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/Sources

Volatiles• 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 Oil

RT: 14.48 - 24.30

15 16 17 18 19 20 21 22 23 24

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

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: 9.69E5

TIC 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+3

Q-TOF

901.4

891.7

902.3

900.6

891.2

892.6

LCQ

R = 0.88

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

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