Transcript GC and GC-MS

GC and GC-MS
Gas Chromatography
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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
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Carrier Gas, N2 or He, 1-2 mL/min
Injector
Oven
Column
Detector
Syringe
Injector
Detector
Gas tank
Column
Oven
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)
Syringe
Syringe
Injector
Injector
He
He
Purge valve
closed
GC column
GC column
Purge valve
open
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 3060 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
Open Tubular Capillary Column
Mobile phase
(Helium)
flowing at 1
mL/min
0.32 mm ID
Liquid
Stationary
phase
15-60 m in length
0.1-5 mm
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
C18:1
C18:2
C18:0
C16:1
RT (min)
Polar column
C18:2
C18:1
C16:0
C18:0
C16:1
Non-polar column
RT (min)
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
220C
160C
50C
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Time (min)
60
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
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A general detector for organic compounds
Very sensitive (10-13 g/s)
Linear response (107)
Rugged
Disadvantage: specificity
ECD
• Ultra-sensitive detection of halogencontaining 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
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Sample introduction
Source (ion formation)
Mass analyzer (ion sep.) - high vac
Detector (electron multiplier tube)
Sample Introduction/Sources
Volatiles
• Probe/electron impact (EI),Chemical ionization (CI)
• GC/EI,CI
Involatiles
• 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
EI Source
Under high vacuum
filament
70 eV e-
To mass
analyzer
GC column
anode
repeller
Acceleration
slits
EI process
M+*
• M + e-
f1
f2
f3
f4
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 Oil
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
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R = m/z/Dm/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
594.3
MVVTLIHPIAMDDGLR
594.7
C78H135N21O22S2+3
Q-TOF
595.0
601.3
595.3
601.0
601.7
m/z
901.4
100
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90
891.7
LCQ
891.2
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65
R = 0.88
902.3
892.6
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900.6
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5
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m/z
602.0
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 reflectron
http://www.rmjordan.com/tt1.html
Sector instruments
http://www.chem.harvard.edu/mass/tutorials/magnetmovie.html
FT-ICRMS
• http://www.colorado.edu/chemistry/chem5
181/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