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 sudheerkumar kamarapu 9

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

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


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 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 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.


ADVANTAGES:

• µg quantities of the solute can be detected

• Stable

• Responds to most of the organic compounds

• Linearity is excellent

• DA: destroy the sample


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.

SUDHEERKUMAR KAMARAPU

35

http://en.wikipedia.org/wiki/GC_ion_detector

http://en.wikipedia.org/wiki/Photon

http://en.wikipedia.org/wiki/UV

http://en.wikipedia.org/wiki/Ion

http://en.wikipedia.org/wiki/Gas_chromatograph

http://en.wikipedia.org/wiki/Ion

http://en.wikipedia.org/wiki/Signal_(information_theory)

http://en.wikipedia.org/wiki/Electric_current

http://en.wikipedia.org/wiki/Signal_(information_theory)

PHOTO IONIZATION DETECTOR

SUDHEERKUMAR KAMARAPU

36

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 sudheerkumar kamarapu 37

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 sudheerkumar kamarapu 38

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

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


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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