Gas chromatography mass spectrometry

1. Gas Chromatography -Mass Spectrometry Hina Qaiser MS-1 1 st Semester Department Of Biotechnology Lahore College For Women University

2. GC-MS is an integrated composite analysis Instrument Combining GC which is excellent in its ability for separation with mass spectrometry ideal in identification and elucidate structure of separated component . Gas Chromatography -Mass Spectrometry

3. Introduction Gas chromatography-mass spectroscopy (GC-MS) is a hyphenated analytical technique exquisitely sensitive but also specific and reliable GC can separate volatile and semi-volatile compounds with great resolution, but it cannot identify them. MS provide detailed structural information on most compounds such that they can be exactly identified, but can’t readily separate them. 3

4. Therefore, marriage of both instruments have been proposed shortly after the development of GC in the mid-1950s. we obtain both qualitative and quantitative information of our sample in a single run within the same instrument Today computerized GC/MS instruments are widely used in environmental monitoring ,in the regulation of agriculture and food safety , and in the discovery and production of medicine. Continued......

5. Principle of GC-MS

6. Instrumental Layout GC-MS comprise following major block

7. GC-MS Instrument The insides of the GC-MS, with the column of the gas chromatograph in the oven on the right.

8. Gas Chromatography Gas chromatography leads to Separation of volatile organic compounds Separation occurs as a result of unique equilibrium established between the solutes and the stationary phase (the GC column) An inert carrier gas carries the solutes through the column

9. Basic Components: Carrier Gas Gas Controls The Injector The Column Two Groups: Packed Column Capillary Column The Oven The Detector (Mass Spectrometer) Continued......

10. Sample Preparation 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 to extensive.

11. Carrier Gas Requirements of a carrier gas Inertness Suitable for the detector High purity ( Better than 99.995%Better than 99.9995% for Mass Spec). Easily available Cheap Should not cause the risk of fire Should give best column performance

12. Soap Bubble Meter Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate Flow regulators / Flow meters D eliver the gas with uniform pressure/flow rate Rota meter Placed before column inlet It has a glass tube with a float held on to a spring. The level of the float is determined by the flow rate of carrier gas Flow meters

13. Injection Devices A GC syringe penetrates a septum to inject sample into the vaporization camber Instant vaporization of the sample, 280  C Carrier gas transports the sample into the head of the column Purge valve controls the fraction of sample that enters the column

14. Purpose of Injection Deposit the sample into the column in the narrowest band possible The shorter the band at the beginning of the chromatographic process - tall narrow peaks Gives maximum resolution and sensitivity Therefore type of injection method and operating conditions is critical in obtaining precise and accurate results

15. Split or Splitless Most common method of Injection into Capillary Columns Most commonly misunderstood also! Same injector hardware is used for both techniques Electronically controlled Solenoid changes Gas Flow to determine Injector function. Split Injection Mechanism by which a portion of the injected solution is discarded. Only a small portion (1/1000 - 1/20) of sample goes through the column Used for concentrated samples (>0.1%) Can be performed isothermal ly Fast injection speed Injector and septa contamination not usually noticed Splitless Injection Most of the sample goes through the column (85-100%) Used for dilute samples (<0.1%) Injection speed slow Should not be performed isothermally Controlled by solenoid valve Requires careful optimisation

16. Splitless (100:90) vs. Split (100:1) Injector Syringe Injector Syringe Purge valve open Purge valve closed GC column GC column He He

17. On Column Injection All of the sample is transferred to the column Needle is inserted directly into column or into insert directly above column Trace analysis Thermally labile compounds e.g Pesticides, Drugs High molecular weight

18. Material of Construction Columns

19. Capillary Column Characteristics Stationary Phases Choice of phase determines selectivity Hundred of phases available Many phases give same separation Same phase may have multiple brand names Stationary phase selection for capillary columns much simpler Like dissolves like Use polar phases for polar components Use non-polar phases for non-polar components

20. Choosing a Column Internal Diameter Smaller ID’s Good resolution of early eluting compounds Longer analysis times Limited dynamic range Larger ID’s Have less resolution of early eluting compounds Shorter analysis times Sufficient resolution for complex mixtures Greater dynamic range

21. Film Thickness Amount of stationary phase coating Affects retention and capacity Thicker films increase retention and capacity Standard capillary columns typically 0.25µm 0.53mm ID ( Megabore ) typically 1.0 - 1.5µm The maximum amount that can be injected without significant peak distortion Column capacity increases with :- film thickness temperature internal diameter stationary phase selectivity If exceeded, results in :- peak broadening asymmetry leaking Column Capacity

22. Length effects Increasing the column length increases the resolution Doubling the column length increases resolution by the factor of 1.4 but also increases the analysis time Long column are employed when sample contains large number of componnets length L = 30 m is the most common column used for many analyses (drugs, pesticides, PAHs)

23. Interfacing GC with Spectroscopic Methods Elutes from column collected as separate fractions after being detected - composition measured by Mass Spectrometry. 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 .

24. 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.

25. Jet Separator Two capillary tubes aligned with a small space between them. (1 mm) A vacuum is created between the two tubes using a 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. 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

26. Basic Mass Spec.Theory Mass Spec. is a Microanalytical Technique used to obtain information regarding structure and Molecular weight of an analyte Destructive method i.e sample consumed during analysis In all cases some form of energy is transferred to analyte to cause ionisation In principle each Mass Spectrum is unique and can be used as a “fingerprint” to characterise the sample GC/MS is a combination technique that combines the separation ability of the GC with the Detection qualities of Mass Spec.

27. Basic GCMS Theory Sample injected onto column via injector GC then separates sample molecules Effluent from GC passes through transfer line into the Ion Trap/Ion source Molecules then undergo electron /chemical ionisation Ions are then analysed according to their mass to charge ratio Ions are detected by electron multiplier which produces a signal proportional to ions detected

28. Electron multiplier passes the ion current signal to system electronics Signal is amplified Result is digitised Results can be further processed and displayed Basic GCMS Theory

29. Types of Ionisation

30. Electron Ionisation 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

31. 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 carbcations .

32. Electron Ionisation

33. 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

34. Six Processes Occur in Mass Spectrometer. Electrons are fired at the gaseous molecules…these knock off other electrons from some of the molecules… M + electron M + + 2e - Gaseous ions are accelerated by passing through an electric field. At this stage they can be traveling at up to 2 x 10^5 m/sec. (about 1/1000 the speed of light. They then pass through an electrostatic analyzer, which selects the ions of kinetic energy within a narrow range by using an electric field. The fast moving ions now pass through the poles of electromagnet, where they are deflected. The deflected ions pass through a narrow slit and are collected on a metallic plate connected to an amplifier. For a given strength of magnetic field, only ions of a certain mass pass through the slit and hit the collector plate. As the ions hit the plate they cause a current to flow through the amplifier. The more ions there are , the larger the current.

35. The equation governing the deflection of ions in the magnetic field is as follows: r= Where r= radius of circular path in the magnetic field m= mass of ion V=accelerating voltage e=electrical charge on the ion B=strength of strength

36. Accelerating voltage V is kept constant Radius of the curvature is prop to Inversely prop to B To obtain a mass spectrum, the current through the elctromagnet is changed at a steady rate. Causes the magnetic field B to change its strength & hence allows ions of different Mass/Charge value to pass successively through the slit. Mass spectrum produced plotting (ion current) against (electromagnetic current), which is equivalent to (relative abundance) against (mass/charge (m/e) ratio).

37. 3 Main Ways In Which Mass Spectrometry Is Applied To Determine The Structures Of Organic Compounds There are three main ways in which mass spectrometry is applied to the determination of the structures of organic compounds. By measuring the relative heights of the molecular ion (M) peak and the (M+1) peak we can determine the number of carbon atoms in a molecule. 2. By measuring the accurate mass of a molecular ion we can determine its molecular formula. 3. By identifying the fragments produced when an ion breaks up inside a mass spectrometer we can often piece together the structure of the parent molecule.

38. Interpretation of Mass Spectra The MS of a typical hydrocarbon, n- decane is shown above. The molecular ion is seen as a small peak at m/z = 142 . Notice the series ions detected that correspond to fragments that differ by 14 mass units, formed by the cleave of bonds at successive -CH2- units

39. REFRENCES   Chaintreau A. Simultaneous Distillation–Extraction: From Birth to Maturity – Review Flavour and Fragrance Journal 2001; 16(2) 136-148.   Flotron V, Houessou JK, Bosio A, Delteil C, Bermond A, Camel V. Rapid Determination of Polycyclic Aromatic Hydrocarbons in Sewage Sludges Using Microwave-Assisted Solvent Extraction. Comparison with Other Extraction Methods. Journal of Chromatography. A 2003; 999(1-2) 175-84.   Rice SL, Mitra S. Microwave-Assisted Solvent Extraction of Solid Matrices and Subsequent Detection of Pharmaceuticals and Personal Care Products ( Ppcps ) Using Gas Chromatography–Mass Spectrometry. Analitica Chimica Acta 2007; 589 125-132.   Hubschmann HJ. Handbook of GC-MS: Fundamentals and Applications. 2d Ed. Weinheim : Wiley-VCH; 2009.   Laskin J, Lifshitz C. Principles of Mass Spectrometry Applied to Biomolecules . New York: John Wiley and Sons; 2006.