Gas Chromatography

Gas Chromatography  In gas chromatography (GC), the sample is vaporized and injected onto the head of a chromatographic column.  A chromatographic technique in which the mobile phase is a gas.  Elution is brought about by the flow of an inert gaseous mobile phase.  Sample should be converted to vapor state (if it is not already a gas).  Separation occurs as the vapor constituents equilibrate between carrier gas and the stationary phase.  Measuring the retention time and comparing this time with that of a standard of a pure substances make it possible to identify the peak.  Since the area of the peak is proportional to the concentration, and so the amount of the substance can be quantitatively determined.  The peak height can be compared with a calibration curved prepared in a same manner. 1 Instrumentation for GC Carrier Gas Supply  Carrier gases, which must be chemically inert, include helium, argon, nitrogen, carbon dioxide, and hydrogen.  Associated with the gas supply are pressure regulators, gauges, and flow meters.  The carrier gas system often contains a molecular sieve to remove water or other impurities. 3 The mobile phase gas supply  Usually an inert gas that available in pure form such as argon, helium or nitrogen.  A highly dense gas is more effective.  The choice of gas determine by the type of detector. Functions:  To bring along gas and injected compounds throughout the column up to detector.  To provide equilibration between the carrier gas and the stationary phase for compounds separation. Volatile Sample  It is a compound that easily evaporated because of their low molecular weight.  In GC, the sample constituents need to be volatiled in order to move through the column.  Nonvolatile solutes will condense on the column, degrading the column’s performance.  Compounds from the mono terpenoids group (limonene, linalool, camphor, menthol etc.) are volatile. Sample introduction We will consider four methods of sample introduction:  Split injection  Splitless injection  Direct on-column injection  Programmed temperature vaporisation (PTV) 5 Sample Injector System  By using a micro-syringe to inject a liquid or gaseous sample through a silicone rubber diaphragm or septum into a flash vaporizer port located at the head of the column.  Sample Introduction must consider three rules:  All constituents injected into GC must be volatile.  The analyte must be present at an appropriate concentration.  Injecting the sample must not degrade the separation (thermally stable). 6 Stationary Phase  Chemically bonded (as opposed to coated) are the most stable.  Less polar polysiloxanes functionalised with methyl, phenyl, trifluoropropyl are common.  Polar phases include polyethylene glycols (less thermally stable) Mobile phase: Hydrogen (best), helium or nitrogen – very little scope for optimisation by mobile phase change Desirable properties for the immobilized liquid phase in a gas liquid chromatographic column include  low volatility  thermal stability  chemical inertness  solvent characteristics such that k’ and alpha values for the solutes to be resolved fall within a suitable range. 7 The Columns  Columns vary in length from less than 2 m to 50 m or more  In order to fit into and oven for thermostating, they are usually formed as coils having diameters of 10 to 30 cm.  Commonly used columns are packed columns and capillary columns. Packed Column  About 1-10 m long and 0.2-0.6 cm in diameter.  Short columns made of glass and longer columns made of stainless steel or can also made of Teflon. Functions of GC Columns  To contain the stationary phase and the passing way of the mobile phase.  The site where the separation of analyte occurs.  To provide analysis in terms of resolution, sensitivity and retention time. Detectors A. Flame Ionization Detector (FID)  It is one of the most widely used and generally applicable detectors for gas chromatography.  The effluent from the column is mixed with hydrogen and air and then ignited electrically.  The most sensitive and widely used detector for organic compounds.  Eluent burnt in a hydrogen fuelled flame  Leads to release of electrons, dependant on the concentration.  Requires own thermostat at temperature > column oven Advantages: Robust, sensitive, semi-universal, wide linear range Disadvantages: Non-selective, destructive, has same response to all organic compounds B. Thermal Conductivity Detector (TCD)  It is based upon changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules. 9 C. Thermoionic Detector (TID)/nitrogen/phosphors detector  It is selective toward organic compounds containing phosphorous and nitrogen.  It is similar in structure to the flame detector.  Eluent burnt in a hydrogen fuelled flame  Leads to release of electrons, dependant on concentration  Requires own thermostat at temperature > column oven Advantages: Robust, sensitive, selective to N or P, wide linear range, make-up gas not required  It is 10–100 times more sensitive than FID Disadvantages: needs frequent renewal and careful calibration, destructive Function of the detector  To respond to compounds analyzed.  To automatically detect the sample as it emerges from the column. 10 D. Electron Capture detector  Senses reduction in standing current  Normally operated at 300 0C Advantages: Extremely sensitive (1 pg), Selective for halogens, nitro groups, peroxides, quinones, nondestructive Disadvantages: Radioactive, limited range, easily contaminated E. Mass selective detector  Use mass spectrometry (EI or CI)  Focus on monitoring specific molecular ions quantitatively, although simple spectra are also possible Advantages: Extremely sensitive, and selective by mass Disadvantages: Most easily interfaced with a low flow rate system. Characteristics Of The Ideal Detector 1. Adequate sensitivity 2. Good stability and reproducibility. 3. A linear response to analyses that extends over several orders of magnitude. 4. A temperature range from room temperature to at least 400 0C. 5. A short response time that is independent of flow rate. 6. High reliability and ease of use. 7. Similarity in response toward all analyses. 8. Nondestructive of sample. Applications of GC  Widely used for the analysis of diverse array of samples in environmental, clinical, pharmaceutical, biochemical, forensic, food science and petrochemical laboratories. 12