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GC and GC-MS

GC and GC-MS. Gas Chromatography. Function Components Common uses Chromatographic resolution Sensitivity. Function. Separation of volatile organic compounds Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species

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GC and GC-MS

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  1. GC and GC-MS

  2. Gas Chromatography • Function • Components • Common uses • Chromatographic resolution • Sensitivity

  3. Function • Separation of volatile organic compounds • Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species • Separation occurs as a result of unique equilibria established between the solutes and the stationary phase (the GC column) • An inert carrier gas carries the solutes through the column

  4. Components • Carrier Gas, N2 or He, 1-2 mL/min • Injector • Oven • Column • Detector

  5. Syringe Injector Detector Gas tank Column Oven

  6. Injector • A GC syringe penetrates a septum to inject sample into the vaporization camber • Instant vaporization of the sample, 280 C • Carrier gas transports the sample into the head of the column • Purge valve controls the fraction of sample that enters the column

  7. Syringe Syringe Injector Injector Purge valve closed Purge valve open Splitless (100:90) vs. Split (100:1) He He GC column GC column

  8. Split or splitless • Usually operated in split mode unless sample limited • Chromatographic resolution depends upon the width of the sample plug • In splitless mode the purge valve is close for 30-60 s, which means the sample plug is 30-60 seconds • As we will see, refocusing to a more narrow sample plug is possible with temperature programming

  9. Open Tubular Capillary Column 0.32 mm ID Mobile phase (Helium) flowing at 1 mL/min Liquid Stationary phase 0.1-5 mm 15-60 m in length

  10. Oven • Programmable • Isothermal- run at one constant temperature • Temperature programming (gradient) - Start at low temperature and gradually ramp to higher temperature • More constant peak width • Better sensitivity for components that are retained longer • Much better chromatographic resolution

  11. Typical Temperature Program 220C 160C 50C 0 60 Time (min)

  12. Detectors

  13. Flame Ionization Detector (ng) High temperature of hydrogen flame (H2 +O2 + N2) ionizes compounds eluted from column into flame. The ions collected on collector or electrode and were recorded on recorder due to electric current.

  14. FIDs • Effluent exits column and enters an air/hydrogen flame • The gas-phase solute is pyrolized to form electrons and ions • All carbon species are reduced to CH2+ ions • These ions collected at an electrode held above the flame • The current reaching the electrode is amplified to give the signal

  15. FID • A general detector for organic compounds • Very sensitive (10-13 g/s) • Linear response (107) • Rugged • Disadvantage: specificity

  16. Thermal Conductivity Detector Properties • Responds to all compounds • Adequate sensitivity for many compounds • Good linear range of signal • Simple construction • Signal quite stable provided carrier gas flow rate, block temperature and filament power are controlled • Nondestructive detection • Are not sensitive enough for capillary columns.

  17. Thermal Conductivity Detector

  18. Thermal Conductivity Detector Measures the changes of thermal conductivity due to the sample (mg). Sample can be recovered. He and H gas is usually the carrier gas At constant electrical power, the temp of the device depends on the thermal conductivity (TC) of the surrounding gas. The filament is a Pt, gold or tungsten wire’Twin detectors are usually used There is a bridge circuit and arranged so that the TC of the carrier gas is cancelled. The filament may loose heat by radiation to a cooler surface and by conduction to the molecules coming into contact with it. When a compound elutes, the thermal conductivity of the carrier gas and compound gas is lowered, and the filament in the sample column becomes hotter than the other control column. Its resistance increased, and this imbalance between control and sample filament resistances is measured by a simple gadget and a signal is recorded

  19. Electron Capture Detector (picogram)

  20. Electron Capture Detector ECD detects ions exiting from the gas chromatographic column by the anode electrode. 3H or 63Ni emits  particles. Ionization of the carrier gas: N2 (Nitrogen carrier gas) +  (e) = N2+ + 2e These N2+ establish a “base line” X (F, Cl and Br) containing sample +  (e)  X- Electronegativespecies capture electron Ion recombination : X- + N2+ = X + N2 The “base line” will decrease and this decrease constitutes the signal. Insecticides, pesticides, vinyl chloride, and fluorocarbons. Halogens, lead, phosphorous, nitro groups, silicone and polynuclear aromatics. Adv-Does not consume sample as in case of FID.

  21. Mass Spectrometry

  22. “Mass spectrometry is the branch of science dealing with all aspects of mass spectroscopes and the results obtained with these instruments.’’ 1. It measures mass better than any other technique. 2. It can give information about chemical structures., molecular wt. etc. • To identify, verify, and quantify: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers etc

  23. How does it work? • Gas-phase ions are separated according to mass/charge ratio and sequentially detected

  24. A gas phase molecule subjected to energy* greater than the ionization energy • electron can be removed which results in the formation of a molecular ion:

  25. Parts of a Mass Spec • Sample introduction • Source (ion formation) • Mass analyzer (ion sep.) - high vac • Detector (electron multiplier tube)

  26. Ion source:makes ions Working of Mass Spectrometer Sample Mass analyzer: separates ions Mass spectrum:presents information

  27. Mass Spectrometer Block Diagram High Vacuum System Ion source Mass Analyzer Data System Inlet Detector

  28. Ionization Techniques Electron Impact

  29. Chemical Ionization

  30. Mass analyzers separate ions based on their mass-to-charge ratio (m/z) • Operate under high vacuum (keeps ions from bumping into gas molecules) • Actually measure mass-to-charge ratio of ions (m/z) • Key specifications areresolution, mass measurement accuracy,andsensitivity. • Several kinds like, quadrupole, time-of-flightand ion traps etc. are mostly used.

  31. Quadrupole Mass Analyzer • Has four parallel metal rods. • Lets one mass pass through at a time. • Can scan through all masses or sit at one fixed mass.

  32. Summary: acquiring a mass spectrum Ionization Mass Sorting (filtering) Detection Ion Source Ion Detector Mass Analyzer • Form ions • (charged molecules) Sort Ions by Mass (m/z) • Detect ions 100 75 Inlet • Solid • Liquid • Vapor 50 25 0 1330 1340 1350 Mass Spectrum

  33. EI, CI • EI (hard ionization) • Gas-phase molecules enter source through heated probe or GC column • electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions • EI spectra can be matched to library stds • CI (soft ionization) • Higher pressure of methane leaked into the source • Reagent ions transfer proton to analyte

  34. EI Source Under high vacuum filament 70 eV e- To mass analyzer GC column anode Acceleration slits repeller

  35. EI process M+* • M + e- f1 f2 f4 f3 This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam

  36. Mass Analyzers • Low resolution • Quadrupole • Ion trap • High resolution • TOF time of flight • Sector instruments (magnet) • Ultra high resolution • ICR ion cyclotron resonance

  37. APPLICATIONS • Pharmaceutical analysis • Bioavailability studies • Drug metabolism studies, pharmacokinetics • Characterization of potential drugs • Drug degradation product analysis • Screening of drug candidates • Identifying drug targets • Bio-molecule characterization • Proteins and peptides • Oligonucleotides • Environmental analysis • Pesticides on foods • Soil and groundwater contamination • Forensic analysis/clinical

  38. INTERFACING • A> Interfacing GC with MS • 1. Molecular jet separator (all-glass) : Ryhage, 1966 • 2. Membrane separator : Llewellyn, 1966 • 3. Effusion separator : Watson-Bieman, 1964 • 4. Open split coupling • 5. Capillary direct interface • B> LC-MS interface • 1. Moving belt wire interface • 2. Thermospray • 3. Atmospheric pressure inlet (API) • 4. Electrospray • 5. Particle beam • 6. Dynamic FABMM

  39. GC-MS Interface

  40. PROBLEMS IN COMBINING HPLC AND MS HPLC • Liquid phase operation • 25 - 50 deg. C • No mass range limitations • Inorganic buffers • 1 ml/min eluent flow is equivalent to 500 ml/min of gas MS • Vacuum operation • 200 - 300 deg. C • Up to 4000 Da for quadrupole MS • Requires volatile buffers • Accepts 10 ml/min gas flow

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