Gas Chromatography & GC-MS

submitted by: Submitted To: Himanshu Sachdeva Dr. Rakesh kr. MarwahaRoll no.1705 Assistant Professor

Dptt. Of Pharmaceutical Sc. MDU, Rohtak1

Contents: Introduction to GC Types of GC and Principles Instrumentation of GC Derivatisation Applications of GC Introduction to GC-MS Interfaces used in GC-MS Interpretation Application

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GAS CHROMATOGRAPHY

• Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition.

• The development of GC as an analytical technique was discovered by Martin and Synge 1941

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Gas Chromatography:• Gas Solid Chromatography (GSC)• Gas Liquid Chromatography (GLC) In both cases, gas is used as mobile phase and

solid and liquid is used as stationary phase respectively.

Gas Solid Chromatography: is not widely used because limited no of stationary phase available. GSC is used only in case, where there is a less solubility of solutes in stationary phase

Principle of separation is ADSORPTION4

GAS Liquid Chromatography: Principle: The principle of separation in GLC is PARTITION. Gas is

used as mobile phase and liquid which is coated on a solid support is used as a stationary phase.

The mixture of components to be separated is converted to vapour and mixed with gaseous mobile phase . The component which is more soluble in stationary phase travel slower and eluted later and the components which are less soluble travel faster and eluted first. Hence the components are separated according to their partition co-efficient.

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CRITERIA FOR GLC:

1.Volatility: Unless a compound is volatile, it can not be mixed with mobile phase. So can not be separate out.

2.Thermostability: The compounds which are in solid or liquid form, first need to be converted to gaseous form so they have to be heated to a higher temperature. At that temperature, the compound have to be thermostable.

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GAS CHROMATOGRAPHIC APPARTUS

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PRACTICAL REQUIREMENTS

• Carrier gas• Flow regulator and flow meter• Injection devices• Columns• Temperature control device• Detectors • Recorders and Integrators

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CARRIER GAS: • The choice of carrier gas determines the

efficacy of chromatographic separation. Most widely used are hydrogen, helium, nitrogen and argon.

• The gas must be inert, less expensive, suitable for detector, high purity, should not cause the risk of fire.

• As carrier gas is compressible, gases are stored under high pressure in cylinder and used when required.

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Flow Regulators: As carrier gas are stored under high pressure,

flow regulators are used to deliver the gas with uniform pressure or flow rate. Flow meter are used to measure the flow rate of carrier gas

Eg.RotameterSoap bubble meter

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Sample Introduction • The sample is usually introduced in form of

solution (~0.5 ml) into the injector• Injector has dual role, provides an inlet for the

sample and vaporizes the sample and mixes it with mobile phase

• Modes of injection vary according to their types• Direct Vaporisation injector• Cold On-Column Injector• Split/Splitless Injector

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Direct vaporization injection

In direct flash vaporization injection, a liquid sample is injected via a syringe into a heated injection port. The sample is rapidly vaporized in the injection port, then transferred to the column

Cold on-column injection• The sample is injected directly on column and

vaporisation occurs after the injection• Needle penetrates the column or precolumn kept at

4oC before raising it to normal operating temperature• Useful for thermolabile components

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• It is designed to maintain the constant flow of carrier gas and to control the amount of sample enters into the column

• If the split vent is closed, via a computer-controlled split valve, then all of the sample introduced into the injector vaporizes and goes on (into) the column(splitless mode). If the split vent is open then most of the vaporized sample is thrown away to waste via the split vent and only a small portion of the sample is introduced to the column.(split mode)

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GC – Split/Splitless Injections

GC – Split/Splitless Injections

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Temperature Control Device

Repeaters: Are used in GC to concert the sample into vapour form and mix with mobile phase

Thermostatically controlled oven: Since partition coefficient as well as solubility of a solute depends upon temperature, temp. maintaining in a column is highly essential for efficient separation

• Isothermal programming• Linear programming

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COLUMNS Column is one of the important part of GC

which decides the separation efficiency. Columns are made up of glass and stainless steel.

Classification of columns: Depending on its use:• Analytical column• Preparative column Depending on its nature:

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Column – Types Packed column: Are available in packed manner

commercially and hence are called as Packed column. Different columns ranging from non polar to polar are available.

Open tubular/ Capillary Column: They are made up of long capillary tubing of 30-90 meters in length and have diameter of 0.025 to 0.075 cm. These are made up of stainless steel and are in the form of a coil. The inner wall of the capillary is coated with stationary phase liquid in the form of a thin film.

More sample can not be loaded18

Support Coated Open Tubular Column: these columns are made by depositing a micron size layer of support material on the inner wall of the capillary column and then coated with a thin film of liquid phase.

These columns also have low resistance to flow of carrier gas but offers the advantage of more sample load or capacity.

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GC – DetectorsDetectors are most important part of GC instrumentProperties of a good detector:• Applicable to wide range of sample• High sensitivity to even small concentration• Rapidity of response• Linearity• Non destructive to sample• Simple and easy to maintainTYPES OF DETECTOR• Thermal conductivity detector• Flame ionisation detector• Electron Capture Detector 20

Flame Ionization Detector (FID)

The carrier gas used with this type of detector can be hydrogen.

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Thermal Conductivity Detector (TCD)The principle is based upon the thermal conductivity difference between carrier gas and sample components. TCD has two platinum wire of uniform dimension which form a Wheatstone bridge

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Electron Capture Detector (ECD)The ECD has two electrodes. One of the electrode is treated with a radioactive isotope which emits e- as it decays(anode)

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Recorders: are used to record the responses obtained from detector after amplfication. They record the baseline and all the peaks obtained with respect to time.

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Derivatisation of sample: It is a technique of treatment of the sample to

improve the process of separation by column or detection by detector.

Precolumn derivatisation: this is done to improve some properties of the sample for separation by column. By this technique, the components are converted to more volatile and thermostable derivative. Moreover improved separation and less tailing will be seen after such treatment. eg. Silylation, acylation and alkylation.

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• Postcolumn derivatisation: It is done to improve the response shown by detector. The components may not be detected by detector unless derivatisation is done. The component may be converted in such a way that their ionisation or affinity toward electron is increased.

eg. Tagging with chlorine can improve response on an ECD detector.

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Application Of GC:• Qualitative analysis• Checking the purity of a compound• Presence of impurity• Quantitative analysis• Isolation of drug or metabolites in urine• Speed – very fast, minutes• Sensitivity – high sensitivity, (ppm range)• Simplicity – simple to operate and understand• Resolution – high resolution of closely related

compounds

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GC-MS

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GC-MS • GC-MS is an advance analytical

instrumental technique that combines the physical separation capability of GC with the mass analysing capability of MS.

• Gas chromatography coupled with mass spectrometry is a versatile tool to separate, quantify and identify unknown substances.

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Gas Chromatography-Mass Spectrometry (GC-MS)

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COUPLING OF GC TO MS

• output of the GC must be reduced to vacuum of 10-5 to 10-6 atm The interface b/w GC-MS play an important role in the overall efficiency of instrument• Both system are heated at 200-300⁰C, both deal with compound in vapor state.• Only one problem is that the atmospheric pressure

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Types of Interfaces: there are four types of interfaces available. These are-

• JET SEPREATOR• PERMSELECTIVE MEMBERANE• MOLECULAR EFFUSION• DIRECT INTRODUCTION

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JET SEPARATOR: In these separators, the GC flow is introduced into an evacuated chamber through a restricted capillary. Light particle dispersed away

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Permselective membrane interface: it is made of a silicone-rubber membrane that transmits organic non-polar molecules

and acts as a barrier for (non-organic) carrier gases.

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The molecular effusion /Watson-Biemann interface: is based on the molecular filtering of the gas effluent by means of a porous glass fritted tube.

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DIRECT INTRODUCTION: In this method capillary column is directly inserted into MS ionisation chamber

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Mass Spectrometry

Sample Introduction → Ionization →

Mass Analysis → Ion Detection/Data Analysis

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Ionization• Electron Impact (EI)• Chemical Ionization (CI)• Field Desorption (FD)• Fast Atom Bombardment (FAB)• Laser Desorption (LD)• Electrospray Ionization (ESI)• Matrix Assisted Laser Desorption (MALDI)

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Interpretation of Result: Through GC a CHROMATOGRAM is obtained Through MS a SPECTRUM is obtained GC-MS gives a 3D graph which has both chromatogram

and spectrum to each separated components in the chromatogram.

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Chromatogram generated by GC While the instrument run, the

computer generated a graph from signal called chromatogram

X-axis show the RT Y-axis show the intensity of the

signal

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Mass Spectrum

• The computer record a graph for each scan called spectrum

• The mass spectrum is essentially a fingerprint for the molecule and can be used to identify the compound.

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Application of GC-MS

Environmental monitoring and clean upCriminal ForensicLaw enforcementanti doping test Food, beverages, and perfume analysismedicines

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Conclusion:• Gas chromatography coupled with mass

spectrometry is a versatile tool to separate, quantify and identify unknown substances.

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References: Braun.R, Introduction To Instrumental Analysis,

Second Edition, PharmaMed Press, Hyderabad, Page no. 251-270.

Chatwal.G.R , Anand .S.K, Instrumental method Of Chemical Analysis, Himalaya Publishing House, Fifth Edition-2012, New Delhi, Page no. 420-449.

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