gc mpharm sud
TRANSCRIPT
Gas Chromatography
Presented By - Sudheer kumar kamarapu Assistant professor Sri Shivani college of Pharmacy
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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)
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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
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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:
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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.
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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.
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Peaks and Data
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PARTS OF INSTRUMENT 1. Carrier gas
2. Flow regulators & Flow Meters
3. Injection devices
4. Columns
5. Temperature control devices
6. Detectors
7. Recorders
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Schematic diagram of gas chromatography sudheerkumar kamarapu 9
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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
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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
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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
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Soap Bubble Meter
◊ Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate
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Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
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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
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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
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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
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Columns
• Packed
• Capillary
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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
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Column temperature and temperature program
The column(s) in a GC are contained in an oven, the temperature of which is
precisely controlled electronically.
The rate at which a sample passes through the column is directly proportional to
the temperature of the column. The higher the column temperature, the faster
the sample moves through the column.
A method which holds the column at the same temperature for the entire
analysis is called "isothermal Programming"
Most methods, however, increase the column temperature during the analysis,.
" Gradient Temperature programming - Start at low temperature and gradually
ramp to higher temperature
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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.
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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
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Types of Detectors
1. Thermal Conductivity Detectors (TCD)
2. Flame Ionization Detectors (FID)
3. Photo Ionization Detectors (PID)
4. Argon Ionization Detectors (AID)
5. Electron Capture Detectors (ECD).
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1.Thermal Conductivity Detector
(Katharometer, Hot Wire Detector)
• A TCD detector consists of an electrically-heated wire or thermistor.
• The temperature of the sensing element depends on the thermal conductivity of
the gas flowing around it.
• Changes in thermal conductivity, such as when organic molecules displace some
of the carrier gas, cause a temperature rise in the element which is sensed as a
change in resistance.
• The TCD is not as sensitive as other detectors but it is non-specific and non-
destructive.
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Thermal Conductivity Basics
When the carrier gas is contaminated
by sample , the cooling effect of
the gas changes. The difference in
cooling is used to generate the
detector signal.
The TCD is a nondestructive,
concentration sensing detector. A
heated filament is cooled by the flow
of carrier gas.
Flo
w
Flo
w
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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
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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
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Flame Ionization Detectors (FID)
It also called as Destructive detector (destructive of sample)
The principle involved in the detectors are based upon the
electrical conductivity of carrier gases. At normal temperature
and pressure gases act as insulators, but become conductive
of ions if electrons are present.
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Flame Ionization Detector sudheerkumar kamarapu 32
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
A potential of a few hundred volts is applied.
The resulting current (~10-12 A) is then measured.
The flame ionization detector exhibits a high sensitivity (~10-13
g/s), large linear response range (~107), and low noise.
A disadvantage of the flame ionization detector is that it is
destructive of sample.
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ADVANTAGES:
• µg quantities of the solute can be detected
• Stable
• Responds to most of the organic compounds
• Linearity is excellent
• DA: destroy the sample
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PHOTO IONIZATION DETECTOR
Principle
A PID is an ion detector which uses high-energy photons, typically in the UV range, to produce ions.
As components elute from the GC's column they are bombarded by high-energy photons and are ionized.
The ions produce an electric current, which is the signal output of the detector.
The greater the concentration of the component, the more ions are produced, and the greater the current.
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PHOTO IONIZATION DETECTOR
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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
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Electron-Capture Detectors(ECD)
The electron capture detector is composed of a radioactive
source which emits electrons, a cathode which repels the
electrons, an anode and wire which collects the electrons.
The ECD has two
electrodes with the
column effluent passing
between them. One of the
electrode is treated with a
radioactive isotope which
emits electrons as it
decays.
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These emitted electrons produce secondary
electrons which are collected by the anode, when a
potential of 20V is applied between them. When carrier
gas alone flows through, all the secondary electrons
are collected by the positively polarised electrode.
An important application of the electron-capture
detector has been for the detection and determination
of chlorinated insecticides.
It is insensitive to functional groups such as
amines, alcohols, and hydrocarbons.
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The electron-capture detector is selective in its response
being highly sensitive to molecules containing electronegative
functional groups such as halogens, peroxides, quinones, and
nitro groups.
ADVANTAGE
Highly sensitive
DISADVANTAGE
Used only for compounds with electron affinity
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RECORDERS & INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORS
Record the individual peaks with Rt, height….
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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
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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.
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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
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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 & S is less.
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
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Retention time
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Separation factor
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Resolution
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Resolution
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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
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HETP (length of the column)
(No of theoritical 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
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Efficiency ( No. of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
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
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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,
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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
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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
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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
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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.
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