CM4106 Separation Methods Gas Chromatography: Applications. Hyphenated Techniques

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1 CM4106 Separation Methods Gas Chromatography: Applications. Hyphenated Techniques Dr. Amalia Muñoz Fundación CEAM. Euphore Laboratories

2 Gas Chromatography Qualitative Analysis The characteristic parameter for the identification of a compound is the RETENTION TIME (RT) or the CORRECTED RETENTION TIME (RT ) RT and RT depend on: Temperature Stationary phase (column) Carrier gas Flow Etc. Time elapsed between the dead point and the peak maximum Time elapsed between the injection point and the peak maximum. Each solute has a characteristic retention time. Small fluctuations could derived in wrong identification It is not correct absolute RT data from literature with those obtained experimentally.

3 Gas Chromatography Qualitative Analysis Relative Retention times It is the ratio between the corrected the retention time of the analyte to identify (RT a ) and a substance used as a reference (RT ref ) RT = r RT RT ' a ' ref To obtain the RT r the reference compound must be added to the sample and to the standards The RT of the reference compound should be closed to the analytes.

4 Gas Chromatography Qualitative Analysis Other method The sample is separated into two aliquots. The first aliquot is injected directly A determined amount of a standard is added into the second aliquot and then injected. If we see a new peak in the second chromatogram, we can ensure that this substance is not in the sample while a signal of some of the peaks appear more intense in the second chromatogram that substance exists in the sample

5 Gas Chromatography Qualitative Analysis It could be simpler Using selective detectors: ECD, NPD, PID, etc. Using hyphenated techniques: GC-MS, GC-FTIR, GC-NMR Using two or more detectors

6 Gas Chromatography Qualitative Analysis Two Detectors A single injection on two GC detectors yields two sets of data in one half of the time Detector A Detector A Injector Injector Detector B Detector B Parallel Dual Detector Configuration Post Column Split Configuration Inyector Detector A Detector B Series Configuration (Detector A must be non-destructive)

7 Gas Chromatography Quantitative Analysis 3 important stages in a GC analysis, 1. The preparation of the sample. 2. The development of the separation and the production of the chromatogram 3. The processing of the data and the presentation of the results. Each stage is equally important and if not carried out correctly the results will be neither precise nor accurate. Sample preparation can be Simple: involving no more that diluting a known weight of sample with mobile phase Much more complex: including an extraction procedure followed by derivatization and then dilution. For some samples the preparation can be the most time consuming and difficult part of the whole analysis.

8 Gas Chromatography Quantitative Analysis Chromatogram The response must be linear Concentration Mass The response factor of each compound is different for each compound Parameters that can be used: Peak Height Peak Area

9 Gas Chromatography Quantitative Analysis Chromatogram Area Normalization PeakAreag % g = 100 areas The sum of the areas of all the peaks corresponds to 100% of the solutes separated. Only true if: All the compounds are eluted Same sensitivity % PeakAreag f g g = 100 ( area f ) i i As the compounds usually do not have the same sensitivity a correction factor should be applied Calibration curve f g = mass area

10 Gas Chromatography Quantitative Analysis Chromatogram Internal Standard An internal standard is a compound, not present in the sample, that is added in a constant amount to samples and calibration standards. The peak of compound must not overlap with the peaks of the analytes. 6 SI Method Area compound/ area SI y = x R 2 = y = 0.497x R 2 = mass compound/ mass SI Advantages: manual injection Disadvantages: To analyse great number of analytes To find a good IS compound A compound B Lineal (compound A) Lineal (compound B)

11 Gas Chromatography Quantitative Analysis Chromatogram External Standard Area compound y = x R 2 = ES Method y = x R 2 = mass compound compound A compound B Lineal (compound A) Lineal (compound B) Advantages: simpler than IS. Disadvantages: Sample injection reproducibility Preferable Automatic injection or sample valve

12 Gas Chromatography DERIVATIZATION Derivatization is the process of chemically modifying a compound to produce a new compound which has properties that are suitable for analysis using a GC WHY? To permit analysis of compounds not directly amenable to analysis due to, for example, inadequate volatility or stability Improve chromatographic behavior or detectability. Derivatization is a useful tool allowing the use of GC and GC/MS to be done on samples that would otherwise not be possible in various areas of chemistry such as medical, forensic, and environmental

13 Gas Chromatography DERIVATIZATION Increases volatility (i.e. sugars): Eliminates the presence of polar OH, NH, & SH groups Derivatization targets O,S, N and P functional groups (with hydrogens available Increases detectability, I.e. steroids/ cholesterol Increases stability Enhances sensitivity for ECD (Electron Capture Detection). The introduction of ECD detectable groups, such as halogenated acyl groups, allows detection of previously undetectable compounds in some cases: derivatization can also be used to decrease volatility to allow analysis of very low molecular weight compounds, to minimize losses in manipulation and to help separate sample peaks from solvent peak.

14 Gas Chromatography DERIVATIZATION Comments Advantages Disadvantages Silylation Readily volitizes the sample - Wide variety of compounds -Large number of silylating reagents available -Easily prepared -Moisture sensitive -Organic solvents must be aprotic (no protons available) -WAX type columns cannot be used Acylation -Used as the first step to further derivatizations or as a method of protection of certain active hydrogens. -Reduces the polarity of amino, hydroxyl, and thiol groups and adds halogenated functionalities. -Increased detectability by ECD -Derivatives are hydrolytically stable - Increased sensitivity by adding molecular weight -Acylation can be used as a first step to activate carboxylic acids prior to esterfication (alkylation) -Difficult to prepare. - Reaction products often need to be removed before analysis - Moisture sensitive - Reagents are hazardous and odorous Alkylation -Reduces molecular polarity by replacing active hydrogens with an alkyl group. - modify compounds with acidic hydrogens, such as carboxylic acids and phenols. -Reagents containing fluorinated benzoyl groups can be used for ECD -Wide range of alkylation reagents. -Reaction conditions can vary from strongly acidic to strongly basic - Some reactions can be done in aqueous solutions - Alkylation derivatives are generally stable -Limited to amines and acidic hydroxyls - Reaction conditions are frequently severe - Reagents are often toxic Formation of perfluoroderivatives Reagents containing fluorinated benzoyl groups can be used for ECD Wide range of application Easy to prepare Selectivity GC-Quiral Derivatiz.

15 Gas Chromatography DERIVATIZATION Some examples Derivatization Reaction Common Derivatizing Agent Methylation of carboxylic acids Oxime formation of carbonyl functionality Diazomethane, methanol/sulfuric acid PFBHA N-hexyl carbonate, carbamate, and ester formation from hydroxylic, aminic, and carboxylic functionality N-hexyl chloroformate Heptafluorobutyramide formation from aromatic amines Heptafluorobutyramide And much more

16 On-line combination of a chromatographic separation technique with a sensitive and Element-specific detector Chromatographic Techniques Spectroscopic Techniques Separation of analytes Necessity of pure analytes Low security in identification High identification level Hyphenated techniques provide the analyst with structural information on the components present in complex mixtures. This information may be sufficient to identify components

17 Chromatograph Column Interface Spectrometer Column Chromatograph Non-Destructive Detector Interface Spectrometer Chromatograph Detector Column Interface Non-Destructive Spectrometer Chromatograph Column Interface 1 Interface 2 Non-Destructive Spectrometer Spectrometer Chromatograph Column Interface 1 Interface 2 Spectrometer Spectrometer

18 Common hyphenated techniques Gas Chromatography GC-MS GC-FTIR-MS GC-FTIR GC-AAS Mass Spectrometry Infrared Spectroscopy Emission and Absorption Atomic Spectroscopy Nuclear Resonance Spectrometry LC-MS LC-FTIR LC-ICP Liquid Chromatography LC-NMR

19 GC/MS Provide Information about the chemical composition of the analyte Molecular analysis Isotopic analysis Trace analysis Elemental analysis Surface analysis Mass Spectrometer High Detection efficiency and specificity of molecular recognition Molecules are ionized (broken down) into electrically charged particles called ions with a specific mass and charge. Due to that, the speed and direction of the ion may be changed with an electric or magnetic field.

20 GC/MS

21 GC/MS Mass Spectrometer Components GC Column/MS Interface Ionizations Source: for example Electron Impact (EI) or Chemical Ionization (CI) Mass Analyzer: for example Magnetic Sector, Quadrupole, Ion Trap, Time of Flight Mass Detector Software/Data display

22 GC/MS Mass Spectrometer Ionization methods Should provide a high ionization efficiency and high stability with minimum kinetic energy distribution and minimum angular dispersion of the ion produced The ion source should produce: Intact molecular ions MW (for MW information) Fragment ions (for structural information) Control over the internal energy transferred to the molecule for control over the degree of fragmentation Should be possible to couple with various types of chromatographs

23 GC/MS Mass Spectrometer Ionization methods Electron Impact, 70 ev Analytes elute here A + B + C + ABC

24 Ionization method Electron Impact (EI) Chemical Ionization (CI) Type of analysis Molecular Molecular Ionization agent Electrons ( 70 ev) Gaseous ions. (Reagent gas: CH 4, NH 3, NF 3, N 2 O) GC/MS Mass Spectrometer Pressure Characteristics Application 10-5 Torr 1 Torr Reproducible spectra, extensive fragmentation Molecular ions and controllable fragmentation VOC; structural elucidation VOC; MW determination Desorption Ionization (DI) Molecular Energetic particles, photons Torr Intact molecular ions from high-mass compounds Condensed phase, high-mass compounds; MW and structure determination Spray Ionization (SI) Molecular Electrical, thermal, and pneumatic energy Torr Intact molecular ions from high-mass compounds Solutions of high-mw compounds; used with LC/MS to determine MW s and structures Glow- Discharge Ionization (GD) Elemental Plasma Torr Very stable, reproducible spectra Elemental analysis of solids Inductively Coupled Plasma (ICP) Elemental Plasma Atmosp. pressure High ionization efficiency Elemental analysis of solutions

25 GC/MS Mass Spectrometer Mass Analyzer Function: to measure the mass-tocharge ratios of ions (m/z), thus providing a mesa to identify them. Depend on the interactions of charged particles with electric or magnetic fields

26 GC/MS Mass Spectrometer Mass Analyzer Quadrupole: Orthogonal DC and RF planes (x/y) are used to separate ion passing through a highly evacuated tube. As an analyte mass fragment pass trough the mass analyzer, the matched x/y field at that instant a very small part of one scan- allows only specific fragment exit into the mass detector. The duration of an entire scan is usually less than 1 second (e.g. 0.6 sec). The m/z range can be adjusted by the analyst MS scanning process occurs very quickly, so each scan includes a measure of all the amounts of each different mass fragment during that scan

27 GC/MS Mass Spectrometer Mass Analyzer Ion trap It is an three-dimensional analog of the quadrupole mass filter. Because forces operate in 3 directions, the electric fields can be used store ions in an electrical bottle. It consist of two end-cap electrodes of hyperbolic cross section that normally are operated at ground potential. A rotationally symmetric electric quadrupole field is generated At a given voltage, ions of a specific mass range are held oscillating in the trap. Initially, the electron beam is used to produce ions and after a given time the beam is turned off. All the ions, (except those selected by the magnitude of the applied rf voltage) are lost to the walls of the trap, and the remainder, continue oscillating within the trap. The potential of the applied rf voltage is then increased, and the ions sequentially assume unstable trajectories and leave the trap via the aperture to the sensor. The ions exit the trap in order of their increasing m/z values.

28 GC/MS Mass Spectrometer Mass Analyzer Time-of-Flight: A beam of ions is accelerated through a known potential V, and the time taken to reach a detector at a distance d, in a linear fligh tube, is measured. If all ions fall through the same potential, V, their velocities must be inversely proportional to the square roots of their masses. The source must be pulsed in order to avoid simultaneous arrivals of ions of different mass-to-charge ratios.

29 GC/MS Mass Spectrometer Method Quantity measured Mass/Charg e range (Da/charge) Resolution at 1000 (Da/charge) (mass peak witdh) Mass Measurement Accuracy at 1000 Da/charge Dynamic range (number of order of magnitude of concentration over which response varies linearly) Operating Pressure Sector Magnet Momentum/ charge < 5 ppm Time of flight Flight time % Quadrupole ion trap frequency % Quadrupole Filters for m/z % Cyclotron Resonance frequency < 10 ppm

30 GC/MS Mass Spectrometer Mass Detector and data generation One scan includes all the mass detector current at each m/z ratio allowed to exit the mass analyzer. A chromatographic peak ( around10 seconds wide) can be scanned 16 or 17 times. Each scan generates an individual mass spectrum which is saved in the computer s hard drive

31 Coupling GC/MS Direct Introduction Interfaces for GC/MS Jet separator Permselective membrane Molecular effusion Direct coupling Interfaces for LC/MS Total solvent elimination before ionization Partial Solvent elimination before ionization Solvent introduction

32 Coupling GC/MS JET SEPARATOR The GC flow is introduced into an evacuated chamber through a restricted capillary. At the capillary tip a supersonic expanding jet of analyte and carrier molecules is formed and its core area sampled into the mass spectrometer. In an expanding jet, high molecular mass compounds are concentrated in the core flow whereas the lighter and more diffusive carrier molecules are dispersed away, in part through collisions. Thus, sampling of the core flow produces an enrichment of the analyte. J. Abian. JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 34, (1999) The jet interface is very versatile, inert and efficient, despite disadvantages of reduced efficiency with more volatile compounds and potential plugging problems at the capillary restrictor.

33 PERMSELECTIVE MEMBRANE Coupling GC/MS It is made of a silicone-rubber membrane that transmits organic non-polar molecules and acts as a barrier for (non-organic) carrier gases. Despite being a very effective enrichment procedure, it also suffers from discrimination effects with more polar analytes and produces significant band broadening of their chromatographic peaks

34 Coupling GC/MS MOLECULAR EFFUSION It is based on the molecular filtering of the gas effluent by means of a porous glass frit. The column effluent passes through a fritted tube situated in a vacuum chamber. Small molecules traverse the microscopic pores in the tube walls and are evacuated whereas high molecular mass molecules are transferred to the ion source. Drawbacks : High dead volume added and high surface area. Shows discrimination effects in the case of smaller molecules

35 Coupling GC/MS DIRECT INTRODUCTION Capillary columns use optimum flow-rates of gas of about 1-2 ml min -1, instead of more than ml min -1 used with packed columns, allowing all the effluent to be directed to the mass spectrometer. This is usually done through a direct coupling where the column exit is introduced into the ion source without a capillary restriction. Extensively used in analytical laboratories. Enrichment interfaces have become unnecessary for GC/MS

36 Coupling LC/MS DIRECT LIQUID INTRODUCTION (DLI) Use of the solvent as the reagent gas in a CI source A major problem in the introduction of liquids through a capillary is that the high vacuum in the ion source produces rapid evaporation of the liquid inside the capillary, eventually leading to flow stoppage through freezing of the solvent. The use of restricted capillaries and the heating of the capillary in part solved this problem Maximum flow-rates accepted by DLI interfaces are in the range μl min -1 and are best suited for micro- and nanobore chromatography (<1 mm i.d.column) Obsolete

37 Coupling LC/MS THERMOSPRAY In the presence of a volatile buffer in the solvent, ions are obtained without any other ionizing source The liquid flowing through the hot capillary is partially evaporated so that an ultrasonic spray of vapour and charged microdroplets is obtained at the probe exit. Ions present in the source are transferred into the mass spectrometer through the ion cone aperture while the main portion of the residual vapours is captured by the high-conductance vacuum line and purged by a rotary pump. Charged microdroplets in the spray are the result of the rapid breakdown of the liquid surface during vaporization and the statistical distribution of electrolyte ions in the droplets. Hence, an equal mixture of positive and negatively charged droplets is formed from a neutral solution. Gas-phase ions are produced in the source from these microdroplets as a result of several processes including ion desolvation and ion evaporation from the charged microdroplets, as well as gas-phase CI processes TO VACUUM

38 Coupling LC/MS THERMOSPRAY The evaporation of neutral molecules from the initial charged droplet produces a reduction in droplet size and a charge density increment. This excess of electrical energy can produce either fragmentation of the droplets into smaller droplets or desorption of ions into the gas phase by ion evaporation. Alternatively, gas-phase ions can be produced by simple desolvation of liquid-phase ions. These processes produce a plasma of reagent ions mainly derived from the ammonium acetate buffer in the solution. If the proton affinity is relatively high, the analyte will keep its charge or will take it from the reagent plasma. If the analyte proton affinity is low relative to other reagent plasma molecules, it will not be ionized or ionmolecules adducts will be observed. TSP ionization causes simple soft ionization spectra containing mostly molecular information TO VACUUM

39 Coupling LC/MS Atmospheric Pressure ionization Ionization of the column effluent is carried out at atmospheric pressure by any of several procedures including a radioactive source, electrical discharges and high voltage electric fields. The ions produced are continuously sampled through a small aperture and pass into the spectrometer where they are mass analysed. Introduce only a small amount of solvent into the low-pressure region of the MS. Two main commercial source types: Atmospheric Pressure Chemical Ionization (APCI) Electrospray (ESI) source

40 Coupling LC/MS APCI An atmospheric pressure vaporization chamber in which 1-2 μl of a liquid sample is injected through a septum. A hot carrier gas (N 2 ) is introduced into the chamber to help vaporization and to transport the analyte to an area close to a radioactive beta source ( 63 Ni). In this area gas ionization occurred, producing an atmospheric pressure reagent plasma that gave rise to analyte ions through ion- molecule reactions. Ions a then transferred to the mass spectrometer through a small aperture and mass analysed Analysis of medium- and low-polarity compounds or when relatively non-polar solvents have to be used.

41 Coupling LC/MS ESI The liquid sample is introduced into a high potential chamber through the capillary and at the exit an electrically induced spray of charged microdroplets is produced. Ions in these droplets enter the gas phase through evaporative processes (similar to TSP). The ions in the spray are captured through a glass capillary restrictor where they are conducted into the low vacuum area of the mass spectrometer. To promote droplet desolvation, the ionization chamber is continuously supplied with a countercurrent flow of dry nitrogen.