GAS CHROMATOGRAPHY

INTRODUCTION & INSTRUMENTATION

M.ARCHANA M.PHARM 1 ST YEAR QUALITY ASSURANCE

INTRODUCTION CHROMATOGRAPHY:
The term chromatography covers those separation techniques in which separation is based upon the partition or distribution or adsorption of the analytes between two phases (stationary phase and mobile phase) in a dynamic process .

 One of the phase is fixed bed of large surface area while the

other is a fluid which moves through , or over the surface of, the fixed phase.
The components of the mixture must be of molecular

dimensions, which requires that they be in solution or in vapour state.

The relative affinity of the solute for each of the phases must

be reversible to ensure that mass transfer occurs during the chromatographic separation.
The fixed phase is called Stationary Phase, & the other is

termed the Mobile Phase.

Gas Chromatography
Gas chromatography is a technique for

separation of volatile substances by percolating gas stream over a stationary phase. Stationary phase - liquid or solid held in column Mobile phase - gas

Gas Solid Chromatography

Gas Liquid Chromatography

GAS SOLID CHROMATOGRAPHY

If the stationary phase is solid we speak of GAS-

SOLID CHROMATOGRAPHY. This depends upon the adsorptive properties of the column packing to separate samples primarily gases.
Common packing used are silica gel, molecular

sieves and charcoal

Principle:

The principle of separation is adsorption.

GSC is quite limited in acceptability due to the following reasons:

1)Excessive retention of active gases on solid surface 2)Tailing of elution peaks

3)Non-linear adsorption isotherms

GAS-LIQUID CHROMATOGRAPHY:
If the stationary phase is liquid we speak of GAS-

LIQUID CHROMATOGRAPHY Principle: The principle of separation is partition. Gas is used as mobile phase. Liquid which is coated on to a solid support is used as stationary phase.
As the components have finite solubility in

stationary (fixed) liquid phase , distribute themselves between this phase and the mobile gas phase in accordance with equilibrium law.
Elution is then carried out by forcing an inert gas through

the column.

 The rate of movement of various components along the column

depends upon their tendency to dissolve in stationary liquid phase. Components having a negligible solubility in stationary phase move rapidly through the column, while those components whose distribution coefficient favours the solvent liquid phase move with low rate. Ideally bell shaped elution curves are obtained. Partition ratio is simply the ratio of solute in stationary phase to amount of solute in mobile phase Amount of solute in liquid phase K= Amount of solute in gas phase

ADVANTAGES:

1.Speed 2.Resolution 3.Qualitative analysis 4.Quantitative analysis 5.Sensitivity 6.Simplicity

Filters/Traps

Data system
H

RESET

Regulators

Syringe/Sampler Inlets

Detectors
Gas Carrier Hydrogen Air

gas

Column

system inlet column detector data system

Schematic Diagram of Gas Chromatography

INSTRUMENTAL COMPONENTS CARRIER GAS SAMPLE INJECTION SYSTEM

COLUMNS
SOLID SUPPORT STATIONARY LIQUID PH ASES DETECTORS ELECTRONIC/CHART

RECORDERS

 The main function of carrier gas is to carry the vapourised

sample onto the column, which contains the stationary phase material.
Carrier gas must be chemically inert. Commonly used gases

include nitrogen, helium, argon & Carbon-di-oxide.

A high pressure gas cylinder serves a source of carrier gas.
The flow rate of the carrier gas is carefully controlled by

means of pressure regulators & flow meters to keep the flow rate during analysis.
The choice of the carrier gas is often dependent upon the type

of detector which is used.

The carrier gas used in GC Gas Helium Nitrogen Application Comments

Oxygen Argon

General carrier gas Expensive or make up gas General gas or make Cheap not good for up gas capillary column as it gives long run time Combustion gas for Not normally used some FID Carrier gas for TCD Determination of helium
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Contd

Hydrogen

Air

Carrier gas for capillary column and combustion gas for FID Combustion gas for FID,

Cheap, explosive

Cheap, readily available Better linearity & selectivity than N2 but poor detection limit

Argon/methane Make up & packed column carrier gas for ECD

 Make up Gas:

Most detector require 30-40 m2/min total gas flow rate for best sensitivity and peak. To supplement carrier gas flow, make up gas is added at the column exit to obtain total gas flow of 30-40 m2/min into detector.

Pressure & flow regulators

High pressure is preferred as it decreases the analysis time. The carrier gas flow is maintained by: a. Needle valve b. Pressure controller c. Mass flow controllers
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a.

b.
c.

Needle Value:- It is the simplest method of control and operated manually. Pressure controller:- The device is used to maintain the constant pressure at the inlet of the column. Mass flow controllers:- These are complex device, which contain an automatic control mechanism.

FLOW METERS :
SOAP BUBBLE METER

ROTAMETER

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 The sample injection system, called the Injection port, is generally

heated to high temperature to vapourise quickly the sample to be analysed.

Most general methods employed for liquid or solids is the syringe

technique. Solids are dissolved in suitable solvents & liquids are injected as such or diluted with the suitable solvents.
For gases, a gas tight syringe of 0.5-10 ml capacity or a sample loop

may be used.
The carrier gas is conducted from the gas reservoir to a sample port

injector.
Since solutes to be chromatographed must be in the vapour state, the

injection port is heated to a temperature, which will ensure rapid vapourisaton but not thermal degradation.

 The temperature of the sample port is usually above 500C

higher than the boiling point of the least volatile component of the sample.
The injection port is placed such that the sample is introduced

directly into the carrier gas.

The port is constructed of a heavy mass that is maintained at

an elevated temperature & contains a pliable septum through which samples are injected.
The

port is designed for instantaneous injection & vapourisation of sample so that the sample is introduced immediately into the column.

 For optimum column efficiency, the sample should not too large, &

should be introduced onto the column as a Plug of vapour. Slow injection of large samples causes b& broadening & loss of resolution.
For packed columns sample size ranges from tenths of a L upto 20

L. Capillary columns, on the other hand, need much less sample, typically around 10-3 L.
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 Split injection: routine method 0.1-1 % sample to column remainder to waste Splitless injection: all sample to column best for quantitative analysis only for trace analysis, low [sample] On-column injection: for samples that decompose above boiling

point - no heated injection port column at low temperature to condense sample in narrow b& heating of column starts chromatography

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 Column is the heart of Chromatography. Column tubing can

be made from copper, stainless steel, aluminum & glass in a straight, bent or coiled form.
In the column, the different solutes in the vapourised samples

are separated from each other by virtue of their different interactions with the column packing.
Columns vary from a few inches to about 100 m. Longer

column lengths give more theoretical plates & resolution. Therefore larger sample sizes may be injected.
Conditioning of column is accomplished by baking at

prescribed temperature, by flushing nitrogen for 6-12hrs at highest permissible temperature.

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COLUMNS
Packed

Capillary

Types of Columns in GC
Packed-bed column
(d > 2 mm, packing particle from 100 to 250 micron)

. Micro-packed column
(d < 1 mm, less than 0.3)

Packed capillary column

(d < 0.6 mm, packing particle 5-20 micron)

Wall coated open tubular columns (WCOT)

Thin layer of stationary phase coated directly on the wall of the tube

Support coated open tubular (SCOT)

Liquid phase + glass powder or particle support

Porous layer open tubular column (PLOT)

Particle support

Two types of column are commonly employed in GLC:

PACKED COLUMN CAPILLARY COLUMN

PACKED COLUMNS
The column container for packed columns is usually stainless

steel or copper or glass tube packed with either a solid substrate or liquid coating on an inert solid
Most packed columns are 1.5-10 m in length & have an

internal diameter of 2-4 mm.

 Contain a finely divided, inert, solid support material

(commonly based on diatomaceous earth) coated with liquid stationary phase.

Advantages: More concentrated samples may be analyzed;

more solvent may be injected; can be used in preparative applications.

Disadvantage: Analyte separation is less favorable;

unsuitable for trace analysis.

 Fused silica, glass and stainless steel are the primary tubing

materials.
The column may be 0.2 - 0.7 mm i.d. and 10 - 100 m long

Stationary phase is a liquid that is bond to the inner surface of

the column.
Fused silica tubing has recently become the preferred type

because it produces flexible inert columns.

Capillary columns are constructed of three parts - fused silica

tubing, polyimide coating and stationary phase

 They are available in different types: WALL COATED OPEN TUBULAR (WCOT): consists of a

capillary tube whose walls are coated with liquid stationary phase

SUPPORT COATED OPEN TUBULAR (SCOT):

The inner walls of the capillary is lined with a thin layer of support material such as Diatomaceous earth, onto which the stationary phase has been adsorbeb

POROUS LAYER OPEN TUBULAR COLUMN (PLOT): the

inner walls of thee capillary is lined with a thin layer of Particle support.

SCOT columns are less efficient than

WCOT columns. But all these are much more efficient than the packed columns
Gas-liquid Chromatography:

- Classical packed column, & WCOT, - Partition mechanism

GAS SOLID CHROMATOGRAPHY :

- Classical packed column, & PLOT, - Adsorption mechanism

 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.

In general, the column temperature is selected to compromise

between the length of the analysis & the level of separation

240 200

Temp (deg C)

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

A method which holds the column at the same temperature for

the entire analysis is called "isothermal."

In Gradient there is an increase in the column temperature

during the analysis, the initial temperature, rate of temperature increase (the temperature "ramp") & final temperature is called the "temperature program.

 The purpose of solid support is to provide support to the thin,

uniform film of liquid phase.

The solid support must have 2 primary requirements: It must

be a poor adsorbent & it must be a finely divided porous substance having a large surface area.
In addition other requirements include: Chemical

Inertness- Minimum of Chemical & Adsorptive Interaction with the sample. Heat Stability Sufficient Mechanical Strength- should not crush on heating.

 Large Surface area- 1 to 20 sq.m/g. Regular Shape, Uniform Size- for efficient packing. Readily wetted by the liquid phase
Commonly used solid support: Diatomaceous earth, grounded

fire brick, glass beads, fluorinated resins & polyaromatic resins.

Physical adsorption of polar analyte species on silicate surface

of solid support may occurs hence support materials should be deactivated by silanization with dimethylchlorosilane.
Acid washing prior to silanization removes impurities (metal

oxides)

 REQUIREMENTS:
1. 2. 3. 4.

5.

Low volatility Thermally stable Chemically inert Solvent characters for solutes must be different Good solvent for component of sample
The immobilized liquid must generate different distribution coefficient for different solutes. The ratios must not be extremely large nor small.

 Advantages: Liquid phases are available in great variety thus allowing

suitable selectivity for separation. Liquid stationary phases are available in well defined purity enabling reproducible retention time to be produced. The amount of stationary phases on the column can be varied to suit either analytical or preparative separations

FILM THICKNESS:
Vary from 0.1-5 m. Thick films- highly volatile analytes Thin films -low volatile analytes

2. Types of liquid stationary phases

a. Hydrocarbon & Perfluorocarbon stationary phases (nonpolar)
Squalane (C30H62); Apiezon; Apolane-87 (C87H167)

b. Ether & ester stationary phases

Poly-ethers (e.g., poly-phenyl ethers), phthalate esters

c. Liquid organic-salts stationary phases

Alkyl ammonium or alkylphosphonium with nucleophilic anions (such as BF4-)

d. Poly (siloxane) stationary phases -poly dimethyl siloxane -poly phenyl methyl siloxane -poly phenyl methyl dimethyl siloxane

 BONDED AND CROSS LINKED STATIONARY

PHASES: -Inhibit bleeding

-long lasting stationary phase that can be rinsed

Bonding:

Attaching a monomolecular layer of stationary phase to silica surface by chemical reaction. To incorporate a peroxide into original liquid.

Cross linking:

CHIRAL STATIONARY PHASES:

- used for separation of enantiomers

eg: amino acid chiral phases

REFERENCES:
William Kemp, Organic Spectroscopy, third edition Instrumental methods of Chemical Analysis (Analytical

Chemistry), by Dr. B.K. Sharma, page no.: C-208 C-217.

Instrumental methods of Chemical Analysis (Analytical

Chemistry), by G.R. Chatwal and S.K. Anand, page no.: 2.691 2.696.
Text book OF Pharmaceutical Analysis, 3rd edition, by Dr. S.

Ravi Sankar, page no.: 17 18 to 17 20.