GAS CHROMATOGRAPHY-MASS SPECTROSCOPY [GC-MS]

THIS PRESENTATION GIVES A DETAIL ACCOUNT ON THE GC-MS WITH ITS INTRODUCTION, BASIC PRINCIPLE OF BOTH COMBINED AND INDIVIDUALLY WITH ITS INSTRUMENTATION, APPLICATION AND EXAMPLES, MAKES EASY TO COLLECT ALL THE DATA AT A PLACE ACCORDING TO THE M.PHARM SYLLABUS S PER PCI

1. GAS CHROMATOGRAPHY – MASS SPECTROSCOPY (GC-MS) (Advanced Spectral Analysis) 1

2. CONTENTS : Introduction - Hyphenated Techniques Introduction – Gas Chromatography and Mass Spectroscopy Principle - Gas Chromatography-Mass Spectroscopy (GC-MS) Interfaces of GC-MS Ionization Techniques Mass Analyzers Data Handling Applications References 2

3. HYPHENATED TECHNIQUE : It is define as the combination or h yphenation between Spectroscopic and separation (chromatographic) technique is known as Hyphenated Technique. Spectroscopic + Chromatographic Hyphenation Hyphenated Technique Hyphenated techniques combines chromatographic and spectral method to exploit the advantages of both. Chromatography produces pure or nearly pure fractions of chemical components in a mixture. Spectroscopy produces selective information for identification using standards or library spectra. 3

4. GAS CHROMATOGRAPHY : Gas Chromatography – “It is a process of separating components from the given crude drug by using a gaseous mobile phase.” It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid . INSTRUMENTATION PARTS OF GC : Carrier Gas Sample Injection System Separation Column Detectors Amplification Recorder 4

5. MASS SPECTROSCOPY : The mass spectroscopy is an technique in which, the compound under investigation is bombarded with beam of electrons which produce an ionic molecule or ionic fragments of the original species. The mass spectrometer is an instrument in which the substance in gaseous or vapour state is bombarded with a beam of electrons, to form a positively charged ions (cations) which are further sorted according to their mass to charge ratio to record their masses and their abundances. INSTRUMENTAION PARTS OF MS : Sample Handling System Ionization Chamber Ion Separator or Mass Analyzer Ion Collector, Detector and Read Out System Vacuum System 5

6. GAS CHROMATOGRAPHY – MASS SPECTROSCOPY (GC-MS) : GC-MS, which is a hyphenated technique developed from coupling of GC and MS, was th e first of its kind to become useful for research and development process. Mass spectra obtained by this hyphenated technique offer more structural information based on the interpretation of fragmentations. The fragment ions with different relative abundances can be compared with library spectra. Compounds that are adequately volatile, small, and stable in high temperature in GC conditions can easily be analyzed by GC-MS. In GC-MS, a sample is injected into the injection port of GC device, vaporized, separated in the GC column, analyzed by MS detector, and recorded. The time elapsed between injection and elusion is called “retention time” ( tR ) . 6

7. NEED OF GC-MS : It is important type of technique is used for the separation of organic and in organic compounds and it is having ability for separation high molecular weight hydrocarbons. It is important type of technique is used for separation and identification of volatile compounds. It is important for determination of fragmentation pattern of compounds. It is also important for determination of protein, peptides, amino acid, nucleic acid, as well as naturally or biological compounds. It is one of the powerful technique is used for qualitative and quantitative analysis . ADVANTAGES OF GC-MS : It is important for identification of compound . It can p rovides sensitive response to most analytes . It is important to provide information of particular or specific class of compound. It can provide information of structure or different structure of compound . It is having high resolution and separation capacity . It is time saving technique, having a high resolution capacity. It is important determination of molecular weight as well as fragmentation pattern of compound. Good Accuracy and Precision. It is simple, rapid, reproducible technique . 7

8. Gas c h r om a t og r ap h y Mass spectrometry GC-MS Separates mixture of components into individual Identifies molecules based on their mass A chemical analysis technique combining two instruments to provide for powerful separation & identification . WORKING OF GAS CHROMATOGRAPHY - MASS SPECTROSCOPY [ GC-MS] : 8

9. COUPLING OF GC TO MS: 9 GC Atmospheric density heated (200-300 ∘ C) Interfaces MS High vacuum (10 -6 torr) heated The interface b/w the GC & MS is an important role to play in the overall efficienc y of the instrument. Both system are heated (200 -300 ∘C) both deal with compounds in the vapor state. Only one problem is that the atmospheric pressure output of the GC must be reduced to vacuum of 10 -5 – 10 -6 torr for the MS inlet .

10. INTERFACES OF GC-MS : The interfaces between the GC and MS has an important role play in overall efficiency of the instrument. It must be capable of providing an inert pathway from the column to the ion source without loss of chromatographic resolution, while at the same time removing of carrier gas and reducing the pressure from about one atmosphere at the column outlet to 10 -5 – 10 -6 mmHg in the ion source. It is hardly surprising that the separator has been a major cause of technical problems encountered in GC/MS. 10

11. WITSON-BIEMANN EFFUSION SEPARATOR : The Witson-Biemann Effusion Separator consists of a sintered glass tube, the surrounded of which are evacuated. The carrier gas, usually helium, passes preferentially through the sintered glass and the effluent is concentrated by a factor of up to 100. Two stage separator may enrich the effluent by a factor of 400 and be capable of dealing with glass flow rates in order of 20-60 ml/min. 11

12. RYHAGE JET SEPARATOR : The Ryhage Jet Separator is based upon the differing rates of diffusion of different gases in an expanding supersonic jet stream. The heavier (sample) compound concentrates in the center of the gas jet. The gases pass at high speed through an orifice aligned with a second orifice, and then on to the ion source, while the carrier gas is pumped away. Usually a Ryhage separator is two stage, although all glass single stage units are used, particularly in combination with quadrupole mass spectrum. 12

13. LLWELLYN-LITTLEJOHN SEPARATOR : in the Llwellyn -Littlejohn separator separation of organic molecules from carrier gas molecules is achieved by means of the selective permeability of an elastomer membrane. Permeability is a function the solubility of the gas molecules in the membrane and their ability to diffuse through it. Gases such as hydrogen, helium argon and nitrogen having a very low solubility and high diffusion rate, pass through the membrane much more slowly than organic vapors where the revers is true. The vapor is therefore concentrated and the remaining carrier gas expelled into the atmosphere. The concentrated is further enriched by passes through a second semi-permeable membrane. Enrichment of the organic phase by a factor of greater than 10 -5 has been achieved. 13

14. IONIZATION TECHNIQUES : 14

15. ELECTRON IMPACT : In the Electron Impact (EI) process, electrons are emitted from a heated filament ( usually made of tungsten or rhenium) and are accelerated across the source by using an appropriate potential (5-100V) to achieve the required electron energy (sufficient to ionize the molecule ). 15

16. The most common form of ionization is EI. Electrons are produced by tungsten filament. These electrons accelerated towards the ion source chamber. The electrons require an energy equal to the voltage b/w the filament & ion source chamber. A proportion of electron beam will strike the electron trap producing trap current. A permanent magnet is positioned across the ion chamber to produce a magnetic flux in parallel to the electron beam. A (+) ve ion repelled voltage & (-) ve ion excitation voltage works to gather to produce an electric field in the source chamber. Such that ions leaves through ion exit slit. The ions are directed through the various focusing & centering lenses are focused on to the source exit slit . 16

17. CHEMICAL IONIZATION : Chemical ionization involves the ionization of a reagent gas, such as methane at relatively high pressure (~1 mbar) in a simple electron impact source. Once produced, the reagent gas ions collide with the analyte molecules producing ions through gas phase reaction processes such as proton transfer. In CI a reagent gas methane or ammonia or isobutene are introduced into the mass spectrometer . The reagent gas will interact with the electron to produce radical electrons . 17

18. NEGATIVE CHEMICAL IONIZATION : In NICI a reagent gas is used & the electrons collide with it so that their energies are reduced to 10Ev. Molecules with a high affinity for electrons are able to capture these low energy thermal electrons . This is known as NICI but it does not involved in the formation of a chemical adduct . 18

19. MASS ANALYZERS : They deflects ions down a curved tubes in a magnetic fields based on their kinetic energy determined by the mass, charge and velocity. The magnetic field is scanned to measure different ions. 19

20. QUADRUPOL : A Quadrupole mass filter consists of four parallel metal rods with different charges Two opposite rods have an applied + potential and the other two rods have a - potential The applied voltages affect the trajectory of ions traveling down the flight path For given D C and A C voltages , only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path. 20

21. ION TRAP : It uses an electric field for the separation of the ion by mass to charge ratios. The electric field in the cavity due to the electrodes causes the ions of certain m/z values to orbit in the space . The ion trap mass analyzer operates by similar principles where it consists of circular ring electrode Plus two end caps that form a chamber. Here AC or DC power along RF potential is applied between the cups and the ring electrode. T here the ions en t e r ing into the cha m b e r are tra p p ed by electromagnetic fields and they o s c i llates in concentric trajectories. This process is called resonant ejection . 21

22. TIME OF FLIGHT (TOF) : TOF mass analyzer is based on simple idea that the velocities of two ions are created by uniform electromagnetic force applied to all the ions at same time, causing them to accelerate down a flight tube. Lighter ions travels faster and strike the detector first so that the m/z ratio of ions is detected . TOF Analyzers separate ions by time without the use of an electric or magnetic field. In a crude sense, TOF is similar to chromatography, except there is no stationary/mobile phase, instead the separation is based on the kinetic energy and velocity of the ions . 22

23. FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR) : Uses a magnetic field in order to trap ions into an or bit inside of it. In this analyzer there is no separation that occurs rather all the ions of a particular range are trapped inside, and an applied external electric field helps to generate a signal . 23

24. DATA HANDLING : All the mass spectrometers now employ computer control of same functions and also use a computerized display and output. The amount of data generated even by a fairly modest mass spectrometer is very large indeed, a single run may store data for upto 100 fragments from each type of molecule and if, GCMS analyses is being performed, a complete mass spectrum is generated and stored every sec for upto 90 min 24

25. APPLICATION : Analysis of Natural Products and Traditional Herbal Medicine Identification of Metabolite Bio analysis / Bioequivalence Studies ADME (Absorption, Distribution, Metabolism, and Excretion) Screening Dissolution Testing Method Development / Validation Forced Degradation Studies Impurity Profiling Manufacturing / QA / QC Analysis of amino acid Determination of pesticides Pharmacokinetic studies Biotechnology Clinical research Biochemical analysis 25

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