Gas Chromatography & Other Methods Lecture Notes PDF

11/10/2023 Gas Chromatography & other methods vevox.app 126-545-180 Nikola P. Chmel L4 CH3F2 1 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Normal phase chromatography (colu...

11/10/2023 Gas Chromatography & other methods vevox.app 126-545-180 Nikola P. Chmel L4 CH3F2 1 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Normal phase chromatography (column, TLC) Reverse phase chromatography (HPLC) Gas chromatography Size exclusion chromatography Ion exchange chromatography Lecture 2 Lecture 3 Lecture 4 3 1 11/10/2023 Gas Chromatography In gas chromatography a gaseous analyte is transported through the column by a gaseous mobile phase, called the carrier gas. For gases and compounds with boiling points below about 250 C Cross section of open tubular column Solute dissolved in liquid phase bonded to the surface of the column In gas-liquid partition chromatography, the stationary phase is a non-volatile liquid coated on the inside of the column or on a fine solid support. In gas-solid adsorption chromatography, the analyte is adsorbed directly on solid particles of stationary phase. Solute adsorbed on surface of stationary phase 4 Samples GC applications: Separation of volatiles Gases Small organic molecules (bp usually < 300°C) Trace analysis Analysis of very complex mixtures 6 2 11/10/2023 Capillary columns Typical number of plates: 25,000-200,000 Typical lengths 15-100 m 8 Van Deemter curves For capillary columns that have no packing the A term is zero and it becomes the Golay equation H = B/u + u [CM+CS] Low values of u: larger number of mass transfer between phases high degree of longitudinal diffusion 10 3 11/10/2023 Temperature programming 11 New Technology Conventional – 35 min. Ultrafast – 35 s RTX1701 5m 0.1mm 0.1μm 40°C to 200°C at 5°C/s He @ 1 mL/min 1 μL split 100:1 12 4 11/10/2023 Properties of Selected Gas Chromatography Detectors Approximate Limit of Detection (gs-1) Approximate Linear Range Comments Thermal conductivity (TCD) 10-5-10-6 103-104 Universal detector -measures changes in heat conduction Flame ionization (FID) 10-12 106-107 Universal detector -measures ion currents from pyrolysis Electron capture (EC or ECD) 10-14 102-103 Selective detector for compounds containing atoms with high electron affinities Flame photometric (FPD) 10-13 102 Selective detector for compounds containing S,P Nitrogen-phosphorus 10-8-10-14 105-107 Selective for N,P containing compounds Photoionisation (PID) 10-8-10-12 105 Universal (some selectivity due to identity of gas in lamp) Hall Detector 10-11 105 Specific detector for compounds which contain halogen, S, or N Mass spectrometer (MS) 10-12 variable, depends on MS Universal Fourier-transform infrared (FTIR) 10-10 102 Polar molecules Type 14 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Normal phase chromatography (column, TLC) Reverse phase chromatography (HPLC) Gas chromatography Size exclusion chromatography Ion exchange chromatography Lecture 2 Lecture 3 Lecture 4 15 5 11/10/2023 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Cross section of open tubular column Solute adsorbed on surface of stationary phase Adsorption Chromatography Normal phase chromatography (TLC, flash column) Solute dissolved in liquid phase bonded to the surface of the column Partition Chromatography Reverse phase chromatography (HPLC) Gas chromatography (GC) 17 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Large molecules are excluded Mobile ions held near cations that are covalently attached to stationary phase Anion-exchange resin; only anions can be attracted to it Ion-exchange Chromatography Small molecules penetrate pores of particles Size-exclusion Chromatography 18 6 11/10/2023 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Large molecules are excluded Mobile ions held near cations that are covalently attached to stationary phase Anion-exchange resin; only anions can be attracted to it Ion-exchange Chromatography Small molecules penetrate pores of particles Size-exclusion Chromatography 19 Size exclusion Chromatography (SEC) Also called: Molecular exclusion chromatography Steric exclusion chromatography Gel filtration chromatography (MP water): desalting Gel permeation chromatography (MP organic solvent) Molecules are separated according to their size Small molecules penetrate the small pores in the stationary phase, while large molecules do not. Large molecules are eluted first. 20 7 11/10/2023 Applications Typical SEC applications: analysis of synthetic polymers and oligomers coal derived substances lipids proteins cellulose derivatives crude oil alkanes SEC methods can also be used to: study processes which lead to change of hydrodynamic volume hydrolysis refolding of proteins polymerisation aggregation etc 21 Size Exclusion Chromatography Molecules are separated according to their size Big molecules cannot penetrate the small pores in the stationary phase. They are eluted by a volume of solvent equal to the volume of mobile phase Vt V0 Kav = Vr – V0 Vt – V0 V0: volume of the mobile phase (void volume) Vr: retention volume for a solute Vt : total volume of the column, r2 x length 22 8 11/10/2023 Size Exclusion Chromatography Molecules are separated according to their size Big molecules: Vr = V0; Kav = 0 Small molecules: Vr  Vt; Kav  1 Kav = Vr – V0 Vt – V0 V0: volume of the mobile phase (void volume) Vr: retention volume for a solute V0 found by e.g. Blue Dextran 2000 Vt : total volume of the column, r2 x length 23 Stationary Phase Polymers of glucose: Cellulose Dextran (If cross-link is glycerin: Sephadex) Polymers of agarose Polyacrylamides  Crossed-linked gels Small pore size Exclude molecules with MM  700  Crossed-linked gels Big pore size Exclude molecules with MM  108 The finer the particle, the greater the resolution, the slower the flow rate of the column Particles with different pore sizes can be mixed to give a wider molecular size separation range In general, two types of molecules can be separated provided that there is a 10% difference in their molecular sizes 24 9 11/10/2023 Columns Unlike the other modes of liquid chromatography, the separation comes from the stationary phase only. The mobile phase should have no effect as long as the sample is well dissolvable. Usual size of SEC columns: 7-8 mm diameter (analytical) 20-25 mm diameter (preparative) Separation is carried out on the pores which typically equals 40% of the total column volume. Length 20-60 cm Packing: As a result long columns, or several columns are required. porous silica cross-linked organic gels 25 Columns Selecting SEC columns: Handling SEC columns: Separation range selected carefully A column set should be always run in the same mobile phase (calibration, life of the column) Small particle size (5µm) provides more theoretical plates Small particle size more sensitive to contamination Small particle size can result on shear degradation of large polymers Combination of packings with different separation range can be achieved by: columns of different porosity or mixed bed columns Chemical nature of column packing crucial Should not be operated in backward position No air bubbles in columns during injection and assembly Always use a pre-column when in doubt about the quality of the sample Handbook of Size Exclusion Chromatography and Related Techniques: Revised And Expanded By Chi-san Wu Edition: 2, illustrated, revised Published by CRC Press, 2004 694 pages 26 10 11/10/2023 GPC polymers - Universal Calibration 28 GPC polymers - Universal Calibration In 1967, Benoit showed that the calibration for polymers of different types can be merged into one single curve based on hydrodynamic volume 29 11 11/10/2023 GPC polymers - Universal Calibration Molecular size difference between a linear chain and a branched chain polymer with the same molecular weight At the same molecular weight branched polymers have lower hydrodynamic volume, higher density and therefore lower intrinsic viscosity Universal Calibration Principle underestimates branching! 31 Molecular Mass Determination Caution: Branching Possible adsorption due to occasional charges on the gel Other adsorption mechanisms (silica must be coated with a hydrophilic phase to minimise solute adsorption) 32 12 11/10/2023 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Large molecules are excluded Mobile ions held near cations that are covalently attached to stationary phase Small molecules penetrate pores of particles Anion-exchange resin; only anions can be attracted to it Ion-exchange Chromatography Size-exclusion Chromatography 33 Ion-exchange chromatography Retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Anion exchanger: positively charged groups on the SP attract solute anion. Cation exchangers: covalently bound, negatively charged sites attract solute cations. Ion-exchange resins: RESINS: amorphous particles of organic material Examples: Polystyrene resins HC Cross-linking HC CH 2 HC CH 2 CH 2 Styrene Divinylbenzene cross-linking affects rigidity and porosity Divinylbenzene 34 13 11/10/2023 Ion-exchange selectivity Competition of Na+ and Li+ for anionic sites in cation-exchange resin R- R-Na+ + Li+ Selectivity coefficient K= R-Li+ + Na+ [R-Li+][Na+] [R-Na+][Li+] Describes the relative selectivity of the resins for Li+ and Na+ In general, ion exchangers favour the binding of ions of:  charge  hydrated radius  polarizability 35 Gradient elution Changes in ionic strength or pH C+ A+ + + + B+ + + + Strength of interaction with the resin: A-  B-  CBy  [C-], B- is eventually displaced and moves down the column. At higher [C-] the anion A- is also eluted 37 14 11/10/2023 Applications Converting a salt into another Pre-concentration of trace components of a solution. Chelex 100 Resin − N (CH2CO2H)2 Aminodiacetic acid Purification of water: obtention of Deionized Water Separation of proteins 38 Recap Schematic of a typical GC instrument Capillary columns GC applications The principle behind size exclusion Typical applications of SEC The principle of ion exchange Typical applications of IEX 39 15