gcms

Gas Chromatography-Mass Spectroscopy (GC/MS) :

Gas Chromatography-Mass Spectroscopy (GC/MS) Presented by : Ms. Sarita M. Nehatkar M.Pharm (QA) Govt . college of Pharmacy, Amravati . .

Covered points::

Covered points: Chromatography Gas chromatography Introduction Advantages Technology development status Interfacing GC with spectroscopic method Instrumentation Purge and trap GCMS Types of ionisation Detectors Analysis GC tendom MS Applications references

What is Chromatography?:

What is Chromatography? Chromatography is a method used by scientists for seperating organic and inorganic compounds so that they can be analysed and studied. 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).

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 history of mass spectroscopy (MS) started in 1912 when Thomson obtd . The mass spectra of compounds such as O2 ,N2 ,CO,CO2 ,and COCl2.These findings were based on the earlier discovery of positive ions by Goldstein 1886 and the deflection of ions in a magnetic field by Wien 1898 . . 2

History of GC - MS:

History of GC - MS The use of a mass spectrometer as the detector in gas chromatography was developed during the 1950s by Roland Gohlke and Fred McLafferty . These sensitive devices were bulky, fragile, and originally limited to laboratory settings. The development of affordable and miniaturized computers has helped in the simplification of the use of this instrument, as well as allowed great improvements in the amount of time it takes to analyze a sample. In 1996 the top-of-the-line high-speed GC-MS units completed analysis of fire accelerants in less than 90 seconds, whereas first-generation GC/MS would have required at least 16 minutes. This has led to their widespread adoption in a number of fields.

Technology Development Status:

Technology Development Status The first general application of molecular mass spectrometry occurred in the early 1940s in the petroleum industry for quantitative analysis of hydrocarbon mixtures in catalytic crackers. Recently, manufacturers of GC/MS instruments have significantly reduced their overall size and increased durability. This allows what was once a laboratory bench-top instrument to perform field analysis.

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.

Theory of GC :

Theory of GC 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 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).

Theory of GC:

Theory of GC 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. A detector is used to monitor the outlet stream from column; thus the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.

GC Step by Step :

GC Step by Step Carrier Gas Injector Column Capillary Stationary Phase Detectors Mass Spectrometer

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)

Advantages of GC over other ..:

Advantages of GC over other .. Fast analysis with good precision and accuracy. - Typically minutes (even sec.) - Can be automated. High seperation power and complex mixture can be seperated Into its components. High sensitivity (sample used in micro gm. Qty.) Cost of equipment as compare to HPLC is less. Allows on-line coupling. Ex. To MS. Sensitive detectors (easy ppm , often ppb). Highly accurate quantification (1-5% RSD).

Introduction of GC-MS:

Introduction of GC-MS Gas chromatography-mass spectroscopy (GC-MS) is one of the so-called hyphenated analytical technique As the name implies, it is actually two techniques that are combined to form a single method of analyzing mixtures of chemicals Gas chromatography separates the components of a mixture and mass spectroscopy characterizes each of the components individually. By combining the two techniques, an analytical chemist can both qualitatively and quantitatively evaluate a solution containing a number of chemical.

Introduction of GC-MS :

Introduction of GC-MS In order for a compound to be analysed by GC/MS it must be sufficiently volatile and thermally stable . In addition, functionalised compounds may require chemical modification ( derivatization ), prior to analysis, to eliminate undesirable adsorption effects that would otherwise affect the quality of the data obtained.

Principle of GC-MS:

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 virtue of their relative 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.

General Uses:

General Uses Identification and quantitation of volatile and semivolatile organic compounds in complex mixtures. Determination of molecular weights and (sometimes) elemental compositions of unknown organic compounds in complex mixtures. Structural determination of unknown organic compounds in complex mixtures both by matching their spectra with reference spectra.

Common Applications:

Common Applications Quantitation of pollutants in drinking and wastewater using official U.S. Environmental Protection Agency (EPA) methods. Quantitation of drugs and their metabolites in blood and urine for both pharmacological and forensic applications. Identification of unknown organic compounds in hazardous waste dumps. Identification of reaction products by synthetic organic chemists. Analysis of industrial products for quality control.

Samples :

Samples State Organic compounds must be in solution for injection into the gas chromatograph. The solvent must be volatile and organic (for example, hexane or dichloromethane). 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 .

Interfacing GC with Spectroscopic Methods:

Interfacing GC with Spectroscopic Methods

Interfacing GC with Spectroscopic Methods -:

Interfacing GC with Spectroscopic Methods - Elutes from column collected as separate fractions after being detected - composition measured by Mass Spectrometry or IR. Limitation - small ( micromolar ) composition of the solute. Procedure still useful for qualitative analysis of multi-component.

Interfacing GC with Spectroscopic Methods:

Interfacing GC with Spectroscopic Methods GC equipment can be directly interfaced with rapid-scan Mass Spectrometers. The flow rate is usually small enough to feed directly into the ionization chamber of the Mass Spectrometer. Packed columns use a jet separator, which removes the carrier gas for the analyte .

Interfacing GC with Spectroscopic Methods:

Interfacing GC with Spectroscopic Methods Increase momentum of heavier analyte molecules so that 50% or more go into the skimmer. Lighter helium molecules are deflected by vacuum and pumped away. Use to identify components present in natural and biological systems. odor/flavor of foods – pollutants.

GC-MS Instrument:

GC-MS Instrument A GC installation. Schematic of a gas chromatograph. A commercial gas chromatograph with a mass spectrometry system for detection (Model GCMS 5973 manufactured by Agilent Technologies). The instrument shown here is equipped with an auto-sampler.

Precaution to be taken..:

Precaution to be taken.. Analyte must not condense in the interface Analyte may not decompose before entering the mass spectrometer ion source The gas load entering the ion source must be within pumping capacity of the mass spectrometer

Column Types:

Column Types Capillary Columns Packed Columns

Capillary columns (open tubular):

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 . ID-0.25 to 0.53mm, with stationary phase film thickness of 0.1 to 2 microm Also available column according to applied stationary phase are… WCOT,SCOT, PLOT, WSCOT .

Contd..:

Contd.. The polarity of the stationary phase liquid characterized by no. of parameters. A more polar stationary phase is applied for the analysis of more polar compounds. But more polar stationary phase generally is more prone to column bleeding, 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.

Capillary Columns:

Capillary Columns

Packed Columns:

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).

Packed Columns:

Packed Columns Although the performance of packed columns is more modest than capillary columns, they are still usually employed for many routine analyses. Easy to manufacture and with a large choice of stationary phases available, they are not however, well adapted to trace analyses. Enrichment devices are used. Jet Separators are most common.

Packed Columns:

Packed Columns

Jet Separator:

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.

Jet Separator:

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. Prone to leaks.

Jet Separator:

Jet Separator

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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 (e.g. 5% phenyl polysiloxane ). 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.

Instrumentation :

Instrumentation These two components, used together, allow a much finer degree of substance identification than either unit used separately. B’coz …. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector ) detects multiple molecules that happen to take the same amount of time to travel through the column ( i.e. have the same retention time) which results in two or more molecules to co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). 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.

Purge and Trap GC-MS:

Purge and Trap GC-MS For the analysis of volatile compounds a Purge and Trap -- to introduce samples. The target analytes are extracted and mixed with water and introduced into an airtight chamber. An inert gas such as Nitrogen (N 2 ) is bubbled through the water; this is known as purging. The volatile compounds move into the headspace above the water and are drawn along a pressure gradient (caused by the introduction of the purge gas) out of the chamber. The volatile compounds are drawn along a heated line onto a 'trap'. The trap is a column of adsorbent material at ambient temperature that holds the compounds by returning them to the liquid phase. The trap is then heated and the sample compounds are introduced to the GC-MS column via a volatiles interface, which is a split inlet system.

Definition of Terms:

Definition of Terms Molecular ion The ion obtained by the loss of an electron from the molecule Base peak The most intense peak in the MS, assigned 100% intensity M + Symbol often given to the molecular ion Radical cation + ve charged species with an odd number of electrons Fragment ions Lighter cations formed by the decomposition of the molecular ion. These often correspond to stable carbocations .

Types of Ionisation:

Types of Ionisation Electron impact ionisation Chemical Ionisation

Electron Ionisation(1) :

Electron Ionisation(1) Sample of interest vaporised into mass spec. Energy sufficient for Ionisation and Fragmentation of analyte molecules is acquired by interaction with electrons from a hot Filament. 70 eV is commonly used. Source of electrons is a thin Rhenium wire heated electrically to a temp where it emits free electrons.

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. In an electron impact mass spectrometer, a high energy beam of electrons is used to displace an electron from the organic molecule to form a radical cation known as the molecular ion . If the molecular ion is too unstable then it can fragment to give other smaller ions. The collection of ions is then focused into a beam and accelerated into the magnetic field and deflected along circular paths according to the masses of the ions. By adjusting the magnetic field, the ions can be focused on the detector and recorded.

Electron Ionisation:

Electron Ionisation

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.

Chemical Ionisation(2):

Chemical Ionisation(2) First - electron ionization of CH 4 : CH 4 + e -  CH 4 + + 2e - Fragmentation forms CH 3 + , CH 2 + , CH + Second - ion-molecule reactions create stable reagent ions: CH 4 + + CH 4  CH 3 + CH 5 + CH 3 + + CH 4  H 2 + C 2 H 5 + CH 5 + and C 2 H 5 + are the dominant methane CI reagent ions

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. Additionally one may find a magnetic sector mass spectrometer, however these particular instruments are expensive and bulky and not typically found in high-throughput service laboratories. Other detectors may be encountered such as time of flight (TOF), tandem quadrupoles (MS-MS).

Quadrupole Mass Ion Filter:

Quadrupole Mass Ion Filter

Ion Trap :

Ion Trap

Time of Flight -TOF:

Time of Flight -TOF

Triple Quadrupole mass spectrometer:

Triple Quadrupole mass spectrometer

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.

1)Full scan MS:

1)Full scan MS When collecting data in the full scan mode, a target range of mass fragments is determined and put into the instrument's method. An example of a typical broad range of mass fragments to monitor would be m/z 50 to m/z 400. The determination of what range to use is largely dictated by what one anticipates being in the sample while being cognizant of the solvent and other possible interferences. A MS should not be set to look for mass fragments too low or else one may detect air (found as m/z 28 due to nitrogen), carbon dioxide ( m/z 44) or other possible interferences. Additionally if one is to use a large scan range then sensitivity of the instrument is decreased due to performing fewer scans per second since each scan will have to detect a wide range of mass fragments. Full scan is useful in determining unknown compounds in a sample. It provides more information than SIM when it comes to confirming or resolving compounds in a sample. During instrument method development it may be common to first analyze test solutions in full scan mode to determine the retention time and the mass fragment fingerprint before moving to a SIM instrument method

2)Selected ion monitoring :

2)Selected ion monitoring In selected ion monitoring (SIM) certain ion fragments are entered into the instrument method and only those mass fragments are detected by the mass spectrometer. The advantages of SIM are that the detection limit is lower since the instrument is only looking at a small number of fragments (e.g. three fragments) during each scan. More scans can take place each second. Since only a few mass fragments of interest are being monitored, matrix interferences are typically lower. To additionally confirm the likelihood of a potentially positive result, it is relatively important to be sure that the ion ratios of the various mass fragment sare comparable to a known reference standard .

GC-tandem MS :

GC-tandem MS When a second phase of mass fragmentation is added, for example using a second quadrupole in a quadrupole instrument, it is called tandem MS (MS/MS). MS/MS can sometimes be used to quantitate low levels of target compounds in the presence of a high sample matrix background. The first quadrupole (Q1) is connected with a collision cell (q2) and another quadrupole (Q3). Both quadrupoles can be used in scanning or static mode, depending on the type of MS/MS analysis being performed. Types of analysis include product ion scan, precursor ion scan, Selected Reaction Monitoring (SRM) (sometimes referred to as Multiple Reaction Monitoring (MRM)) and Neutral Loss Scan.

GC-tandem MS :

GC-tandem 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.

Contd..:

Contd.. GC/MS is a technique that can be used to separate volatile organic compounds (VOCs) and pesticides . Portable GC units can be used to detect pollutants in the air, and they are currently used for vapor intrusion investigations. However other uses of GC or MS, combined with other separation and analytical techniques, have been developed for radionuclides , explosive compounds such as Royal Demolition Explosive (RDX) and Trinitrotoluene (TNT) , and metals.

Contd..:

Contd.. 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.. Security A post-September 11 development, explosive detection systems have become a part of all US airports . These systems run on a host of technologies, many of them based on GC-MS. There are only three manufacturers certified by the FAA to provide these systems, [ citation needed ] one of which is Thermo Detection (formerly Thermedics ), which produces the EGIS , a GC-MS-based line of explosives detectors. The other two manufacturers are Barringer Technologies, now owned by Smith's Detection Systems, and Ion Track Instruments, part of General Electric Infrastructure Security Systems.

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.

Contd..:

Contd.. Medicine Dozens of congenital metabolic diseases also known as Inborn error of metabolism are now detectable by newborn screening tests, especially the testing using gas chromatography-mass spectrometry. GC/MS can determine compounds in urine even in minor concentration. These compounds are normally not present but appear in individuals suffering with metabolic disorders. This is an increasingly becoming a common way to diagnose IEM for earlier diagnosis and institution of treatment eventually leading to a better outcome. It is now possible to test a newborn for over 100 genetic metabolic disorders by a urine test at birth based on GC/MS. In combination with isotopic labeling of metabolic compounds, the GC-MS is used for determining metabolic activity . Most applications are based on the use of 13 C as the labeling and the measurement of 13 C/ 12 C ratios with an isotope ratio mass spectrometer ( IRMS ); an MS with a detector designed to measure a few.

Nuts and Bolts…Relative Costs:

Nuts and Bolts…Relative Costs Although in principle GC-MS experiments can be performed on magnetic sector instruments, in practice almost all GC-MS today is done on quadrupole or ion trap instruments. These instruments are relatively inexpensive and are simple to control by a computer. The major factor influencing the cost of a quadrupole - or ion-trap–based GC-MS system is the ionization methods available on the instrument and the mass range of the mass spectrometer. Simple quadrupole or ion trap instruments that use only electron impact ionization and have a mass range of 20 to 700 cost about $50,000. Those capable of both positive and negative chemical ionization and with mass ranges of 20 to 2000 cost about $200,000. Operating costs include instrument maintenance, GC carrier gases and columns, and spare parts. In most laboratories, these costs are about 5% of the instrument cost per year.

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[1]. 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.

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