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. Injector
Syringe
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. Splitless (100:90) vs. Split (100:1)
Injector
Syringe
Purge valve
open
closed 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 s, which means the sample plug is 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 Program60
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-
Electronegative species 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
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. Working of Mass Spectrometer
Ion source: makes ions
Mass analyzer: separates ions
Mass spectrum: presents information
Sample

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 are resolution, mass measurement accuracy, and sensitivity.
Several kinds like, quadrupole, time-of-flight and ion traps etc. are mostly used.
Mass analyzers separate ions based on their mass-to-charge ratio (m/z)

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
Inlet
Ionization
Mass Analyzer
Mass Sorting (filtering)
Ion
Detector
Detection
Ion Source
• Solid
• Liquid
• Vapor
Detect ions
Form ions
(charged molecules)
Sort Ions by Mass (m/z)
1330
1340
1350
100 75 50 25
Mass Spectrum

33. EI, CI EI (hard ionization) CI (soft 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+* f1 f2 f4 f3 M + e-
This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam

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

38. 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

39. INTERFACING A> Interfacing GC with MS B> LC-MS interface
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

40. GC-MS Interface

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