Apat 2013 gc workshop 2

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GC workshop at the National Symposium for Advances in Pharmaceutical Analysis (APAT 2013). St Peter's Institute of Pharmaceutical Sciences, Hanamkonda, Warangal, AP, India.

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Apat 2013 gc workshop 2

  1. 1. http://analysciences.com 1
  2. 2. AnalySys Sciences 2 Instrumental in your success http://analysciences.com http://analysciences.com
  3. 3. An Analytical chemist … … tries to answer only two questions. Given a sample … What is it? Qualitative analysis How much is it? Quantitative analysis
  4. 4. The evolution of analysis 1900‟s Manual titration 1 mg 10-3 0.001 gm 1920‟s TLC 1 µg 10-6 0.000001 1960‟s GC 1 ng 10-9 0.000000001 1980‟s HPLC 1 pg 10-12 0.0000000001 1990‟s GC-MS 1 fg 10-15 0.000000000000001 2008 LCMS 1 ag 10-18 0.000000000000000001 2013 FTMS 1 zg 10-21 0.000000000000000000001
  5. 5. Analytical Chemistry – The road ahead Increased use of hyphenated techniques, like LC-MS, GC-FTIR & LC-NMR. Lower limits of detection. “Walk-away” automation. Intuitive software and data handling. Increasing use single-point control systems via the Internet.
  6. 6. The Analytical Pharmacist in the 21st century Full-time analytical chemist. Part-time software engineer and EDP specialist. AND…a knowledge of software platforms, data handling techniques and preferably, basic electronics.
  7. 7. Chromatography … An introduction 7http://analysciences.com From: The Universal Etymological Dictionary, 1731
  8. 8. Chromatography … since Biblical times. So Moses brought Israel from the Red Sea, and they went out in the wilderness of Shur …and found no water. And when they came to Marah, they could not drink of the waters of Marah, for they were bitter; And the people murmured against Moses, saying, What shall we drink? And he cried unto the Lord and the Lord shewed him a tree, which when he had cast into the waters, the waters were made sweet. Exodus, Chapter 15 §22–25 (King JamesVersion). Source: Article by Leslie Ettre. Ion exchange chromatography?
  9. 9. 110 years of modern chromatography March 21, 1903 At the Warsaw Society of Natural Scientists, Russian botanist, Mikhail Semenovich Tswett presented the first official lecture on chromatographic separation. 9 Tswett, MS (1906) Physico-chemical studies on chlorophyll adsorptions. Berichte der Deutschen botanischen Gesellschaft, 24, 316-23 Tswett, MS (1906) Adsorption analysis and chromatographic method. Application to the chemistry of chlorophyll. Berichte der Deutschen botanischen Gesellschaft, 24, 385 http://www.life.uiuc.edu/govindjee/Part2/34_Krasnovsky.pdf http://web.lemoyne.edu/~giunta/tswett.html http://analysciences.com
  10. 10. When a chlorophyll solution in petrol ether is filtered through the column of an adsorbent …then the pigments will be separated from the top down in individual colored zones…the pigments which are adsorbed stronger will displace those which are retained more weakly. http://analysciences.com 10
  11. 11. "Like light rays in the spectrum, the different components of a pigment mixture, obeying a law, are separated on the calcium carbonate column and can thus be qualitatively and quantitatively determined. I call such a preparation a chromatogram and the corresponding method the chromatographic method." http://analysciences.com 11
  12. 12. Gas chromatography – the pioneers. Erika Cremer, Univ of Innsbruck, Austria, 1944, developed the theory and use of gas chromatography. She was assisted by her PhD student, Fritz Prior. http://analysciences.com 12
  13. 13. Chromatography is … “…a method in which the components of a mixture are separated on an adsorbent column in a flowing system". M.Tswett http://analysciences.com 13 A separation involving a mobile phase, a stationary phase, and the sample. The sample undergoes a series of interactions between these two phases, resulting in separation of its components. Sample components elute in increasing order of interaction.
  14. 14. What interaction? Adsorption …analyte in mobile phase adsorbed onto stationary phase. Equilibration between the mobile and stationary phase results in separation. http://analysciences.com 14
  15. 15. Partition …thin film of a liquid stationary phase formed on a solid support. Solute molecules partition between the mobile phase and stationary phase. http://analysciences.com 15
  16. 16. Ion-exchange Ion-ex resin is used to covalently attach anions or cations onto it. Solute ions of the opposite charge are attracted to the resin. Example: Purification of hard water. http://analysciences.com 16
  17. 17. Affinity specific interaction between a solute molecule and a molecule that is immobilized on a stationary phase. eg. purification of immunoglobulins. http://analysciences.com 17
  18. 18. Size Exclusion a porous gel separates molecules by size. Example: Purification of enzymes or proteins. http://analysciences.com 18
  19. 19. Mobile phase Gas Gas-solid (Adsorption) Gas-Liquid (Partition) Liquid TLC /Planar chromatography Column chrom HPLC Supercritical fluid SFC 19http://analysciences.com Chromatography – Modes
  20. 20. Mobile phase Stationary phase Eluate collection Sample introduction Detection Chromatography – the system 20 Stationary phase is packed into a column, or … In the form of a thin layer coated on a glass or aluminium plate or … In the form of a thick sheet of paper.
  21. 21. A typical chromatogram http://analysciences.com 21 Y axis = Detector response (usually in millivolts) X axis = retention time (or volume) A symmetrical peak is known as a Gaussian peak.
  22. 22. Some boring equations http://analysciences.com 22
  23. 23. Retention Volume / Time Volume of mobile phase required to elute a particular analyte from the stationary phase. Time taken by an analyte to elute from the stationary phase. VR = tR x Fc tR = Retention time Fc = Flow rate http://analysciences.com 23
  24. 24. Retention Time Dead Time/volume Retention time / retention volume taken by an unretained solute to elute from the system. Represents the combined volume of tubings, detector flow cell, injector loop, column volume. Relative (corrected) retention time 0R Rt t t   http://analysciences.com 24
  25. 25. Partition Co-efficient (Distribution / Adsorption co-efficient) M sC K C  http://analysciences.com 25 CS = concentration of the analyte in the stationary phase. CM = concentration in the mobile phase Analytes in a sample mixture will separate in a chromatographic system only if their K values are significantly different.
  26. 26. Partition Ratio (Capacity Factor) Measure of the time spent by a solute in the mobile phase, with respect to the stationary phase. For baseline separation, K’ > 2 http://analysciences.com 26
  27. 27. Relative retention (Selectivity / separation factor) For baseline separation, a > 1.5 2 1 k k a    http://analysciences.com 27
  28. 28. Selectivity Depends on •Nature of the two phases •Column temperature http://analysciences.com 28
  29. 29. Resolution For baseline separation, Rs >2 2 1 1 2 2 R R s t t R w w         http://analysciences.com 29
  30. 30. Peak Width (4s) http://analysciences.com 30
  31. 31. Tailing factor (Asymmetry/ Skew factor) BC As CA  http://analysciences.com 31
  32. 32. Tailing factor - 2 http://analysciences.com 32
  33. 33. System Suitability Parameters USP Plate count > 2000 plates/meter Tailing factor < 2 Resolution > 2 Partition ratio > 2 Relative retention > 1.5 Precision / repeatability RSD </= 1% for n >/= 5 http://analysciences.com 33
  34. 34. Chromatography Theories or… why a chromatography column will not do what it’s told.. http://analysciences.com 34
  35. 35. Plate theory Martin and Synge (1941) Nobel in Chemistry, 1952 for “their invention of partition chromatography”. Chromatography column assumed to be similar to a distillation column. Separation occurs across a series of theoretical plates. Higher number of theoretical plates improves column performance. http://analysciences.com 35
  36. 36. Plate theory explained A distillation column is used for fractional distillation of liquid mixtures. Higher surface area inside the column improves distillation efficiency. This is done by putting in a series of glass plates, with each plate containing glass beads or similar packing material. A chromatographic column is similar to a distillation column. The packing inside the column is considered similar to the packing inside a distillation column. There are no real plates inside, hence „theoretical plates‟. Hence, height equivalent to a theoretical plate (HETP). Higher number of plates, higher separation efficiency. http://analysciences.com 36
  37. 37. Rate theory Dr JJ van Deemter (1956) Plate theory does not explain band spreading and peak broadening. Does not take into account packing material properties, mobile phase flow rate and column geometry. Rate theory takes into account various factors that cause chromatographic peak broadening and reduction of separation efficiency. 37
  38. 38. van Deemter Equation linear velocity ( flow rate) C H A B       38 van Deemter took into account several factors that can affect HETP and column performance. He formulated a mathematical equation that defined the relationship between various chromatographic factors and HETP. This equation made it possible to numerically calculate column performance, design better chromatography stationary phases and improve separation efficiency.
  39. 39. A term – Multipath effect or Eddy diffusion Analyte molecules take different paths through the packing, leading to band broadening To reduce eddy diffusion, reduce stationary phase particle size. However, backpressure will increase. In GC, backpressure is not a major issue. http://analysciences.com 39
  40. 40. B term Longitudinal diffusion / wall effect Distortion of the mobile phase front, due to varying velocity across the column, especially at the column wall To reduce wall effect, increase flow rate http://analysciences.com 40
  41. 41. C term – mass transfer resistance Analytes remain trapped in stagnant pockets in the packing. To improve mass transfer, decrease mobile phase flow rate. http://analysciences.com 41
  42. 42. Van Deemter plot http://analysciences.com 42 What does it mean? In practical terms, it means that for a given stationary phase and for a given chromatography column or plate, there is one optimal mobile phase flow rate. Increasing or decreasing flow rate might have an adverse effect on performance. For example: For an HPLC column with 4.6mm internal diameter and 150mm length, packed with 5u, spherical particles, the optimal flow rate is 1ml/min.
  43. 43. HETP Height Equivalent to a theoretical plate 2 2 4 16 2 5.54 R R L H t L H t s s             http://analysciences.com 43
  44. 44. Plate Count 2 2 16 4 25 5 R R t t s s             2 5.54 2 R L N H t s        http://analysciences.com 44
  45. 45. Plate count – what it means. The plate count gives you an idea of the efficiency and separating power of a column. Higher plate count for a given column implies better performance (but does not guarantee it !) Plate count is affected by: Nature of sample Flow rate Detector flow cell volume Dead volume Temperature Detector settings / Data system settings. Injector reproducibility, etc… Be wary when comparing plate counts!! http://analysciences.com 45
  46. 46. A typical chromatogram http://analysciences.com 46 Y axis = Detector response (usually in millivolts) X axis = retention time (or volume)
  47. 47. Quantitation in Chromatography Area (height) under the peak is proportional to the injected amount. Proportionality constant is the response factor. http://analysciences.com 47
  48. 48. How is peak area determined? Integration Data system sub-divides peak into small rectangles, calculates area of each, and adds them up. http://analysciences.com 48
  49. 49. Quantitation – External standards Inject known concentrations of the analyte using reference standards. Analyse the test sample under the same conditions. Plot a calibration curve of analyte concentration v/s peak area (or height). http://analysciences.com 49
  50. 50. Internal Standards Chemically similar to the analyte. Added to the sample and external standards. Same amount added to both. Accounts for variations in injection volume and other system variables. Provides better precision. http://analysciences.com 50
  51. 51. Gas Chromatography 51 http://analysciences.com
  52. 52. Gas Chromatography Mobile phase is a gas Used for volatile, heat stable samples only. eg. Petroleum products, volatile oils, perfumeries. … Or analytes that can be converted to volatile derivatives, eg. amino acid silyl derivatives, fatty acid methyl esters. 52http://analysciences.com
  53. 53. Why GC? Minimal sample prep. Fast analysis time. High separation efficiency. Easier to automate. Easier to upgrade to hyphenated methods like GC-MS. Lower capital costs and running costs. Given a choice between HPLC and GC, choose GC! http://analysciences.com 53 Restricted to analytes that are volatile and thermo-stable … or to analytes that can be derivatised.
  54. 54. Carrier gas Filters/traps Injector Detector Column oven Column Data system GC Schematics
  55. 55. http://analysciences.com 55 GC – Mobile phases / Carrier gases.
  56. 56. GC – Mobile phases Helium is commonly used as a carrier gas. Nitrogen is also used. Hydrogen is becoming a popular alternative to helium. Gases are stored in high-pressure cylinders. Gas flow is controlled by regulators. Sometimes nitrogen and helium generators are used instead of cylinders. http://analysciences.com 56
  57. 57. Hydrogen as carrier gas. H2 has low viscosity and high diffusivity. Hence, faster analysis times. Much cheaper than helium. Lower cost- per-analysis. Helium is extracted from natural gas. Process is very expensive. Not eco-friendly. Acute shortage of Helium. H2 can be cheaply produced using H2 generators. http://analysciences.com 57
  58. 58. Gas manifolds Gas manifolds are used to purify and dehumidify the gases before they enter the GC. Dust filters, moisture traps, silica gel pellets and molecular sieves are used. 58 http://analysciences.com
  59. 59. Sample Introduction http://analysciences.com 59
  60. 60. Injector ports Samples are injected through sealed, heated injection ports. Injection volumes are very small, usually less than 5 μl. Injectors should accurately deliver the vaporised sample on to the head of the GC column. 60http://analysciences.com
  61. 61. Packed column injector http://analysciences.com 61 Injector septum provides a leak-tight seal. Injector liner protects the inlet seal from dirt and contaminants. Inlet seal protects the GC column. Injector body is heated by a programmable heater system.
  62. 62. Used with capillary columns. Injects small sample volumes. (<1μl) Splits the injection volume into smaller volumes, by adjusting the split ratio. http://analysciences.com 62
  63. 63. PTV injector Programmable temperature vaporising injector. Used for large sample volumes and thermo-labile compounds Instantly vaporises sample, upto 3000C Highly reproducible and accurate. http://analysciences.com 63
  64. 64. Injector septa Septa ensure a leak-tight seal at the injection port. Available in various materials – teflon, rubber and silicone. http://analysciences.com 64
  65. 65. Sampling Valves Used for continuous, reproducible injection of gaseous samples. Can be configured in several ways: •Multiple column switching •Detector switching •Automated air sampling http://analysciences.com 65
  66. 66. Injector liners Glass liners are used inside the injector body. Protect the injector from sample debris. http://analysciences.com 66
  67. 67. GC – sample injection syringes Septum piercing needle. Available in various volumes, from 1ul to 100ul. Can be automated. http://analysciences.com 67
  68. 68. Autosamplers Two types: Carousel XYZ samplers Can automate many tasks: Simple injection Sample prep/derivatisation/filtration/ dilution/heating/cooling /weighing. http://analysciences.com 68
  69. 69. Autosamplers – pros & cons Low cost-per-analysis. Reagent & solvent consumption is reduced. High reproducibility. Reliable results. 24/7/365 operation. Chemist is free of repetitive manual tasks. High capital costs. http://analysciences.com 69
  70. 70. GC – Stationary phases & columns http://analysciences.com 70
  71. 71. Packed columns 71http://analysciences.com Made of SS, glass or copper tubing, filled with porous packing material, which may be coated with a viscous liquid phase. Packed columns contain a finely divided, inert, solid support material (usually based on diatomaceous earth ) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
  72. 72. Packed columns – phases. The packing usually consists of an inert porous material such as Celite (a diatomaceous earth), or calcined Celite (in the form of powdered fire brick) or a synthetically polymeric resin. Glass beads and molecular sieves are also used. http://analysciences.com 72
  73. 73. Packed columns - Kieselguhr Packings are treated with dimethylchlorosilane to remove active silanols. Washed with HCl to remove trace metals. http://analysciences.com 73 Diatomaceous earth or kieselguhr is soft, sedimentary rock that contains fossilised remains of diatoms (hard- shelled algae). It consists of 80-90% silica, and small amounts of alumina and iron oxide. It crumbles easily into a fine, white powder. Celite is a brand name, owned by World Minerals Inc, a division of Imerys Filtration. Chromosorb W = Untreated celite Chromosorb P = Calcined celite Chromosorb S = Celite calcined with sodium carbonate.
  74. 74. Packed columns – Molecular sieves Molecular sieves are synthetic zeolites (complex alumino-silicates of sodium, potassium or calcium) of various pore sizes, usually 4 Å or so. Used for separation of fixed gases like CO, CO2, CH4, Ar, H2, O2. http://analysciences.com 74
  75. 75. Packed columns – Polymeric packings Macroporous, spherical, ultrapure resins. Used for difficult separations in gas chromatography. Eg. Separation of H2S and H2O. Separation of gas mixtures. HayeSep is a popular brand. http://analysciences.com 75
  76. 76. Capillary columns Made from fused silica. Have an internal diameter of a few tenths of a millimeter, usually 0.32mm and 0.53 mm. Length between 3m to 30m. Capillary columns are more efficient than packed columns. Much higher plate counts >30,000 plates per meter. http://analysciences.com 76
  77. 77. Capillary columns - 2 Liquid stationary phase is coated or chemically bonded to the inner wall of the capillary. Most common phases: Polysiloxanes Polyethylene glycols. http://analysciences.com 77
  78. 78. http://analysciences.com 78
  79. 79. Separation mechanisms in GC Partition: Analyte partitions between the carrier gas and a viscous stationary phase. Adsorption: Analyte adsorbs/desorbs between the carrier gas and a solid stationary phase. http://analysciences.com 79
  80. 80. GC - Detection systems http://analysciences.com 80
  81. 81. Thermal Conductivity Detector Detector cell contains a heated filament with an applied current. As carrier gas containing solutes passes through the cell, a change in the filament current occurs. The current change is compared against the current in a reference cell. The difference is measured and a signal is generated. (Wheatstone bridge principle). Selectivity: All compounds except for the carrier gas Sensitivity: 5-20 ng Linear range: 105-106 Temperature: 150-250°C http://analysciences.com 81
  82. 82. Flame Ionisation Detector Analytes are burned in a hydrogen-air flame. Carbon containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Selectivity: Compounds with C-H bonds. Sensitivity: 0.1-10 ng. Linear range: 105-107 Gases: Combustion - hydrogen and air; Makeup - helium or nitrogen. Temperature: 250-450°C. http://analysciences.com 82
  83. 83. Electron Capture Detector Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons, causing a reduction in current. The amount of current loss is indirectly measured and a signal is generated. Selectivity: Halogens, nitrates and conjugated carbonyls. Sensitivity: 0.1-10 pg Temperature: 300-400°C http://analysciences.com 83
  84. 84. Pulsed discharge ionisation detector (PDID) Pulsed DC discharge creates a plasma by ionising helium gas inside the detector body. Charged helium plasma in turn ionises analytes eluting from the GC column. This results in a current that is proportional to the amount of the analyte. http://analysciences.com 84
  85. 85. PDID - Advantages Universal, non-destructive detector. Very sensitive, can detect analytes in the femtogram level (10-15). Good alternative to electron-capture detector for pesticides and halogenated compounds, since it is non-radioactive. More sensitive than FID, and can be used in settings where a flame is not safe (like petroleum and gas analyses.) http://analysciences.com 85
  86. 86. Flame Photometric Detector. Uses a photomultiplier tube to detect spectral lines of analytes, as they are burned in a flame. (like in a flame photometer). Especially useful for sulfur and phosphorus compounds. http://analysciences.com 86
  87. 87. Photoionisation detector UV lamp ionises analytes from the GC column eluent. Useful for volatile organic compounds like polyaromatic hydrocarbons and inorganic species that are ionised in UV light. Used for environmental pollutants. http://analysciences.com 87
  88. 88. Inside the GC 88http://analysciences.com GC columns are mounted in an oven. Oven temperature can be programmed. Better separations are achieved with temperature programming.
  89. 89. Temperature programming – why. In GC, analytes are separated according to boiling point and polarity. Molecules with low boiling point will elute early from the GC column. Compounds with high boiling point will elute later. Analytes interact with the GC column. If the column is non-polar, analytes with high polarity will travel faster through the column while more non-polar compounds will be retained.
  90. 90. Isothermal GC Isothermal GC is not a good choice for samples containing analytes with varying boiling points. For example, petroleum products, silylated amino acids, methylated fatty acids. In an isothermal GC analysis, the column temperature is constant. Fast eluting compounds may then appear as overlapping peaks and late eluting compounds will have long retention time and broad peak shape. http://ull.chemistry.uakron.edu/chemsep/slide.php?Chapt er=/chemsep/GC/&Last=100&Slide=56
  91. 91. Temperature programming By varying column temperature over time, analytes with different boiling points can be separated. Analysis time can be optimised. http://ull.chemistry.uakron.edu/chemsep/slide.php?Chapt er=/chemsep/GC/&Last=100&Slide=56
  92. 92. www.chem.agilent.com
  93. 93. Temperature ramping http://analysciences.com 93

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