GC and GC-MS
Transcript of 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
• 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
Components
• Carrier Gas, N2 or He, 1-2 mL/min• Injector• Oven• Column• Detector
Gas tank
Oven
Column
Injector
Syringe
Detector
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
Splitless (100:90) vs. Split (100:1)
Injector
Syringe
Injector
Syringe
Purge valveopen
Purge valveclosed
GC column GC column
HeHe
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
0.32 mm ID
Liquid Stationary phase
Mobile phase (Helium) flowing at 1 mL/min
Open Tubular Capillary Column
15-60 m in length
0.1-5 m
FSOT columns
• Coated with polymer, crosslinked– Polydimethyl soloxane (non-polar)– Poly(phenylmethyldimethyl) siloxane (10%
phenyl)– Poly(phenylmethyl) siloxane (50% phenyl)– Polyethylene glycol (polar)– Poly(dicyanoallyldimethyl) siloxane– Ploy(trifluoropropyldimethyl) siloxane
Polar vs. nonpolar
• Separation is based on the vapor pressure and polarity of the components.
• Within a homologous series (alkanes, alcohol, olefins, fatty acids) retention time increases with chain length (or molecular weight)
• Polar columns retain polar compounds to a greater extent than non-polar– C18 saturated vs. C18 saturated methyl ester
C16:0
C18:0
C18:1C18:2
C16:1
C16:0
C18:0
C18:1
C18:2
C16:1
RT (min)
RT (min)
Polar column
Non-polar column
Oven• Programmable• Isothermal- run at one constant
temperature• Temperature programming - 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– Peak refocusing at head of column
Typical Temperature Program
Time (min)0 60
50C
220C
160C
Detectors
• Flame Ionization Detectors (FID)• Electron Capture Detectors (ECD)• Electron impact/chemical ionization (EI/CI)
Mass spectrometry
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
FID
• A general detector for organic compounds• Very sensitive (10-13 g/s)• Linear response (107)• Rugged• Disadvantage: specificity
ECD
• Ultra-sensitive detection of halogen-containing species
• Pesticide analysis• Other detectors besides MS
– IR– AE
Mass Spectrometry
What kind of info can mass spec give you?
• Molecular weight• Elemental composition (low MW with high
resolution instrument)• Structural info (hard ionization or CID)
How does it work?
• Gas-phase ions are separated according to mass/charge ratio and sequentially detected
Parts of a Mass Spec
• Sample introduction• Source (ion formation)• Mass analyzer (ion sep.) - high vac• Detector (electron multiplier tube)
Sample Introduction/SourcesVolatiles• Probe/electron impact (EI),Chemical ionization (CI)• GC/EI,CIInvolatiles• Direct infusion/electrospray (ESI)• HPLC/ESI• Matrix Assisted Laser Adsorption (MALDI)Elemental mass spec• Inductively coupled plasma (ICP)• Secondary Ion Mass Spectrometry (SIMS)
– surfaces
EI, CI• EI (hard ionization)
– Gas-phase molecules enter source through heated probe or GC column
– 70 eV 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 (mtorr)
– Reagent ions transfer proton to analyte
To massanalyzer
filament
70 eV e-
anoderepeller Acceleration
slits
GC column
EI SourceUnder high vacuum
EI process
• M + e- M+*
f1 f2 f3f4
This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam
Ion Chromatogram of Safflower OilRT: 14.48 - 24.30
15 16 17 18 19 20 21 22 23 24Time (min)
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Rel
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RT: 20.82AA: 3547389BP: 67
RT: 21.04AA: 665791BP: 55
RT: 16.04AA: 304398BP: 74
RT: 21.90AA: 291543BP: 28RT: 16.84
AA: 78898BP: 28
NL: 9.69E5TIC F: {0,0} + c EI det=350.00 Full ms [ 25.00-510.00] MS ICIS evanssaf
CI/ ion-molecule reaction
• 2CH4 + e- CH5+ and C2H5
+
• CH5+ + M MH+ + CH4
• The excess energy in MH+ is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation
EI spectrum of phenyl acetate
Mass Analyzers• Low resolution
– Quadrupole– Ion trap
• High resolution– TOF time of flight– Sector instruments (magnet)
• Ultra high resolution– ICR ion cyclotron resonance
Resolution
• R = m/z/m/z• Unit resolution for quad and trap• TOF up to 15000• FT-ICR over 30000
– MALDI, Resolve 13C isotope for a protein that weighs 30000
– Resolve charge states 29 and 30 for a protein that weighs 30000
High vs low Res ESI
• Q-TOF, ICR– complete separation of the isotope peaks of a
+3 charge state peptide– Ion abundances are predictable– Interferences can be recognized and
sometimes eliminated
• Ion trap, Quad– Unit resolution
MVVTLIHPIAMDDGLR594.3594.7
595.0
601.3
595.3601.0
601.7
602.0
m/z
C78H135N21O22S2+3Q-TOF
901.4
891.7
902.3
900.6
891.2
892.6
LCQ
R = 0.88
m/z
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Quadrupole Mass Ion Filter
Ion Trap
Time of Flight -TOF
Where:
•mi = mass of analyte ion •zi = charge on analyte ion •E = extraction field •ti = time-of-flight of ion •ls = length of the source •ld = length of the field-free drift region •e = electronic charge (1.6022x10-19 C)
TOF with reflectronhttp://www.rmjordan.com/tt1.html
Sector instrumentshttp://www.chem.harvard.edu/mass/tutorials/magnetmovie.html
FT-ICRMS
• http://www.colorado.edu/chemistry/chem5181/MS_FT-ICR_Huffman_Abraham.pdf
Mass accuracy
• Mass Error = (5 ppm)(201.1001)/106 = 0.0010 amu
• 201.0991 to 201.1011 (only 1 possibility)• Sector instruments, TOF mass analyzers• How many possibilities with MA = 50
ppm?with 100 ppm?
Exact Mass Determination
• Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF)
• C9H15NO4, FM 201.1001 (mono-isotopic)• Mass accuracy = {(Mass Error)/FM}*106
• Mass Error = (5 ppm)(201.1001)/106 = 0.0010 amu