GC Theory and application PDF

EFFICIENCY Efficiency is a factor that is typically used to describe peak width. High Efficiency - narrow peaks The term that is generally used to describe column efficiency is “number of theoretical plates” or N...

EFFICIENCY Efficiency is a factor that is typically used to describe peak width. High Efficiency - narrow peaks The term that is generally used to describe column efficiency is “number of theoretical plates” or N N = L/H Where: L = column length H = plate height (both in the same units) 1 Importance of Theoretical Plates (N) Peak heigth vs. Plate number 2 N can be measured from the peaks on a chromatogram.. 2  t  2  t r 2   t N = 16 r N = 5.54 w w b  1/2   ( ) = 5. 55 r  w1   2  The value of N is greatly dependent on the value of H. The value of H depends primarily on four factors: 1) the velocity of the mobile phase, 2) eddy diffusion or multipath diffusion, 3) the diffusion of the compound in the mobile phase 4) the transfer of the compound between the stationary phase and the mobile phase. 3 Theoretical Plate Height H = A + B/ + (Cs + Cm)   = the average linear mobile phase velocity A is a term expressing multipath diffusion B/ is the term for longitudinal diffusion Cs is the mass transfer term in the stationary phase Cm is the mass transfer term in the mobile phase 4  Van Deemter plots ▪A plot of plate height vs. average linear velocity of mobile phase. The effect of the three terms in the van Deemter equation are shown graphically in figure Predicts that there will be an optimum velocity that gives a minimum value for (H) and thus, a maximum efficiency. Such plots are of considerable use in determining the 5 optimum mobile phase flow rate. 7  Why use GC ?  Highly Sensitive  High Speed of Resolution  Short Analysis Time  Small samples (L or g needed)  Wide Choice of Stationary Phase  Wide Choice of Sensitive Detectors  Highly accurate quantification (1-5 % RSD)  Ease of Operation GOALS  To separate mixtures of organic compounds  To detect individual analytes  To identify & quantitate using reference standards  Preparative work 8 9 Configuration of a GC System General Overview  Configuration of a GC System General Overview Gas A gas chromatograph consists of source A regulated and purified carrier gas source, which moves the sample through Sampler Sampler the instrument Inlet An inlet, which also acts as a vaporizer for liquid samples A column, in which the time separation Column occurs A detector, which responds to the components as they elute from the Detector column by changing its electrical output Output: Data interpretation of some sort Output Configuration of a GC System Gas source & PC system purifiers Injection port Detector Column 13  Carrier Gas 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) Flow rate increases N2 < He < H2 - Solutes diffuse rapidly → smaller mass transfer term Diffusion coefficients follow: H2 > He > N2  Columns  Heart of the separation process  Can be classified by packed and capillary type TYPE OF COLUMNS 1. Packed Columns - Length 0.5~20 m - I. D. 2~4 mm 2. Capillary columns -Length 10~100 m -I.D. 0.1~0.53 mm 22 wall—coated support-coated porous-layer open tubular open tubular open tubular 23 http://toolboxes.flexiblelearning.net.au/demosites/series5/508/laboratory/studynotes/Graphics/capillary.gif 24  ADVANTAGES DISADVANTAGES Economic Se o material de enchimento não for colocado na coluna de forma compacta e uniforme, os High load capacity espaços vazios resultantes funcionarão como Higher sample quantity câmaras de diluição da amostra. Packed Low efficiency Slow Analysis Higher lengh =>narrow peaks Lower quantity of stationary phase =>Higher efficiency Lower sample processment Capacity. Separation of complex mixtures Capillary Fast saturation. Fast Analysis Higher separation Low sample quantity Higher sensibility/detection limits Split is needed 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 N, where N increases directly with column length Easy to generate long (10s of meters) lengths of narrow columns to maximize resolution Choosing column dimensions Rs 28  Column choice is governed by three important requirements: Optimization targets Separation power, Sample capacity and Speed of the column. Important separation parameters are: Retention, measured by the retention factor k Selectivity, measured by the selectivity factor α 29 Plate number N Influence of stationary phase polarity on the selectivity for a specific test sample 30 Film thickness and retention (temperature programmed analysis) 31 FEATURES THICK-FILM COLUMNS Advantages: High k-values for volatile compounds High sample capacity Highly inert, even for polar compounds (acids, bases) Higher efficiencies. Disadvantages: Higher analysis time More bleeding Lower optimum gas velocity 32 Resolution as function of column length 33 1.) Open Tubular Columns Increasing Resolution Decrease tube diameter Increase resolution Increase Column Length Increase resolution 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 35 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  Colunas não polares ✓ BP1; OV1; SE30  Colunas mediamente polares ✓ BP5; OV-3; SE52  Colunas polares ✓ BP20; carbowax20M, DBwax Polaridade dos solutos Forno de colunas  A temperatura da coluna é uma variável importante ✓ necessidade de controlo rigoroso (± 0,1 C) / controlo dos tempos de retenção o gamas entre - 50 e + 450 C ✓ depende do p.e. da amostra e do grau de separação necessário o o aumento de temperatura diminui a separação mas diminui drasticamente o tempo de eluição o para amostras com importante gama de pontos de ebulição deve usar-se um programa de temperaturas  Programa de temperaturas ✓ Isotérmica ✓ Rampa contínua ✓ Rampa por patamares o último patamar bem acima do p.e. do menos volátil – Até 50 C 20 Programa de temperaturas ✓ Temperatura do Injector: o 30 a 50 C acima do menos volátil Isotérmica a 45 ºC ✓ Temperatura do Detector o : 30 a 50 C acima do menos volatil ✓ Temperatura da coluna Isotérmica a 145 ºC o Iniciar a 10-20 C abaixo do mais volátil – Temperaturas muito baixas trazem dificuldades de estabilização o Subir em rampa se a escala das volatilidades abrange toda a gama o Terminar a rampa a cerca de 20 C acima do mais volátil o Usar várias rampas se houver várias famílias de Temperatura programada: 30 -180 compostos (com diferenças significativas de volatilidade) ✓ As colunas não polares podem ser aquecidas a mais de 300 C ✓ As colunas polares raramente podem ser submetidas a temperaturas acima de 250 C  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 TFIM TEMPERATURA R TINI tINI tFIM TEMPO Temperature gradient improves resolution while also decreasing retention time  3.) Packed Columns Greater sample capacity Broader peaks, longer retention times and less resolution - Improve resolution by using small, uniform particle sizes Open tubular column Packed column  Comparison of Packed and Capillary Column http://origin-ars.els-cdn.com/content/image/1-s2.0-S0021967399007256-gr3.gif Capillary Column http://lipidlibrary.aocs.org/gc_lipid/05_gc_fa/Figure5-01.png Packed Column 43 GC detection principles The detector visualises the result of the chromatographic separation by registering whether 'something' or 'nothing' elutes from the column. Essential features of a detector Universal/selective Sensitivity Low noise level Linearity Accuracy (reproducibility) Maintenance Speed Reliability Ease ofuse Cost (purchase and operation) The DETECTOR 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 Area of Gaussian peak = 1.064  peak height  w 1 2 Peak area increases proportional to Peak Area concentration of standard if unknown/standard have the identical retention time → same compound Concentration of Standard Linear range is the area between the minimum detectable concentration and the upper limit of the linear range, Configuring a GC System Common Detectors  Overview of the main GC detectors Detector Operating principle Linearity Carrier gas Thermal conductivity (TCD) Thermal conductivity of compound vs. reference 104 H2, He, N2 Thermal dissociation Flame ionisation (FID) and ionization 106 H2, He, N2 Reduce background N2, Ar, CH4, Electron capture (ECD) 104 electron current by ionization of compound He + make-up gas Nitrogen-phosphorus (NPD) Ionization by thermo-ionization source 105 H2, He, N2 P: 104 Flame photometric detector (FPD) Chemiluminescence H2, He, N2 S: 103 Mass spectrometer Mass separation He (MS) of ionised molecule fragments Configuring a GC System DETECTORS ~ 15 equipam cromatógrafos comerciais 4 respondem pela maior parte das aplicações DCT TCD DIC FID Detector por Detector por Condutividade Ionização em Térmica Chama DCE ECD EM MS Detector por Detector Es- Captura de pectrométrico de Eletrons Massas DETECTORES Classificação UNIVERSAIS: Geram sinal para qualquer substância eluida. SELETIVOS: Detectam apenas substâncias com determinada propriedade físico-química. ESPECÍFICOS: Detectam substâncias que possuam determinado elemento ou grupo funcional em suas estruturas Detectors 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 Detectors Electron Capture Detector Sensitive to halogen-containing and other electronegative compounds Based on the capture of electrons by electronegative atoms - Compounds ionized by -rays from radioactive 63Ni Extremely sensitive (~ 5 fg/s) Steady current (flow of electrons) disrupted by compounds with high electron affinity  Response vs sensitivity is the degree to which a detector produces a change in signal when a sample component passes through the detector Selectivity vs specificity Detection limit Detection limits for several commonly used GC detectors Minimum detectable amount Detector [ng] FID 0.1 TCD between 1 and 10 ECD 0.001 MS (full scan mode) 0.1 MS (SIM mode, 1 ion) 0.001 Introduction Which Separation Technique for Which Compound? 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) 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 TYPICAL GC APPLICATIONS