gas chromatography · 1.packed column: columns are available in a packed manner s.p for glc:...

Gas Chromatography

Gas Chromatography Presented By -

Dr. Sohail Ayub Department of Civil Engineering AMU Aligarh India Email – sohailayub@rediffmail.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

Temperature Control

• Isothermal • Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (d

eg C

)

Instrumentation - Oven

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

Measures the changes of thermal conductivity due to the sample (g). Sample can be recovered.

1.Thermal Conductivity Detector (Katharometer, Hot Wire Detector)

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.

Flow

Flow

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

Thermal Conductivity Detector

� 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 - 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

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

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

C

C

C

Det

ecto

r R

espo

nse

Retention Time

14

16

18

Sample Concentration (mg/ml)

2

4

6

8

10

0.5 1.0 1.5 2.0 2.5 3.0

T h e c o n te n t % o f C f a t t y a c id s =C

C + C + C

= th e c o n te n t % o f C f a t ty a c id s1 4

1 4

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane 1.6 min = RT

Res

pons

e

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

GC Retention Time on SE-30

Unknown compound RT= 4 min on SE-30

GC Retention Time on SE-30

Hexane RT= 4.0 min on SE-30

Retention Times

Advantages of Gas Chromatography

• Very good separation

• Time (analysis is short)

• Small sample is needed - 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.