded as a
sequence of peaks as they leave the column.
Chromatographic Separation

– Deals with both the stationary phase and


the mobile phase.
• Mobile – inert gas used as carrier.
• Stationary – liquid coated on a solid or a solid
within a column.
Chromatographic Separation
• Chromatographic Separation
– In the mobile phase, components of the sample are
uniquely drawn to the stationary phase and thus,
enter this phase at different times.
– The parts of the sample are separated within the
column.
– Compounds used at the stationary phase reach the
detector at unique times and produce a series of
peaks along a time sequence.
Chromatographic Separation
(continued)
– The peaks can then be read and analyzed by a
forensic scientist to determine the exact
components of the mixture.
– Retention time is determined by each component
reaching the detector at a characteristic time.
Chromatographic Analysis

– The number of components in a sample is


determined by the number of peaks.
– The amount of a given component in a
sample is determined by the area under
the peaks.
– The identity of components can be
determined by the given retention times.
Peaks and Data
PRACTICAL REQUIREMENTS
• Carrier gas
• Flow regulators & Flow meters
• Injection devices
• Columns
• Temperature control devices
• Detectors
• Recorders & Integrators
CARRIER GAS
» Hydrogen
better thermal conductivity
disadvantage: it reacts with unsaturated
compounds & inflammable
» Helium
excellent thermal conductivity
it is expensive
» Nitrogen
reduced sensitivity
it is inexpensive
Requirements of a carrier gas
 Inertness
 Suitable for the detector
 High purity
 Easily available
 Cheap
 Should not cause the risk of fire
 Should give best column performance
Flow regulators & Flow meters
 deliver the gas with uniform pressure/flow
rate
 flow meters:- Rota meter & Soap bubble
flow meter
Rota meter
 placed before column inlet
it has a glass tube with a float held on to a
spring.
the level of the float is determined by the
flow rate of carrier gas
Soap Bubble Meter
◊ Similar to Rota meter & instead of a float,
soap bubble formed indicates the flow rate
Injection Devices
 Gases can be introduced into the column by
valve devices
 liquids can be injected through loop or
septum devices
COLUMNS
• Important part of GC
• Made up of glass or stainless steel
• Glass column- inert , highly fragile
COLUMNS can be classified
 Depending on its use
1. Analytical column
1-1.5 meters length & 3-6 mm d.m
2. Preparative column
3-6 meters length, 6-9mm d.m
Depending on its nature
1.Packed column: columns are available in
a packed manner
S.P for GLC: polyethylene glycol, esters,
amides, hydrocarbons, polysiloxanes…
2.Open tubular or Capillary column or
Golay column
 Long capillary tubing 30-90 M in length
 Uniform & narrow d.m of 0.025 - 0.075 cm
 Made up of stainless steel & form of a coil
 Disadvantage: more sample cannot loaded
3.SCOT columns (Support coated open
tubular column
 Improved version of Golay / Capillary
columns, have small sample capacity
 Made by depositing a micron size
porous layer of supporting material on
the inner wall of the capillary column
 Then coated with a thin film of liquid
phase
Columns

• Packed

• Capillary
Equilibration of the column
 Before introduction of the sample
 Column is attached to instrument &
desired flow rate by flow regulators
 Set desired temp.
 Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control Devices
 Preheaters: convert sample into its vapour
form, present along with injecting devices
 Thermostatically controlled oven:
temperature maintenance in a column is
highly essential for efficient separation.
Two types of operations
 Isothermal programming:-
 Linear programming:- this method is
efficient for separation of complex mixtures
Instrumentation - Oven
Temperature Control
• Isothermal • Gradient

240

200

Temp (deg C)
160

120

80

40

0
0 10 20 30 40 50 60
Time (min)
DETECTORS
 Heart of the apparatus
The requirements of an ideal detector are-
 Applicability to wide range of samples
 Rapidity
 High sensitivity
 Linearity
 Response should be unaffected by
temperature, flow rate…
 Non destructive
 Simple & inexpensive
1.Thermal Conductivity Detector
(Katharometer, Hot Wire Detector)

Measures the changes of thermal conductivity due


to the sample (µ g). Sample can be recovered.
Thermal Conductivity Basics
When the carrier gas is contaminated
The TCD is a nondestructive, by sample , the cooling effect of
concentration sensing detector. A the gas changes. The difference in
heated filament is cooled by the flow cooling is used to generate the
of carrier gas. detector signal.
Flow

Flow
Thermal Conductivity Detector
When a separated compound elutes from the
column , the thermal conductivity of the
mixture of carrier gas and compound gas is
lowered. The filament in the sample column
becomes hotter than the control column.
The imbalance between control and sample
filament temperature is measured by a simple
gadget and a signal is recorded
� Measures heat loss from a hot filament –

� filament heated to const T


• 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 goes
down, T of filament will rise. (resistance of
the filament will rise).
Relative Thermal Conductivity
Compound Relative Thermal
Conductivity
Carbon Tetrachloride 0.05
Benzene 0.11
Hexane 0.12
Argon 0.12
Methanol 0.13
Nitrogen 0.17
Helium 1.00
Hydrogen 1.28
Advantages of Katharometer
 Linearity is good
 Applicable to most compounds
 Non destructive
 Simple & inexpensive
Disadvantages
 Low sensitivity
 Affected by fluctuations in temperature and
flow rate
 Biological samples cannot be analyzed
Flame Ionization Detector
 Destructive detector
 The effluent from the column is mixed with H
& air, and ignited.
 Organic compounds burning in the flame
produce ions and electrons, which can
conduct electricity through the flame.
 A large electrical potential is applied at the
burner tip
 The ions collected on collector or electrode
and were recorded on recorder due to
electric current.
FIDs are mass sensitive rather than
conc. sensitive
ADVANTAGES:
• µg quantities of the solute can be
detected
• Stable
• Responds to most of the organic
compounds
• Linearity is excellent

• DA: destroy the sample


FID
Argon ionization detector
 Depends on the excitation of argon atoms to a
metastable state, by using radioactive energy.
Argon→ irradiation Argon + e- →collision Metastable
Argon→ collision of sub. → Ionization →↑Current
ADVANTAGES
1.Responds to organic compounds
2.High sensitivity
DISADVANTAGES
1.Response is not absolute
2.Linearity is poor
3. Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR
The detector consists of a cavity
that contains two electrodes and a
radiation source that emits β -
radiation (e.g.63Ni, 3H)
The collision between electrons and the
carrier gas (methane plus an inert gas)
produces a plasma containing electrons
and positive ions.
• If a compound is present that contains
electronegative atoms, those electrons are
captured and negative ions are formed, and
rate of electron collection decreases

• The detector selective for compounds with


atoms of high electron affinity.
• This detector is frequently used in the
analysis of chlorinated compounds
• e.g. – pesticides, polychlorinated biphenyls
ADVANTAGE
 Highly sensitive
DISADVANTAGE
 Used only for compounds with electron
affinity
RECORDERS & INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORS
Record the individual peaks with Rt, height….
Derivatisation of sample
 Treat sample to improve the process of
separation by column or detection by detector.
 They are 2 types
 Precolumn derivatisation
Components are converted to volatile & thermo
stable derivative.
Conditions - Pre column derivatisation
 Component ↓ volatile
 Compounds are thermo labile
 ↓ tailing & improve separation
Post column derivatisation
 Improve response shown by detector
 Components ionization / affinity towards
electrons is increased
Pretreatment of solid support
 To overcome tailing
 Generally doing separation of non polar
components like esters, ethers…
Techniques: 1. use more polar liquid S.P
2. Increasing amt. of liquid phase
3.Pretreatment of solid support to remove
active sites.
Parameters used in GC
Retention time (Rt)
It is the difference in time b/w the point of
injection & appearance of peak maxima. Rt
measured in minutes or seconds
(or) It is the time required for 50% of a
component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is
required to elute 50% of the component from
the column.
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to
be separated.
If more difference in partition co-efficient b/w two
compounds, the peaks are far apart & S
Is more. If partition co-efficient of two compounds are
similar, then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a
chromatogram is measured by resolution
It is the measure of both column & solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
 An imaginary unit of the column where
equilibrium has been established between
S.P & M.P
 It can also be called as a functional unit of
the column
HETP – Height Equivalent to a Theoretical
Plate
 Efficiency of a column is expressed by the
number of theoretical plates in the column or
HETP
 If HETP is less, the column is ↑ efficient.
 If HETP is more, the column is ↓ efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion
which arises due to packing of the column
B = Molecular diffusion, depends on flow rate
C = Effect of mass transfer,depends on flow rate
u = Flow rate
Efficiency ( No. of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w 2

N = no. of theoretical plates


Rt = retention time
W = peak width at base
 The no. of theoretical plates is high, the
column is highly efficient
 For G.C the value of 600/ meter
Asymmetry Factor
 Chromatographic peak should be
symmetrical about its centre
 If peak is not symmetrical- shows Fronting or
Tailing
 FRONTING
Due to saturation of S.P & can be avoided by
using less quantity of sample
 TAILING
Due to more active adsorption sites & can be
eliminated by support pretreatment,
Asymmetry factor (0.95-1.05) can be
calculated by using the formula AF=b/a
b & a calculated at 5% or 10% of the peak
height
ADVANTAGES OF G.C
Very high resolution power, complex mixtures can be
resolved into its components by this method.
Very high sensitivity with TCD, detect down to 100
ppm
It is a micro method, small sample size is required
Fast analysis is possible, gas as moving phase- rapid
equilibrium
Relatively good precision & accuracy
Qualitative & quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic Analysis

– The number of components in a sample is


determined by the number of peaks.
– The amount of a given component in a
sample is determined by the area under
the peaks.
– The identity of components can be
determined by the given retention times.
Applications of G.C
• G.C is capable of separating, detecting &
partially characterizing the organic
compounds , particularly when present in
small quantities.
1, Qualitative analysis
Rt & RV are used for the identification &
separation
2, Checking the purity of a compound
Compare the chromatogram of the std. & that
of the sample
3, Quantitative analysis
It is necessary to measure the peak area or
peak height of each component
4, used for analysis of drugs & their
metabolites.
Semi-Quantitative Analysis of Fatty Acids

C18
10

C16 8

Peak Area
6
Response
Detector

C14 4

0.5 1.0 1.5 2.0 2.5 3.0


Sample Concentration (mg/ml)
Retention Time

C1
4
The content %of C14fatty acids = ∗1
0
C + C +1
C
6 1
8
1
4

= the content %of C fatty acid


14 s
Tentative Identification of Unknown
Compounds
Mixture of known compounds

Response
Octane Decane
1.6 min = RT
Hexane

GC Retention Time on Carbowax-20 (min)


Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)


Retention Times

RT= 4.0 min on SE-30

Response
Hexane

GC Retention Time on SE-30

RT= 4 min on SE-30


Response

Unknown compound

GC Retention Time on SE-30


Advantages of Gas Chromatography

• Very good separation


• Time (analysis is short)
• Small sample is needed - µ l
• Good detection system
• Quantitatively analyzed
How a Gas Chromatography
Machine Works

– First, a vaporized sample is injected onto the


chromatographic column.
– Second, the sample moves through the
column through the flow of inert gas.
– Third, the components are recorded as a
sequence of peaks as they leave the column.