LECT 11 Gas Chromatography PDF

KAEDAH DAN INSTRUMENTASI BIOLOGI MARIN MMB3123 LECTURE 11 – GAS CHROMATOGRAPHY GAS CHROMATOGRAPHY  A sample of organic compounds is injected into an inert gaseous mobile phase(known as the carrier gas) for separation, qualitative(identification) and quant...

KAEDAH DAN INSTRUMENTASI BIOLOGI MARIN MMB3123 LECTURE 11 – GAS CHROMATOGRAPHY GAS CHROMATOGRAPHY  A sample of organic compounds is injected into an inert gaseous mobile phase(known as the carrier gas) for separation, qualitative(identification) and quantitative(conc. determination) analysis.  The sample is carried through a packed or capillary column and the components in the sample are separated based on their ability to distribute between the stationary phase and the mobile phase. GC analytical technique 1. Volatile compounds (organics) such as petroleum hydrocarbons, pesticides, organic compounds…… 2. Separation processes i. Different adsorption with column packing materials. ii. Different Boiling point (Temp programming System). iii. Different in weight (Molecular weight)  Sample injected (A, B & C) CB A Carrier gas Initial column temp set at 60oC The boiling points for A=70oC B=100oC & C= 150oC. If we increase the column temperature from 60oC to 200oC at the rate of 10oC/min, the results of the chromatogram will be Retention Time, RT A B C Peak height Time, min. INJECTION PORT  Comparison Of column Capillary column Packed column Capillary Column (Shimadzu 2010) YOUTUBE LINKS FOR ADDITIONAL INFO ON GC  https://www.youtube.com/watch?v=4Xaa9WdXVTM&ab _channel=khanacademymedicine  https://www.youtube.com/watch?v=uD-29- mV3N0&ab_channel=AgilentTechnologies TYPES OF GC COLUMN (0.01 –0.053cm) GC SEPARATION  The common inert mobile phase are Ne, Ar , and N2 which have the advantage of being chemically inert toward both the sample and the stationary phase. The mobile phase velocity is usually within 25-150ml/min where as the flow rates for capillary columns are within 1-25ml/min.  The chromatography columns. 1.Packed Columns. A packed column is constructed from glass, stainless steel, copper or aluminum and typically 2-6 meters long with a internal diameter of 2-4 mm.The column is filled up with a particulate solid with diameter ranging between 30-350 m. For more efficiency in separation, the smaller the solid particle the better will be the separation.  2. Capillary Columns Capillary, or open tubular columns are constructed from fused silica coated with a protective polymer. Column may be up to 100 meters length with diameter of approximately 150-300 m.  The Stationary Phases Selectivity in gas chromatography is influenced by the choice of stationary phase. Elution order in GC is determined by the solute’s boiling point and to a lesser degree, by the solute’s interaction with the stationary phase. Solutes with different of boiling points are easily separated. Two solutes with the same boiling points could be separated if the stationary phase reacts with one of the solutes. Weight of the molecule.  In general non-polar solutes are more easier separated with a non-polar stationary phase. While polar solutes are easier separated by the polar stationary phase.  Stationary phases should be chemically inert, thermally stable and low volatility.  Sample injection 1. Sample must be volatile. 2. The analytes must be present at an appropriate concentration. 3. Injection of the sample must not be degradable in the column.  The Temperature Control (Temp Programming). Controlling the temp is critical for good separation in GC.The column is packed inside a thermo-stated oven. In an isothermal separation, the column is maintained at a constant temperature. Normally, the temp is set slightly below that for the lowest boiling solute and increase gradually by a program known as the temperature programming.  DETECTORS FOR GC 1. Thermal Conductivity Detector This Detector (TCD) is based on the mobile phase’s thermal conductivity. Helium is the mobile phase in choice for the TCD because of its high TC. When a solute is eluted from the column, the TC of the mobile phase decreases. Thus, there is a change in conductivity. TCD gives a signal for any solute which has TC differs from that of helium. 2. Flame Ionization Detector (FID) Combustion of organic compounds in an H2/air flame results in a flame rich in electrons and ions. If a potential of about 300 v is applied across the flame, a small electricity is produced. Thus, a signal is produced. FID is used only for organic compounds. For inorganic compounds such as CO2, no signal can be detected. The sample is destroyed because of burning, and the sensitivity is 2-3 orders of magnitude lower than the TCD. 3. Electron Capture Detector (ECD) The ECD consists of a beta emitter (electron) such as 63Ni. The emitting electrons ionize the mobile phase which is usually N2, resulting in production of additional electrons which give rise to electricity current. When the eluted solute pass through the ECD, the electric current in the ECD decrease and a signal is obtained. ECD is a selective detector which is especially for solutes with electronegative functional groups such as halogens and nitrogen groups. Therefore ECD is used for pesticides analysis. Example of Chromatogram-Petroleum Hydrocarbon Analysis  Petroleum hydrocarbons are extracted from sea water and fractionated into 1. Aliphatic hydrocarbon & 2. Aromatic hydrocarbon fractions using Column Chromatography. GC-FID with capillary column(30meters) and temperature programming with oven temperature hold for 20 min at 70 oC, then increase to 300oC with a rate of 8oC/min and hold at 300 oC for 30 min. The whole process will take about 1.2 hours.  Aliphatic Hydrocarbons FID2 B, (JOHNSON\SIG11155.D) pA 250 C23 C24 C22 C27 C26 200 C21 C28 C19 C29 150 C18 100 C17 50 ISTDi C32 C8 C20 C16 C10 C9 0 10 20 30 40 50 60 70 min  Polyaromatic Hydrocarbons FID2 B, (JOHNSON\SIG11026.D) pA 120 100 BkF 80 Pyr 60 40 BbF ISTDIII IP BaA 20 ISTDII ISTDI 0 10 20 30 40 50 60 70 min Qualitative & Quantitative Analysis 1. Use Reference Standard Material- such as EPA 16 Aromatic Hydrocarbons. The RSM is analyzed with the GC under the same condition set for the analysis. The RT value and the intensity of peak are recorded. These values will be used for the Qualitative & Quantitative Analysis Retention Value(RT) – 11.9 minutes AH External Standard FID2 B, (JOHNSON\SIG11151.D) C10 pA C15 600 C11 C9 C25 C20 C30 C12 500 C13 C14 400 C24 C22 C23 C28 C26 C16 C21 C27 C29 C32 C17 C19 C18 300 C8 200 100 0 10 20 30 40 50 60 PAH External Standard FID2 B, (JOHNSON\SIG11032.D) Na Ace Acy pA 450 400 350 300 250 200 Flu 150 FI AnPh 100 BbF BaA Chyrs Pyr dBahA BaP IP 50 BghiP BkF 0 10 20 30 40 50 60 2. Use the GC-MS analysis. Each solute undergo characteristic fragmentation into smaller ions. The mass spectrum of ion intensity as a function of mass to charge ratio will provide qualitative information for identification of the compound. https://www.youtube.com/watch?v=cBXgSPO3pzw&ab_channel =AromaticPlantResearchCenter  GC-MS (Identification of Organic Compounds) The effluent from GC is introduced into the mass spectrometer’s ionization chamber where most of the carrier gas(Mobile phase) are removed. In the ionization chamber, all molecules are ionized and they are separated by their mass to charge ratio. Each solute is undergo characteristic fragmentation into smaller ions. The mass spectrum of ion intensity as a function of mass to charge ratio will provide qualitative information for identification of the compound. A selective monitoring detector is used to detect the specific mass-to-charge ratio produced electricity current. GC MS(Shimadzu 2010) GC-MS FLOW DIAGRAM Anthracene Spectrum TOC (Total Organic Carbon) Analyzer Total Organic Carbon Analyzer (TOC)  Analysis of organic substances in - Water - solid including tissues and sediment IR light absorption Organic + O2 CO2 600oC Infrared Analysis Gas Chromatograph – Mass Spectrometer GC – MS 3D VIEW END