Content
General aspects of GC
analysis of volatiles
Introduction: food volatiles, principles of GC separation
Main parameters for the optimisation of volatile analysis
columns
carrier gas
samples inlet systems
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temperature programming
detectors
Identification
retention time, Kovats and retention indices
spectral data (GC/MS)
Multidimensional GC, GCxGC
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What are the volatile compounds ?
• Physically it is rather imprecise term, because there
is no precise boundary in the boiling temperature
between ‘volatile’ and ‘non-volatile’ compounds
• For foods: the compounds which are present in
detectable concentrations in headspace (both in
instrumental and sensory analysis) may be
considered as volatile compounds
• Depends on the matrix (food), compound
concentration, physical and chemical properties
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Why to analyse volatile compounds?
• Responsible for food aroma (also off-odours deffects)
• May indicate about desirable and undesirable
changes (food spoilage)
• May indicate about reactions taking place in foods
• May provide scientific information for the control of
food flavour during processing/storage
• May provide scientific information for designing food
flavourings
• Therefore, we need about volatiles as much
information as possible
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Main steps in volatile analysis
•
•
•
•
•
•
Sample preparation
Isolation
Preliminary fractionation
Concentration
Separation (GC)
Identification (GC-MS other specroscopic
techniques)
• Quantification
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Sample preparation
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GC analysis
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Gas chromatography - definition
Extract of volatiles
• Large number of compounds
• Different chemical properties (functional
groups, reactivity, etc.)
• Different physical properties (boiling
temperature, flashpoint, polarity, etc.)
• Different concentrations
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GC – main techniques for the
analysis of volatiles
Gas-liquid chromatography (GLC), or simply gas
chromatography (GC), is a type of
chromatography in which the mobile phase is a
carrier gas, usually an inert gas such as helium
or an unreactive gas such as nitrogen, and the
stationary phase is a microscopic layer of
liquid or polymer on an inert solid support,
inside glass or metal tubing, called a column.
Martin and Synge for the developments in
GC separation were avarded Nobel prize in
1952 (chemistry)
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The principle of chromatographic separation
When the components moving through the column an equilibrium is
formed between components, which are adsorbed by the column
stationary phase and the components which dissolve in column mobile
phase:
Component separation in GC
The time, during which a component is retained in the column is called
retention time (tR) in analytical chromatography and retention factor Rf
in preparative chromatography
High K – the compound moves fast – low tR (high Rf )
Low K – the compound moves slowly – high tR (low Rf)
Stationary Phase
Collect/
Detect
Components
mobile phase
Component
mobile phase
mobile phase
mobile phase
mobile phase
Component
Component
Inject/
Add
Sample
mobile phase
Stationary Phase
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Principal scheme of gas chromatography
Blue molecules are better soluble in a liquid (or
less volatile) than green molecules
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Column effectiveness
Are the peaks separated efficiently?
If no how to improve the separation
Separation – Does the peak always reach baseline (0)?
Peak shape: is it Gaussian ?
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Composition (quantitative
information) – identification of
the components
Analysis of volatile
compounds by GC
Separation
Trennzahl TZ = number of baseline separated peaks
between one pair of system homologues (Kaiser)
⎡ t R ( B ) − t R ( A) ⎤
TZ = ⎢
⎥ −1
⎢⎣ w0 , 5 ( A ) + w0 , 5 ( B ) ⎥⎦
Content (quantitative) measurement of component
concentration
A and B – two members of homologue, differ in one methylene group
The number of peaks which may be recorded between the two contiguous
homologues members
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Configuration of a GC
Data acquisition and processing
Injector
Sample
Carrier gas
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Essential parameters for the
optimisation of GC analysis
Carrier gas
Sample inlet
Systems
Detector
GC oven
Columns
Analytical column
Injection
techniques
Analysis temperature program
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Detection system
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Packed Column
Column technology
Separation of volatile compounds
proceeds in column, therefore selection
of a right column is a crucial step in the
GC analysis of volatile
• Packed columns vs open tubes
• Stationary phases
• Testing
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Characterisation of the column by
1) Amount and weight of stationary phase
2) Type of carrier particles
3) Dimensions
e.g. 1% OV-1 on Chromosorb W
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Types of WCOT
Very pure fused silica tube
material coated outside with
polyimide to protect the column
and to give flexibility
Stationary phase is just on the inner wall
of the tube, there is a free gas flow and
no „EDDY DIFFUSION“ , that means a
drastically increase in length is possible
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„Non reactive“ patricles
(different shapes and sizes possible)
Stationary Phase
Capillary Columns
Wall Coated Open Tubes WCOT
Carrier gas
Carrier gas flow
Glass tube (2-4 mm i.d.)
Lenght 2-4 m
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WCOT are classified by
1) Inner diameter:
MICROBORE 250 µm inner diameter and less (down to 10 µm!!!)
high speed, high resolution types with low capacity
MEDIUMBORE 320 µm id
a good compromise between capacity and performance
MEGABORE 530 or 750 µm id
„working horses“ for high sample amount and capacity
2) Film Thickness:
Normal film thickness: 0.25 – 0.5 µm
Thin Film : down to 1 nm (nanometer)
Thick Film: up to 10 µm
Length of column: standard size 10-50 m
Longest column (Guiness books of records) Chrompack: 2.2 km
For high speed analysis: 1 or 2 m with small inner diameter and thin film
(limited by data acquisition!)
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Comparison- packed vs. capillary column
Polarity of stationary phase
Packed column
3 m length, 2 mm id
OV1
Apolar
-
+
Polar
Capillary column
35 m length,
0.28 mm id, OV1
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Stationary phases
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Sample: the mixture consisting of 3 components is
eluting through the column
Cross linking of stationary phase
Due to the cross linking a chemical bonding between the
surface and the stationary phase is achieved. Due to this
effect the temperature range, the stability and the life time of
the column is increased. The cross linking is made by
radiation with γ-rays
OH
O
bp 207 °C
bp 212 °C
OH
OH
OH
OH
OH
OH
bp 177 °C
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
For which compound retenion time will be the lowest (moves fastly)?
For which compound retenion time will be the highest (moves slowly)?
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The answer
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Will move most fast, the lowest Rt, because it is pure
hydrocarbon and does not form strong intermolecular bonds
with stationary phase
O
OH
Will move more slowly, medium RT; may form partial
hydrogen bonds (donor of electron pair) with stationary
phase – some intermolecular interactions
Will move most slowly, the highest RT; may form effective
hydrogen bonds (donor of electron pair and hydrogen) with
stationary phase – strong intermolecular interactions
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Temperature range of stationary phases
OH
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Lower Temperature: is given by the melting point of the
phase (-60°C to +40°C depending on the polarity)
Upper Temperature: is given by the vapour pressure of the
stationary, liquid (!) phase. The vapour pressure increases in
an exponential manner with the temperature!!!!!
Maximum upper Working Temperature:
is defined as the temperature where the column can be used
for 6 month and loosing 50% of the stationary phase
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Influence of film thickness
Column bleeding
Abundance
15 m/0.3 mm, SE-52
Ion 207.00 (206.70 to 207.70): SB_REKL.D
3000
Temp. program:
2800
2600
50°C (1´) at 10°C/min to 310°C (5´)
2400
2200
20 oC programmed
2 oC/min to 80 C
2000
1800
1600
1400
1200
1000
Bleeding
800
600
400
200
0
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Time-->
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Influence of polarity-FAME
Capacity of capillary columns
Abundance
TIC: FSSTD.D
21.21
21.48
380000
360000
340000
320000
300000
280000
19.11
260000
21.14
240000
220000
200000
180000
HP-5 MS
order of elution:
unsatuated
BEFORE saturated
160000
140000
120000
25.66
23.16
22.98
100000
27.32
24.98
80000
16.51
23.65
23.40
23.35
60000
25.43
27.52
40000
20000
0
16.00
17.00
18.00
19.00
20.00
21.00
22.00
23.00
24.00
25.00
26.00
27.00
Time-->
C 22:6
C 24:1
C 24:0
C 22:0
C 22:1
C 20:2
C 14:1
30
C 20:3
C 20:4
C 18:3
C 18:0
C 18:1
C 20:0
C 20:1
40
C 16:1
C 14:0
50
C 18:2
HP-WAX
unsatuated
AFTER
saturated
60
C 16:0
FID1 A, (G:\DATA\FSME\FSMESTA.D)
pA
20
10
7.5
10
12.5
15
17.5
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20
22.5
25
27.5
min
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Special columns for the separation of
enantiomers: carvone
Comparison
of columns
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Special columns for the separation of
enantiomers: limonene
Fresh citrus,
orange like
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Cyclodextrin columns
Harsh,
turpentine-like,
lemon note
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Enantioselective separation of linalool
Composition of enatiomers in natural
and synthetic oils (authenticity testing)
natural
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synthetic
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Carrier gases
Carrier gas
Mainly used: Nitrogen A: inexpensive D: slow separation see van-Deemter
Hydrogen A: good separation D: explosive mixtures with air/oxygen
Helium
A: similar separation behaviour like H2, Excellent for GC/MS
D: price, extra gas cylinder
Mobile GC phase
Should be inert – do
not react with the
majority of organic
compounds
Purity:
Main impurities:
Usually pressure range
10-100 psi
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4.6 means 99.996% less than 0.004 % impurities
5.0 means 99.999%
Water
Oxygen
Mineral Oil
Always try to use metal tubes instead of plastic materials (reduces background
signal and avoids contamination
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Van Deemter equation
Comparison of different carrier gases
Describes the relation between the linear gas velocity and the HETP
(Height Equivalent of a Theoretical Plate)
A theoretical plate is the length of the column where a balance between the
mobile and the stationary phase is reached
HETP
N2
He
H2
20
50
Constant flow
Constant pressure
systems!
Viscosity of gases
increase with the
temperature due to
higher movements of
gas molecules
Linear velocity [cm/s]
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Effect of carrier gas
on separation
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Injector (injection systems)
The sample is injected
with microsyringe (μL)
The amount of injected
sample depends on the
column and desirable
separation
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Inlet systems
Split-splitless injector
Compounds for GC must be volatile and MUST not
decompose during vaporisation!!
Compounds were injected
(~ 1 µL = 1 mg, if density is 1
g/mL) and vaporized at
temperatures between 200300°C
2 different modes of
injection for different
samples and concentration
ranges
Split mode: Concentration
of compounds of interest
relatively high
Split ration 1:5 – 1:1000
Splitless mode:
for
trace analysis
All sample compounds are
transferred to the column
Samples for standard injection devices must be either liquid
by itself (e.g. essential oil) or should be dissolved in an
appropriate solvent
SPLIT-SPLITLESS INJECTOR
COOL ON-COLUMN INJECTOR
PTV
DIRECT INJECTION
INLET FOR SOLID SAMPLES
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Switching the Split Valve in the Splitless
Mode
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Improper purge valve setting
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On column injector
Advantage: suitable for thermolabile
compounds
Comparison
OC - SSL
Disadvantage: Samples have to be
“clean”
Avoid any contamination with non
volatiles (Sugar, minerals…)
ONLY for trace compounds
HANDLING OF SYRINGE IS VERY
IMPORTANT !
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a) isothermal 45oC
Gradient
b) isothermal 145oC
240
200
Temp (deg C)
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Effect of temperature
Temperature control
Isothermal
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160
120
c) Programmmed from
30oC to 180oC
80
40
0
0
10
20
30
40
50
60
Time (min)
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Solvent vapours during injection
Temperature program
syringe
Carrier gas
septum
Septum purge
Split vent
Different types of liners
Single Double
Jennings
Split
taper
taper
GC column
Deactivated glass wool
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Choosing the proper liner
Vapor volumes of solvents
Flow calculator
There are three liner characteristics that must be
considered for each application:
• Liner volume
• Liner treatments or deactivation
• Any liner design feature that might affect
carrier gas flow through the inlet or sample
vaporization
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Temp. Injector: 250 °C; Column Head pressure: 138 kPa01
If the vapour volume is too great for the liner, reproducibility
and sensitivity can be compromised, due to backflash or loss
of the sample into the septum purge or split lines.
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Test mixture for activity
How to test the system?
To maintain a proper GC system it is necessary to check it on a
regular base
WHAT is regular?
Depends on the type of analysis (polar, non polar, dirty samples, high
number of injections, different methods for different boiling ranges and
different analytes.....)
Check it with a test mixture
Record the performance
Keep a log book with the history of ever column (date of purchase, date of
installation, approximate number of injections, types of samples....)
n-Alkanes from C9 - C16
α-Pinene
β-Pinene
p-Cymene
1,8-Cineole
Menthol
Dodecanoic acid methyl ester
Acenaphtene
Dodecanole
Inject 1 µL splitless of this test mixture diluted in diethyl ether
or n-pentane at a concentration of 10 mg/L (injected amount
of each compound is 10 nanogram)
Even test every new column you buy!
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Test mixture for activity
Liner
Lineraktiv
active
Abundance
GC-Parameters:
HP5-MS 30 m, 0,25mm, 0,25 µm df
200000
Dirty liner with glass
wool
Dirty liner without
glass wool
?
?
100000
20.00 [min]
10.00
Abundance
Liner desaktiviert
Liner
deactivated
Temp. program:
30°C (0 min)
with 5,3°C/min to 250°C
Injection 1µL splitless
Injector: 250°C
CHP: 0,81 bar constant pressure
300000
200000
deactivation
process ensures
high performance !
100000
10.00
20.00 [min]
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Cleaning of liners
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Rinsing a column
Remove liner from injector
Remove glass wool plug
Immerse liner in a mixture of 30 % H2O2 & H2SO4 conc.
ATTENTION VERY CORROSIVE!!!!!!!
Vacuum hose to pump
Pasteurpipette
GC column
Wash carefully with distilled water followed by acetone
Dry in an oven at 300°C
Side of Detector!!!
Immerse the liner in pure Hexamethyldisilazane (HDMS)
Put it back into the hot oven for a couple of minutes
Remove and install still warm into the injector
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30 mL Headspace Vial
with septum at the
screw cap
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4 mL Vial
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Rinsing a column
Recipe for column deactivation
Remove GC column from GC
Immerse the detector end in the vial with the rinsing solvent
Puncture the septum of the 30 ml vial and put the other column end
into it
Suck each of the following solvents through the column (in this order)
1) Methanol
2) Ethyl acetate
3) Hexane
After sucking the solvent install the column (with the detector side!!!)
in the injector
Apply carrier gas at normal flow rates for a couple of minutes at room
temperature
Ramp the oven at 2°C/min to the maximum oven temperature
required
Hold it for 20 min then cool down
Test the column
ATTENTION: Only chemically bonded column can be rinsed!
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Syringes and Injection Techniques
Plunger
Sample
Install the column in the injector (with the detector end!) and apply
carrier gas for a couple of minutes at room temperature.
Reduce carrier gas pressure to 0.1 bar and close the open end with a
septum
Increase oven temperature to 200°C (15`) and then to 300°C for 30`
Afterwards cool down to room temperature
Rinse once with a few mL of Dichloromethane and condition the column
as described
K. Grob, Journal of HRC & CC 5, (1982) 13, 349-354
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Syringes and injection techniques
Type A: Hot needle technique
Type A Standard 5 or 10 µL
Needle
Rinse column as described previously
Suck a 10 % solution of Diphenyltetramethyldisilazane (DPTMDS) until
5 -10 % of the column is filled (start at the detector end!!!)
Glass body
Rinse the syringe several times with a solvent (should be the same as
used for sample)
Push the plunger to the end, solvent remains in the needle, move plunger
back until you see the air plug
Fill a little bit more than desired volume of sample (watch for bubbles!)
Adjust exactly the desired volume, wipe off solvent from needle and push
plunger back until you can see the air in the glass body
Type B small volumes 1 µL
Minimum 50 nanoliter
Sample
Tungsten wire
Sample
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Air
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Solvent
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Syringes and injection techniques
Type B: Filled, cold needle technique
Atosamplers
Clean the syringe several time with a solvent
The samples are
in the bottles with
silicone septa
Fill and discard with sample
Pump several time with sample and adjust desired volume
Push the plunger a little back
Put the syringe in the injector and depress immediately the plunger to
prevent peak splitting
Air
Tungsten wire
Sample
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Solvent effect
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Improper solvent effect
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Wrong solvent
Detector
The solvent (MeOH) is not wetting the surface of the non polar
stationary phase and forms droplets where the analytes are dissolved
Two main types:
1. Simple (TCD/FID)
– indicate ”when”
and ”how”)
TIC
2. Spectroscopic –
provide information
about compound
structure
Extracted Ion 119 (p-Cymene)
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Flame ionisation detector
Sensitivity
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TCD
FID
EC or ECD
FPD
FTIR
MS
UV, NMR
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ECD
FID response (signal)
Sensitive towards halides,
peroxides, and nitro groups.
Highly sensitive, but not always linear
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NPD
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Identification of volatiles
Chromatogram
Retention time, min
• RT or related data (RI, KI)
–RTx = RTr at the same GC
parameters (very unreliable)
–RTx = RTr at 2-3 columns (polar and
apolar) – better, but still not
sufficient for positive identification
–Spiking – additional prove
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GC parameters
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Identification
Many compounds have the same RT
Addition of reference compound
(”spiking”)
Assuming that the peak
is isopentane?
Adding
reference
compound purepentane
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The peak
increases –
supports
assumption
The peak
changes – surely
not isopentane
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Compilations of retention indices
Isothermal analysis
Programmed analysis
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Linear RI
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MS libraries
Willey
NIST
NBS
EPA
GC-MS of pepper extract
Coupling of GC and MS
21.23
?
8000000
8.75
The effluents from the GC column are moved via
interface to the mass spectrometer (mass detector)
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10.41
23.40
TIC
5000000
7.49
18.98
7
1000000
24.55
CH3
27.35
1
24.43
10.00
16.00
20.00
24.00 [min]
6
(R ) - (+)
2
3
5
4
10
8
H3C
m/z
fragments
9
CH2
X compound
Reference in library
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Compilations of RI (KI) and MS
Comparisson of 2 mass spectra
Abundance
#25411: .BETA.-PINENE
93
9500
9000
8500
8000
7500
7000
6500
6000
5500
5000
4500
4000
3500
41
69
3000
136
2500
77
2000
1500
β-Pinene
121
1000
53
107
500
63
36
0
30
40
50
60
82
70
80
90
100
110
120
130
140
m/z-->
Abundance
#25416: .GAMMA.-TERPINENE
93
9500
9000
8500
8000
7500
7000
6500
6000
5500
5000
4500
136
4000
3500
3000
77
2500
121
43
2000
γ-Terpinene
1500
1000
105
51
500
58
65
38
0
30
40
115
82 87
50
60
70
80
90
100
110
128
120
130
140
m/z-->
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Identification of volatiles
• RT or related data (RI, KI)
• Spectroscopic data (mass
spectra)
Rimantas Venskutonis
Types of Mass Spectrometers
• Quadrupole
• Ion Trap
–MS pattern X = MS pattern reference
= not sufficient
• RIx=RIr; MSx=MSr most reliable
data for a positive identification
Department of Food Technology, Kaunas University of Technology
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
• Time of Flight
• Fourier Transform MS
• Magnetic Sector
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
23
Diffusion pump
Components of MS
to MS
Vacuum system (rough pump + high vacuum
pump)
to rough pump
Ion source
need a rough vacuum
5*10-3 torr
Analyser, separation according to mass and
charge
Detector
Diffusion pump oil (mineral oil with very high boiling point) is heated at the
bottom, pumped to the top and injected over the circular openings.
Vacuum is generated by diffusion from areas of higher gas partial pressure
to areas with lower gas partial pressure.
Data acquisition and handling
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Turbomolecular pump
Based on the principle that single gas particles collide with
the very fast moving areas of the rotor. By this collision they
get an impulse to in the direction of the rough pump
Turning speed of the rotor: 16.000-70.000 U/min
1 Stator
2 Rotor
3 Motor
4 Connection to MS
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Electron Impact Ionisation (EI)
Electrons are accelerated with 70 V (Energy = 70eV)
These electrons can remove another electron from a
neutral, non charged molecule and form a single charged
radical
e- + M -> M.+ + 2eThis radical can undergo an decay under the formation of
more fragments. Under well defined conditions this
fragmentation pattern is characteristic for a molecule
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
24
Comparison of two different MS
Scheme of an ion source
Abundance
#83538: Tetradecane (CAS) $$ n-Tetradecane $$ Isotetradecane
43
9500
9000
8500
8000
7500
Tetradecane
57
7000
6500
6000
5500
5000
71
4500
4000
3500
3000
29
85
2500
2000
1500
1000
99
500
113
127
141
0
20
30
40
50
60
70
80
90
155
169
198
100 110 120 130 140 150 160 170 180 190 200
m/z-->
Abundance
#87359: Pyrene (CAS) $$ .beta.-Pyrene $$ Benzo[def]phenanth
202
9500
9000
8500
8000
7500
7000
Pyrene
6500
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
101
500
0
26
20
30
50
40
50
63
60
75
70
80
88
113
126 137
150
163
175
187
90 100110120130140150160170180190200210
m/z-->
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Ion trap
Quadrupole-MS
Quadrupole rods (permanent magnet) are overlayed with
a radio frequency to filter the desired masses.
Only at certain times a particle at a defined mass-tocharge ratio (m/z) can reach the detector
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
25
Resolution of mass spectrometer
Scan or SIM?
R = m/Δm
Scan: For the identification of unknown samples. Gives a
spectrum which can be interpreted or identified via a mass
spec library
ATTENTION: Time of acquisition MUST with the
chromatographic resolution, that mean at least 10 points over
the peak for quantitative information
High resolution MS (e.g. magnetic sector MS) up 100.000
for the exact determination of molecular weight
Low resolution MS (Quadrupole): Constant resolution over the
total mass range, it is possible to resolve one mass from
another (e.g. 501 – 502)
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Not resolved substances
SIM: (selected ion monitoring):
The substance is already known, reduces the information, but
increases the sensitivity drastically
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Quantification
Abundance
GC peak area is proportional to the amount of the compound
TIC: KOLBEN2S.D
450000
400000
Pak area may be easily measured by the integrator
350000
300000
250000
200000
150000
100000
50000
0
27.20
27.40
27.60
27.80
28.00
28.20
28.40
28.60
28.80
29.00
Time-->
Abundance
Abundance
Scan 3911 (28.329 min): KOLBEN2S.D
104
12000
Scan 3921 (28.400 min): KOLBEN2S.D
105
11500
11000
11500
195
11000
10500
210
10500
10000
10000
9500
9500
9000
9000
8500
8500
8000
91
8000
7500
7500
7000
7000
6500
118
6500
6000
6000
5500
165
5500
5000
77
5000
212
4500
4500
4000
4000
180
77
3500
3500
91
3000
3000
2500
2500
2000
39
51
152
128
1500
65
1500
222
231 244
0
60
80
100
120
140
41
165
65
1000
500
40
194
183
141
1000
m/z-->
51
2000
160
180
200
220
115 128 141152
234
224
246
500
0
240
40
60
80
100
120
140
160
180
200
220
240
m/z-->
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
26
Quantitative analysis
Quantification
Peak areas of separated components are being summed up and
the content of each components is expressed in percentage
• Absolute calibration
– GC analysis of the samples containing different
concentration of the relevant reference compound,
determination of peak areas and preparation of
calibration curves
• Internal standard
– Measured amount of reference compound is added to
the analysed mixture and according to the obtained
chromatogram calibration curve is preparaed
• Internal normalisation
– Based on assumption that the sum of the areas and/or
heights of all peaks is 100 %, while individual peak area
and/or heights constitutes a definite part in the sum
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Use of internal standard (IS)
Absolute calibration
peak area
X
ω i = k A ,i
Y
Z
Ai × 100
a
Ai – peak area of the component i
kA,i – correction coefficient of the
component i which depends on
detector sensitivity
a- sample amount in mg or ml
Component content
X, Y and Z the curves of absolute
calibration of each component
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Rimantas Venskutonis
Lavender
essential
oil
RFmiA=m x Ai/miA
RF= analyte response in
relation to IS
Ai ir A = IS and
analyte peak areas
mi and m = weight of IS and
analyte
Lavender
essential
oil with IS
methyldecanoate
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
27
Analysis of peppermint oil on two GC
columns; C10-C18 alkane mixture is added
Apolar OV-1
Multidimensional GC
Polar Carbowax 20M
J.V.Hinshaw, 2004
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Threedimensional GC
J.V.Hinshaw, 2004
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
28
Transfer of a fraction to another column
‘Heartcut’ of peppermint essential oil
Polar column
Apolar column`
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Comprehensive 2D
gas chromatography
(GCXGC)
The M are heated and cooled
out of phase with each other.
Peaks are trapped at the M1
when it is cooled down. The
M2 is also cooled down and
then the M1 is heated. The
The trapped peaks move to
the cooled M2 zone along
with any material that leaks
through while the M1 is hot.
When the M1 is cooled off
again, the two trapping zones
are effectively isolated from
each other. The M2 is heated,
and the peaks trapped within
are released into the C2
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Cryogenic modulation
J.V.Hinshaw, 2004
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
29
GCxGC
Department of Food Technology, Kaunas University of Technology
GCxGC
Rimantas Venskutonis
GCxGC in the analysis of cheese volatiles
Gogus F.et al. Analysis of the volatile components of cheddar
cheese by direct thermal desorption-GC × GC-TOF/MS) J. Sep.
Sci., 2006, 29, 1217-1222
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Multidimensional gas chromatography
(MD-GCO)
Isolated from Cheddar cheese using direct thermal desorption (DTD) and
analysed using comprehensive 2-D GC (GC x GC) coupled with TOF MS
(TOF/MS). In total 12 aldehydes, 13 acids, 13 ketones, 5 alcohols, 3
hydrocarbons and 9 miscellaneous compounds were identified at desorption
temperatures of 100, 150, 200 and 250°C using mature Cheddar cheese. A
temperature of 150°C was found to be optimum for the DTD of volatiles from
mature Cheddar cheese. The major components were acetic acid, butanoic
acid, 3-hydroxy-2-butanone and 2,3-butanediol. A DTD temperature of
150°C was used to observe the effect of maturation (mild, medium or
mature) on the volatiles of Cheddar cheese. The major components of the
volatiles of mild, medium and mature Cheddar cheese were almost the
same. However, their percentage compositions were found to change with
the stage of maturity. DTD is simple, fast and requires only a small amount
of sample (approximately 10 mg) and works well with comprehensive GC x
GC-TOF/MS. Comprehensive GC also separated a number of components
which remained overlapped on the single column, such as octane and
hexanal.
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
Department of Food Technology, Kaunas University of Technology
Rimantas Venskutonis
30