Gas chromatography GC

A separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column.
Separating mixtures of gases or volatile materials based primarily on their physical properties.

1. Gas Chromatography

2. To every one who has helped us with support, new books, hard/soft ware And over the internet . S pecial thanks to Thermo Thanks

3. Contents Introduction Theory Hardware Analysis Applications

4. Introduction

5. Experience has shown that maximum value can be derived from a scientific instrument if there is one person who has a major responsibility for the instrument. Thermo recommend that you designate a key operator to manage the operation and maintenance of the TRACE system. also recommend that the key operator receive training . Declaration

6. A separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column. Separating mixtures of gases or volatile materials based primarily on their physical properties. It gives both: Quantitative, Qualitative Gas Chromatography

7. Theory

8. Theory Components of mixture carried in the mobile phase are differentially attracted to the stationary phase and thus Move through the stationary phase at different rates. Chromatographic separation involves the use of:

9. Boiling points is The number one factor to consider in separation of compounds on the GCs. Differences in polarity of the compounds is only important if you are separating a mixture of compounds which have widely different polarities. Column temperature , the polarity of the column, flow rate, and length of a column are constant . Generally

10. Hardware

11. Hardware Manual Injection. Automatic Injections. Injectors Columns Oven Detectors Carrier Gas

12. Sample Preinjection Liquid introduction by syringe Most commonly used technique Different syringe types manual and automatic Other techniques and devices Sampling valve (gas or liquids) Head-space (liquids or solids) Purge and trap (water) Thermal desorption (solids) SPME (vapours, liquids or solids) Pyrolizer (solids)

13. Manual Injection Micro Syringes Are used to introduce a known volume of a liquid or gas samples. Adaptor Can be used to help control the volume injected.

14. Syringe injection Samples should be injected as a plug. Rapid and consistent injection is necessary in order to obtain Acceptable precision Injection Volume Liquids 0.1-10 µl is typical Gases 0.5- 5 ml is typical

15. Hot Needle injection Draw sample into syringe barrel. Draw 2-3 ul air into barrel. Inset needle into injection port and allow to heat for a few seconds. Rapidly inject sample and withdraw the needle. This insure that all sample is injected and the hot needle assists in solvent volatilization.

16. Normal injection Rinse the syringe with your sample at least twice . Draw up the suggested amount of sample into the syringe. Pull up about 1-2 ul of air. This will give a signal showing the beginning of the elution. Insert the needle through the injection port and septum in one movement. Quickly push the plunger.

17. Auto Injector AI 3000 Automatic Injection Sample loading Sample capacity: 8 Vials Max. vial capacity: 2 ml Injections/vial: 0-99 Syringes Standard sampling: 10 µl Micro volume sampling: 5 µl Injection parameters Max volume: 5 µl Min. Volume : 0.1 µl Increments: 0.1 µl steps Viscosity Delay: Yes/No

18. Auto Sampler AS 3000 Automatic Injection Sample loading Sample capacity: 105 Vials Max. vial capacity: 2 ml Injections/vial: 0-99 Syringes Standard sampling: 10 µl Micro volume sampling: 5 µl Injection parameters Max volume: 5 µl Min. Volume : 0.1 µl Increments: 0.1 µl steps Viscosity Delay: Yes/No

19. Large sample vial capacity Max 2 trays installed Simultaneously 1 , 2 and 2.5 ml vials (up to 300 vials) Syringe size: 5, 10, 100 and 250 µ l. Self recognized syringes and trays Washing station: 4 x 10ml or 2 x 100ml; multiple Solvent rinsing supported Rapid Mode: Allows to perform cleaning operations during GC run or cooling time. Suitable for Ultra Fast GC requirements, eliminating dead times between successive runs. Auto Sampler TriPlus

20. Head Space Head-space gas analysis Volatiles reach equilibrium at STP An aliquot of headspace is total in liquid and gas phase Conc . in gas proportional Conc. In liquid phase With headspace matrix left behind Clean and gentle chromatography Representative of sample

21. Determination of residual volatile solvents in pharmaceutical. Blood alcohol analysis. Determination of volatile hydrocarbons in waste water. Determination of di-acetyl and dichetones in beers. Monomers in polymers determination. Determination of out-gassing solvents from packages. Flavor profiles in drinking beverages or foods (cheese ). Head-Space Applications

22. Cold and Trap A llows the analysis of trace of compounds in large volumes of gaseous samples. The column can be cooled down to -150 °C by the action of the liquid nitrogen, So to trap (i.e. to reconcentrate) the volatile compounds contained in the sample. When the trapping is completed, the tube is heated with a fast temperature programming rate (°C/s), reaching temperatures up to 400 °C, so to transfer the trapped compounds into the analytical column.

23. GC Basic Components

24. The injection port Is a hollow, heated, glass-lined cylinder The injector is heated so that all components in the sample will be vaporized. 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. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. 1. Injector

25. Vaporizing. The liquid sample is evaporated prior to be transferred to the separation column. Split Spliless: SSL (permanently hot) Programmed Temperature Vaporizer: PTV Direct: PKD, PPKD (permanently hot, low resolution columns) Nonvaporizing. The liquid sample evaporates into the separation column (or a precolumn) Cold On Column: OC (permanently cool) Injection Techniques

26. Septa Ensure optimal performance of your GC instrument with bleed and temperature. Made of low-bleed silicone, have excellent mechanical properties, are ideal for demanding GC and GC-MS applications, and may be used reliably up to 400 °C. Septum must be replaced at least after 200 injections.

27. Liner Types

28. Liners Applications

29. Quick Liner Selections

30. Injector Types Split/ Splitless Injector On-Column Injector High Oven Temperature On-Column Injector Large Volume On-Column Injector Packed Column Injector Purged Packed Injector Programmable Temperature Vaporizing Injector

31. Split mode The split vent is open, part of the sample go into the column. When analyzing high concentration or neat samples. Yields the sharpest peaks if the split gas is properly mixed. Standard for capillary columns . 1. Split/ Splitless Injector ( vaporising injector)

32. Splitless mode The split vent is closed, most of the sample go into the column. When analyzing low concentration or diluted samples. Splitless times of ~ 1 minute are typical. Standard for capillary columns .

33. Non- vaporising injectors The sample is transferred as a liquid directly inside the column under the oven temperature control. The sample evaporation takes place inside the column. Sample doesn’t come in contact with any “column-external” device No vaporization step at a temperature above that of the column 2. On-Column Injector

34. Primary cooling Permanently active fan Keep the injector head at room temperature independently from oven temperature Secondary cooling Temporary stream of compressed air Avoid evaporation from the needle even at oven T close or slightly higher than BP Reduces the length of the flooded zones Avoid liquid backflow reducing the vapor pressure at the plug front More e efficient and rapid cooling of the injector base after a temperature ramp Cooling Time, The amount of time the secondary cooling stays on after the start the injection.

35. The OCI is the same as the regular cold on column except that a cooling jacket is installed in the GC oven around the head of the column. This allows a cold on column injection with a high oven temp. Temperature (°C) . The temp. checkbox checked for this option to be used. This specifies cooling jacket temp. during injection. Duration (min) the duration of the jacket cooling from the start of the run. 3. High Oven Temperature On-Column Injector

36. 4. Large Volume on column injector LV-SL injection overcomes the limitation of the maximum sample volume to 1-2 µL of classical splitless injection by exploiting the Concurrent Solvent Recondensation technique (CSR ). CSR technique allows injection of large volumes by combining a restricted evaporation rate with an accelerated sample transfer granted by the pressure surge generated by solvent evaporation and by the quick solvent recondensation in a precolumn. LV-SL has the following advantages: It is simpler because it allows injections of up to 50 µL in a conventional split/splitless injector without any special tuning of operating parameters; It is robust versus sample by-products or contaminants and extremely suitable for food matrices.

37. The PKD is used for injections with the sample vaporizing directly in the column. The PKD standard injector accepts metal or glass packed columns. The injector temperature may range from ambient to 400 °C. Injector temperature is regulated by a temperature controller in the GC CPU board and monitored by a platinum wire sensor. 5. Packed Column Injector

38. The Purged Packed (PPKD) column injector is a packed column injector with a septum purge. The PPKD standard injector accepts wide-bore capillary columns . The sample vaporizes in a liner and enters the wide-bore capillary column . The injector temperature is controllable from 50 °C to 400 °C. You should use high temperature septa with a longer life expectancy. 6. Purged Packed Injector

39. 7. Programmable Temperature Vaporizing Injector Can vary the temp. during injection in both split and Splitless. Can eliminate many of the unwanted effects , such as distillation of the sample within the needle and large vapor clouds inside injector chamber. In Constant Temperature (CT) mode, the PTV functions like a split/ splitless injector. Sample volumes are lower than when using S/SL injector because of the smaller PTV liner volume . Can analyze relatively dirty samples that can not be analyzed using a traditional on-column.

40. Good injector Capable to quantitative accept a broad volatility range . Low discrimination. To h andle dirty and clean matrices. With dirty matrices reduces sample clean-up and preserves the column. With clean matrices boosts sensitivity with LVI techniques . Extremely inert. Able to handle polar/active compounds. Provide optimum band shape.

41. GC Basic Components

42. The column Is where the chromatographic separation of the sample occurs. Several types of columns are available for different chromatographic applications: The heart of the system. It is coated with a stationary phase which greatly influences the separation of the compounds. 2. Column

43. Stationary phase Solid resin packed in a column , or Liquid supported by course paper or Inactive solid over which a mixture passes. Each component of the mixture differs in the way it adheres to this phase and therefore travels along it at a unique rate.

44. There are two types of packing employed in GC, the adsorbents and the supports, on which the stationary phase is coated. There are both inorganic and organic types of adsorbents. Alumina , in an activated form, is used to separate the permanent gases and hydrocarbons up to about pentane. Silica gel It is used for the separation of the lower molecular weight gases and some of the smaller hydrocarbons sulfur gases, hydrogen sulfide, sulfur dioxide and carbon disulfide. Synthetic zeolites used for the separation of hydrogen, oxygen, nitrogen, methane and carbon monoxide and also rare gasses. The Adsorbents

45. POLYSILOXANES The most common stationary phases. They are available in the greatest variety and are the most stable, robust and versatile. The most basic Poly Siloxane is the 100% methyl substituted POLYETHYLENE GLYCOLS They are less stable, less robust and have lower temperature limits than most Poly Siloxane. must be liquids under GC temperature conditions. Types of Stationary Phase

46. There have been a number of materials used as supports for packed GC columns including, Celite (a proprietary form of a diatomaceous earth), fire-brick (calcined Celite), fire-brick coated with metallic silver or gold, glass beads, Teflon chips and polymer beads. Polystyrene beads Inactive Solid Supports

47. Column Types Conventional 1/8-1/4 OD 6-8 feet in length Stainless steel or glass tube Preparative >1/4 OD > 10 feet in length Capillary 0.1- 0.5 ID 10 – 100 meters in length

48. Volatility of compound : Low boiling (volatile) components will travel faster through the column than will high boiling 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. Columns have lower and upper temperature limits. Factors Affecting Column Separations

49. 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.

50. Contaminated column. Damaged stationary phase. Different column temperature, carrier flow rate or column. Large changes in the sample concentration. Improper injector operation. Loss of Separation or Resolution

51. Column Damage Column breakage Column bleed Thermal damage Oxygen damage Chemical damage

52. GC Basic Components

53. The use of a temperature programmed for the column oven influences the separation process significantly and is used for optimization of time and peak separation. The oven must not be opened when the oven temperature is above room temperature. Never turn off the nitrogen flow unless the column and oven are at room temperature. 3. Oven

54. Temperature range 5 °C above ambient to 350 °C Temperature programming - up to six ramps Maximum run time - 999.99 minutes Temperature ramp rates - 0 to 120°C/min The oven accommodates one inlet, one detector, and one column. The Oven Capabilities

55. A multiple-ramp temperature program changes the oven temperature from an initial value to a final temperature, but with various rates, times , and temperatures in between. Multiple ramps can be programmed for temperature decreases as well as increases. Multiple-ramp temperature programs

56. GC Basic Components

57. 4. Detector The part of a gas chromatograph which signals the change in composition of the mixture passing through it.

58. Detector types Electron Capture Detector. Flame ionization Detector. Nitrogen Phosphors Detector. Thermal Conductivity Detector. Flame Photometric Detector. Photo ionization Detector. Electrolytic Conductivity Detector. Mass Spectrometric Detector.

59. Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of current loss is indirectly measured and a signal is generated. Selectivity: Halogens, nitrates, conjugated carbonyls Sensitivity: 0.1-10 pg (halogenated compounds); 1-100 pg (nitrates); 0.1-1 ng (carbonyls) Linear range: 1000-10000 Gases: Nitrogen or argon/methane Temperature: 300-400°C 1. Electron Capture Detector (ECD)

60. Mechanism: Compounds are burned in a hydrogen-air flame. Carbon containing compounds produce ions that are attracted to the collector . The No . 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 He or N2 Temperature: 250-300°C,and 400-450°C for high temp. 2. Flame ionization Detector (FID)

61. 3. Nitrogen Phosphors Detector (NPD) Mechanism: Compounds are burned in a plasma surrounding a rubidium bead supplied with hydrogen and air . Nitrogen and phosphorous 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 : Nitrogen and phosphorous Sensitivity : 1-10 pg Linear range: 104-10-6 Gases: Combustion - hydrogen and air; Makeup - Helium Temperature: 250-300°C

62. Mechanism: A 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. Selectivity: All compounds except for the carrier gas Sensitivity: 5-20 ng Linear range: 105-106 Gases: Makeup - same as the carrier gas Temperature: 150-250°C 4.Thermal Conductivity Detector (TCD)

63. Mechanism: Compounds are burned in a hydrogen-air flame. Sulfur and phosphorous containing compounds produce light emitting species (sulfur at 394 nm and phosphorous at 526 nm). A monochromatic filter allows only one of the wavelengths to pass. A photomultiplier tube is used to measure the amount of light and a signal is generated. A different filter is required for each detection mode. Selectivity: Sulfur or phosphorous containing compounds. Only one at a time. Sensitivity: 10-100 pg (sulfur); 1-10 pg (phosphorous) Linear range: Non-linear (sulfur); 103-105 (phosphorous) Gases: Combustion - hydrogen and air; Makeup - nitrogen Temperature: 250-300°C 5. Flame Photometric Detector (FPD)

64. Mechanism: Compounds eluting into a cell are bombarded with high energy photons emitted from a lamp. Compounds with ionization potentials below the photon energy are ionized. The resulting ions are attracted to an electrode, measured, and a signal is generated. Selectivity: Depends on lamp energy. Usually used for aromatics and olefins (10 eV lamp). Sensitivity: 25-50 pg (aromatics); 50-200 pg (olefins) Linear range: 105-106 Gases: Makeup - same as the carrier gas Temperature: 200°C 6. Photo ionization Detector (PID)

65. Mechanism: Compounds are mixed with a reaction gas and passed through a high temperature reaction tube. Specific reaction products are created which mix with a solvent and pass through an electrolytic conductivity cell. The change in the electrolytic conductivity of the solvent is measured and a signal is generated. Reaction tube temperature and solvent determine which types of compounds are detected. Selectivity: Halogens, sulfur or nitrogen containing compounds. Only one at a time. Sensitivity: 5-10 pg (halogens); 10-20 pg (S); 10-20 pg (N) Linear range: 105-106 (halogens); 104-105 (N); 103.5-104(S) Gases: Hydrogen (halogens and nitrogen); air (sulfur) Temperature: 800-1000°C (halogens), 850-925°C (N), 750-825°C (S) 7. Electrolytic Conductivity Detector (ELCD)

66. Mechanism: Compounds are bombarded with electrons (EI) or gas molecules (CI). then fragmented into characteristic charged ions or fragments. The resulting ions are focused and accelerated into a mass filter . mass filter selectively allows all ions of a specific mass to pass through to the electron multiplier. All of the ions of the specific mass are detected. The mass filter then allows the next mass to pass through while excluding all others. The mass filter scans stepwise through the designated range of masses several times per second. The total number of ions are counted for each scan. The abundance or number of ions per scan is plotted versus time to obtain the chromatogram (called the TIC). A mass spectrum is obtained for each scan which plots the various ion masses versus their abundance or number. Selectivity: compound gives fragments within mass range. Sensitivity: 1-10 ng (full scan); 1-10 pg (SIM) Linear range: 105-106 Gases: None Temperature: 250-300°C (transfer line), 150-250°C (source) 8. Mass Detector

67. Good Detector High sensitivity. Rapidly respond to concentration changes. Large linear range. Stable with respect to noise and drift. Low sensitivity to variation in flow, Pressure and temperature. Possible selectivity. Produces an easily handled signal. A temperature range from room temperature to at least 400 C. Properties of a good detector.

68. GC Basic Components

69. An inert gas, which is used to sweep a mixture to be separated through a gas chromatograph , (helium, hydrogen, or nitrogen). Push the sample through the gas chromatograph column. Clean out the gas chromatograph column after sample analysis. 5. Carrier gas

70. Carrier Gas Control The electronic control of the carrier gas allows also the following operations . Column Evaluation Gas Saver Function Leak Check The Flow mode has four options for the carrier gas control: Constant flow Constant pressure Programmed flow Programmed pressure Split flow control (in ml/min) Septum purge flow control (in mL/min)

71. All pure gases are classified by grade, so you can be certain of purity levels. The first digit of the classification indicates the number of nines purity (for example, 5.0 = 99.999% purity). The second digit is the number following the last nine (for example 4.7 helium has a guaranteed minimum purity of 99.997% and a corresponding maximum impurity level of 0.003% or 30ppm). Gas Purity

72. The carrier gas Ultra-pure and research-grade gases of up to 99.9999% (Grade 6.0) purity. The carrier gas system often contains a molecular sieve to remove water or other impurities. Linear Velocity (u) Is the speed at which the carrier gas or mobile phase travels through the column. The linear velocity is generally expressed in cm/s. The linear velocity is independent of the column diameter while the flow rate is dependent on the column diameter.

73. Helium For carrier gas: 99.995%1 high purity, with less than 1.0 ppm each of water, oxygen, and total hydrocarbons after purification. Use water, oxygen, and hydrocarbon traps. Hydrogen For carrier or detector fuel gas: 99.995%1 high purity, with < 1.0 ppm of total hydrocarbons after purification. Use water, oxygen and hydrocarbon traps. Air For detector fuel gas: 99.995%1 high purity. Air compressors are not acceptable because they do not meet pressure, water, and hydrocarbon requirements. Nitrogen For carrier or make-up gas: 99.995% high purity, with less than 1.0 ppm of total hydrocarbons after purification. Argon 5% Methane For ECD make-up gas: 99.995%1 high purity. Required Gases Purities

74. CAUTION Do not use liquid soap leak detectors to check for leaks. Liquid soap leak detectors may contaminate you system. A mixture of 50% H2O/50% methanol or isopropyl Alcohol may be used as a liquid leak detector. WARNING! Never use liquid leak detectors on or around electronic pneumatic circuits Leak Detection

75. Generators Nitrogen Generator The nitrogen generator can also operate directly from the laboratory compressed air supply. General contaminants are first removed with appropriate filters and adsorbents and the purified air passes over layers of polymeric hollow fiber membranes through which nitrogen selectively permeates. Hydrogen Generator In the Packard Hydrogen Generator, hydrogen is generated electrolytically from pure deionized water. The electrolysis unit uses a solid polymer electrolyte and thus does not need to be supplied with electrolytes, only the deionized water.

76. Hydrogen Hydrogen is a colorless, odorless, highly flammable gas with the molecular formula H2 and an atomic weight of 1.00794, making it the lightest element . Hydrogen is potentially explosive and must be used with extreme care. Hydrogen is a dangerous gas that, when mixed with air , could create an explosive mixture . Hydrogen is a dangerous gas, particularly in an enclosed area when it reaches a concentration corresponding to its lower explosion level (4% in volume ).

77. The data recorder plots the signal from the detector over time. The retention time, is qualitatively indicative of the type of compound. The area under the peaks or the height of the peak is indicative of the amount of each component. Chromatogram

78. RT , is the time it takes for a compound to travel from the injection port to the detector. Thousands of chemicals may have the same retention time, peak shape, and detector response.  For example, under certain conditions, DDT has the same retention time as PCBs (polychlorinated biphenyls). Retention Time (RT)

79. Retention Time Shifts Different column temperature. Different carrier gas flow rate or linear velocity. Leak in the injector, especially the septum. Contaminated column. Change in the sample solvent.

80. Gases - pressures, carrier gas average linear velocity, and flow rates (detector, split vent, septum purge). Temperatures - column, injector, detector . System parameters - purge activation times, detector attenuation, mass ranges, etc. Gas lines and traps - cleanliness, leaks, expiration. Injector consumables - septa, liners, O-rings and ferrules. Sample integrity - concentration, degradation, solvent, storage. Syringes - handling technique, leaks, needle sharpness, cleanliness. Data system - settings and connections. Simple Checks

81. Troubleshooting

82. Troubleshooting categories Baseline disturbances. Irregular peak shapes or sizes. Retention time shifts. Loss of separation or resolution. Quantitation difficulties. Rapid column deteriorations. Ghost peaks . Broad solvent fronts .

83. Troubleshooting Tools An electronic leak detector A flow meter An accurate thermometer A reliable analytical column New syringes Spare septa and high temperature septa Spare ferrules Detector cleaning solutions Spare recorder and electrometer cables Instrument manuals

84. Applications

85. Major Current GC Markets

86. Environmental What is the environmental market? Testing or commercial laboratories Industrial laboratories Government laboratories Research institutes Requirements of the applications Drinking water High sensitivity Waste Sensitivity and selectivity Air Sample introduction

87. Clean water analysis Pollutants in water Halocarbons Acid priority pollutants: phenols, chlorophenols, nitrophenols Pesticides and PCBs Base neutral priority pollutants Polynuclear aromatic hydrocarbons

88. Petrochemical and Gas Large replacement business Refinery Oil Industry Gas suppliers Requirements of the applications Multi-valves applications Fastest possible cycle time QC of gases: sensitivity Customized software Easy to use data handling and reporting

89. Food & Beverages Very extended market field No really regulated methods Ideal market to exploit the TRACE GC modularity Requirements of the applications QC of producers: Sensitivity and rapidity Multi detection capability Correct sample inlet (PTV-OC) Easy to use data handling and reporting

90. Pharmaceutical Highly regulated market (pharmacopeia) Requires Validation package Requirements of the applications Limited instrument requirements SSL/NPD/FID Highly Automated market High sensitivity detectors A unified chromatography software

91. Thanks [email protected]