Gaseous Phase
Chromatography
Gas chromatography
Analytes must be in the gas state
must be volatile, boiling point < 200°C
moderately polar solutes
polar solutes after derivatisation
= to modify the solute to render it volatile
must not be thermolabile
not for fragile solutes
Interactions in GC
Solute
Solubility in the SP
Volatility
No interactions with MP
SP
MP
Liquid
Gas
Competitive factors:
Solubility in the SP vs. Volatility
Separation in GC
Solute 2
Less volatile
Solute 1
More volatile
Stationary phase
Mobile phase
Analysis time
Separation in GC
Non polar stationary phase
Cannot establish interactions other than London forces
Mobile phase
Stationary phase
Mobile phase
Solute 1, more volatile: spends more time in the mobile phase
Solute 2, less volatile: spends more time in the stationary phase
Separation in GC
Polar stationary phase
Can establish polar interactions with polar solutes
Solute 1 and 2
Same volatility
Mobile phase
Stationary phase
Mobile phase
Solute 1, less polar: cannot interact with the stationary phase
Solute 2, more polar: can interact with the stationary phase
Effect of Temperature
Solubility of a gas in a liquid decreases as T goes up
= Solutes are less dissolved in the stationary phase when T goes up
retention can be reduced by increasing T column
Increased temperature will reduce retention
but all components may not be affected to the same extent
Effect of Temperature
Retention
time
The degree of reduction of retention
time with increasing T is not the
same for each component
 Separation may improve or
degrade for any pair of peaks
Oven temperature
Effect of Temperature
Retention
time
The degree of reduction of retention
time with increasing T is not the
same for each component
 Separation may improve or
degrade for any pair of peaks
 Elution order may vary
Oven temperature
Effect of Temperature
Temperature programming
Temperature held constant during the entire analysis: isothermal
Temperature varied during the analysis: gradient analysis
tr
Carbon number
Retention time increases exponentially with the number of carbon
As tr increases, width decreases, making detection impossible
Effect of Temperature
Temperature programming
Final temperature
Initial temperature
Ramp (°C/min)
More complex programs are possible
Example: Environmental issues
Analysis of Polynuclear Aromatic Hydrocarbons
Increasing number of
aromatic rings
=
Decreasing volatility
Extract injected in hexane
Column: BPX5 (SGE) 25 m X 0.22 mm X 0.15 µm
temperature programmed from 50°C to 310°C at 5°C/min
MS detection
Example: Environmental issues
Analysis of Polynuclear Aromatic Hydrocarbons
Isomers can be separated
(different bp)
Extract injected in hexane
Column: BPX5 (SGE) 25 m X 0.22 mm X 0.15 µm
temperature programmed from 50°C to 310°C at 5°C/min
MS detection
Example: Environmental issues
Fenthion
Analysis of Pesticides
68
66
64
62
60
58
44
42
40
38
36
34
32
30
28
26
Trifluralin
Hexachlorobutadiene
Dichlorvos
46
24
22
20
18
16
14
Azinphos ethyl
48
Parathion
50
Atrazine
52
Lindane
54
Fenitrothion
Hexachlorobenzene
Simazine
56
12
10
8
6
4
2
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (min)
26
28
Extract injected in hexane
Column: BPX5 (SGE) 25 m X 0.22 mm X 0.15 µm
temperature programmed from 50°C to 310°C at 5°C/min
MS detection
30
32
34
36
38
40
42
44
46
Example: Derivatisation
Fatty Acids Methyl Esters (FAME)
Polar = High bp
Less polar = bp
is easily reached
O
Free Fatty Acid
HO
O
Fatty acid Methyl Ester
H3CO
Example: Derivatisation
Fatty Acids Methyl Esters (FAME)
Increasing number of carbon atoms
= Decreasing volatility
GC analysis of a complex mixture of natural fatty acids
(as methyl ester derivatives) on a packed column
Data obtained with 2m x 4mm glass column packed with 15% (w/w) EGSSY stationary phase
Carrier gas, nitrogen at 50 mL/min.; column temperature 194ºC
"Gas Chromatography and Lipids" by William W. Christie, published by the Oily Press, Bridgwater in 1989
Example: Isomer separation
Linolenic acid
O
9c
HO
12c
O
9c
HO
12t
O
9t
12t
9t
12c
HO
O
HO
WCOT column (30 m x 0.3 mm i.d.) coated with SS-4™
carrier gas: Nitrogen ; operating temperature 190ºC.
"Gas Chromatography and Lipids" by William W. Christie, published by the Oily Press, Bridgwater in 1989
Apparatus
First instrumental chromatographic method developed commercially
Relatively easy to produce a stable flow and pressure for the mobile phase
Compressed
gas cylinder
of carrier
gas
Pressure
regulator
Sample
injector
Detector
Valve
Computer
Column
Filters
Oven
Choice of carrier gas
Carrier gas is Nitrogen, Helium or Hydrogen
Carrier gas need to be extremely pure
-No water
-No oxygen
-No hydrocarbons
Impurities possibly deteriorating the stationary phase
Causing high baseline noise of the detector
Filters
Sample injector
Purpose of injection:
- To evaporate the sample
- To introduce it in the column
Sample injector
Syringe to introduce a known volume of
gas or liquid sample (0.1 to 10 μL injected)
Septum maintains seal
Liner provides an area for vaporisation
All non-volatile materials and degradation
products end up here
Split gas is to reduce total sample size
Part of the sample immediately goes to
waste and doesn’t enter the column
Gas expansion
When a liquid is injected, the vaporisation causes an expansion
= large increase in volume
1 μL liquid
100-1000 μL gas
Higher boiling point species
- take more time to volatilize
- don’t expand as much
Column
The column is the heart of the separation process
Vast number of materials available
Classified by dimensions and packing type
Packed
Open
Silica tubing filled with
particles of stationary phase
Silica is covered with a
layer of stationary phase
The layer can be simply coated or
chemically bound to the silica
Coated = simple coating on the inside of the fused silica tube
Bonded = chemically bound via a silane bond
Column
Major choice when selecting a stationary phase is do you want
a non-polar
moderately polar
polar column
?
Non polar columns can solve about 80% of all problems
Column
Phase polarity examples
Non-polar
methyl silicone
Best for non polar compounds
Intermediate
methyl silicone
/ phenyl silicone
(20-50% phenyl)
Best for mixed samples
Polar
polyethylene glycol
Best for polar compounds
For complex samples, pick the column that best reflects the overall
polarity of your sample
Detection
Objectives of the detection
Identification of the solute
Quantification
Comparison with a standard
Use of a specific detector
Detection
Comparison with a standard
Pre-requisites:
Knowing the identity of the analytes
Having standard solutions
Katharometer or Thermal Conductivity Detector (TCD)
Signal depends on the thermal
conductivity and specific heat of
the gas evolving from the column
Both of these parameters change
in the presence of a solute vapor
Non destructive
Extremely flow and pressure
sensitive
Must be carefully thermostated
Universal
Flame Ionisation Detector (FID)
Sample components enter at the base of the
detector, mix with H2 and enter the flame
Production of ions in the flame result in a
current that can be measured
Response is based on the number of carbon
Halogen and oxygen reduce combustion
Nearly universal (very rare exceptions)
Most useful GC detector available
Simplest, easiest and most reliable
Electron Capture Detector (ECD)
Radioactive source (63Ni) produces β particles
Electrophores (species containing halogens,
nitriles, nitrates, conjugated double bonds and
organometallics) absorb β particles, reducing the
current
Specific: sample must contain a gas-phase
electrophore
Non destructive
Excellent trace analysis for halogenated
compounds (pesticides), nitro group compounds
(explosives), conjugated double bonds
GC-Mass Spectrometry coupling
Sample
MS
GC
Chromatogram
Mass Spectra
GC-MS coupling
To each chromatographic peak, a mass spectrum is associated
Identification of the eluted analytes
GC-MS coupling
799
Mass spectrum of one
eluted analyte
232
797
801
400
399
401
220
299
%
139
795
234
803
151
402
398
218
132
311
265297
320
959
461
392
531
390
266
961
640
638
0
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
GC-MS coupling
Use of a spectrum library to identify the analyte
GC-MS coupling: difficulties encountered
Difference of pressure:
GC is under pressure
MS must be in void (10-4 – 1 Pa)
Mobile phase:
Must be compatible with the chosen ionisation mode
Interface:
Quantitative transmission of the analytes
No degradation of the solutes
No deterioration of the chromatographic separation
Suppressing the mobile phase
Acquisition time:
GC is very rapid: the mass spectrum must be rapidly acquired
GC-MS coupling
Simplest approach is to split the flow
Flow from
chromatograph
Only allow what the MS can handle
to enter
MS
Waste
This can result in only 1/1000 or
less of the sample to enter the MS
(not good for trace work!)
Electron Impact Mass Spectrometer (EI-MS)
Column effluent
Repeller
Coil
70 eV trap
Analyser
Chemical Ionisation Mass Spectrometer (CI-MS)
Column effluent + reactant gas
Repeller
Coil
Pump
Emission current
Comparison EI / CI
More sensitive
O2N
OH
H
H
More specific
Example: Toxicology
RT: 1.69 - 2.33
NL:
5.92E4
AA: 13499
100
m/z=
473.5474.5 MS
Genesis
100fg on
col
98
96
94
Relative Abundance
92
90
88
86
84
82
80
78
76
74
1.70
1.75
1.80
1.85
1.90
1.95
2.00
2.05
Time (min)
2.10
2.15
2.20
2.25
2.30
Low limits of detection allow the identification of THC
in very low concentrations in hair
Example: Environmental issues
Analysis of Volatile Organic Ccompounds (VOC)
11.92
100
90
11.77
80
13.36
14.35
Relative Abundance
70
12.81
14.66
9.00
60
15.25
50
17.40
9.90
18.29
7.90
7.29
40
30
5.42
8.44
17.29
10.43
10.59
6.07 7.09
6.62
20
4.28
4.19
10
16.37
2.90 3.59
0
0
2
18.92
4
6
8
10
Time (min)
12
14
16
18
20
Example: Environmental issues
PAK100 #10123-10127 RT: 94.38-94.41 AV: 5 SB: 63 93.90-94.10, 94.77-95.07 NL: 1.19E6
T: {0,0} + c EI det=500.00 Full ms [ 80.00-520.00]
300.2
100
Analysis of Polynuclear
Aromatic Hydrocabons (PAH)
90
Relative Abundance
80
70
150.1
149.1
60
50
40
148.1
30
298.2
301.2
20
10
99.3
147.0
123.1
0
100
150.9
163.0
191.0
150
207.1
296.2
246.2 270.2 281.1
200
250
302.2
327.1
300
m/z
RT: 10.31 - 100.01
100
NL:
1.35E7
TIC MS
PAK100
90
Relative Abundance
80
70
60
50
40
30
20
10
15
20
25
30
35
40
45
50
55
Time (min)
60
65
70
75
80
85
90
95
100
Example: essential oils
Lemon essential oil
Column: SLB-5MS (30 m x 0.25 mm, 0.25 μm film thickness)
Carrier gas: Helium, 32.4 cm/s
Split ratio 1/10
Temperature program: 40 to 250°C, 3°C/min
Crupi et al., Food Chemistry, (2007) in press
2D- GC
Use of two columns to resolve a sample
Current column technology is very near the theoretical limit
However, it is still not possible to resolve all components in a complex
mixture
Basic assumption is that no column can resolve all components of interest
Coupling two columns can help achieve complex separations
2D- GC
Examples
Tobacco smoke: over 1000 peaks identified, each actually contains 2 or
more components
PCBs: 207 species but only about 180 resolved
Coffee: over 600 components identified
2D- GC
1D-analysis
2D-analysis
Chiral fragrances
1. α-Pinene
2. β-pinene
3. Limonene
4. Menthone
5. Isomenthone
6. Menthol
7. Isomenthol
8. Pulegone
9. menthyl acetate
10. Sabinene
11. Linalol
12. α-terpineol
13. terpinen-4-ol.
Chiral separations
Enantiomers have
Identical volatility
MP cannot separate them
and
Identical polarity
Usual SP cannot separate them
Need for a stationary phase that would be able to distinguish
enantiomeric species
Chiral stationary phase
Relies on the 3-point rule
Can interact with SP
A
A
B
B
C
C
A
C
B
Cannot interact with SP
A
B
C
3 different interaction sites
Chiral separation in GC
Lemon essential oil
Crupi et al., Food Chemistry, (2007) in press
2D-chiral GC
Strawberry flavour
Two-dimensional contour plot of
direct solvent injection of a
strawberry mix, highlighting the
separation of linalool
enantiomers.
EtTBS-β-CD (20 m x 0.25 mm x 0.25 μm)
Cyclosil-B (26 m x 0.25 mm x 0.25 μm)
Hydrogen carrier gas
Detection: FID
Williams et al., J. Chromatogr. B, 817 (2005) 97-107
Choice of carrier gas
No interaction with the mobile phase:
Choice of the carrier gas will not affect
K
k
α
But different viscosities of gases affect Dm
N
partition coefficient
retention factor
selectivity
diffusion coefficient
efficiency (thin peaks)
Choice of carrier gas
The Van Deemter curve
N2
dispersion
He
H2
10 20
40
90
Linear velocity of mobile phase
(cm/s)
N2 generates the lowest dispersion (at the expense of speed)
He provides better speed with only small increase in dispersion
H2 is the best : high flow rate with little loss of resolution
Sample injector
Purpose of injection:
- To evaporate the sample
- To introduce it in the column
T injector > T column + 50°C
Temperature focusing
To reduce the band width of the injected
peak, solutes condense at start of
column at low T
Column
Column
Packed
Capillary
Length, m
ID, mm
Flow, mL/min
Head pressure, psi
Total plates
Film thickness, um
0.5 – 5
2–4
10 – 60
10 – 40
4000
1 – 10
5 – 100
0.1 – 0.7
0.5 – 15
3 – 40
250 000
0.1 – 8
Major difference: smaller ID and longer length for capillaries
Compounds remain in the column longer while still retaining good peak shape
Longer columns allow for better efficiencies resolution = f(length1/2)
Better efficiencies allow for better sensitivity