GAS CHROMATOGRAPHY GAS CHROMATOGRAPHY PHAM VAN HUNG, PhD PHAM VAN HUNG, PhD Function Function   Separation of volatile organic compounds Separation of volatile organic compounds   Volatile Volatile – – when heated, when heated, VOCs VOCs undergo a phase undergo a phase transition into intact gas transition into intact gas - - phase species phase species   Separation occurs as a result of unique Separation occurs as a result of unique equilibria equilibria established between the solutes and the established between the solutes and the stationary phase (the GC column) stationary phase (the GC column)   An inert carrier gas carries the solutes through An inert carrier gas carries the solutes through the column the column Application  Uses  Separation and analysis of organic compounds  Testing purity of compounds  Determine relative amounts of components in mixture  Compound identification  Isolation of pure compounds (microscale work)  Similar to column chromatography, but differs in 3 ways:  Partitioning process carried out between Moving Gas Phase and Stationary Liquid Phase  Temperature of gas can be controlled  Concentration of compound in gas phase is a function of the vapor pressure only.  GC also known as Vapor-Phase Chromatography (VPC) and Gas-Liquid Partition Chromatography (GLPC). Filters/Traps Air Hydrogen Gas Carrier Column Components Components   gas system gas system   inlet inlet   column column   detector detector   data system data system Data system Syringe/Sampler Inlets Detectors Regulators H RESET Schematic Diagram of Gas Chromatography Carrier Gas Carrier Gas - - Supply Supply   Carrier gases, which must be chemically inert, Carrier gases, which must be chemically inert, include helium, nitrogen, and hydrogen. include helium, nitrogen, and hydrogen. Associated with the gas supply are pressure Associated with the gas supply are pressure regulators, gauges, and flow meters. In addition, regulators, gauges, and flow meters. In addition, the carrier gas system often contains a molecular the carrier gas system often contains a molecular sieve to remove water or other impurities. sieve to remove water or other impurities.   Must be at a constant flow rate so that retention Must be at a constant flow rate so that retention times & retention volumes may be equated times & retention volumes may be equated Injector Injector   A GC syringe penetrates a septum to inject A GC syringe penetrates a septum to inject sample into the vaporization camber sample into the vaporization camber   Instant vaporization of the sample, 280 Instant vaporization of the sample, 280 ° ° C C   Carrier gas transports the sample into the head Carrier gas transports the sample into the head of the column of the column   Purge valve controls the fraction of sample that Purge valve controls the fraction of sample that enters the column enters the column Split or Split or splitless splitless   Usually operated in split mode unless sample Usually operated in split mode unless sample limited limited   Chromatographic resolution depends upon the Chromatographic resolution depends upon the width of the sample plug width of the sample plug   In In splitless splitless mode the purge valve is close for 30 mode the purge valve is close for 30 - - 60 s, which means the sample plug is 30 60 s, which means the sample plug is 30 - - 60 60 seconds seconds   As we will see, refocusing to a more narrow As we will see, refocusing to a more narrow sample plug is possible with temperature sample plug is possible with temperature programming programming Column Configurations Column Configurations   Two general types of columns are encountered Two general types of columns are encountered in gas chromatography, in gas chromatography, packed packed and and open open tubular tubular , or , or capillary capillary . .   Chromatographic columns vary in length from Chromatographic columns vary in length from less than 2 m to 50 m or more. They are less than 2 m to 50 m or more. They are constructed of stainless steel, glass, fused silica, constructed of stainless steel, glass, fused silica, or Teflon. In order to fit into an oven for or Teflon. In order to fit into an oven for thermostating thermostating , they are usually formed as coils , they are usually formed as coils having diameters of 10 to 30 cm. having diameters of 10 to 30 cm. Columns Columns • Packed • Capillary 0.32 mm ID Liquid Stationary phase Mobile phase (Helium) flowing at 1 mL/min Open Tubular Capillary Column Open Tubular Capillary Column 15-60 m in length 0.1-5 μm Polar vs. Polar vs. nonpolar nonpolar   Separation is based on the vapor pressure and Separation is based on the vapor pressure and polarity of the components. polarity of the components.   Within a homologous series ( Within a homologous series ( alkanes alkanes , alcohol, , alcohol, olefins, fatty acids) retention time increases with olefins, fatty acids) retention time increases with chain length (or molecular weight) chain length (or molecular weight)   Polar columns retain polar compounds to a Polar columns retain polar compounds to a greater extent than non greater extent than non - - polar polar   C18 saturated vs. C18 saturated methyl ester C18 saturated vs. C18 saturated methyl ester C16:0 C18:0 C18:1 C18:2 C16:1 C16:0 C18:0 C18:1 C18:2 C16:1 RT (min) RT (min) Polar column Non-polar column Oven Oven   Programmable Programmable   Isothermal Isothermal - - run at one constant temperature run at one constant temperature   Temperature programming Temperature programming - - Start at low Start at low temperature and gradually ramp to higher temperature and gradually ramp to higher temperature temperature   More constant peak width More constant peak width   Better sensitivity for components that are retained Better sensitivity for components that are retained longer longer   Much better chromatographic resolution Much better chromatographic resolution   Peak refocusing at head of column Peak refocusing at head of column Typical Temperature Program Typical Temperature Program Time (min) 0 60 50°C 220°C 160°C Detection Systems Detection Systems Characteristics of the Ideal Detector: Characteristics of the Ideal Detector: The The ideal detector for gas chromatography has the ideal detector for gas chromatography has the following characteristics: following characteristics: 1. Adequate sensitivity 1. Adequate sensitivity 2. Good stability and reproducibility. 2. Good stability and reproducibility. 3. A linear response to solutes that extends over 3. A linear response to solutes that extends over several orders of magnitude. several orders of magnitude. 4. A temperature range from room temperature 4. A temperature range from room temperature to at least 400 to at least 400 o o C. C. Characteristics of the Ideal Detector Characteristics of the Ideal Detector 5. A short response time that is independent of 5. A short response time that is independent of flow rate. flow rate. 6. High reliability and ease of use. 6. High reliability and ease of use. 7. Similarity in response toward all solutes or a 7. Similarity in response toward all solutes or a highly selective response toward one or more highly selective response toward one or more classes of solutes. classes of solutes. 8. Nondestructive of sample. 8. Nondestructive of sample. Detectors Detectors   Flame Ionization Detectors (FID) Flame Ionization Detectors (FID)   Electron Capture Detectors (ECD) Electron Capture Detectors (ECD)   Electron impact/chemical ionization (EI/CI) Electron impact/chemical ionization (EI/CI) Mass spectrometry Mass spectrometry FIDs FIDs   Effluent exits column and enters an Effluent exits column and enters an air/hydrogen flame air/hydrogen flame   The gas The gas - - phase solute is phase solute is pyrolized pyrolized to form to form electrons and ions electrons and ions   All carbon species are reduced to CH All carbon species are reduced to CH 2 2 + + ions ions   These ions collected at an electrode held above These ions collected at an electrode held above the flame the flame   The current reaching the electrode is amplified The current reaching the electrode is amplified to give the signal to give the signal FID FID   A general detector for organic compounds A general detector for organic compounds   Very sensitive (10 Very sensitive (10 - - 13 13 g/s g/s ) )   Linear response (10 Linear response (10 7 7 ) )   Rugged Rugged   Disadvantage: specificity Disadvantage: specificity ECD ECD   Ultra Ultra - - sensitive detection of halogen sensitive detection of halogen - - containing containing species species   Pesticide analysis Pesticide analysis   Other detectors besides MS Other detectors besides MS   IR IR   AE AE SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS C C C Detector Response Retention Time 14 16 18 Peak Area (cm ) Sample Concentration (mg/ml) 2 4 6 8 10 0.5 1.0 1.5 2.0 2.5 3.0 2 The content % of C fatty acids = C C + C + C 100 ∗ 14 1816 14 = the content % of C fatty acids 14 14 TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS Response GC Retention Time on Carbowax-20 (min) Mixture of known compounds Hexane Octane Decane 1.6 min = RT Response Unknown compound may be Hexane 1.6 min = RT Retention Time on Carbowax-20 (min) Response GC Retention Ti me on SE-30 Unknown compound RT= 4 min on SE-30 Response GC Retention Ti me on SE-30 Hex an e RT= 4.0 min on SE-30 Retention Times GLC ADVANTAGES 1. Very good separation 2. Time (analysis is short) 3. Small sample is needed - μl 4. Good detection system 5. Quantitatively analyzed DISADVANTAGES OF GAS CHROMATOGRAPHY Material has to be volatilized at 250C without decomposition. R C OH CH 3 OH H 2 SO 4 O R C O CH 3 O CH 2 O C R CH O C R CH 2 O C R O O O CH 3 OH O R C O CH 3 CH 3 ONa Fatty Acids Methylester Reflux + 3 Volatile in Gas Chromatography Volatile in Gas Chromatography ++ Gas Chromatogram of Methyl Esters of Fatty Acids . purge valve is close for 30 - - 60 s, which means the sample plug is 30 60 s, which means the sample plug is 30 - - 60 60 seconds seconds   As we will. C18 saturated vs. C18 saturated methyl ester C 16: 0 C18:0 C18:1 C18:2 C 16: 1 C 16: 0 C18:0 C18:1 C18:2 C 16: 1 RT (min) RT (min) Polar column Non-polar column Oven Oven   Programmable Programmable   Isothermal Isothermal - - run