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?What is Gas Chromatography?

• It is also known as…It is also known as…– Gas-Liquid Chromatography (GLC)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?What is Gas Chromatography?

• The father of modern The father of modern gas chromatography gas chromatography is Nobel Prize winner is Nobel Prize winner John Porter MartinJohn Porter Martin, , who also developed who also developed the first liquid-gas the first liquid-gas chromatograph. chromatograph. (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

– First,First, a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column. .

– Second,Second, the sample moves through the the sample moves through the column through the flow of inert gas.column through the flow of inert gas.

– Third,Third, the components are recorded as a the components are recorded as a sequence of peaks as they leave the column.sequence of peaks as they leave the column.

Chromatographic SeparationChromatographic Separation

– Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase. . • Mobile Mobile – inert gas used as carrier.– inert gas used as carrier.• StationaryStationary – liquid coated on a solid or a solid – liquid coated on a solid or a solid

within a column.within a column.

Chromatographic SeparationChromatographic Separation

• Chromatographic SeparationChromatographic Separation– In the mobile phase, components of the sample are In the mobile phase, components of the sample are

uniquely drawn to the stationary phase and thus, uniquely drawn to the stationary phase and thus, enter this phase at different times. enter this phase at different times.

– The parts of the sample are separated within the The parts of the sample are separated within the column.column.

– Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence. peaks along a time sequence.

Chromatographic Separation Chromatographic Separation (continued)(continued)

– The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixture.components of the mixture.

– Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic time.reaching the detector at a characteristic time.

Chromatographic AnalysisChromatographic Analysis

– The number of components in a sample is The number of components in a sample is determined by the number of peaks.determined by the number of peaks.

– The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks. the peaks.

– The identity of components can be The identity of components can be determined by the given retention times.determined by the given retention times.

Peaks and DataPeaks 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

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould 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 meterplaced 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 lengthUniform & narrow d.m of 0.025 - 0.075 cmMade up of stainless steel & form of a coilDisadvantage: 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 operationsIsothermal 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 (

deg

C)

Instrumentation - Oven

DETECTORSHeart 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 (µ 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.

Flo

w

Flo

w

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 ConductivityCompound 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 KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple & inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological 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

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS & INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord 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 derivatisationComponent ↓ volatileCompounds are thermo labile↓ tailing & improve separation

Post column derivatisation

Improve response shown by detectorComponents ionization / affinity towards

electrons is increased

Pretreatment of solid supportTo 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 rateˣ

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 baseThe no. of theoretical plates is high, the

column is highly efficientFor 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 AnalysisChromatographic Analysis

– The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks..

– The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks. the peaks.

– The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention 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

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mg/ml)

2

4

6

8

10

0.5 1.0 1.5 2.0 2.5 3.0

The content % of C fatty acids =C

C + C + C100∗

14

181614

= the content % of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane1.6 min = RT

Res

pons

e

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

Res

pons

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 4.0 min on SE-30

Retention TimesRetention Times

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 How a Gas Chromatography Machine WorksMachine Works

– FirstFirst, a vaporized sample is injected onto the , a vaporized sample is injected onto the chromatographic columnchromatographic column. .

– SecondSecond, the sample moves through the , the sample moves through the column through the flow of inert gas.column through the flow of inert gas.

– ThirdThird, the components are recorded as a , the components are recorded as a sequence of peaks as they leave the column.sequence of peaks as they leave the column.