GC –MS &LC-MS : GC –MS &LC-MS Vidhyakalaivani.M M.Phil Biotechnology 12MZBE06 Chromatography: Chromatography Chromatography is the science which is studies the seperation of molecules based on differences in their structure and/or composition. Chromatography was first developed and defined by the Russion Botonist Mikhail Tswett in 1903 . He produced a colourful seperation of plant pigments using a column of calcium carbonate(chalk). Mass spectrometry : Mass spectrometry Charged molecules or molecular fragments are generated in a high vacuum or immediately prior to a sample entering a high vacuum using a variety of methods for ion production. magnetic fields to enable the determination of their molecular weight (m/z).The history of mass spectroscopy (MS) started in 1912 when Thomson obtd . Why we combine these two? : Why we combine these two? Combining the two processes reduces the possibility of error, as it is extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore, when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically lends to increased certainty that the analyte of interest is in the sample. History of Gas Chromatography and Mass Spectroscopy : History of Gas Chromatography and Mass Spectroscopy Gas Chromatography was first described in 1952 by James and martin with the separation of a mixture of small carboxylic acids . The power of GC was substantially enlarged by the introduction of open capillary columns in 1958 by Golay . The introduction of the fused-silica capillary column in 1976 by Dandeneau and Zerner can be considered as a breakthrough in the development of GC . The use of a mass spectrometer as the detector in gas chromatography was developed during the 1950s by Roland Gohlke and Fred McLafferty . PowerPoint Presentation: GC-MS : Gas chromatography & mass spectrometry LC-MS :Liquid chromatography & mass spectrometry What is gas chromatography?: What is gas chromatography? It is an instrumental method for the seperation and identification of chemical compounds. Gas chromatography is a chromatographic technique that can be used to separate volatile organic compounds. The organic compounds are seperates due to differences in their partitioning behaviour between the mobile phase and the stationary phase in the column. PowerPoint Presentation: Principle of GC-MS The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas (usually helium). The sample flows through the column and the compounds comprising the mixture of interest are separated by interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase ). The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions. Components: Components The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer . Carrier Gas, N 2 or He, 1-2 mL/min Injector Oven Column Detector Carrier Gas : Carrier Gas Inert Helium (hydrogen/ nitrogen). Choice dictated by detector, cost, availability Pressure regulated for constant inlet pressure( below 0.3MPa). Flow controlled for constant flow rate ~20ml/min for packed column ~1ml/min for open capillary column Chromatographic grade gases (high purity) PowerPoint Presentation: Samples State Organic compounds must be in solution for injection into the gas chromatograph. The solvent must be volatile and organic. Amount Depending on the ionization method, analytical sensitivities of 1 to 100 pg per component are routine. Preparation Sample preparation can range from simply dissolving some of the sample in a suitable solvent . Purge and Trap (Aqueous and Soils / Volatiles Preparation): Purge and Trap (Aqueous and Soils / Volatiles Preparation) Courtesy of Environmental Conservation Laboratories, Inc. Sonication (Soils, Solids / Semivolatiles): Sonication (Soils, Solids / Semivolatiles) Courtesy of Environmental Conservation Laboratories, Inc. PowerPoint Presentation: Solid Phase Extraction (Aqueous / Semivolatiles) Courtesy of Stanford Laboratory Cartridge Sample PowerPoint Presentation: GC – typical set up Typical GC setup – courtesy of Waters, Inc . PowerPoint Presentation: Column Types Capillary Columns Packed Columns PowerPoint Presentation: Capillary columns (open tubular) Made of the highest purity fused silica obtained with an external polyimide coating Length-10 to 100 m -depends on application. For fast analysis shorter column are applied e.g.-for heat sensitive and for high boiling compound. Large columns are required for high resolution seperation . PowerPoint Presentation: Contd.. A more polar stationary phase is applied for the analysis of more polar compounds. So least polar column- CP- Sil 5 and CP- Sil 8 applied. For high resolution more polar stationary phase have to be applied. In GC-MS low bleed columns are applied. PowerPoint Presentation: Capillary Columns PowerPoint Presentation: Packed Columns These columns, less commonly used today, have diameter of 1.6 to 9.5mm and a length of between 1–3m. Manufactured from steel or glass, the internal wall of the tube is treated to avoid catalytic effects with the sample. They can withstand a carrier gas flow rate within the range 10–40 mL /min. They contain an inert and stable porous support on which the stationary phase can be impregnated or bounded (between 3 and 20 per cent). PowerPoint Presentation: Packed Columns PowerPoint Presentation: Jet Separator Two capillary tubes aligned with a small space between them. (1 mm) A vacuum is created between the two tubes using a rotary pump. The GC effluent enters the vacuum region, those molecules which continue in the same direction enter the second capillary tube and continue to the ion source. PowerPoint Presentation: Jet Separator The carrier gas molecules are more easily diverted from the linear path by collisions. The analyte molecules are much larger and carry more momentum. The surface of the separator must be inactive and a reasonably even temperature. PowerPoint Presentation: packed columns capillary columns GC-MS Instrument: GC-MS Instrument The insides of the GC-MS, with the column of the gas chromatograph in the oven on the right. Instrumentation: Instrumentation The gas chromatograph utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties. The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules take different amounts of time (called the retention time) to come out of (elute from) the gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio . Types of Ionisation: Types of Ionisation Electron impact ionisation Chemical Ionisation Electron Ionisation: Electron Ionisation The physics behind mass spectrometry is that a charged particle passing through a magnetic field is deflected along a circular path on a radius that is proportional to the mass to charge ratio, m/e. Chemical ionisation: Chemical ionisation Used to confirm molecular weight. Known as a “soft” ionisation technique. Differs from EI in that molecules are ionised by interaction or collision with ions of a reagent gas rather that with electrons. Common reagent gases used are Methane , Isobutane and Ammonia. Reagent gas is pumped directly into ionisation chamber and electrons from Filament ionise the reagent gas. Types of Mass Spectrometer Detectors: Types of Mass Spectrometer Detectors The most common type of mass spectrometer (MS) associated with a gas chromatograph (GC) is the quadrupole mass spectrometer, sometimes referred to by the Hewlett-Packard (now Agilent ) trade name " Mass Selective Detector" (MSD). Another relatively common detector is the ion trap mass spectrometer. Other detectors may be encountered such as time of flight (TOF), tandem quadrupoles (MS-MS). Triple Quadrupole mass spectrometer: Triple Quadrupole mass spectrometer Time of Flight -TOF: Time of Flight -TOF Analysis: Analysis A mass spectrometer is typically utilized in one of two ways: Full Scan or Selective Ion Monitoring (SIM). The typical GC/MS instrument is capable of performing both functions either individually or concomitantly, depending on the setup the particular instrument. GC/MS Data Processing Important to Review Peak Integration: GC/MS Data Processing Important to Review Peak Integration Chromatogram (maximum information content) Final Report GC/MS Applications: Applications Environmental Monitoring and Cleanup GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The cost of GC-MS equipment has decreased significantly, and the reliability has increased at the same time, which has contributed to its increased adoption in environmental studies. There are some compounds for which GC-MS is not sufficiently sensitive, including certain pesticides and herbicides, but for most organic analysis of environmental samples, including many major classes of pesticides, it is very sensitive and effective. Criminal Forensics : Criminal Forensics GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. The analysis of fire debris using GC-MS is well established, and there is even an established American Society for Testing Materials (ASTM) standard for fire debris analysis. GCMS/MS is especially useful here as samples often contain very complex matrices and results, used in court, need to be highly accurate. Contd..: Contd.. Law Enforcement GC-MS is increasingly used for detection of illegal narcotics, and may eventually supplant drug-sniffing dogs. It is also commonly used in forensic toxicology to find drugs and/or poisons in biological specimens of suspects, victims, or the deceased. Food, Beverage and Perfume Analysis Foods and beverages contain numerous aromatic compounds , some naturally present in the raw materials and some forming during processing. GC-MS is extensively used for the analysis of these compounds which include esters , fatty acids , alcohols , aldehydes , terpenes etc. It is also used to detect and measure contaminants from spoilage or adulteration which may be harmful and which is often controlled by governmental agencies, for example pesticides . Contd..: Contd.. Astrochemistry Several GC-MS have left earth. Two were brought to Mars by the Viking program . Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-MS. The Huygens probe of the Cassini-Huygens mission landed one GC-MS on Saturn's largest moon, Titan .The material in the comet 67P/ Churyumov-Gerasimenko will be analysed by the Rosetta mission with a chiral GC-MS in 2014. What is LC-MS?: What is LC-MS? It is the combination of liquid chromatography and the mass spectrometry. In LC-MS we are removing the detector from the column of LC and fitting the column to interface of MS. In the most of the cases the interface used in LC-MS are ionization source. PowerPoint Presentation: MOBILE PHASE COLUMN ION SOURCE MASS ANALYSER ION CLLECTION SYSTEM VACCUM SYSTEM DATA HANDLING SYSTEM EFFUENT Sequence of efforts taken place during the development of modern liquid chromatography: Sequence of efforts taken place during the development of modern liquid chromatography To reduce separation time to achieve fast analysis . ( From hours to minutes) HOW? The solutes must move faster through stationary phase MEANS By increasing the mobility of the liquid mobile phase. PowerPoint Presentation: The MIGRATION VELOCITY of the liquid mobile phase should be HIGH. We can achieve faster migration velocities of liquid mobile phase by- 1.Applying vacuum at the other end of the chromatographic column 2.Applying high pressure on the liquid mobile phase. Application of higher pressure on liquid mobile phase seems to be an easier and more practical way for achieving fast analysis. Sample preparation: Sample preparation Sample preparation generally consists of concentrating the analyte and removing compounds that can cause background ion or suppress ionization. Example of sample preparation include:- (1) on –column concentration to increase analyte concentration. (2) desalting to reduce the sodium and potassium adduct formation that commonly occurs in electro spray. (3) filtration to separate a low molecular-weight drug from proteins in plasma, milk, or tissue. PowerPoint Presentation: COlUMN Column type:- Specialized mode:- The use of di-functional or tri-functional silanes to create bonded groups with two or three attachement points leading to phases with higher stability in low or higher pH and lower bleed for LCMS Most widely used columns for LCMS are:- (1) fast LC column. the use of short column. (15-50mm) (2) Micro LC column. the use of large column. ( 20-150mm) CHROMATOGRAPHIC COLUMN: CHROMATOGRAPHIC COLUMN Glass is normally the choice for chromatographic column material due to its chemical inertness towards stationary phase, mobile phase and solutes being separated. We could not increase the pressure of a glass column? Hence glass will not be able to withstand high pressures of liquid mobile phase because glass has relatively small mechanical strength. PowerPoint Presentation: Which IDEAL MATERIAL should be selected in place of glass? STAINLESS STEEL due to its chemical inertness & mechanical strength. PowerPoint Presentation: 48 Previous condition :- Liquid mobile phase moving across the stationary phase in a glass column under atmospheric pressure. Present condition :- Liquid mobile phase with high migration velocity moving across the stationary phase in a stainless steel column under high pressure.. Will the column separation efficiency of the latter be good? NO With the increase in the liquid mobile phase velocity under high pressures, solute molecules will not get enough time to interact with the stationary phase and separation efficiency will be reduced and we will not be able to achieve good separation of solutes. : With the increase in the liquid mobile phase velocity under high pressures, solute molecules will not get enough time to interact with the stationary phase and separation efficiency will be reduced and we will not be able to achieve good separation of solutes. 49 How can we achieve good column separation efficiency?: How can we achieve good column separation efficiency? 50 By reducing the Particle size of the stationary phase support. 2. By reducing the width of chromatographic column. We use Narrow bore (3mm, id) SS columns filled with small particles of stationary phase support (5µ) will give us high separation efficiency with low analytical time when liquid mobile phase moves through the column under high pressures of the order of 500 –5000 psi. : We use Narrow bore (3mm, id) SS columns filled with small particles of stationary phase support (5µ) will give us high separation efficiency with low analytical time when liquid mobile phase moves through the column under high pressures of the order of 500 –5000 psi. 51 Can the physically coated stationary phases used in GC be used in narrow bore SS columns through which liquid mobile phase moves under high pressures? : Can the physically coated stationary phases used in GC be used in narrow bore SS columns through which liquid mobile phase moves under high pressures? 52 Obviously… NO Because under high pressures of liquid mobile phase, physically coated liquid stationary phases will be removed physically or by dissolution in liquid mobile phase. We need physically&chemically stable stationary phase. : Because under high pressures of liquid mobile phase, physically coated liquid stationary phases will be removed physically or by dissolution in liquid mobile phase. We need physically&chemically stable stationary phase. 53 Chemically treated inert (not strictly) particles have free silinol group at their surface.: Chemically treated inert (not strictly) particles have free silinol group at their surface. 54 Silica particle CCCCCC Si - OH Si - OH Si - OH Free silinol groups Early attempts were then made to chemically bonded long chain aliphatic alcohols with the free silinol groups present on the surface of silica particles. : Early attempts were then made to chemically bonded long chain aliphatic alcohols with the free silinol groups present on the surface of silica particles. 55 Si - OH + HO - R - H 2 O Si - O - R However these chemically bonded phases with Si - O - C bonds are stable only in acidic media. : However these chemically bonded phases with Si - O - C bonds are stable only in acidic media. 56 We cannot restrict our separation work always in acidic media: We cannot restrict our separation work always in acidic media 57 In basic media Si– O- C bond undergoes hydrolysis. Si - O - R basic medium Si - O H + R - OH We therefore would like to use chemical bonding with ‘ Si ’ of free silinol groups which will be hydrolytically stable over a wide acidic to basic pH range. : We therefore would like to use chemical bonding with ‘ Si ’ of free silinol groups which will be hydrolytically stable over a wide acidic to basic pH range. 58 Attempts were then made to carry out chemical reactions with substituted silane ( Si H4) having suitable long chain of hydrocarbons attached to it. : Attempts were then made to carry out chemical reactions with substituted silane ( Si H 4 ) having suitable long chain of hydrocarbons attached to it. 59 PowerPoint Presentation: 60 Si - OH 1 2 18 + Cl – Si – C – C C - H CH 3 CH 3 H H H H H H Substituted siliane 1 2 18 – Si – C – C C - H CH 3 CH 3 H H H H H H Si - O -H Cl This chemically bonded phase ( Si-O-Si bond ) is therefore mechanically strong and hydrolytically stable over a wide pH range (2-9). : This chemically bonded phase ( Si-O-Si bond ) is therefore mechanically strong and hydrolytically stable over a wide pH range (2-9). 61 PowerPoint Presentation: 62 When the liquid mobile phase is moving under high pressures through chromatographic columns, introduction of a sample on to the column will require some specially designed injection system. Introduction of sample PowerPoint Presentation: 63 The most popular and commonly used injector system, is the syringe –loop injector type . It is a fixed volume universal injector which allows introduction of micro litres of samples on to the chromatographic column. PowerPoint Presentation: 64 Thus so far with we have achieved the following attributes with LC Fast Analysis Small Sample Size Versatility Efficiency Non-destructive Interface: Interface It is difficult to interface a liquid chromatography to a mass-spectrometer cause of the necessity to remove the solvent. The commnly used interface are:- (1) Electrospray ionization (ESI) (2) Thermospray ionization (TSI) (3) Atmospheric pressure chemical ionization (APCI) (4) Atmospheric pressure photoionization(APPI) (5) Partical beam ionization. Electron spray ionization: Electron spray ionization Particle beam ionization : Particle beam ionization NOW WHAT IS LEFT?: NOW WHAT IS LEFT? PowerPoint Presentation: 69 DETECTORS: As soon as the solutes are eluted they should be detected and quantitated with sensitive and specific or universal detectors. PowerPoint Presentation: 70 With specific and universal detectors we have achieved the following attributes: High sensitivity High reliability and accuracy PowerPoint Presentation: 71 Thus in place of glass column liquid chromatography now we have 1 4 5 2 3 1- Pumping system 2- Universal injector 3- Column 4- Detector 5- Recorder Mass Analyser: Mass Analyser 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. Types of mass analyzer:- (1) Quadrapole mass filter. (2) time of flight (3) Ion trap (4) Fourier transform ion cyclotron resonance (FT-ICR or FT-MS) Ion trap: Ion trap PowerPoint Presentation: 74 1.Pumping system capable of giving pulseless flow of liquid mobile phase under high pressures of the order of 500 to 5000psi. * This makes fast analysis . PowerPoint Presentation: 75 2.Universal injector which allows to introduce samples in microlitres . * This reduces sample size PowerPoint Presentation: 76 3.Narrow bore SS columns with suitable chemically bonded phases with small particle size (3-5µ), desired polarity and pore size and hydrolytically stable over a wide acidic to basic pH range. * This gives high separation efficiency , high versatility in separating solutes with diverse nature. PowerPoint Presentation: 77 4.Sensitive and Specific or Universal detector * This gives high sensitivity , reliability and accuracy of detection and quantitation of solutes. PowerPoint Presentation: 78 5.Computing system allows to record and compute chromatographic results. PowerPoint Presentation: 79 All the above factors add “High Performance” to normal Liquid Chromatography. It is therefore known as “ H igh P erformance L iquid C hromatography” abbreviated as HPLC. Appplication of LC-MS: Appplication of LC-MS Molecular weight determination Determining the molecular weight of green fluorescent proteins Structural determination e.g. structural determination of ginsenoside . Pharmaceutical application e.g. identification of bile acids metabolites. Biochemical application e.g. rapid protein identification using capillary lc / ms / ms. PowerPoint Presentation: Food application e.g. identification of aflatoxin in food determination of vitamin D 3 in poultry feed supplement using MS 3 Environmental application e.g. detection of phenyl urea herbicides, detection of low level of carbaryl in food. PowerPoint Presentation: GC Computer MS GC-MS PowerPoint Presentation: LC Computer MS LC-MS GC-MS LC-MS: GC-MS LC-MS Volatile or stable compounds can be used as samples. All gases are determined. Mobile phase used is gas. inner wall of the column is lined with a thin layer of support material such as diatomaceous earth. Non-volatile compounds can be used as samples. Can be used for all salts & macromolecules. Mobile phase used is liquid. a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane References: References Principles and Intrumentation of gas chromatography-mass spectroscopy by W. M. A. Niessen , hyphen MassSpec Counsultancy,Leiden,The Netherlands. gas chromatography-mass spectroscopy from Wikipedia ,The free encyclopedia. Gas chromatography by U. A. Devkate Sir. Encyclo pedia of Chromatography – Jack Cazes Gas chromatography by Ian A. Fowlis 2 nd Edition hollas_J.M ._ modern _Spectroscopy Handbook of Instrumental Technique for Analytical Chemistry by Frank Settle. Moderm Instrumentation Methods and Techniques by Francis Rouessac and Annick Rouessac . Instrumental methods of Analysis by Willard,Merritt,Dean,Settle,7 th Edition. PowerPoint Presentation: 10. W. Paul & H. Steinwedel ; Zeitschrift für Naturforschung , 8A; 1953 , p448. 11. W. Paul; Agewandte Chemie - International Edition , 29; 1990 , p739. 12. G. C. Stafford et al.; International Journal of Mass Spectrometry and Ion Processes , 60; 1984 , p85 and Analytical Chemistry , 59; 1987 , p1677. PowerPoint Presentation: Thank U!