Gas chromatography by Devi manozna


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gives a miniature explantion of important points in gas chromatography

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Gas chromatography by Devi manozna

  2. 2. INTRODUCTION “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of the phases constituting a of large surface area, the other being a that percolates through or along the stationary bed.” (Ettre & Zlatkis, 1967, “The Practice of Gas Chromatography) Chromatography dates to 1903 in the work of the Russian scientist, Mikhail Semenovich Tswett. German graduate student Fritz Prior developed solid state gas chromatography in 1947.
  3. 3. CHROMATOGRAPHY MEANS………. It is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.
  4. 4. BASED ON THE PRINCIPLE OF SEPARATION, IT IS CLASSIFIED AS: ADSORPTION CHROMATOGRAPHY: In this the components of mixture are separated based on their relative affinities towards the stationary phase. This method is used for a larger quantity of mixtures . (e.g: TLC,CC,GSC) PARTITION CHROMATOGRAPHY: In this the components of mixture are separated based on the relative solubilities or partition co-efficient. This method is used for a smaller quantity of mixture. This method is an accurate method than the GSC. (e.g) GLC, HPLC).
  6. 6. Gas Chromatography Gas Chromatography is a chromatographic technique in which the mobile phase is a gas. GC is currently one of the most popular methods for separating and analyzing compounds. This is due to its high resolution, low limits of detection, speed, accuracy and reproducibility. GC can be applied to the separation of any compound that is either naturally volatile (i.e., readily goes into the gas phase) or can be converted to a volatile derivative. This makes GC useful in the separation of a number of small organic and inorganic compounds.
  7. 7. FATHER OF GC Mr.A.J MARTIN 1903 - Mikhail Tswett separated plant pigments using paper chromatography liquid-solid chromatography 1930’s - Schuftan & Eucken use vapor as the mobile phase gas solid chromatography Archer John Porter Martin, who was awarded the Nobel Prize for his work in developing liquid–liquid (1941) and paper (1944) chromatography, laid the foundation for the development of gas chromatography and he later produced liquid-gas chromatography (1950). Erika Cremer laid the groundwork, and oversaw much of Prior's work.
  8. 8.  In gas chromatography (GC), the sample is vaporized and injected onto the head of a chromatographic column. Elution is brought about by the flow of an inert gaseous mobile phase.  The mobile phase does not interact with molecule of the analyte; its only function is to transport the analyte through the column.  Gas-liquid chromatography is based upon the partition of the analyte between a gaseous mobile phase and a liquid phase immobilized on the surface of an inert solid.
  9. 9. Factors which influence the GC separation Volatility of compound: Low boiling (volatile)components will travel faster through the column than high boiling point components. Polarity of compounds: Polar compounds will move more slowly, especially if the column is polar. Column temperature: Raising the column temperature speeds up all the compounds in a mixture.
  10. 10. Column packing polarity: Usually, all compounds will move slower on polar columns, but polar compounds will show a larger effect. Flow rate of the gas through the column: Speeding up the carrier gas flow increases the speed with which all compounds move through the column. Length of the column: The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation.
  11. 11. Separations To detector Time 1 Time 2 Time 3
  13. 13. Schematic diagram of gas chromatograph
  14. 14. INSTRUMENTATION A. Carrier gas B. Flow regulator C. Injector D. Column E. Detector F. Integrator G. Display system - printer/monitor Thermostated oven Integrator
  15. 15. Working Principle The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column. First, a vaporized sample is injected onto the chromatographic column. Second, the sample moves through the column through the flow of inert gas. Third, the components are recorded as a sequence of peaks as they leave the column.
  16. 16. Chromatographic Separation In the mobile phase, components of the sample are uniquely drawn to the stationary phase and thus, enter this phase at different times. The parts of the sample are separated within the column. Compounds used at the stationary phase reach the detector at unique times and produce a series of peaks along a time sequence.
  17. 17. Chromatographic Analysis The number of components in a sample is determined by the number of peaks. The amount of a given component in a sample is determined by the area under the peaks. The identity of components can be determined by the given retention times.
  18. 18. MOBILE PHASE (GAS)
  19. 19. CARRIER GAS  The main purpose of a carrier gas is to carry the sample through the column. Secondarily, it should be providing a suitable matrix for the detector to measure the sample component.  This is the mobile phase and should be pure gas so as not to react with the column or analyte. Gas is usually He, Ar, N2, or H2. Choice will depend on the type of detector used. He and H2 give better resolution (smaller plate height) than N2. Pressure is also important and as expected the system comes with regulators. Can you find where in GC equations that are dependent on pressure?
  20. 20. POINTS TO REMEMBER FOR CARRIER GAS Oxygen and water impurities can chemically attack the liquid phase of the column and destroy it. Trace water can absorb the column contaminants and produce high detector background or ghost peaks. Trace hydro carbons can cause high detector background with FID’s and limit detect ability
  21. 21. REQUIREMENTS OF A CARRIER GAS  Inertness  Suitable for the detector  High purity(minimum of 99.995%)  Easily available  Cheap  Should not cause the risk of fire  Should give best column performance
  22. 22. SUB CLASSIFICATION OF GAS CHROMATOGRAPHIC TECHNIQUES (depending on stationary phase)  GSC : gas solid chromatography In this type the stationary phase used would be a solid .  GLC : gas liquid chromatography In this type the stationary phase used would be liquid .
  23. 23. 1.) Gas-solid chromatography (GSC) - same material is used as both the stationary phase and support material Magnified Pores in activated carbon
  24. 24. Solids usually , traditionally run in packed columns These solid should have small and uniform particle size (example : 80/100 mesh range) SOMECOMMON GC ADSORBENTS : •Silica gel •Activated alumina •Zeolite molecular sieves •Carbon molecular sieves •Porous polymers •Tenax polymers Some of these solids have been coated on the inside wals of capillary columns and these are called as SUPPORT COATED OPEN TUBULAR COLUMNS (SCOT)
  25. 25. ADVANTAGES: - long column lifetimes - ability to retain and separate some compounds not easily resolved by other GC methods ‚ geometrical isomers ‚ permanent gases DISADVANTAGE: - very strong retention of low volatility or polar solutes - catalytic changes that can occur on GSC supports - GSC supports have a range of chemical and physical environments ‚ different strength retention sites ‚ non-symmetrical peaks ‚ variable retention times
  26. 26. 2.) Gas-liquid chromatography (GLC) - stationary phase is some liquid coated on a solid support. - over 400 liquid stationary phases available for GLC many stationary phases are very similar in terms of their retention properties - material range from polymers (polysiloxanes, polyesters, polyethylene glycols) to fluorocarbons, molten salts and liquid crystals
  27. 27. The Stationary Phase Desirable properties for the immobilized liquid phase in a gas-liquid chromatographic column include: (1) low volatility (ideally, the boiling point of the liquid should be at 100oC higher than the maximum operating temperature for the column) (2) Thermal stability (3) chemical inertness (4) solvent characteristics such that k` and  values for the solutes to be resolved fall within a suitable range. The retention time for a solute on a column depends upon its distribution constant which in turn is related to the chemical nature of the stationary phase.
  28. 28. The Stationary Phase To have a reasonable residence time in the column, a species must show some degree of compatibility (solubility) with the stationary phase. Here, the principle of “like dissolves like” applies, where “like” refers to the polarities of the solute and the immobilized liquid. Polar stationary phases contain functional groups such as –CN,--CO and –OH. Hydrocarbon-type stationary phase and dialkyl siloxanes are nonpolar, whereas polyester phases are highly polar. Polar solutes include alcohols, acids, and amines; solutes of medium polarity include ethers, ketones, and aldehydes.
  29. 29. Water Carboxylic acids Amides Alcohol/amines Esters/aldehydes/ketone s Ethers Halides Aromatic hydrocarbons Olefins Aliphatic hydrocarbons Polar Non-polar Polarity of Stationary Phase
  30. 30. CHOICE OF CARRIER GAS: 31 Advantages Disadvantages Hydrogen  Cheap  Gives the most time efficient separation  Still very efficient at high gas velocities i.e.. 60 cm/ sec  Can form an explosive mixture with air  Some industries in some countries have regulated AGAINST the use of hydrogen  is a reductive gas Helium  Very inert, will not react with analytes  Gives a very time efficient separation  Non flammable  Expensive  A non-replenishable resource Nitrogen  Cheap  Very inert, will not react with analytes  Non flammable  Very slow velocity to achieve good efficiency  Narrow range for maximum efficiency
  31. 31. Flow regulators & Flow meters 18-Jan-1532 Flow regulators – To deliver the gas with uniform pressure or flow rate. Flow meter – To measure the flow rate of carrier gas. TYPES OF FLOW METERS 1. Rota meter . 2. Soap bubble flow meter.
  32. 32. 1. ROTAMETER 18-Jan-1533
  33. 33. 34 Soap bubble flow meter Aqueous solution of soap or detergent  soap bubble meter  soap bubbles formed indicates the flow rate. Glass tube with a inlet tube at the bottom.  Rubber bulb-----store soap solution  When the bulb is gently pressed of soap solution is converted into a bubble by the pressure of a carrier gas &travel up
  34. 34. 35 inlet tube
  35. 35. Sample injection systems
  36. 36. Inlets are points of sample introduction Ideal sample inlets for column type : 1. PACKED COLUMNS :  Flash vaporizer  on- column 2. CAPILLRY COLUMNS:  Split injector  Splitless injector  On-column
  37. 37. Mode of Injections
  38. 38. SPLITLESS INJECTOR In this case the sample has to be diluted in a volatile solvent and around 1-5ml is injected in the heated injection port. Septum perge is essential in split less injection ADVANTAGES OF SPLITLESS INJECTION •Improved sensitivity over a slit injector . DIS –ADVANTAGES OF SPLITLESS INJECTOR •its time consuming •Initial temperature and time of opening the split valve needs to be optimized . •Not well suited for volatile compounds (boiling points of peaks of interest to be about 30 degrees centigrade higher than the solvent)
  39. 39. SPLIT INJECTOR It’s the oldest , simplest and easiest injection technique. ADVANTAGES OF SPLIT INJECTION •High resolution seperations •Neat samples can be introduced •Dirty samples can be introduced by putting a deactivated glass wool plug in the liner to trap non volatile components. DIS-ADVANTAGES •Trace analysis is limited. •Process sometimes discriminates between high molecular weight solutes so that the sample entering the column is not represntative of sample injected .
  40. 40. OTHER TYPES OF INLETS •DIRECT INJECTION : It involves in injecting a small sample into a glass liner where vapors are carried directly into the column . •ON-COLUMN INJECTION : It deals with inserting precisely aligned needle into the capillary column and making injections inside the column. •FLASH VAPOURIZER : It involves in heating the port to a temperature well above the boiling point to ensure rapid volatilization . •STATIC HEADSPACE : it concentrates the vapour over a solid or liquid sample (best chosen for residual solvent analysis)
  41. 41. Sample Injection System  Column efficiency requires that the sample be of suitable size and be introduced as a “plug” of vapor; slow injection of oversized samples causes band spreading and poor resolution.  The most common method of sample injection involves the use of microsyringe to inject a liquid or gaseous sample through a self-sealing, silicone- rubber diaphragm or septum into a flash vaporizer port located at the head of the column (the sample port is ordinarily about 50oC above the boiling point of the least volatile component of the sample).
  42. 42. Sampling place
  43. 43. 46
  44. 44. COLUMNS
  45. 45. GC Columns Capillary columnsPacked columns •Typically a glass or stainless steel coil. •1-5 total length and 5 mm inner diameter. • Filled with the st. ph. or a packing coated with the •Thin fused-silica. •Typically 10-100 m in length and 250 mm inner diameter. •St. ph. coated on the inner surface. •Provide much higher separation eff. •But more easily overloaded by too much sample.
  46. 46. GAS CHROMATOGRAPHIC COLUMNS Open tubular Columns Open tubular, or capillary, columns are of two basic types ,namely  WALL—COATED OPEN TUBULAR (WCOT) : These are simple capillary tubes coated with a thin layer of the stationary phase. In support-coated open tubular columns, the inner surface of the capillary is lined with a thin film (~30 m) of a support material, such as diatomaceous earth. This type of column holds several times as much stationary phase as does a wall-coated column and thus has a greater sample capacity.
  47. 47.  SUPPORT-COATED OPEN TUBULAR (SCOT) : These are surface coated open tubular columns. These are no longer available in fused silica  PLOT : These are the porous layer open tubular column which is less than 5% of all gas chromatographic use these days
  48. 48. Packed Columns Packed columns are fabricated from glass, metal (stainless steel, copper, aluminum), or Teflon tubes that typically have lengths of 2 to 3 m and inside diameters of 2 to 4 mm. These tubes are densely packed with a uniform, finely divided packing material, or solid support, that is coated with a thin layer (0.05 to m) of the stationary liquid phase. In order to fit in a thermostating oven, the tubes are formed as coils having diameters of roughly 15 cm.
  49. 49. 3.SCOT COLUMNS (SUPPORT COATED OPEN TUBULAR COLUMN)  Improved version of Golay / Capillary columns, have small sample capacity  Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column  Then coated with a thin film of liquid phase
  50. 50. capillary PACKED • Packed • Capillary
  51. 51. Solid Support Materials The most widely used support material is prepared from naturally occurring diatomaceous earth, which is made up of the skeletons of thousands of species of single-celled plants (diatoms) that inhabited ancient lakes and seas. Such plants received their nutrients and disposed of their wastes via molecular diffusion through their pores. As a consequence, their remains are well-suited as support materials because gas chromatography is also based upon the same kind of molecular diffusion.
  52. 52. COLUMNS: Columns in GC are two types 1) packed column 2) capillary column Packed column (capillary column) Open tubular column 56
  53. 53. Liquid phase Solid support coated with liquid phase Porous Adsorbent Porous Layer Open Tubular (PLOT) Wall-coated Open Tubular (WCOT) Support-coated Open Tubular (SCOT) Types of open tubular column: 57
  54. 54. Typical gc dual column
  55. 55. The oven Inside here Column 59
  56. 56. TEMPERATURE CONTROL DEVICES  Preheaters: Convert sample into its vapor form, present along with injecting devices  Thermostatically controlled oven: Temperature maintenance in a column is highly essential for efficient separation.  Two types of operations:  Isothermal programming:- this method is generally used for all compounds  Linear programming:- this method is efficient for separation of complex mixtures
  57. 57. Temperature Control • Isothermal • Gradient 0 40 80 120 160 200 240 0 10 20 30 40 50 60 Time (min) Temp(degC) Instrumentation - Oven
  58. 58. Column Ovens  Column temperature is an important variable that must be controlled to a few tenths of a degree for precise work. Thus, the column is ordinarily housed in a thermostated oven. The optimum column temperature depends upon the boiling point of the sample and the degree of separation required.  Roughly, a temperature equal to or slightly above the average boiling point of a sample results in a reasonable elution time (2 to 30 min). For samples with a broad boiling range, it is often desirable to employ temperature programming, whereby the column temperature is increased either continuously or in steps as the separation proceeds.
  59. 59. The thermostated oven serves to control the temperature of the column within a few tenths of a degree to conduct precise work. The oven can be operated in two manners: isothermal programming or temperature programming. In isothermal programming, the temperature of the column is held constant throughout the entire separation. The optimum column temperature for isothermal operation is about the middle point of the boiling range of the sample. However, isothermal programming works best only if the boiling point range of the sample is narrow. If a low isothermal column temperature is used with a wide boiling point range, the low boiling fractions are well resolved but the high boiling fractions are slow to elute with extensive band broadening. If the temperature is increased closer to the boiling points of the higher boiling components, the higher boiling components elute as sharp peaks but the lower boiling components elute so quickly there is no separation.
  61. 61. DETECTION SYSTEMS Characteristics of the Ideal Detector: The ideal detector for gas chromatography has the following characteristics: 1. Adequate sensitivity 2. Good stability and reproducibility. 3. A linear response to solutes that extends over several orders of magnitude. 4. A temperature range from room temperature to at least 400oC.
  62. 62. Characteristics of the Ideal Detector 5. A short response time that is independent of flow rate. 6. High reliability and ease of use. 7. Similarity in response toward all solutes or a highly selective response toward one or more classes of solutes. 8. Nondestructive of sample.
  63. 63. Few mostly used detectors 1) FID ( flame ionization detector ) 2) TCD ( thermal conductivity detector ) 3) ECD ( electron capture detector ) 4) FPD ( flame photometric detector ) 5) HID ( helium ionization detector ) 6) NPD ( nitrogen-phosphorus detector ) 7) PID ( photo ionization detector ) 8) TID ( thermionic ionization detector ) 9) CCD ( catalytic combustion detector ) 10) NPD/DELCD ( combination NPD and dry electrolytic conductivity detector ) 11) FID/DELCD ( combination FID and dry electrolytic conductivity detector ) 12) FID/FPD ( combination FID and FPD ) 13) Dual FPD ( dual wavelength FPD for both sulfur and phosphorus ) 14) FID dual FPD ( dual FPD plus FID combination )
  64. 64. 1. Flame Ionization Detector (FID)
  65. 65.  Effluent from the column is passes through a small burner fed H2 and air.  Combustion of the organic compounds flowing through the flame creates charged particles (ionic intermediates are responsible for generating a small current between the two electrodes).  The burner, held at ground potential acts as one of the electrodes.  The second electrode called as a collector, is kept at a positive voltage & collects the current that is generated.  Signal amplified by electrometer that generate measurable voltage. How does FID works?
  66. 66. 1. FID  Advantages  Rugged  Sensitive (10-13 g/s)  Wide dynamic range (107)  Signal depends on number of C atoms in organic analyte - mass sensitive not concentration sensitive.
  67. 67.  Disadvantages  Weakly sensitive to carbonyl, amine, alcohol & amine groups.  Not sensitive to non-combustibles analyte such as H2O, CO2, SO2, NOx.  Destructive method.
  68. 68. 2. Thermal Conductivity Detector (TCD)
  69. 69. 2. TCD  A universal detector.  Has a moderate sensitivity.  Less satisfactory with carrier gas whose conductivities closely resemble those of most sample components.
  70. 70.  Consists of an electrically heated source whose temperature at constant electric power depends on the thermal conductivity of the surrounding gas.  The electrical resistance of this element (fine platinum, gold or tungsten wire or thermistor) depends on the thermal conductivity of the gas.  Operating principles relies on the thermal conductivity of the gaseous mixture.  The thermal conductivity affects the resistance of the thermistor as a function of temperature. How does TCD works?
  71. 71.  Twin detectors are normally used One located ahead of sample injection chamber and the other immediately beyond the column or alternatively, the gas stream can be split.  When the solutes elutes from the column there is a change in the composition of the mobile phase thus in the thermal conductivity.  this results in a deviation from thermal equilibrium, causing a variation in the resistance of one the filament.  this variation is proportional to the concentration of the analyte, provided its concentration in the mobile phase is low. How does TCD works?
  72. 72. 2. TCD  Advantages  Simple  Large linear dynamic range  Responds to both organic and inorganic species  Nondestructive; permits collection of solutes after detection.  Disadvantage  Relatively low sensitivity.
  73. 73. 3. Electron Capture Detector (ECD)
  74. 74.  Sample elute from a column is passed over a radioactive β emitter, usually nickel-63.  An electron from the emitter causes ionization of carrier gas (often N2) and the production of a burst of electrons.  In the absence of organic species, a constant standing of current.  In the presence of organic molecules containing electronegative functional groups that tend to capture electrons, the current decreases markedly. How does ECD works?
  75. 75. 4.) Electron Capture Detector (ECD) - radiation-based detector - selective for compounds containing electronegative atoms, such as halogens Process - based on the capture of electrons by electronegative atoms in a molecule - electrons are produced by ionization of the carrier gas with a radioactive source ‚ 3H or 63Ni - in absence of solute, steady stream of these electrons is produced - electrons go to collector electrode where they produce a current - compounds with electronegative atoms capture electrons, reducing current
  76. 76.  Most widely used for environmental samples  Advantages  Selectively responds to halogen-containing organic compounds such as pesticides and polychlorinated biphenyls.  Highly sensitive towards halogens, peroxides, quinones and nitro groups.  Disadvantages  Insensitive to functional groups such as amines, alcohols and hydrocarbons.
  77. 77. 3.) Nitrogen-Phosphorus Detector (NPD) - used for detecting nitrogen- or phosphorus containing compounds - also known as alkali flame ionization detector or thermionic detector Process - same basic principal as FID - measures production of ions when a solute is burned in a flame - ions are collected at an electrode to create a current - contains a small amount of alkali metal vapor in the flame - enhances the formation of ions from nitrogen- and phosphorus- containing compounds Alkali Bead
  79. 79. 3.) Nitrogen-Phosphorus Detector (NPD) advantages: - useful for environmental testing ‚ detection of organophosphate pesticides - useful for drug analysis ‚ determination of amine-containing or basic drugs - Like FID, does not detect common mobile phase impurities or carrier gases - limit of detection: NPD is 500x better than FID in detecting nitrogen and phosphorus- containing compounds - NPD more sensitive to other heterocompounds, such as sulfur-, halogen-, and arsenic- containing molecules disadvantage: - destructive detector - NPD is less sensitive to organic compounds compared to FID
  80. 80. Atomic Emission Detectors (AED) The atomic emission detector is available commercially. In this device the eluent is introduced into a microwave-energized helium plasma that is coupled to a diode array optical emission spectrometer. The plasma is sufficiently energetic to atomize all of the elements in a sample and to excite their characteristic atomic emission spectra.
  81. 81. Thermionic Detectors (TID) The thermionic detector is selective toward organic compounds containing phosphorus and nitrogen. Its response to a phosphorus atom is approximately ten times greater than to a nitrogen atom and 104 to 106 larger than a carbon atom. Compared with the flame ionization detector, the thermionic detector is approximately 500 times more sensitive to phosphorus- containing compounds and 50 times more sensitive to nitrogen bearing species. These properties make thermionic detection particularly useful for detecting and determining the many phosphorus-containing pesticides.
  82. 82. Element-selective detectors 18-Jan-1588 It includes :  Thermionic ionization detector (TID).  Give selective response to phosphorus and /or nitrogen containing compounds.  Use- For determination of pesticides like malathion and parathion.  Flame photometric detector  For the determination of compounds containing sulphur and phosphorus.
  83. 83. RECORDERS & INTEGRATORS 18-Jan-1589 RECORDERS Used to record the responses obtained from detectors after amplification. Retention time can be found out. INTEGRATORS Record the individual peaks with retention time , height and width of peaks , peak area , percentage of area etc:-
  84. 84. Analysis of Output  Less than ideal spectral peaks may indicate less than ideal analytical procedures or equipment. The technician can readily observe whether the output exhibits unsatisfactory results. Ideally, the spectral peaks should be symmetrical, narrow, separate (not overlapping), and made with smooth lines. GC evidence may be suspect if the peaks are broad, overlapping, or unevenly formed. If a poorly shaped peak contains a steep front and a long, drawn-out tail, this may indicate traces of water in the specimen.  The GC technician should inject the specimen into the septum rapidly and smoothly to attain good separation of the components in a specimen. If the technician injects the specimen too slowly, the peak may be broad or overlap. A twin peak may result from the technician hesitating during the injection. A smoothly performed injection, without abrupt changes, should result in a smoothly formed peak. A twin peak may also indicate that the technician injected two specimens consecutively.
  88. 88. ADVANTAGES OF G.C  Very high resolution power, complex mixtures can be resolved into its components by this method.  Very high sensitivity with TCD, detect down to 100 ppm  It is a micro method, small sample size is required  Fast analysis is possible, gas as moving phase- rapid equilibrium  Relatively good precision & accuracy  Qualitative & quantitative analysis is possible
  89. 89. DIS ADVANTAGES Response Factor The size of a spectral peak is proportional to the amount of the substance that reaches the detector in the GC instrument. No detector responds equally to different compounds. Results using one detector will probably differ from results obtained using another detector. Therefore, comparing analytical results to tabulated experimental data using a different detector does not provide a reliable identification of the specimen. A "response factor" must be calculated for each substance with a particular detector. A response factor is obtained experimentally by analyzing a known quantity of the substance into the GC instrument and measuring the area of the relevant peak. The experimental conditions (temperature, pressure, carrier gas flow rate) must be identical to those used to analyze the specimen. The response factor equals the area of the spectral peak divided by the weight or volume of the substance injected. If the technician applies the proper technique, of running a standard sample before and after running the specimen, determining a response factor is not necessary.
  90. 90. Worn Septum An injection port septum should last between 100 and 200 injections. Higher injection port temperatures shorten the septum's lifespan. A leaking septum adversely affects the GC instrument's sensitivity. If a portion of the specimen leaks back out of the septum, the amount of the specimen is not recorded. This event makes any eventual quantitative result erroneous. If air should leak into the injection port through a worn septum, the oxygen and water contained in air may skew the results. Any oxygen may react with the specimen components. If this happens, the GC instrument will provide results indicating the presence of this unintended reaction product, instead of the original compounds present in the specimen vial. Any water in the column adversely affects the GC instrument's ability to separate components.
  91. 91. Injection Port Temperature The temperature of the GC injection port must be high enough to vaporize a liquid specimen instantaneously. If the temperature is too low, separation is poor and broad spectral peaks should result or no peak develops at all. If the injection temperature is too high, the specimen may decompose or change its structure. If this occurs, the GC results will indicate the presence of compounds that were not in the original specimen. Residual Impurities Ideally, all components of a specimen elute completely from the GC column. If any substance remains inside the column, the substance may elute during subsequent analyses with other specimens. This may result in an unexpected peak in the output. The peak produced should be broad.
  92. 92. Carrier Gas If the GC instrument uses hydrogen for the carrier gas, the technician must consider whether the hydrogen may react with any of the compounds present in the specimen. If the hydrogen does react, a broad peak will result. When using a thermal conductivity detector, care should be taken as a false peak may occur if the carrier gas's thermal conductivity is in the range of the thermal conductivity of any compound in the specimen. An unstable carrier gas flow rate may produce a drifting baseline and false broad peaks. A carrier gas should be pure. Regular changing of the gas filter should prevent significant impurities. Crucial Factors GC analysis is highly reliable if the instrument is properly maintained, the technician follows proper procedures, and the interpretation of the results is competent. While some factors rarely affect GC analysis, some factors are absolutely essential for the use of reliable GC evidence. In all cases a technician must process a standard sample containing a verified composition identical to the presumed contents of the collected specimen. This standard sample
  93. 93. Any output from the collected specimen that does not match the standard sample is inconclusive. If tabulated reference data exists for the relevant conditions, the specimen data must match the reference data. If advance notice of GC testing is available, an adverse party should observe the procedure. If a retained consultant or the knowledgeable attorney observes the technician's use of the GC instrument, important information can be recorded. The technician's preparation of the specimen and the subsequent injection can be observed for errors or malfunctioning equipment. The observer should record the instrument's make, model, serial number, injection temperature, column temperature, carrier gas flow rates and pressure, identify the type of detector used, and observe any manipulation of the data by use of a computer. Ensure that the technician properly starts measuring the time at injection and records the time of elution. Any discrepancy in the time will produce an erroneous retention time. If the procedure can not be observed, the adverse party should seek all pertinent information (experimental
  94. 94. Headspace gas chromatography analysis  Headspace GC (HSGC) analysis employs a specialized sampling and sample introduction technique, making use of the equilibrium established between the volatile components of a liquid or solid phase and the gaseous / vapor phase in a sealed sample container. Aliquots of the gaseous phase are sampled for analysis.
  95. 95. Pyrolysis gas chromatography  Pyrolysis GC (PGC) is used principally for the identification of non-volatile materials, such as plastics, natural and synthetic polymers, drugs and some microbiological materials. The thermal dissociation and fragmentation of the sample produces a chromatogram which is a fingerprint for that sample. The small molecules produced in the pyrolysis reaction are frequently identified using a GC-MS system and information on molecular structure for identification is also obtained.
  96. 96. Food analysis  Analysis of foods is concerned with the assay of lipids, proteins, carbohydrates, preservatives, flavours, colorants and texture modifiers, and also vitamins, steroids, drugs and pesticide residues and trace elements. Most of the components are non- volatile and although HPLC is now used routinely for much food analysis, GC is still frequently used. For examples, derivatization of lipids and fatty acid to their methyl esters(FAMEs), of proteins by acid hydrolysis followed by esterification (N-propyl esters) and of carbohydrates by silylation to produce volatile samples suitable for GC analysis.
  97. 97. Food and Cancer  Chemicals that can cause cancer have a wide variety of molecular structures and include hydrocarbons, amines, certain drugs, some metals and even some substances occurring naturally in plants and molds. In this way, many nitrosamines have carcinogenic properties and these are produced in a number of ways such as cigarette smoke. GC can be used to identify these nitro-compounds in trace quantities.
  98. 98. Drugs  There are still numerous GC applications involving both quantitative and qualitative identification of the active components and possible contaminants, adulterants or characteristic features which may indicate the source of the particular sample. Forensic analysis frequently users GC to characterize drugs of abuse, in some cases the characteristic chromatographic fingerprint gives an indication of the source of manufacture of the sample or worldwide source of a vegetable material such as cannabis.  Analytical procedures, chromatographic methods and retention data are published for over 600 drugs, poisons and metabolites. These data are extremely useful for forensic work and in hospital pathology laboratories to assist the identification of drugs.
  99. 99. Metal chelates and inorganic materials  Although inorganic compounds are generally non- volatile, GC analysis can be achieved by converting the metal species into volatile derivatives. Only some metal hydrides and chlorides have sufficient volatility for GC.  Organometallics other than chelates, which can be analyzed directly, include boranes, silanes, germanes, organotin and lead compounds.
  100. 100. Environmental analysis  Environmental pollution is an age-old trademark of man and in recent years as technology has progressed, populations have increased and standards of living have improved. So the demands on the environment have increased, with all the attendant problems for the ecosystems. Combustion of fossil fuel, disposal of waste materials and products, treatment of crops with pesticides and herbicides have all contributed to the problems. Technological developments have enabled man to study these problems and realize that even trace quantities of pollutants can gave detrimental effects on health and on the stability of the environment. There is a vast amount of literature on the use of GC for studying a wide variety of these problems.
  101. 101.  Every year many new substances are synthesized that differ radically from the natural products that exist in biosystems. The Environmental Protection Agency is empowered to control water pollution and the production, use and disposal of toxic chemicals. It follows that detailed studies must be made of their effect on the environment and their method of movement through the ecosystem. Many of the compounds are not biodegradable and will thus progressively pollute the environment. There are a number of tragic examples of which DDT (dichlorodiphenyltrichloroethane) and the PCBs (polychlorinated biphenyls) are well known instances. The materials of interest are present in environmental samples at very low concentrations and are often to be found among a myriad of other compounds from which they must be separated and identified. It follows that GC, with its inherent high sensitivity and high separating power, is one of the more commonly used techniques in the analysis of environmental samples.
  102. 102. Derivatisation of sample Treat sample to improve the process of separation by column or detection by detector. They are 2 types Precolumn Derivatisation Components are converted to volatile & thermo stable derivative. Conditions - Pre column derivatisation Component ↓ volatile Compounds are thermo labile
  103. 103. Derivatisation Post column Derivatisation  Improve response shown by detector  Components ionization / affinity towards electrons is increased Pretreatment of solid support To overcome tailing Generally doing separation of non polar components like esters, ethers… Techniques: 1. use more polar liquid S.P 2. Increasing amt. of liquid phase 3.Pretreatment of solid support to remove active sites.
  104. 104. Kovat’s retention index Kovat’s retention index (also known as Kovat’s index, retention index; plural retention indices) is a concept used in gas Chromatography to convert retention times into system- independent constants. The index is named after the Hungarian-born Swiss chemist Ervin Kováts who outlined this concept during the 1950s while performing research into the composition of the essential oils . The retention index of a certain chemical compound is its retention time normalised to the retention times of adjacently eluting n-alkanes
  105. 105. While retention times vary with the individual chromatographic system (e.g. with regards to column length, film thickness, diameter, carrier gas , Velocity and pressure, and void time), the derived retention indices are quite independent of these parameters and allow comparing values measured by different analytical laboratories under varying conditions. Tables of retention indices can help identify components by comparing experimentally found retention indices with known values
  106. 106. The method takes advantage of the linear relationship between the values of and the number of carbon atoms in a molecule. The value of Kovat’s index is usually represented by I in mathematical expressions. Its applicability is restricted to organic compounds. For isothermal chromatography, the Kovat’s index is given by the equation Where: Kovat’s retention index, the number of carbon atoms in the smaller n- alkane,
  107. 107. For temperature programmed chromatography, the Kovat’s index is given by the equation Where: Kovat’s retention index the number of carbon atoms in the smaller n- alkane, the number of carbon atoms in the larger n- alkane, the retention time.
  108. 108. PARAMETERS USED IN GC  Retention time (Rt)  Retention volume (Vr)  Separation factor (S)  Resolution (R)  Theoretical Plate
  109. 109. Parameters used in GC Retention time (Rt) It is the difference in time b/w the point of injection & appearance of peak maxima. Rt measured in minutes or seconds (or) It is the time required for 50% of a component to be eluted from a column Retention volume (Vr) It is the volume of carrier gas which is required to elute 50% of the component from the column. Retention volume = Retention time ˣ Flow rate
  110. 110. Separation factor (S) : Ratio of partition co-efficient of the two components to be separated. If more difference in partition co-efficient b/w two compounds, the peaks are far apart & S Is more. If partition co-efficient of two compounds are similar, then peaks are closer Resolution (R) : The true separation of 2 consecutive peaks on a chromatogram is measured by resolution It is the measure of both column & solvent efficiencies R= 2d W1+W2
  111. 111. •Requires only very small samples with little preparation •Good at separating complex mixtures into components •Results are rapidly obtained (1 to 100 minutes) •Very high precision •Only instrument with the sensitivity to detect volatile organic mixtures of low concentrations •Equipment is not very complex (sophisticated oven) • •Fast analysis,Typically minutes (even sec.) Can be automated • Small samples (µl or µg needed) • High resolution Record: N ~ 1.3 x 106 •• Reliable, relatively simple and cheap (~ $20,000) • Non-destructive and Allows on-line coupling, e.g. to MS • Sensitive detectors (easy ppm, often ppb) • Highly accurate quantification (1-5% RSD) ADVANTAGES OF GAS CHROMATOGRAPHY
  112. 112. DISADVANTAGES OF GC • Limited to volatile samples – T of column limited to ~ 380 °C – Need Pvap of analyte ~ 60 torr at that T – Analytes should have b.p. below 500 °C • Not suitable for thermally labile samples • Some samples may require intensive preparation – Samples must be soluble and not react with the column • Requires spectroscopy (usually MS) to confirm the peak identity
  113. 113. ADVANCES IN GC - MINI GC