GC-MSGas Chromatography-Mass Spectrometry An Hybrid technique which couples the powerful

separation potential of gas chromatography with the

specific characterization ability of mass spectroscopy.

Supporting & Servicing Excellence

• GC History• What is GC• Key Components• Separation Process• GC Theory• Carrier Gas• Injectors• Columns

Overview

GC History

• Development of GC (1941) by Martin and Synge

• Theory of Capillary GC (1957) by Golay

• Capillary GC Instruments (1977)

• Fused Silica Capillary Columns (1979)

What is GC?• GC is a Separation Technique• Sample is usually a complex mixture we

require to separate into constituent components.

• Why: usually to quantify some or all components e.g. Pharmaceuticals, Environmental pollutants, etc

• Occasionally as a qualitative tool

What is the sample?

• Usually a mixture of several components• Sample usually introduced as a liquid• Components of interest (analytes) usually in

low concentrations (<1% to ppb levels)• Samples dissolved in volatile solvent

Comaparison: GC & HPLC HPLC

• non-volatile samples

• thermally unstable compounds

• macromolecules

• inorganic and ionic samples

• More complex interface to Mass Spec .

GC

•volatile & thermally stable

•rapid analysis

•good resolution

•easily interfaced to Mass Spec

Key components of GC • Hardware to introduce the sample• Technique to separate the sample into components• Hardware to detect the individual components.• Data Processing to process this information.

Basic Block Daigram!

Separation Process• Sample is introduced into system via hot, vaporising

injector. • Typically 1ul injected• Flow of “Carrier Gas” moves vaporised sample (i.e. gas)

onto column• Column is coated with wax type material with varying

affinity for components of interest • Components are separated in the column based on this

affinity.• Individual analytes are detected as they emerge from the

end of the column through the Detector.

Example Chromatogram (Capillary)

1 2 3 4 5

Minutes

-87

0

250

500

750

mVolts0.5

41

0.754

1.113

1.474

2.038

2.853

3.210

4.463

5.320

5.562

c:\star\examples\level4.run File:Channel:

Last recalc:

c:\star\examples\level4.runA = TCD Results25/07/1993 18:35

WI:2 WI:4

Time Inject Point

Detector Response

•

1

2

4

6

3

5

7

8

10

9

11

12

13

14

15

16 17 18

1. -HCH2. -HCH3. -HCH4. Heptachlor5. -HCH6. Aldrin7. Heptachlor epoxide8. Endosulfan I9. 4,4’-DDE

10. Dieldrin11. Endrin12. 4,4’-DDD13. Endosulfan II14. 4,4’-DDT15. Endrin aldehyde16. Endosulfan sulfate17. Methoxychlor18. Endrin ketone

Analysis of Halogenated Pesticides

2ppb in Water2ppb in Water

Chromatogram

GC Step by Step • Carrier Gas• Injector• Column

– Capillary– Stationary Phase

• Detectors– Mass Spectrometer

Carrier Gas Inert

Helium

Choice dictated by detector, cost, availability

Pressure regulated for constant inlet pressure

Flow controlled for constant flow rate

Chromatographic grade gases (high purity)

Column Types

Packed Columns

Length: <2m

Diameter: 1/8” & ¼” OD

Capillary Columns

Length: 10m to 100m

Diameter: 180um, 250um, 320um & 530um I.d

• Capillary Column Flow

– 250 um 1 ml/min

– 320 um 1.5 ml/min

– 530um up to 2.0 ml/min

Typical column flow rates

Purpose of Injection• Deposit the sample into the column in the narrowest band

possible

• The shorter the band at the beginning of the chromatographic process - tall narrow peaks

• Gives maximum resolution and sensitivity

• Therefore type of injection method and operating conditions is critical in obtaining precise and accurate results

Splitless injector Design

“Unique”Dual Split Vent design

•Improved Precision

•More Efficient Sweep

Graphite/Viton Seal

•Reduced Sample Contact

Shortened Capillary Guide

•Minimal Cold Spots

•Minimal Upswept Volume

Large Internal Volume

•Minimum Solvent Tailing

Cross Section of PTV Injector

Modern Temperature Programmable Injector (Varian 1079)

Programmable Temperature Vapourising Injector

Split & Splitless Injection• Most common method of Injection into Capillary

Columns• Most commonly misunderstood also!• Same injector hardware is used for both

techniques• Electronically controlled Solenoid changes Gas

Flow to determine Injector function.

Split Injection• Mechanism by which a portion of the injected solution is discarded.

• Only a small portion (1/1000 - 1/20) of sample goes through the column

• Used for concentrated samples (>0.1%)

• Can be performed isothermally

• Fast injection speed

• Injector and septa contamination not usually noticed

Splitless Injection• Most of the sample goes through the column (85-100%)

• Used for dilute samples (<0.1%)

• Injection speed slow

• Should not be performed isothermally

• Solvent focusing is important

• Controlled by solenoid valve

• Requires careful optimisation

On Column Injection• All of the sample is transferred to the column

• Needle is inserted directly into column or into insert directly above column

o Trace analysis

o Thermally labile compounds e.g Pesticides, Drugs

o Wide boiling point range

o High molecular weight

Large Volume Injection• To enhance sensitivity in Envoirnmental applications.

• Uses 100µL syringe: Inject up to 70 µl

• Very slow injection with injector temperature a few degrees below solvent boiling point, split open, flow at about 150 mls/ min

• Solvent vents out of split vent, thus concentrating the analytes

• Close split

• Fast temperature ramp to top column temperature +20°C

• Column programming as per sample requirements

Columns

Material of Construction• Metal (1957)

• Glass (1959)

• Fused Silica (1979)

• Aluminium Clad (1984)

• Inert Metal (1990)

Capillary Column Characteristics• Length (10M - 50M)

• Internal Diameter (0.1mm - 0.53mm)

• Liquid Stationary Phase

• Film Thickness (0.1um - 5um)

• Polarity (Non-polar - Polar)

Stationary PhasesChoice of phase determines selectivity

Hundred of phases available

Many phases give same separation

Same phase may have multiple brand names

Stationary phase selection for capillary columns much simpler

Like dissolves likeUse polar phases for polar componentsUse non-polar phases for non-polar components

Column Bleed Bleed increases with film thickness

Polar columns have higher bleed

Bleed is excessive when column is damaged or degraded Avoid strong acids or bases Adhere to manufacturer’s recommended temperature limits Avoid leaks

Choosing a Column• Internal Diameter

• Film Thickness

• Length

• Phase

Internal Diameter, Smaller ID’s• Good resolution of early eluting compounds

• Longer analysis times

• Limited dynamic range

ID Effects - larger ID’s• Have less resolution of early eluting compounds

• Shorter analysis times

• Sufficient resolution for complex mixtures

• Greater dynamic range

Film Thickness Amount of stationary phase coating

Affects retention and capacity

Thicker films increase retention and capacity

Thin films are useful for high boilers

Standard capillary columns typically 0.25µm

0.53mm ID (Megabore) typically 1.0 - 1.5µm

The maximum amount that can be injected without significant peak

distortion

Column capacity increases with :- film thickness temperature internal diameter stationary phase selectivity

If exceeded, results in :- peak broadening asymmetry leading

Column Capacity

Length effects - isothermal analysis• Retention more dependant on length

• Doubling column length doubles analysis times

• Resolution a function of Square Root of Length

• Gain 41% in resolution

• Is it worth the extra time and expense?-

Length effects - programmed analysis

• Retention more dependant on temperature

• Marginally increases analysis times

• Run conditions should be optimised

Summary - Effect of ID, Film Thickness, and Length

ID • Choice based on

capacity and resolution • Use 0.25mm for MSDs • Use 0.32mm for

split/ splitless & DI • Use 0.53mm for DI & • purge & trap

Film Thickness • Thick film for low

boilers • Thin film for high

boilers • Thicker films for larger

ID's

Length Gain in resolution is

not double Isothermal: tR L Programmed: tR is

more dependent on temperature

Detectors

• Basic Mass Spectrometry Theory• Types of Ionisation - Electronic Ionisation - Chemical Ionisation• Interpretation of Mass Spectra• Ion Trap Theory• Components of the Ion Trap

Overview

Ion Trap Mass Spectrometry

Basic Mass Spec.Theory• Mass Spec. is a Microanalytical Technique used to obtain information

regarding structure and Molecular weight of an analyte

• Destructive method ie sample consumed during analysis

• In all cases some form of energy is transferred to analyte to cause ionisation

• In principle each Mass Spectrum is unique and can be used as a “fingerprint” to characterise the sample

• GC/MS is a combination technique that combines the separation ability of the GC with the Detection qualities of Mass Spec.

Basic GCMS Theory(1)• Sample injected onto column via injector• GC then separates sample molecules• Effluent from GC passes through transfer line into

the Ion Trap/Ion source• Molecules then undergo electron /chemical

ionisation• Ions are then analysed according to their mass to

charge ratio• Ions are detected by electron multiplier which

produces a signal proportional to ions detected

Basic GCMS Theory(2)

• Electron multiplier passes the ion current signal to system electronics

• Signal is amplified• Result is digitised• Results can be further processed and

displayed

Types of Ionisation

• Electron impact ionisation• Chemical Ionisation

Definition of TermsMolecular ion

The ion obtained by the loss of an electron from the molecule

Base peak The most intense peak in the MS, assigned 100% intensity

M+ Symbol often given to the molecular ion

Radical cation

+ve charged species with an odd number of electrons

Fragment ions

Lighter cations formed by the decomposition of the molecular ion.  These often correspond to stable carbcations.

Electron Ionisation(1) • Sample of interest vaporised into mass spec• Energy sufficient for Ionisation and Fragmentation

of analyte molecules is acquired by interaction with electrons from a hot Filament

• 70 eV is commonly used• Source of electrons is a thin Rhenium wire heated

electrically to a temp where it emits free electrons

Electron Ionisation

Electron Ionisation• The physics behind mass spectrometry is that a charged particle

passing through a magnetic field is deflected along a circular path on a radius that is proportional to the mass to charge ratio, m/e.  In an electron impact mass spectrometer, a high energy beam of electrons is used to displace an electron from the organic molecule to form a radical cation known as the molecular ion. If the molecular ion is too unstable then it can fragment to give other smaller ions.  The collection of ions is then focused into a beam and accelerated into the magnetic field and deflected along circular paths according to the masses of the ions. By adjusting the magnetic field, the ions can be focused on the detector and recorded.

Chemical ionisation• Used to confirm molecular weight• Known as a “soft” ionisation technique• Differs from EI in that molecules are ionised by interaction

or collision with ions of a reagent gas rather that with electrons

• Common reagent gases used are Methane , Isobutane and Ammonia

• Reagent gas is pumped directly into ionisation chamber and electrons from Filament ionise the reagent gas

Chemical Ionisation(2)• First - electron ionization of CH4:

– CH4 + e- CH4+ + 2e-

• Fragmentation forms CH3+, CH2

+, CH+

• Second - ion-molecule reactions create stable reagent ions:– CH4

+ + CH4 CH3 + CH5+

– CH3+ + CH4 H2 + C2H5

+ • CH5

+ and C2H5+ are the dominant methane CI reagent

ions

Chemical Ionisation(3)• Form Pseudomolecular Ions (M+1)

– CH5+ + M CH4 + MH+– M+1 Ions Can Fragment Further to Produce a Complex CI

Mass Spectrum

• Form Adduct Ions– C2H5+ + M [M + C2H5]+ M+29 Adduct– C3H5+ + M [M + C3H5]+ M+41 Adduct

• Molecular Ion by Charge Transfer– CH4+ + M M+ + CH4

• Hydride Abstraction (M-1)– C3H5+ + M C3H6 + [M-H]+

» Common for saturated hydrocarbons

EI vs CI for Cocaine analysis• EI Spectrum of Cocaine• Extensive Fragmentation• Molecular Ion is Weak at m/z 303

Methane CI of CocainePseudomolecular Ion and Fragment Ions

Proton Affinity• Proton Affinity Governs CI Susceptibility • The higher the affinity the more tightly

bound the proton is to the parent species• The greater the difference in proton

affinities between the analyte and reagent gas the more energy transferred to the protonated molecule –more fragmentation

Interpretation of Mass Spectra(1)

Intepretation of Mass Spectra(2)•The MS of a typical hydrocarbon, n-decane is shown above.The molecular ion is seen as a small peak at m/z = 142. 

•Notice the series ions detected that correspond to fragments that differ by 14 mass units, formed by the cleave of bonds at successive -CH2- units

Interpretation of Mass Spectra(3)

Interpretation of Mass Spectra(4)•The MS of benzyl alcohol is shown above. •The molecular ion is seen at m/z = 108. •Fragmentation via loss of 17 (-OH) gives a common fragment seen for alkyl benzenes at m/z = 91. •Loss of 31 (-CH2OH) from the molecular ion gives 77 corresponding to the phenyl cation.• Note the small peaks at 109 and 110 which correspond to the presence of small amounts of 13C in the sample (which has about 1% natural abundance).

Determining Isotope Patterns in Mass Spectra

•Mass spectrometers are capable of separating and detecting individual ions even those that only differ by a single atomic mass unit. •As a  result molecules containing different isotopes can be distinguished. •This is most apparent when atoms such as bromine or chlorine are present (79Br : 81Br, intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained. •The intensity ratios in the isotope patterns are due to the natural abundance of the isotopes. •"M+1" peaks are seen due the the presence of 13C in the sample.

Isotope Patterns 2,Chloropropane

•Examples of haloalkanes with characteristic isotope patterns. •The first MS is of 2-chloropropane.•Note the isotope pattern at 78 and 80 that represent the M and M+2 in a 3:1 ratio. •Loss of 35Cl from 78 or 37Cl from 80 gives the base peak a m/z = 43, corresponding to the secondary propyl cation. •Note that the peaks at m/z = 63 and 65 still contain Cl and therefore also show the 3:1 isotope pattern.  

1,Bromopropane

• The second MS is of 1-bromopropane.• Note the isotope pattern at 122 and 124 that

represent the M amd M+2 in a 1:1 ratio.• Loss of 79Br from 122 or 81Br from 124 gives

the base peak a m/z = 43, corresponding to the propyl cation.

• Note that other peaks, such as those at m/z = 107 and 109 still contain Br and therefore also show the 1:1 isotope pattern.  

ION TRAP THEORY

• Ionize analytes within the ion trap– Use energetic electrons to ionize

• Store ions and continue to ionize until the optimum trap capacity is reached– Optimum ion time calculated by software

• Increase the voltage on the Ring Electrode of the ion trap to scan ions out in order from low to high mass– This voltage-time relationship called the EI/MS

Scan Function• Store the mass-intensity information as a mass

spectrum

Gate

Filament

Ring Electrode

Trapped IonsAnalytes + He Carrier Gas

Gate

Filament

Ring Electrode

Trapped IonsAnalytes + He Carrier Gas

Filament

Trapped Ions

CARRIER GAS

Ring electrode

Gate

Electron Ionization Happens Inside the Ion Trap

Mass Spectrum of Toluene

Mass Spectrum of Caffeine

Mass Spectrum of Glycerin

Mass Spectrum of Cholesterol

Mass Spectrum of Aspirin