Under the guidance of Mr. CH NARASIMHA RAJU BH.

,
M.Pharm., A.I.C, (Ph.D).,

Associate Professor,
Department of Pharmaceutical analysis, A.S.N. Pharmacy college

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 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 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.

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
240 200
Temp (deg C)

Gradient

160
120 80 40 0 0 10 20 30 Time (min) 40 50 60

DETECTORS
Heart

of the apparatus The requirements of an ideal detector areApplicability 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

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).

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: g 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.
irradiation

Argon

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 - 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 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

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

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,

References
1. Braun Robert D., Introduction to Instrumental Analysis, chapter no.26, first edition, year2006, pharma book syndicate, page no. 890 - 928. 2. Christian Gary D., Analytical Chemistry, chapter no.17, fifth edition, year2001, library of congress cataloging in publication data, page no. 535 536. 3. Munson James W., Pharmaceutical Analysis part A, chapter no.1, year2001, Ashish arts, page no. 4 54.
4. Text book of Pharmaceutical Analysis, fourth edition, Dr S. Ravi Sankar17.1 17.20
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