GC and GC-MS Gas Chromatography - PowerPoint Presentation

Slide1

GC and GC-MS

Slide2

Gas Chromatography

Function

Components

Common uses

Chromatographic resolution

Sensitivity

Slide3

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

Slide4

Components

Carrier Gas, N

2

or He, 1-2 mL/min

Injector

Oven

Column

Detector

Slide5

Gas tank

Oven

Column

Injector

Syringe

Detector

Slide6

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

Slide7

Splitless (100:90) vs. Split (100:1)

Injector

Syringe

Injector

Syringe

Purge valve

open

Purge valve

closed

GC column

GC column

He

He

Slide8

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

Slide9

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

m

Slide10

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

Slide11

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

Slide12

C16:0

C18:0

C18:1

C18:2

C16:1

C16:0

C18:0

C18:1

C18:2

C16:1

RT (min)

RT (min)

Polar column

Non-polar column

Slide13

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

Slide14

Typical Temperature Program

Time (min)

0

60

50



C

220



C

160



C

Slide15

Detectors

Flame Ionization Detectors (FID)

Electron Capture Detectors (ECD)

Electron impact/chemical ionization (EI/CI) Mass spectrometry

Slide16

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 CH

2

+

ions

These ions collected at an electrode held above the flame

The current reaching the electrode is amplified to give the signal

Slide17

FID

A general detector for organic compounds

Very sensitive (10

-13

g/s)

Linear response (10

7

)

RuggedDisadvantage: specificity

Slide18

ECD

Ultra-sensitive detection of halogen-containing species

Pesticide analysis

Other detectors besides MS

IR

AE

Slide19

Mass Spectrometry

Slide20

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)

Slide21

How does it work?

Gas-phase ions are separated according to mass/charge ratio and sequentially detected

Slide22

Parts of a Mass Spec

Sample introduction

Source (ion formation)

Mass analyzer (ion sep.) - high vac

Detector (electron multiplier tube)

Slide23

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

Slide24

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

Slide25

To mass

analyzer

filament

70 eV e-

anode

repeller

Acceleration

slits

GC column

EI Source

Under high vacuum

Slide26

EI process

M + e-

M

+*

f

1

f

2

f

3

f

4

This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam

Slide27

Slide28

Ion Chromatogram of Safflower Oil

Slide29

Slide30

CI/ ion-molecule reaction

2CH

4

+ e-

 CH

5

+

and C

2

H5

+CH5

+ + M  MH+ + CH

4The excess energy in MH+

is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation

Slide31

EI spectrum of phenyl acetate

Slide32

Slide33

Mass Analyzers

Low resolution

Quadrupole

Ion trap

High resolution

TOF time of flight

Sector instruments (magnet)

Ultra high resolution

ICR ion cyclotron resonance

Slide34

Resolution

R = m/z/

D

m/z

Unit resolution for quad and trap

TOF up to 15000

FT-ICR over 30000

MALDI, Resolve

13

C isotope for a protein that weighs 30000Resolve charge states 29 and 30 for a protein that weighs 30000

Slide35

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

Slide36

MVVTLIHPIAMDDGLR

594.3

594.7

595.0

601.3

595.3

601.0

601.7

602.0

m/z

C

78

H

135

N

21

O

22

S

2

+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

Slide37

Quadrupole Mass Ion Filter

Slide38

Ion Trap

Slide39

Time of Flight -TOF

Slide40

 

                                                     

Where:

m

i

= mass of analyte ion

z

i

= charge on analyte ion

E = extraction field ti = time-of-flight of ion l

s = length of the source ld = length of the field-free drift region

e = electronic charge (1.6022x10-19 C)

Slide41

TOF with reflectron

http://www.rmjordan.com/tt1.html

Slide42

Sector instruments

http://www.chem.harvard.edu/mass/tutorials/magnetmovie.html

Slide43

FT-ICRMS

http://www.colorado.edu/chemistry/chem5181/MS_FT-ICR_Huffman_Abraham.pdf

Slide44

Mass accuracy

Mass Error = (5 ppm)(201.1001)/10

6

=



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?

Slide45

Exact Mass Determination

Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF)

C

9

H

15

NO

4

, FM 201.1001 (mono-isotopic)

Mass accuracy = {(Mass Error)/FM}*106Mass Error = (5 ppm)(201.1001)/106

=  0.0010 amu