GAS CHROMATOGRAPHY

(GC) SAIMA ALEEM.

M.PHIL.

Course no CHM -754.

COURSE SUPERVISOR: DR RAHAT

GAS CHROMATOGRAPHY 

INTRODUCTION

• Gas chromatography is one of the most versatile and ubiquitous analytical techniques in the laboratory. It is widely used for the determination of organic compounds.

• Complex mixtures can be separate by this technique.

• A gas chromatography is a chemical analysis instrument for separating chemicals in a complex sample.

A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates.

It depending on their various chemical and physical properties and their interaction with a specific column filling called the stationary phase.

As the chemicals exit the end of the column, they are detected and identified electronically. The function of the stationary phase in the column is to separate different

components, causing each one to exit the column at a different time (retention time).

Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, column length and the temperature.

Gas chromatography

In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance" (head) of the column, usually using a microsyringe (or, solid phase microextraction fibers, or a gas source switching system).

As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column.

The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials.

Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times

(retention time). A detector is used to monitor the outlet stream from the column.

The time at which each component reaches the outlet and the amount of that component can be determined.

AUTO SAMPLER• The auto sampler provides the means to

introduce a sample automatically into the inlets.

• Manual insertion of the sample is possible but is no longer common. Automatic insertion provides better reproducibility and time-optimization.

• Different kinds of auto samplers exist. Auto samplers can be classified in relation to sample capacity (auto-injectors vs. auto samplers, where auto-injectors can work a small number of samples), to robotic technologies (XYZ robot vs. rotating robot – the most common), or to analysis:

Liquid.

Static head-space by syringe technology. Dynamic head-space by transfer-line

technology. Solid phase micro extraction (SPME).

CARRIER GAS• In gas chromatography Helium, nitrogen,

Hydrogen or argon gases are used as a carrier gas.

• The choice of gas is depend upon availability, purity required, consumption and the type of detector employed.

• Helium is preferred, when thermal conductivity detectors are used. Because of it high thermal conductivity relative to that of the vapours of most organic compounds.SAFETY

• Free standing gas cylinder must always be supported by means of clamps or chains.

• Waste gases, especially hydrogen, must be vented through an extraction hood.

INJECTORSPacked Column Injectors

In general, the sample injected onto a packed GC column ranges in volume from 0.5 micro liters to 5 micro liter and usually contains the materials of interest at concentrations ranging from 5% v/v to 10% w/v.

A Pack column injector The sample is injected by a hypodermic syringe,

through a silicone rubber septum directly into the column packing or into a flash heater. Although the latter tends to produce broader

peaks it also disperses the sample radially across the column.

Direct injection into the packing constrains the sample into a small volume, but can cool the front of the packing.

The silicone septum is compressed between metal surfaces in such a manner that a hypodermic needle can pierce it, but when it is withdrawn the hole is closed as a result of the septum compression and there is no gas leak.

The glass liner prevents the sample coming in contact with the heated metal wall and thus, reduces the chance of thermal decomposition. The glass liner can be fitted with a separate heater and the volatalization temperature can, thus, be controlled. This "flash heater" system is available in most chromatographs.

By using a syringe with a long needle, the tip can be made to penetrate past the liner and discharge its contents directly into the column packing.

This procedure is called 'on-column injection' and, as it reduces peak dispersion on injection and thus, provides higher column efficiencies, is often the preferred procedure.

Open Tubular Column Injection Systems Due to the very small sample size that must be

placed on narrow bore capillary columns; a split injection system is necessary.

As the sample passes the column opening, a small fraction is split off and flows directly into the capillary column are called a split injector.

Regulating the portion of the carrier gas that passes to waste changes the split ratio. This achieved by an adjustable flow resistance situated in the waste flow line. This device is only used for small diameter capillary columns where the charge size is critical.

The device has certain disadvantages due to component differentiation and the sample placed on the column may not be truly representative. The solutes with the higher diffusivities (low molecular weight) are lost preferentially to those with lower diffusivities (higher molecular weights). Consequently, quantitative analyses carried out using the high efficiency small diameter capillary columns may have limited accuracy and precision, depending on the nature of the sample.

The Split Injection System This problem was partially solved by

using larger diameter columns that would permit on-column injection.

There are also difficulties associated with this type of injector. On injection, the sample breaks up into separate portions, and bubbles form at the beginning of the column causing the sample to be deposited at different

positions along the open tube as the solvent evaporates.

On starting to develop the separation, each local concentration of sample acts as a separate injection.

Column

• The actual components is effected in the Column

• Where the nature of the solid support, type and amount of liquid phase, method of packing length and temperature are important factor in obtaining the desired resolution.

• The Column is enclosed in a thermostatically controlled oven so that its temperature is held constant to within 0.5 C. Thus ensuring reproducible conditions.

• The operating temperature may range from ambient to over 400 C and for isothermal

operation is kept constant during the separation process.

Type of Column

1. Packed columns

• The internal diameter of Column is 2-6 mm.

The outer diameter of Column is 3-10 mm and it is coil for compactness

Packed columns

Qualities of the packed column

• Glass column must be used if any of the sample components are decompose by compact with metal.

• The material chosen as a inaet support should be uniform granular size and capable of being packed in to a uniform bed in a column.

• The surface area of the material should be large so as to promote distribution of the liquid phase as a film and ensure the rapid attainment of equilibrium between the stationary phase and mobile phase.

• The most commonly used supports(e.g celite) are made from diatomaceous materials which can hold liquid phases in amounts exceeding 20% without becoming too sticky to flow freely and can be easily packed.

• To be a good resolution the height is equivalent of the theoretical plate is proportion to the average particle diameter.

• Rapidly increase the pressure due to decrease the particle size.

• It is necessary to increase the pressure for achieving the flow rate through the column

Open columns

• These capillary columns (i.d < 1mm) are increasingly used in GLC.

• It is used for complex mixtures.

• This result from a highly theoretical plate numbers which can be attain with long column of this type for the relatively smaller pressure drop.

• In these capillary columns the stationary phase s coated on the inner wall of the tube,

Here 2 types of capillaries are available.

1. Wall coated open tubular

• Here the stationary phase is directly coated on the inner wall of tubing.

2. Supported coated open tubular

• It have finely divided layer of solid support material deposited on the inner wall of the stationary phase is then coated

• Supported coated open tubular is less efficient then wall coated open tubular.

• Capillary column are made up of thin wall stainless steel.

• The dimension of the column, which is coiled, is 25- 200 m long and 0.2-0.5mm i.d.

• The drawback of this column is that they have low sample capacity than pack column.

DETECTOR

The choice of detector will depend the concentration level of the sample.

The important properties of a detector are as follows

1. SENSITIVITY

• This is usually defined as the detector response (mV) per unit concentration of analyte (mg/ml).

• It is closely related to the detection limit.

Since high sensitivity often give low limit of detection.

• The limit of detection will be rises if the detector produced noise.

• The sensitivity also determines the slop of calibration graph and therefore influences the precision of the analysis.

2 . Linearity

• The linear range of the detector refer to the concentration range over which the signal is directly proportional to the concentration of analyte.

• Linearity in detector response will give linearity of the calibration graph and allow later to be drawn with more certainty.

• With the convex calibration curved, the precision is reduced at higher concentrations where the slope of the curved is much lesser.

• The high linearity range has high advantage but detector small range will be used because of their other qualities, although they will need to be recalibrated over a number of difference concentration ranges.

3. Stability

• The important characteristic of a detector is the extent to which the signal output remains constant with time, assuming there is a constant input.

• Lack of the stability can be exhibited in to two ways.

1. Baseline noise.

2. Drift.

1 .Baseline noise.

• Baseline noise is caused by a rapid random variation in detector output difficult to measure small peaks against the fluctuation background.

2 .Drift

• Drift is often due to factors external to the detector and impose a more fundamental limit on its performance, such as temperature change or column bleed and so is controllable, where as noise is usually due to poor contacts within detector and impose a more fundamental limit on its performance.

• It must be reproducible TYPE OF DETECTORThermal conductivity detector

• The most important of the bulk physical property detector is a thermal conductivity detector which is universal, nondestructive,

concentration – sensitive detector. It is still widely used in gas chromatography.

• Helium and hydrogen are the best carrier gases to use in conjunction with this type of detector. Since their thermal conductivity are much higher than the other gases.

Thermal Conductivity Detector (TCD)

• In the detector 2 pair of match filaments are arranged in a Wheatstone bridge circuit, 2 filaments is opposite arm of the bridge is surrounded by the carrier gas only.

• The other 2 are surrounded by the effluent from the chromatographic column.

• There are 2 types of gas channels through the cell.

1. Sample channels.

2. Reference channels.

• When the pure carrier gas is pass over both the Sample and reference filaments the bridge is balanced but when the vapours emerges from the column, the rate of cooling of the sample filament become changed and the bridge become unbalanced.

• It is used for the detection limit of permanent gases light hydrocarbon and compound which response poorly from the flame ionization detector.

• It is used for the gas chromatography studies of metal chelates, e.g mixture of beryllium, aluminium, gallium and indium

trifluoroacethyacetonates. For many general applications it is replaced by Flame ionization detector.

Ionisation detector

• An important characteristic of the common carrier gases is that they behave as perfect insulators at normal temperature and pressures .The increase conductivity due to the presence of a few charge molecules in the effluent from the column thus provide the high sensitivity which is a feather of the ionisation based detector.

• Ionisation detector in current used include the flame ionisation detector, thermionic ionisation detector, photo ionisation detector and election capture detector each of course employing a different method to generate an ion current.

• The basis of this detector is that the effluent from the column is mixed with hydrogen and burned in air to produce a flame which has sufficient energy to ionize solute molecules having low Ionisation potential.

Flame Ionisation detector

• The ion produced are collect at electrodes and a result ion current measured.

• The burner jet is the negative electrode whilst the anode is usually wire or grid extending into the tip of the flame. Because the sample is destroy in the flame.

• A stream-splitting device is inserted between the column and detector and allows the bulk of the sample to by-pass the detector.

• It is widely applicable for gas chromatography of organic compounds.

• It has high sensitivity, stability, fast response and linearly response with analytes.

Electron capture detector• Most ionisation detectors are based

on measurement of the increase in current which occur when a more readily ionised molecule appears in the gas stream.

• the Electron capture detector is differ from the other ionisation detectors .it exploits the recombination phenomenon, based on Electron capture by compound having affinity for free electron .it only measure the decrease in current.

Electron capture detector

• A beta rays is used to generate slow electron by ionisation of the carrier gas (nitrogen preferred) flowing through the detector.

• The slowly electron migrate to the anode under a fixed potential and give rise to a steady baseline current

• When the electron capturing gas is emerges from the column. It will react with electron by a negative ion of much higher mass with a corresponding reduction in current flow.

• It response due to electron affinity of the eluate molecules. Being sensitive to compound containing halogens, sulphur, anhydrides, nitrites, conjugated carbonyls, nitrates and organo metallic compounds.

• It has high sensitivity. Element sensitive detector

• Element sensitive detector in current used includes “Thermionic Ionisation detector, Flame photometric detector and atomic absorption detector”.

Thermionic Ionisation detector

• It only response to nitrogen containing compound and phosphorus.

• It contains an electrically heated rubidium silicate bead situated a few millimeters above the detector jet tip and below the collector electrode.

• The temperature of beat is maintained at 600-800 degree centigrade.

Flame photometric detector

• It only response to sulphur and phosphorus.

• Hydrogen – rich flame results in the formation of luminescent species that

emit light character of the heteroatom introduced in to the flame.

• When nitrogen is carrier gas, it can be used.

• Hydrogen is introduced at the burner to initiate combustion.

Atomic absorption spectroscopy

• It is used for the separation of organo metallic compound.

TYPES OF CHROMATOGRAPHY

• Gas solid (adsorption) chromatography.

• Gas liquid (partition) chromatography.

The most important one of the two is gas liquid chromatography, used in the foam of capillary column.

GAS-LIQUID CHROMATOGRAPHY

• In gas-liquid chromatography, the mobile phase is a gas such as helium and the stationary phase is a high boiling point liquid absorbed onto a solid.

A flow scheme for gas-liquid chromatography

Injection of the sample

• Very small quantities of the sample that you are trying to analyses are injected into the machine using a small syringe. The syringe needle passes through a thick

rubber disc (known as a septum) which reveals itself again when the syringe is pulled out.

• The injector is contained in an oven whose temperature can be controlled. It is hot enough so that all the sample boils and is carried into the column as a gas by the helium (or other carrier gas).

Column: packed Column.

Retention time

• The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

• Different compounds have different retention times. For a particular compound, the retention time will vary depending on the boiling point of the compound.

• A compound which boils at a temperature higher than the column temperature is going to spend nearly all of its time condensed as a liquid at the beginning of the column. So high boiling point means a long retention time.

• The solubility in the liquid phase. The more soluble a compound is in the liquid phase, the less time it will spend being carried. along by the gas. High solubility in the liquid phase means a high retention time.

• The temperature of the column. A higher temperature will tend to excite molecules

into the gas phase - either because they evaporate more readily, or because they are so energetic that the attractions of the liquid no longer hold them.

• A high column temperature shortens retention times for everything in the column.

• The lower the temperature of the column, the better the separation.

Detector

• There are several different types of detector is use.

• The flame ionisation detector described below is commonly used and is easier to describe and explain than the alternatives.

GAS SOLID CHOROMATOGRAPHY

Comprises all gas chromatographic methods in which the stationary is an active solid (e.g. charcoal, molecular sieves).Separation is achieved by adsorption of the components of a sample.

The retention time is long, due to which it is less used then gas liquid chromatography.

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