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Gas Chromatography Introduction 1.) Gas Chromatography  Mobile phase (carrier gas) is a gas - Usually N2, He, Ar and maybe H2 - Mobile phase in liquid chromatography is a liquid  Requires analyte to be either naturally volatile or can be converted to a volatile derivative - GC useful in the separation of small organic and inorganic compounds  Stationary phase: - Gas-liquid partition chromatography – nonvolatile liquid bonded to solid support - Gas-solid chromatography – underivatized solid particles - Bonded phase gas chromatography – chemical layer chemically bonded to solid support Magnified Pores in activated carbon Zeolite molecular sieve Bonded phase
Gas Chromatography Introduction 2.) Instrumentation  Process: - Volatile liquid or gas injected through septum into heated port - Sample rapidly evaporates and is pulled through the column with carrier gas - Column is heated to provide sufficient vapor pressure to elute analytes - Separated analytes flow through a heated detector for observation
Gas Chromatography Instrumentation 1.) Open Tubular Columns  Commonly used in GC  Higher resolution, shorter analysis time, and greater sensitivity  Low sample capacity  Increasing Resolution - Narrow columns  Increase resolution - Resolution is proportional to , where N increases directly with column length N Easy to generate long (10s of meters) lengths of narrow columns to maximize resolution
Gas Chromatography Instrumentation 1.) Open Tubular Columns  Increasing Resolution Decrease tube diameter Increase resolution Increase Column Length Increase resolution
Gas Chromatography Increase Stationary Phase Thickness Increase resolution of early eluting compounds Also, increase in capacity factor and reduce peak tailing But also decreases stability of stationary phase Instrumentation 1.) Open Tubular Columns  Increasing Resolution
Gas Chromatography Instrumentation 2.) Choice of liquid stationary phase:  Based on “like dissolves like”  Nonpolar columns for nonpolar solutes  Strongly polar columns for strongly polar compounds  To reduce “bleeding” of stationary phase: - bond (covalently attached) to silica - Covalently cross-link to itself
Gas Chromatography Instrumentation 3.) Packed Columns  Greater sample capacity  Broader peaks, longer retention times and less resolution - Improve resolution by using small, uniform particle sizes Packed column Open tubular column
Gas Chromatography Instrumentation 3.) Packed Columns  The major advantage and use is for large-scale or preparative purification  Industrial scale purification maybe in the kilogram or greater range 500 L chromatography column Oil refinery – separates fractions of oil for petroleum products
Gas Chromatography Retention Index 1.) Retention Time  Order of elution is mainly determined by volatility - Least volatile = most retained - Polar compounds (ex: alcohols) are the least volatile and will be the most retained on the GC system  Second factor is similarity in polarity between compound and stationary phase
Gas Chromatography Retention Index 2.) Describing Column Performance  Can manipulate or adjust retention time by changing polarity of stationary phase  Can use these retention time differences to classify or rate column performance - Compare relative retention times between compounds and how they change between columns  Can be used to identify unknowns
Gas Chromatography Retention Index 2.) Describing Column Performance  Retention index based on the difference in the number of carbons (N, n) for linear alkane and corresponding retention times (tr’(unknown), tr’(N),tr’(N)):  Provides a means to compare the performance of different columns              ) n ( t log ) N ( t log ) n ( t log ) unknown ( t log ) n N ( n I ' r ' r ' r ' r 100 Increase in Polarity
Gas Chromatography Temperature and Pressure Programming 1.) Improving Column Efficiency  Temperature programming: - Temperature is raised during the separation (gradient) - increases solute vapor pressure and decrease retention time Temperature gradient improves resolution while also decreasing retention time
Gas Chromatography Temperature and Pressure Programming 1.) Improving Column Efficiency  Pressure Programming: - Increase pressure  increases flow of mobile phase (carrier gas) - Increase flow  decrease retention time  Pressure is rapidly reduced at the end of the run - Time is not wasted waiting for the column to cool - Useful for analytes that decompose at high temperatures Van Deemter curves indicate that column efficiency is related to flow rate
Gas Chromatography Carrier Gas 1.) N2, He and H2 are typical carrier gases  He: - Most common and compatible with most detectors - Better resolution (smaller plate heights) - Solutes diffuse rapidly  smaller mass transfer term  N2: - Lower detection limit for a flame ionization detector - Lower resolution and solute diffusion rates  H2: - Fastest separations - Can catalytically react with unsaturated compounds on metal surfaces - Can not be used with mass spectrometers Forms explosive mixtures with air - Better resolution (smaller plate heights) - Solutes diffuse rapidly  smaller mass transfer term Flow rate increases N2 < He < H2 Diffusion coefficients follow: H2 > He > N2
Gas Chromatography Sample Injection 1.) “Sandwich” Injection  Separate sample with air bubbles and solvent - Air bubble prevents depletion of most volatile compounds before sample injection is complete (barrier between oven and sample during injection) - Solvent is used to pushes out sample, but bubble prevents mixing - Final air bubble pushes out solvent - Gas-tight syringe is required for gas samples - Injection volume is typically 0.1-2 mL
Sample Injection 1.) “Sandwich” Injection  Injection port - Inject rapidly ( < 1s) through septum into evaporation zone - Injector temperature is kept high (350oC) for fast evaporation - Rapid gas flows carries sample to mixing chamber for complete vaporization and complete mixing before entering column Gas Chromatography
Gas Chromatography Sample Injection 2.) Split Injection  Delivers only 0.2-2% of sample to the column - Split ratio of 50:1 to 600:1 (sample discarded)  For samples where analytes of interest are >0.1% of sample - Best resolution is obtained with smaller amount of sample - ≤ 1 mL with ≤ 1 ng of each compound (0.5 mL of gas volume)  Not quantitative, split not constant After mixing, pressure regulator controls the fraction of sample discarded Remainder of the sample is flushed from injector port to column
Gas Chromatography Sample Injection 2.) Splitless Injection  Delivers ~80% of sample to the column  For trace analysis, where analytes of interest are < 0.01% of sample - Large volume (~2 mL) injected slowly (2s)  No mixing chamber or split vent - Injection temperature is lower (220oC) - 40oC below the boiling point of the solvent Lower temperature “traps” solvent in a narrow band at the head of the column Raise temperature to volatize sample and start separation Injecting larger volume, don’t want broad peaks
Gas Chromatography Sample Injection 2.) Splitless Injection  “Solvent trapping” significantly improves the performance of splitless injections - Initial lower temperature of column during injection keeps larger volume into a narrow band - Chromatography is initiated by raising column temperature - Cold trapping – condense solutes in narrow band at the beginning of column by using an initial temperature 150oC below boiling points of solutes of interest Without “Solvent trapping” With “Solvent trapping”
Gas Chromatography Sample Injection 3.) On-column Injection  Delivers ~100% of sample to the column  For samples that decompose above their boiling points  Solution injected directly on column - Warming column initiates chromatography Raise temperature to volatize sample and start separation Lower initial column temperature to prevent sample decomposition
Gas Chromatography Detectors 1.) Qualitative and Quantitative Analysis  Mass Spectrometer and Fourier Transform Infrared Spectrometers can identify compounds as part of a GC system - Compare spectrum with library of spectra using a computer  Compare retention times between reference sample and unknown - Use multiple columns with different stationary phases - Co-elute the known and unknown and measure changes in peak area  The area of a peak is proportional to the quantity of that compound 2 1 064 1 w t peak heigh .    peak Gaussian of Area Peak Area Concentration of Standard Peak area increases proportional to concentration of standard if unknown/standard have the identical retention time  same compound
Gas Chromatography Ohm’s Law: V =IR Based on Ohm’s law, monitored potential (V) or current (I) Changes as resistance (R) of filament changes due to presence of compound Detectors 2.) Thermal Conductivity Detector  Measures amount of compound leaving column by its ability to remove heat - He has high thermal conductivity, so the presence of any compound will lower the thermal conductivity increasing temperature of filament  As heat is removed from filament, the resistance (R) of filament changes - Causes a change in an electrical signal that can be measured  Responds to all compounds (universal) - Signal changes in response to flow rate of mobile phase and any impurities present - Not very sensitive
Gas Chromatography Detectors 3.) Flame Ionization Detector  Mobile phase leaving the column is mixed with H2 and air and burned in a flame - Carbon present in eluting solutes produces CH radicals which produce CHO+ ions - Electrons produced are collected at an electrode and measured  Responds to almost all organic compounds and has good limits of detection - 100 times better than thermal conductivity detector - Stable to changes in flow rate and common mobile phase impurities (O2, CO2,H2O,NH3) Burn sample and measure amount of produced electrons
Gas Chromatography Detectors 4.) Electron Capture Detector  Sensitive to halogen-containing and other electronegative compounds  Based on the capture of electrons by electronegative atoms - Compounds ionized by b-rays from radioactive 63Ni  Extremely sensitive (~ 5 fg/s) Steady current (flow of electrons) disrupted by compounds with high electron affinity
Gas Chromatography Detectors 5.) Mass Spectrometry  Detector of Choice  But Expensive!  Sensitive and provides an approach to identify analytes  Selected ion monitoring – monitor a specific mass/charge (mz) compared to scanning over the complete spectra - Simplifies complex chromatogram - Increases sensitivity by 102-103
Gas Chromatography Detectors 6.) Other Detectors  Respond to limited class of analytes  Modification of previous detectors  Nitrogen-Phosphorous detector - Modified flame ionization detector - Extremely sensitive for compounds containing N and P - Important for drugs and pesticides  Flame photometric detector - Measures optical emission from P (536 nM) , S (394 nM), Pb, Sn, and other select elements after passing sample through flame (flame ioniation detector)  Photionization detector - Uses a ultraviolet source to ionize aromatic and unsaturated compounds, electrons produced are measured (Electron capture detector)  Sulfur/nitrogen chemiluminescence detector - Collects exhaust of flame ionization detector - S and N converted to SO and NO - Mix with O3 form excited state of SO2 (emits blue light) and NO3
Gas Chromatography Sample Preparation 1.) Transform sample into form suitable for analysis  Extraction, concentration, removal of interfering species or chemically transforming (derivatizing) 2.) Solid-phase microextraction  Extract analytes from complex mixture without solvent  Uses a fused-silica fiber coated with stationary phase - Stationary phase similar to those used in GC  Expose Fiber to sample to extract compounds and then inject fiber into GC to evaporate analytes
Gas Chromatography Sample Preparation 3.) Purge and Trap  Removes volatile analytes from liquids or solids, concentrates sample and transfer to GC  Goal is to remove 100% of analyte Bubble purge gas (He) through heated sample to evaporate analytes Analytes are captured on adsorbent column Connect port to GC Heat column to 200oC to transfer analytes to GC
Gas Chromatography Method Development in GC 1.) How to Choose a Procedure for a Particular Problem  Many Satisfactory Solutions  The order in which the decision should be made should consider: 1. Goal of the analysis 2. Sample preparation 3. Detector 4. Column 5. Injection  Goal of the analysis - Qualitative vs. quantitative - Resolution vs. sensitivity - Precision vs. time - Interest in a specific analyte  Sample preparation - Cleaning-up a complex sample is essential - Garbage in  garbage out  Choosing the Detector - Detect a specific analyte(s) or everything in the sample - sensitivity - Identify an unknown (MS, FTIR)
Gas Chromatography Method Development in GC 1.) How to Choose a Procedure for a Particular Problem  Selecting the Column - Consider stationary phase, column diameter and length, stationary phase thickness - Match column polarity to sample polarity - To improve resolution, use a: a. Longer column b. Narrower column c. Different stationary phase  Choosing the Injection Method - Split injection is best for high concentrated samples - Splitless injection is best for very dilute solutions - On-column injection is best for quantitative analysis and thermally instable compounds

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chapter-24.ppty5y54y645654645645645y5t55

  • 1. Gas Chromatography Introduction 1.) Gas Chromatography  Mobile phase (carrier gas) is a gas - Usually N2, He, Ar and maybe H2 - Mobile phase in liquid chromatography is a liquid  Requires analyte to be either naturally volatile or can be converted to a volatile derivative - GC useful in the separation of small organic and inorganic compounds  Stationary phase: - Gas-liquid partition chromatography – nonvolatile liquid bonded to solid support - Gas-solid chromatography – underivatized solid particles - Bonded phase gas chromatography – chemical layer chemically bonded to solid support Magnified Pores in activated carbon Zeolite molecular sieve Bonded phase
  • 2. Gas Chromatography Introduction 2.) Instrumentation  Process: - Volatile liquid or gas injected through septum into heated port - Sample rapidly evaporates and is pulled through the column with carrier gas - Column is heated to provide sufficient vapor pressure to elute analytes - Separated analytes flow through a heated detector for observation
  • 3. Gas Chromatography Instrumentation 1.) Open Tubular Columns  Commonly used in GC  Higher resolution, shorter analysis time, and greater sensitivity  Low sample capacity  Increasing Resolution - Narrow columns  Increase resolution - Resolution is proportional to , where N increases directly with column length N Easy to generate long (10s of meters) lengths of narrow columns to maximize resolution
  • 4. Gas Chromatography Instrumentation 1.) Open Tubular Columns  Increasing Resolution Decrease tube diameter Increase resolution Increase Column Length Increase resolution
  • 5. Gas Chromatography Increase Stationary Phase Thickness Increase resolution of early eluting compounds Also, increase in capacity factor and reduce peak tailing But also decreases stability of stationary phase Instrumentation 1.) Open Tubular Columns  Increasing Resolution
  • 6. Gas Chromatography Instrumentation 2.) Choice of liquid stationary phase:  Based on “like dissolves like”  Nonpolar columns for nonpolar solutes  Strongly polar columns for strongly polar compounds  To reduce “bleeding” of stationary phase: - bond (covalently attached) to silica - Covalently cross-link to itself
  • 7. Gas Chromatography Instrumentation 3.) Packed Columns  Greater sample capacity  Broader peaks, longer retention times and less resolution - Improve resolution by using small, uniform particle sizes Packed column Open tubular column
  • 8. Gas Chromatography Instrumentation 3.) Packed Columns  The major advantage and use is for large-scale or preparative purification  Industrial scale purification maybe in the kilogram or greater range 500 L chromatography column Oil refinery – separates fractions of oil for petroleum products
  • 9. Gas Chromatography Retention Index 1.) Retention Time  Order of elution is mainly determined by volatility - Least volatile = most retained - Polar compounds (ex: alcohols) are the least volatile and will be the most retained on the GC system  Second factor is similarity in polarity between compound and stationary phase
  • 10. Gas Chromatography Retention Index 2.) Describing Column Performance  Can manipulate or adjust retention time by changing polarity of stationary phase  Can use these retention time differences to classify or rate column performance - Compare relative retention times between compounds and how they change between columns  Can be used to identify unknowns
  • 11. Gas Chromatography Retention Index 2.) Describing Column Performance  Retention index based on the difference in the number of carbons (N, n) for linear alkane and corresponding retention times (tr’(unknown), tr’(N),tr’(N)):  Provides a means to compare the performance of different columns              ) n ( t log ) N ( t log ) n ( t log ) unknown ( t log ) n N ( n I ' r ' r ' r ' r 100 Increase in Polarity
  • 12. Gas Chromatography Temperature and Pressure Programming 1.) Improving Column Efficiency  Temperature programming: - Temperature is raised during the separation (gradient) - increases solute vapor pressure and decrease retention time Temperature gradient improves resolution while also decreasing retention time
  • 13. Gas Chromatography Temperature and Pressure Programming 1.) Improving Column Efficiency  Pressure Programming: - Increase pressure  increases flow of mobile phase (carrier gas) - Increase flow  decrease retention time  Pressure is rapidly reduced at the end of the run - Time is not wasted waiting for the column to cool - Useful for analytes that decompose at high temperatures Van Deemter curves indicate that column efficiency is related to flow rate
  • 14. Gas Chromatography Carrier Gas 1.) N2, He and H2 are typical carrier gases  He: - Most common and compatible with most detectors - Better resolution (smaller plate heights) - Solutes diffuse rapidly  smaller mass transfer term  N2: - Lower detection limit for a flame ionization detector - Lower resolution and solute diffusion rates  H2: - Fastest separations - Can catalytically react with unsaturated compounds on metal surfaces - Can not be used with mass spectrometers Forms explosive mixtures with air - Better resolution (smaller plate heights) - Solutes diffuse rapidly  smaller mass transfer term Flow rate increases N2 < He < H2 Diffusion coefficients follow: H2 > He > N2
  • 15. Gas Chromatography Sample Injection 1.) “Sandwich” Injection  Separate sample with air bubbles and solvent - Air bubble prevents depletion of most volatile compounds before sample injection is complete (barrier between oven and sample during injection) - Solvent is used to pushes out sample, but bubble prevents mixing - Final air bubble pushes out solvent - Gas-tight syringe is required for gas samples - Injection volume is typically 0.1-2 mL
  • 16. Sample Injection 1.) “Sandwich” Injection  Injection port - Inject rapidly ( < 1s) through septum into evaporation zone - Injector temperature is kept high (350oC) for fast evaporation - Rapid gas flows carries sample to mixing chamber for complete vaporization and complete mixing before entering column Gas Chromatography
  • 17. Gas Chromatography Sample Injection 2.) Split Injection  Delivers only 0.2-2% of sample to the column - Split ratio of 50:1 to 600:1 (sample discarded)  For samples where analytes of interest are >0.1% of sample - Best resolution is obtained with smaller amount of sample - ≤ 1 mL with ≤ 1 ng of each compound (0.5 mL of gas volume)  Not quantitative, split not constant After mixing, pressure regulator controls the fraction of sample discarded Remainder of the sample is flushed from injector port to column
  • 18. Gas Chromatography Sample Injection 2.) Splitless Injection  Delivers ~80% of sample to the column  For trace analysis, where analytes of interest are < 0.01% of sample - Large volume (~2 mL) injected slowly (2s)  No mixing chamber or split vent - Injection temperature is lower (220oC) - 40oC below the boiling point of the solvent Lower temperature “traps” solvent in a narrow band at the head of the column Raise temperature to volatize sample and start separation Injecting larger volume, don’t want broad peaks
  • 19. Gas Chromatography Sample Injection 2.) Splitless Injection  “Solvent trapping” significantly improves the performance of splitless injections - Initial lower temperature of column during injection keeps larger volume into a narrow band - Chromatography is initiated by raising column temperature - Cold trapping – condense solutes in narrow band at the beginning of column by using an initial temperature 150oC below boiling points of solutes of interest Without “Solvent trapping” With “Solvent trapping”
  • 20. Gas Chromatography Sample Injection 3.) On-column Injection  Delivers ~100% of sample to the column  For samples that decompose above their boiling points  Solution injected directly on column - Warming column initiates chromatography Raise temperature to volatize sample and start separation Lower initial column temperature to prevent sample decomposition
  • 21. Gas Chromatography Detectors 1.) Qualitative and Quantitative Analysis  Mass Spectrometer and Fourier Transform Infrared Spectrometers can identify compounds as part of a GC system - Compare spectrum with library of spectra using a computer  Compare retention times between reference sample and unknown - Use multiple columns with different stationary phases - Co-elute the known and unknown and measure changes in peak area  The area of a peak is proportional to the quantity of that compound 2 1 064 1 w t peak heigh .    peak Gaussian of Area Peak Area Concentration of Standard Peak area increases proportional to concentration of standard if unknown/standard have the identical retention time  same compound
  • 22. Gas Chromatography Ohm’s Law: V =IR Based on Ohm’s law, monitored potential (V) or current (I) Changes as resistance (R) of filament changes due to presence of compound Detectors 2.) Thermal Conductivity Detector  Measures amount of compound leaving column by its ability to remove heat - He has high thermal conductivity, so the presence of any compound will lower the thermal conductivity increasing temperature of filament  As heat is removed from filament, the resistance (R) of filament changes - Causes a change in an electrical signal that can be measured  Responds to all compounds (universal) - Signal changes in response to flow rate of mobile phase and any impurities present - Not very sensitive
  • 23. Gas Chromatography Detectors 3.) Flame Ionization Detector  Mobile phase leaving the column is mixed with H2 and air and burned in a flame - Carbon present in eluting solutes produces CH radicals which produce CHO+ ions - Electrons produced are collected at an electrode and measured  Responds to almost all organic compounds and has good limits of detection - 100 times better than thermal conductivity detector - Stable to changes in flow rate and common mobile phase impurities (O2, CO2,H2O,NH3) Burn sample and measure amount of produced electrons
  • 24. Gas Chromatography Detectors 4.) Electron Capture Detector  Sensitive to halogen-containing and other electronegative compounds  Based on the capture of electrons by electronegative atoms - Compounds ionized by b-rays from radioactive 63Ni  Extremely sensitive (~ 5 fg/s) Steady current (flow of electrons) disrupted by compounds with high electron affinity
  • 25. Gas Chromatography Detectors 5.) Mass Spectrometry  Detector of Choice  But Expensive!  Sensitive and provides an approach to identify analytes  Selected ion monitoring – monitor a specific mass/charge (mz) compared to scanning over the complete spectra - Simplifies complex chromatogram - Increases sensitivity by 102-103
  • 26. Gas Chromatography Detectors 6.) Other Detectors  Respond to limited class of analytes  Modification of previous detectors  Nitrogen-Phosphorous detector - Modified flame ionization detector - Extremely sensitive for compounds containing N and P - Important for drugs and pesticides  Flame photometric detector - Measures optical emission from P (536 nM) , S (394 nM), Pb, Sn, and other select elements after passing sample through flame (flame ioniation detector)  Photionization detector - Uses a ultraviolet source to ionize aromatic and unsaturated compounds, electrons produced are measured (Electron capture detector)  Sulfur/nitrogen chemiluminescence detector - Collects exhaust of flame ionization detector - S and N converted to SO and NO - Mix with O3 form excited state of SO2 (emits blue light) and NO3
  • 27. Gas Chromatography Sample Preparation 1.) Transform sample into form suitable for analysis  Extraction, concentration, removal of interfering species or chemically transforming (derivatizing) 2.) Solid-phase microextraction  Extract analytes from complex mixture without solvent  Uses a fused-silica fiber coated with stationary phase - Stationary phase similar to those used in GC  Expose Fiber to sample to extract compounds and then inject fiber into GC to evaporate analytes
  • 28. Gas Chromatography Sample Preparation 3.) Purge and Trap  Removes volatile analytes from liquids or solids, concentrates sample and transfer to GC  Goal is to remove 100% of analyte Bubble purge gas (He) through heated sample to evaporate analytes Analytes are captured on adsorbent column Connect port to GC Heat column to 200oC to transfer analytes to GC
  • 29. Gas Chromatography Method Development in GC 1.) How to Choose a Procedure for a Particular Problem  Many Satisfactory Solutions  The order in which the decision should be made should consider: 1. Goal of the analysis 2. Sample preparation 3. Detector 4. Column 5. Injection  Goal of the analysis - Qualitative vs. quantitative - Resolution vs. sensitivity - Precision vs. time - Interest in a specific analyte  Sample preparation - Cleaning-up a complex sample is essential - Garbage in  garbage out  Choosing the Detector - Detect a specific analyte(s) or everything in the sample - sensitivity - Identify an unknown (MS, FTIR)
  • 30. Gas Chromatography Method Development in GC 1.) How to Choose a Procedure for a Particular Problem  Selecting the Column - Consider stationary phase, column diameter and length, stationary phase thickness - Match column polarity to sample polarity - To improve resolution, use a: a. Longer column b. Narrower column c. Different stationary phase  Choosing the Injection Method - Split injection is best for high concentrated samples - Splitless injection is best for very dilute solutions - On-column injection is best for quantitative analysis and thermally instable compounds