GC and GC-MS slide.ppt
- 1. Gas chromatography (GC) and Gas chromatography mass spectrometry (GC-MS)
- 2. Gas Chromatography • Function • Components • Common uses • Chromatographic resolution • Sensitivity
- 3. Function • Separation of volatile organic compounds (VOC). • Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species. • Separation occurs as a result of unique equilibria established between the solutes and the stationary phase (the GC column). • An inert carrier gas carries the solutes through the column.
- 5. GC Components 1. Carrier Gas 2. Injector 3. Oven 4. Column 5. Detector
- 6. Carrier gas • Carrier gas for gas chromatography (GC) should be an inert gas that does not react with the sample component. • Carrier gas is an inert gas used to carry samples. • Helium (He), nitrogen (N2), hydrogen (H2), and argon (Ar) are often used. • Successful GC analysis requires careful control of carrier gas.
- 7. Comparison of the three gases
- 8. Brief comparison of Carrier Gas
- 10. How Does Gas Chromatography Work?
- 11. Injector • 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
- 12. Sample Injection Volume • The guidelines for sample injection volumes are as follows. If the injection volume is too large, the peak shape will become deformed, or the injection port will become dirty, leading to problems. • Liquid samples: Approx. 1 to 2 µL • Gas samples: Approx. 0.2 to 1 mL
- 13. Sample Injection Methods Hot Injection • Split: Most of the sample is eliminated as only a portion is injected into the column. • Splitless: Not split, but only for 1 to 2 minutes after injection • Total volume injection (Direct injection): There is no splitting mechanism. Cold Injection • Cold on-column injection (OCI) • Programmed temperature vaporization (PTV) https://www.ssi.shimadzu.com/service-support/faq/gas-chromatography/sample- injection/index.html
- 14. Split Injection Method Notes If the split ratio is small, the peak may inadvertently broaden at the injection port, reducing the separation Only a portion of the injected sample is injected into the column, so the sample vaporized within the insert must be uniform. → Be sure to place silica wool within the glass insert. Depending on the sample, change the amount and positioning of the wool, or the filler within the insert.
- 15. Splitless (100:90) vs. Split (100:1) Injector Syringe Injector Syringe Purge valve open Purge valve closed GC column GC column He He
- 16. Split or splitless • Usually operated in split mode unless sample limited • Chromatographic resolution depends upon the width of the sample plug • In splitless mode the purge valve is close for 30- 60 s, which means the sample plug is 30-60 seconds • As we will see, refocusing to a more narrow sample plug is possible with temperature programming
- 17. Gas Chromatography Columns • Two types of columns are used in gas chromatography: packed columns and capillary columns.
- 18. 0.32 mm ID Liquid Stationary phase Mobile phase (Helium) flowing at 1 mL/min Open Tubular Capillary Column 15-60 m in length 0.1-5 mm
- 19. Packed columns introduction • Short, thick columns made of glass or stainless steel tubes, packed columns have been used since the early stages of gas chromatography. • Packed columns produce broad peak shapes and have low separation performance, but can also handle large sample volumes and are not susceptible to contamination. They are still used today in official analytical methods and for gas analysis.
- 20. Packed columns • Viewing a cross-section image of a packed column reveals a tube filled with a particulate substance called packing. Packed columns have been used throughout the long history of gas chromatography, and many different packed columns have been created for different analytical applications.
- 22. Packed Column info • Stainless steel or glass tube filled with particulate packing material (an adsorbent material, or a support material coated or impregnated with a solid phase). • Internal Diameter: 2 to 4 mm • Length: 0.5 to 5 m (most commonly 2 m) • Packing: Support material with 0.5 to 25 % liquid phase (partition material) or no liquid phase (adsorbent material) • Liquid Phase: Multiple types available
- 24. Capillary columns • Capillary columns produce sharp peak shapes, achieve excellent separation performance, and are suited to high-sensitivity analysis. • In contrast, typical capillary columns consist of a thin, fused silica glass tube with a thin, internal liquid phase coating. Capillary columns were developed after packed columns, and though there are fewer types of capillary columns, their separation performance is dramatically superior to packed columns.
- 25. Capillary Column info • A typical capillary column is a thin, fused silica glass tube, lined with a liquid phase or adsorbent material or having a chemical bonding layer. Thin metal tubes are also sometimes used as capillary columns. • Internal Diameter: 0.1, 0.25, 0.32, 0.53 mm • Length: 5 to 100 m (most commonly 30 m) • Material: Fused silica glass • Liquid Phase: Good separation but less variety than packed columns
- 26. Porous Layer Open Tubular (PLOT) column (contains immobilized porous polymer/alumina, etc.) Wall-coated open-tubular (WCOT) or chemical bonding column (lined with liquid phase or a chemical bonding layer)
- 28. capillary vs packed column gc
- 31. Columns and Stationary Phases
- 33. fused silica open tubular (FSOT) column • Coated with polymer, crosslinked ; – Polydimethyl soloxane (non-polar) – Poly(phenylmethyldimethyl) siloxane (10% phenyl) – Poly(phenylmethyl) siloxane (50% phenyl) – Polyethylene glycol (polar) – Poly(dicyanoallyldimethyl) siloxane – Ploy(trifluoropropyldimethyl) siloxane
- 34. Polar vs. nonpolar • Separation is based on the vapor pressure and polarity of the components. • Within a homologous series (alkanes, alcohol, olefins, fatty acids) retention time increases with chain length (or molecular weight) • Polar columns retain polar compounds to a greater extent than non-polar – C18 saturated vs. C18 saturated methyl ester
- 35. C16:0 C18:0 C18:1 C18:2 C16:1 C16:0 C18:0 C18:1 C18:2 C16:1 RT (min) RT (min) Polar column Non-polar column
- 36. Column Type and Effect on Separation • Packed columns produce broad peaks and capillary columns produce sharp peaks. • In addition, capillary columns produce taller peaks, which allows the detection of lower concentrations (high detection sensitivity). This is the advantage of capillary columns.
- 38. • Sharper peaks provide better separation but also shorter analysis times.
- 39. • Component separation is affected by the following elements.
- 40. Classification of Capillary Column Liquid Phases Type of Solid Phase Polarity Separation Characteristics Application Operational Temperature Range (Approx.) Methyl silicone Non-polar Boiling point order Petroleum, solvents, high boiling point compounds -60 to 360 °C Phenylmethyl Slightly polar | Moderately polar Phenyl groups retain aromatic compounds. Perfumes, environmental compounds, aromatic compounds -60 to 340 °C Cyanopropyl phenol Moderately polar | Strongly polar Effective at separating oxygen-containing compounds, isomers, etc. Agricultural chemicals, PCBs, oxygen- containing compounds *Better to avoid use with FTDs (NPDs) -20 to 280 °C Trifluoropropyl Moderately polar | Strongly polar Specifically retains compounds that contain halogens. Halogen-containing compounds, polar compounds, solvents -20 to 340 °C Polyethylene glycol Strongly polar Strong retention of polar compounds Polar compounds, solvents, perfumes, fatty acid methyl esters 40 to 250 °C
- 41. General Guide to Selecting Polarity • Selecting columns with polar properties that are close to the polarity of the target compounds 1. Analysis of non-polar compounds → Non-polar column 2. Analysis of polar compounds → Strongly polar column • Selection by analytical objective 1. Large difference in boiling point between analytical target compounds → Non-polar column 2. Isomers or other compounds with little difference in boiling points → Strongly polar column
- 42. Guide to Selection of Internal Diameter, Length, and Coating Thicknes • Selection based on required separation 1. High-resolution separation required → Internal diameter: Thin, Length: Long 2. Adequate separation with shorter analysis time → Internal diameter: Thick, Length: Short, Coating thickness: Thin • Selection by analytical objective 1. Analysis of low boiling point compounds → Length: Long, Coating thickness: Thick 2. Analysis of high boiling point compounds → Length: Short, Coating thickness: Thin
- 43. Oven • Programmable • Isothermal- run at one constant temperature • Temperature programming - Start at low temperature and gradually ramp to higher temperature – More constant peak width – Better sensitivity for components that are retained longer – Much better chromatographic resolution – Peak refocusing at head of column
- 44. Typical Temperature Program Time (min) 0 60 50C 220C 160C
- 45. Detectors • Flame Ionization Detectors (FID) • Electron Capture Detectors (ECD) • Electron impact/chemical ionization (EI/CI) Mass spectrometry
- 46. Flam ionization detector (FID) • Effluent exits column and enters an air/hydrogen flame. • The gas-phase solute is pyrolized to form electrons and ions. • All carbon species are reduced to CH2 + ions. • These ions collected at an electrode held above the flame. • The current reaching the electrode is amplified to give the signal.
- 47. FID • Advantage ; 1. general detector for organic compounds 2. Very sensitive (10-13 g/s), normal working range is between 0.1 and 100000 ppm 3. linearity response factors - Linear response (106 to 107) 4. Rugged/durable • Disadvantage: destructive nature, and specific for volatile hydrocarbons and many carbon containing compounds only (cannot detect inorganic substances).
- 49. Electron capture detector (ECD) • To analyse halogenated compounds and is primarily found in the environmental, forensic and pharmaceutical markets. • Pesticide analysis • Up to 1000 times more sensitive than Flame Ionization Detectors
- 50. GC-ECD principle • ECD operates using two electrodes with a current passing between them. • When a sample passes between these two electrodes, the molecules pick up some of the electrons, causing a reduction in the current. • This reduction is recorded as a positive peak in the detection of components.
- 51. Sensitivity for ECD • Advantage ; 1.Exceptional sensitivity for halogenated compounds 2.Non destructive
- 52. Sensitivity for ECD • Disadvantage ; 1.Use radioactive source, need special license for operation. 2.Need several gases for ionization 3.Detector linear range is limited to around three or four orders of magnitude (103 to 104), depending upon the electronegativity of the analyte. 4.susceptible to oxygen and water impurities
- 53. Electron Capture Detector Schematic of a typical ECD detector. Red species are β particles; green species are nitrogen make-up gas molecules; blue species are analyte molecules.
- 54. Flame Photometric Detector (FPD) • Allows sensitive and selective measurements of volatile sulphur and phosphorus compounds. • FPD is highly selective as it detects element- specific light emitted within a hydrogen flame. • Main Applications > 1.Analysis of phosphorus pesticides 2.Analysis of sulfur-based malodors and food odor components 3.Analysis of organic tin in marine products
- 55. Principle of detection used by the FPD • Sulfur compounds, phosphorus compounds, and organic tin compounds each emit light at unique wavelengths when burned. • By passing light through a filter, only light of these unique wavelengths reaches a photomultiplier tube. • The photomultiplier tube then converts the detected light intensity into an electrical signal.
- 57. Flame Thermionic Detectors (FTD) • Selective, high-sensitivity detector for organic nitrogen compounds and inorganic and organic phosphorus compounds. • Does not react to inorganic nitrogen compounds. • Main Applications > 1.Drug analysis 2.Analysis of nitrogen and phosphorus pesticides
- 58. Principle of detection used by the FTD • FTD detects ions by reading the change in ion current gathered at the collector. • When a current is passed through the platinum coil with an alkali source attached to the coil (rubidium salt), the coil increases in temperature, which creates plasma around the alkali source. • Rubidium radicals (Rb*) are generated within this plasma. -Capable of oxidizing CN and organic phosphorus compounds -PO2 reacts with Rb* as shown below, creating ions. • CN + Rb* → CN- + Rb+ PO2 + Rb* → PO2- + Rb+ A current flows when ions gather in the collector.
- 59. Schematic Diagram of FTD
- 60. Thermal Conductivity Detectors (TCD) • TCD can detect all compounds other than the carrier gas. The TCD is mainly used to detect inorganic gas and components that the FID is not sensitive to. • Used for both organic and inorganic compounds detection. • Helium is commonly used as a carrier gas. (N2 and Ar are used to analyze He and H2.) • Main Applications > 1. Water, formaldehyde, formic acid, etc. 2. Analysis of compounds not detectable by the FID
- 61. Principle of detection used by the TCD • TCD detects target components by reading the change in filament temperature caused by the difference in thermal conductivity between the carrier gas and target components. • A direct voltage is applied between A and B.
- 62. Principle of detection used by the TCD A direct voltage is applied between A and B. 1.When only the carrier gas is flowing at a constant flowrate -Each filament maintains a constant temperature and a constant voltage is produced between C and D. Components are eluted from an analysis-side column. 1.A change in filament temperature occurs, which 2.Changes the resistance value, and 3.Changes the voltage between C and D
- 63. How does a thermal conductivity detector TCD work? • It functions by having two parallel tubes both containing gas and heating coils. • The gases are examined by comparing the rate of loss of heat from the heating coils into the gas. • The coils are arranged in a bridge circuit so that resistance changes due to unequal cooling can be measured.
- 64. Schematic Diagram of the TCD
- 65. TCD Analysis Example • When the Thermal Conductivity of the Analytical Target Component is Lower than the Carrier Gas
- 66. TCD Analysis Example • Selection by Analytical Objective • Reference ; • https://www.shimadzu.com/an/service-support/technical- support/analysis- basics/fundamentals/detector2.html#6_3_2_TCD
- 68. What is Mass Spectrometry? • mass spectrometers can be used to identify unknown compounds via molecular weight determination, to quantify known compounds, and to determine structure and chemical properties of molecules • How does a mass spectrometer perform such a feat? Every mass spectrometer consists of at least these three components: • Ionization Source • Mass Analyzer • Ion Detection System
- 69. What kind of info can mass spec give you? • MS provides qualitative and quantitative information about the atomic and molecular composition of inorganic and organic materials. • Molecular weight, Elemental composition (low MW with high resolution instrument), Structural info (hard ionization or CID) • Applications include drug detection, forensic substance identification and environmental analysis etc.
- 70. MASS SPECTROMETER (MS) IS COMMONLY USED AS A GC DETECTOR.
- 71. GC-MS advantages and disadvantages • Advantages ; enhanced sample identification, higher sensitivity, an increased range of analyzable samples, and faster results. • Disadvantages ; not capable of directly analyzing drugs that are nonvolatile, polar, or thermally labile. High cost, Limited sensitivity-detection of trace pollutants
- 73. How does it work? • Gas-phase ions are separated according to mass/charge ratio and sequentially detected • mass spectrometer generates multiple ions from the sample under investigation, it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type.
- 74. Basic Principle of MS Mass spectrometer should always perform the following processes: 1.Produce ions from the sample in the ionization source. 2.Separate these ions according to their mass-to-charge ratio in the mass analyzer. 3.Eventually, fragment the selected ions and analyze the fragments in a second analyzer. 4.Detect the ions emerging from the last analyzer and measure their abundance with the detector that converts the ions into electrical signals. 5.Process the signals from the detector that are transmitted to the computer and control the instrument using feedback.
- 75. Figure: Components of a Mass Spectrometer http://www.premierbiosoft.com/tech_notes/mass- spectrometry.html#:~:text=Basic%20Principle,abundance%20of% 20each%20ion%20type.
- 77. Parts of a Mass Spec • Sample introduction • Source (ion formation) • Mass analyzer (ion sep.) - high vac • Detector (electron multiplier tube)
- 78. Sample Introduction/Sources Volatiles • Probe/electron impact (EI),Chemical ionization (CI) • GC/EI,CI Involatiles • Direct infusion/electrospray (ESI) • HPLC/ESI • Matrix Assisted Laser Adsorption (MALDI) Elemental mass spec • Inductively coupled plasma (ICP) • Secondary Ion Mass Spectrometry (SIMS) – surfaces
- 79. EI, CI • EI (hard ionization) – Gas-phase molecules enter source through heated probe or GC column – 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions – EI spectra can be matched to library stds • CI (soft ionization) – Higher pressure of methane leaked into the source (mtorr) – Reagent ions transfer proton to analyte
- 80. To mass analyzer filament 70 eV e- anode repeller Acceleration slits GC column EI Source Under high vacuum
- 81. EI process • M + e- M+* f1 f2 f3 f4 This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam
- 83. Ion Chromatogram of Safflower Oil
- 85. CI/ ion-molecule reaction • 2CH4 + e- CH5 + and C2H5 + • CH5 + + M MH+ + CH4 • The excess energy in MH+ is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation
- 86. EI spectrum of phenyl acetate
- 88. Mass Analyzers • Low resolution – Quadrupole – Ion trap • High resolution – TOF time of flight – Sector instruments (magnet) • Ultra high resolution – ICR ion cyclotron resonance
- 89. Resolution • R = m/z/Dm/z • Unit resolution for quad and trap • TOF up to 15000 • FT-ICR over 30000 – MALDI, Resolve 13C isotope for a protein that weighs 30000 – Resolve charge states 29 and 30 for a protein that weighs 30000
- 90. High vs low Res ESI • Q-TOF, ICR – complete separation of the isotope peaks of a +3 charge state peptide – Ion abundances are predictable – Interferences can be recognized and sometimes eliminated • Ion trap, Quad – Unit resolution
- 92. Quadrupole Mass Ion Filter
- 93. Ion Trap
- 94. Time of Flight -TOF
- 95. Where: •mi = mass of analyte ion •zi = charge on analyte ion •E = extraction field •ti = time-of-flight of ion •ls = length of the source •ld = length of the field-free drift region •e = electronic charge (1.6022x10-19 C)
- 97. Mass accuracy • Mass Error = (5 ppm)(201.1001)/106 = 0.0010 amu • 201.0991 to 201.1011 (only 1 possibility) • Sector instruments, TOF mass analyzers • How many possibilities with MA = 50 ppm? with 100 ppm?
- 98. Exact Mass Determination • Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF) • C9H15NO4, FM 201.1001 (mono-isotopic) • Mass accuracy = {(Mass Error)/FM}*106 • Mass Error = (5 ppm)(201.1001)/106 = 0.0010 amu
- 99. Advantages and limitations of the various mass analyzers Mass Analyzer Description Advantages Limitations Magnetic Sector Scanning High resolution Expensive and bulky ContinuousHigh dynamic range Slow scan speed High reproducibility High vacuum required High sensitivity Difficult to couple with pulsed ionization techniques and LC Quadrupole Scanning Compact and simple Limited mass range Mass Filter Relatively cheap Low resolution ContinuousGood selectivity (SIM) Little qualitative information Moderate vacuum required → well suited for coupling to LC Time-of-Flight Non- scanning High sensitivity and ion transmission Requires pulsed introduction to MS Pulsed High resolution Requires fast data acquisition Excellent mass range Fast scan speed Ion Trap Trap Small and relatively cheap Limited dynamic range Pulsed High sensitivity Limited ion trap volume Good resolution Limited resolution Compact Requires pulse introduction to MS
- 100. Application of Mass Spectrometry • Analysis of Biomolecules using Mass Spectrometry • Analysis of Glycans • Analysis of Lipids • Analysis of Proteins and Peptides • Analysis of Oligonucleotides
- 101. Analysis of Biomolecules using Mass Spectrometry • Till the1970s, the only analytical techniques which provided similar information were electrophoretic, chromatographic or ultracentrifugation methods. The results were not absolute as they were based on characteristics other than the molecular weight. Thus the only possibility of knowing the exact molecular weight of a macromolecule remained its calculation based on its chemical structure. • The development of desorption ionization methods based on the emission of pre-existing ions such as plasma desorption (PD), fast atom bombardment (FAB) or laser desorption (LD), allowed the application of mass spectrometry for analyzing complex biomolecules.
- 102. Analysis of Glycans • Oligosaccharides are molecules formed by the association of several monosaccharides linked through glycosidic bonds. • The determination of the complete structure of oligosaccharides is more complex than that of proteins or oligonucleotides. • It involves the determination of additional components as a consequence of the isomeric nature of monosaccharides and their capacity to form linear or branched oligosaccharides. • Knowing the structure of an oligosaccharide requires not only the determination of its monosaccharide sequence and its branching pattern, but also the isomer position and the anomeric configuration of each of its glycosidic bonds.
- 103. Analysis of Lipids • Lipids are made up of many classes of different molecules which are soluble in organic solvents. Lipidomics, a major part of metabolomics, constitutes the detailed analysis and global characterization, both spatial and temporal, of the structure and function of lipids (the lipidome) within a living system. • Many new strategies for mass-spectrometry-based analyses of lipids have been developed. The most popular lipidomics methodologies involve electrospray ionization (ESI) sources and triple quadrupole analyzers. Using mass spectrometry, it is possible to determine the molecular weight, elemental composition, the position of branching and nature of substituents in the lipid structure.
- 104. Analysis of Proteins and Peptides • Proteins and peptides are linear polymers made up of combinations of the 20 amino acids linked by peptide bonds. Proteins undergo several post translational modifications, extending the range of their function via such modifications. • The term Proteomics refers to the analysis of complete protein content in a living system, including co- and post-translationally modified proteins and alternatively spliced variants. • Mass Spectrometry has now become a crucial technique for almost all proteomics experiments. It allows precise determination of the molecular mass of peptides as well as their sequences. This information can very well be used for protein identification, de novo sequencing, and identification of post-translational modifications.
- 105. Analysis of Oligonucleotides with GC-MS • Oligonucleotides (DNA or RNA), are linear polymers of nucleotides. These are composed of a nitrogenous base, a ribose sugar and a phosphate group. Oligonucleotides may undergo several natural covalent modifications which are commonly present in tRNA and rRNA, or unnatural ones resulting from reactions with exogenous compounds. • Mass spectrometry plays an important role in identifying these modifications and determining their structure as well as their position in the oligonucleotide. It not only allows determination of the molecular weight of oligonucleotides, but also in a direct or indirect manner, the determination of their sequences.
- 107. Energy and Chemical • Gas chromatographs are used through the entire hydrocarbon value chain, from exploration to final product. They are used to determine the quality, purity, and composition of petroleum products, including crude oil, natural gas, liquefied petroleum gas (LPG), and refined products like diesel fuel, gasoline, or heating oil. • GC also are used to help develop and test alternative energy sources, such as biofuels and batteries.
- 108. Environmental • Labs test drinking water, wastewater, soil, and air for the presence of pollutants that can be harmful to human health. • GC systems give scientists the confidence that they can implement standard methods to meet regulatory requirements and perform untargeted screening to discover new contaminants of concern.
- 109. Food and Beverage • Used to test crops, additives, and processed foods and beverages, from the measurement of pesticide residues and persistent organic pollutants (POPs) in raw materials to the detection and quantitation of leached materials in packaged foods to confirmation of accurate nutrition labeling.
- 110. Forensics • Because the field of forensics is diverse and accurate results are paramount, scientists require versatile and dependable gas chromatography systems. • GC to determine blood alcohol content (BAC), analyze samples for performance-enhancing or recreational drugs, or confirm the identity and potency of seized narcotics.
- 111. Pharmaceutical • Pharmaceutical manufacturers must adhere to strict regulations concerning the amount of residual solvent and reaction by-products that can be present in their products. They also must test for compounds that can leach out of packaging and into pills or other formulations. • Gas chromatography systems help pharmaceutical scientists test for these contaminants to ensure they are below mandated levels.