Gc mpharm sud

Gas Chromatography

Presented By -
Sudheer kumar kamarapu
Assistant professor
Sri Shivani college of Pharmacy
sudheerkumar kamarapu 1

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)


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:


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 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


PARTS OF INSTRUMENT
1. Carrier gas
2. Flow regulators & Flow Meters
3. Injection devices
4. Columns
5. Temperature control devices
6. Detectors
7. Recorders


Schematic diagram of gas chromatography

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


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


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.


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


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).


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.


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

Relative Thermal Conductivity
Relative Thermal
Compound
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 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.


Flame Ionization Detector

 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.

ADVANTAGES:
• µg quantities of the solute can be detected
• Stable
• Responds to most of the organic compounds
• Linearity is excellent

• DA: destroy the sample


35 P HOTO 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.

SUDHEERKUMAR KAMARAPU

36 P HOTO IONIZATION DETECTOR

SUDHEERKUMAR KAMARAPU

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 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.

Electron-Capture Detector

 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.


 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


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 & 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

Retention time


Separation factor


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 (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


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


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.


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