INTRODUCTION TO GAS CHROMATOGRAPHY IN FOOD...
Transcript of INTRODUCTION TO GAS CHROMATOGRAPHY IN FOOD...
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INTRODUCTION TO GAS
CHROMATOGRAPHY IN
FOOD ANALYSIS
“Food Safety, Quality and Nutrition Course”
July 14th, 2014
KAMILA KALACHOVÁ
Application specialist GC–MS
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Gas chromatography (GC)
Separation based on boiling points and structure differences
Separation in column with:
• Stationary (immobile) phase (sorbent or highly boiling liquid)
• Mobile phase (gas)
Suitable for:
• Volatile compounds (at 350°C at least partly in gaseous state)
• Termostabile and non-reactive compounds
• Solids, liquids, volatiles, organic and inorganic permanent gases etc.
Sampling Extraction Clean-up
(fractionation)
Separation & identification
(quantification)
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Gas chromatography (GC) - theory
Analytes are separated between mobile and stationary phase = formation of
equilibration
Only molecules in mobile phase are moving (with the same speed)
Separation is based on different retention of individual analytes in stationary phase
Separation of two compounds occurs when they have different distribution between
stationary and mobile phase
MAIN FACTORS INFLUENCING THE SEPARATION
Physico-chemical properties of analytes
Stationary phase
Temperature (↑ temperature → ↑ molecules in mobile phase → ↓ retention
time and separation)
Column dimensions
Type of carrier gas and its flow rate
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Gas chromatograph
Gas (He, N2, H2)
Trap of air and
moisture residues
Inlet
Sample
Oven
Chromatographic
column
Detector
Data collection
Source: http://hiq.linde-gas.com
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GC at a glance
Separation Sample separation (analytes and matrices) between carrier gas and
stationary phase
1D-GC, quick GC, multidimensional GC
Detection Detection of separated sample components
Conventional detectors (FID, ECD, TCD…), mass spectrometers (MS)
Identification & Quantification Mass spectra libraries, exact mass
Calibration curve, standard addition etc.
Matrix effects
Agilent 7890B GC
Injection Introduction of sample in to GC inlet and its vaporization
Split / splitless, PTV, on-column etc.
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Carrier gas Helium, nitrogen, hydrogen
Defined purity
Traps absorbing moisture, hydrocarbons,
oxygen…
Impurities can:
- react with a sample or a GC column
- plug the capillary
- contaminate the detector
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Choice of carrier gas Nitrogen
High efficiency (low h)
Optimal ū is very low → high retention
times
Very steep curve → small shift of a flow
speed = big shift in the efficiency
Low price
VAN DEEMTER CURVE
Helium
Lower efficiency than N2
Higher optimal ū → similar resolution
in a shorter R.T.
Less steep curve → lower risk of
mistake in R.T.
High price
Hydrogen
The same efficiency as for helium
High optimal ū → lower R.T.
without loss of the efficiency
The best for capillary columns
Price between N2 and He
Risk of explosion
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Sample introduction into GC LIQUID SAMPLES (EXTRACTS )
The choice of optimal injection technique relies on:
Concentration range of target analytes (special requests in ultra trace analysis)
Physico-chemical properties of analytes
Presence of matrix co-extracts in the sample (sample extract)
Hot injection
Split
Splitless (pulsed)
On-column
Cold injection (large volume)
PTV
On-column
On-column-SVE
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Sample introduction into GC GASEOUS SAMPLES (GAS PHASE)
SOLID SAMPLES
Head space (HS) - static or dynamic
Purge and trap
Solid phase micro extraction (SPME) – HS or direct immersion (DI)
Valves
Thermal separation probe
TSP
SPME
Valve system
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Split injection
Different types
of liner
Injection with sample splitting (a portion of the sample
goes on a column)
Small sample volume (0.1–2 µL) is quickly injected into the heated liner
Bigger part of a sample is blown away — smaller part goes on a column (split ratio)
Main factors: internal column diameter and sample concentration
Split ratio ratio of a sample distribution
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Split injection SPLIT RATIO
Based on internal column diameter and analyte concentration in the sample
Determines the amount of sample which goes on the column
Split ratio ratio of sample distribution
Split ratio is influenced by:
Sample volatility
Injected volume
Injection solvent
Injection temperature
Column temperature
Inlet volume
1:200
1:5
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Split injection APPLICATIONS
High analytes’ concentrations
Dirty samples
Columns with very small i.d.
DISADVANTAGES
1) DISCRIMINATION - Injected sample ≠ sample introduced onto the column
- Caused by different volatility of sample components
2) BACK FLUSH - The sample expands 100–1000x during the vaporization
- If the vapor volume is bigger than the volume of the liner
- Loss of the sample, presence of „ghost“ peaks
- Liner type (with glass wool)
- Elevated inlet temperature
- ↑ liner volume
- ↓ injection volume
- ↓ inlet temperature
- ↑ carrier gas flow
- Change of injection solvent
ELIMINATION
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Splitless injection Sample (1 to 5 µL) is completely introduced onto a GC column
Splitless period = time, when sample is applied onto the GC column
SPLIT SPLITLESS
×
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Splitless injection
Source: www.sge.com
ts: 10 s
ts: 60 s
Length of a splitless period relies on :
Solvent properties
Analyte properties
Inlet volume
Injection volume
Injection speed
Carrier gas flow
F
Vt ls 2
Vl…liner volume (mL)
F…carrier gas flow (mL/min)
ts = theoretically 1.5 to 2x time, the
carrier gas needs to go throw the
inlet space
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Splitless injection APPLICATIONS
(Ultra)trace concentration levels of analytes
Quite clean samples
OPTIMIZATION
Splitless period = approx. 2x liner volume / column flow
Solvent boiling point at least 25°C < boiling point of the most volatile analyte
Initial column temperature 25–30°C bellow the solvent boiling point
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Programmable temperature vaporization (PTV)
Large volume injection (1–1000 µL)
Solvent vent mode = solvent is withdraw in the inlet space
STEPS OF PTV
• Sample is injected at the temperature
considerably bellow the solvent boiling point
(e.g. 0–30°C)
• Solvent vapor is vented through the split
capillary
• After solvent blow off, the injector is quickly
heated and analytes are transferred onto the
column
• Large volume injection → sorbent in the liner
→ column protection against solvent clot
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–3.5 0.0 3.5 24.0 Time (min)
50
300
Tem
per
atu
re (°C
)
Co
lum
n f
low
(m
L/m
in)
0
100
Injector temperature
GC oven temperature
Split capillary flow
Vent Splitless period Separation
Carrier gas flow through the GC column
Start PTV
Start GC
Start MS (data collection)
Programmable temperature vaporization (PTV)
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LINERS
Small internal volume→ quick heating
Deactivated surface
Liners with sorbent (glass wool..) →
column protection against solvent clot
Labile analytes (degradation on glass wool..) → baffled liners
PTV liner
Empty PTV liner
PTV liner with glass wool
Source: Godula et al., J. Sep. Sci., 24 (2001) 355–366
Programmable temperature vaporization (PTV)
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ADVANTAGES
↓ discrimination of less volatile compounds
Gentle to termolabile analytes
Possibility of large volume injections (LVI) → ↑ sensitivity, simplified sample
preparation, ↓ sample amount
DISADVANTAGES
Difficult (time consuming) optimization of PTV parameters
• Initial and final inlet temperature
• Inlet heating speed
• Vent time
• Flow and pressure
• Period when analytes are transferred onto the GC column (splitelss perioda)
• Injection volume
• Liner type
Programmable temperature vaporization (PTV)
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On-column injection
Liquid sample is transferred directly onto a GC column (without heating)
APPLICATIONS
Clean diluted sample
Small sample volume
Analytes eluted before the solvent
ADVANTAGES
↓ risk of thermodegradation in the inlet
↓ discrimination of less volatile compounds
DISADVANTAGES
System contamination with non-volatile
components of the sample
↑ risk of backflash, ghost peaks
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Head space (HS)
Volatile sample components diffuse into the gas phase
Partition between HS gas phase and liquid/solid sample
APPLICATIONS
Volatile or semi-volatile organic compounds
Alcohols, residual solvents, flavors, fragrances etc.
ADVANTAGES
Little or no sample preparation
↓ protection of the GC system from non-volatile
sample components (dirty)
Agilent 7697 HS
Source: www.labhut.com
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PACKED COLUMN
Length: 0.6–10 m
Internal diameter: 2–5 mm
Material: glass, steel, cupper, nickel
Stacionary phase: Chromosorb, Carbopack, Tenax, liquid polymers
22
GC columns
CAPILLARY COLUMN
Length: 5–150 m
Internal diameter: 0.1–0.75 mm
Material: silica capillary coated with a polyimide layer
Thickness of stationary phase: 0.1–7 µm
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Capillary column – types WCOT
Wall-coated open tubular
Liquid polymer deposit on the inner
capillary wall
Most Agilent columns
SCOT
Support-coated open tubular
Liquid polymer deposit on the support particles on the inner capillary wall
PLOT
Porous-layer open tubular
Adsorbent particles fixed on the capillary by chemical bound (Al2O3)
Analysis of permanent gases, low molecular hydrocarbons, sulphur gases,
amines, etc.
Stationary
phase
Support with
stationary
phase
Sorbent
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Capillary column – separation mechanisms
DISPERION INTERACTIONS
Universal, primary separation mechanism
Intermolecular gravitation between analyte and stationary phase
↓ analyte vapor tension → ↑ retention in stationary phase
DIPOLE INTERACTIONS
Polar stationary phases
Analytes with dipole moment (heteroatom, -OH, = bound)
HYDROGEN BOUND
Polar stationary phases
Strong bound -OH, -NH
No bound -halogens, hydrocarbons
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Capillary column – stationary phases
HP-1ms = DB-1ms HP-5ms
Polyethylene glycol
DB-17ms
100% dimethylpolysiloxane (5%-phenyl)methylpolysiloxane (50%-phenyl)methylpolysiloxane
(50%-cyanopropylphenyl)methyl polysiloxane
DB-225ms
NON-POLAR MEDIUM POLAR
MEDIUM POLAR POLAR
(50%-cyanopropyl)methyl polysiloxane
DB-23 HP-INNOWax
(35%-trifluoropropyl)methyl polysiloxane
DB-200
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Stationary phases – siloxane chemistry
MEDIUM POLAR NON-POLAR MEDIUM POLAR
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Capillary column – stacionary phases
NON-POLAR PHASE
Compounds separation based on boiling points
High temperature range (usually up to 350–400°C)
Low bleed
Examples: DB-1, DB-5
POLAR PHASE Separation based on specific interactions
Lower temperature range (usually up to 220–280°C)
Higher bleed
Examples: HP-Innowax
MEDIUM POLAR PHASE
Separation efficiency changes with the
temperature
Low temperature: as non-polar phase
High temperature: as polar phase
Examples: DB-17ms, BPX-50
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Capillary columns – diameters, analysis types
Category Internal diameter (mm) Commercially available
columns – i.d.(mm)
Megabore 0.5 0.53
Wide bore 0.3–0.5 0.32; 0.45
Narrow bore 0.2–0.3 0.20; 0.25; 0.28
Microbore 0.2–0.3 0.10; 0.15; 0.18
Sub-microbore 0.1 various
Source: K. Maštovská, S.J. Lehotay (2003) J. Chromatogr. A 1000, 153–180
Type of GC analysis Separation time w1/2 peak
Conventional 10 min 1 s
Fast 1–10 min 200–1000 ms
Very fast 0.1–1 min 30–200 ms
Ultra fast 0.1 min 5–30 ms
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How to speed-up the GC analysis?
( ) 1 + = k u
L t R
Reduce a column length ( L)
Lower a retention factor ( k)
Increase a carrier gas flow ( u)
INCREASE A CARRIER GAS FLOW ( u)
Higher than optimal carrier gas flow
Increase the optimum of carrier gas flow
• Use hydrogen as a carrier gas
• Low pressure GC
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REDUCE A COLUMN LENGTH ( L)
The easiest way, how to speed-up the GC run
Most fast GC analysis is run on short columns (10 m)
Shorter column leads to a lower resolution (R~L)
Possible to compensate a lower R by a mass spectrometry
Analysis of volatile organic compounds (stationary phase: PEG) Source: www.phenomenex.com
60 m 0,25 mm 0,25 µm 10 m 0,10 mm 0,10 µm 20 min 6 min
How to speed-up the GC analysis?
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USE COLUMNS WITH SMALL INTERNAL DIAMETER
Microbore columns (i.d. 0.2–0.3 mm); lower capacity
USE COLUMNS WITH THIN FILM OF STATIONARY PHASE
Decrease the capacity factor (retention) faster GC analysis
QUICK TEMPERATURE PROGRAMMING
Conventional heating in GC oven
Resistance heating of GC column
• Electric current is used for a direct heating of conductive
material (metal) placed in the vicinity of the column
• Quick heating as well as cooling
• Heating speed up to 30°C/s (= 1800°C/min)
How to speed-up the GC analysis?
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Oven temperature program:
60°C (0.5 min),
360°C/min do 90°C,
63.5°C/min do 180°C,
82.9°C/min do 325°C (1.25 min)
Conventional GC–FPD
Fast GC–FPD
Oven temperature program:
60°C (2 min),
10°C/min do 180°C,
2°C/min do 240°C,
15°C/min do 325°C (2 min)
Source: Maštovská et al., J. Chromatogr. A 907 (2001) 235–2456
5 min
52 min
How to speed-up the GC analysis?
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Detectors in GC
MASS SPECTROMETERS CONVENTIONAL DETECTORS
Flame-ionization detector (FID)
Thermal-conductivity detector (TCD)
Electron-capture detector (ECD)
Nitrogen-phosphorus detector (NPD)
Halogen-specific detector (XSD)
Photo-ionization detector(PID)
Flame-photometric detector (FPD)
Pulsed flame-photometric detector (PFPD)
Atomic emission detector (AED)
Electrolytic conductivity detector (ELCD)
Quadrupole (Q)
Ion trap (IT)
Time-of-flight (TOF)
Sector (S)
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Thermal-conductivity
detector (TCD) Compounds that are thermally conductive and
have bigger thermal conductivity than a carrier gas
Flame-ionization detector
(FID) Compounds that can be burned or ionized
Electron-capture detector
(ECD) Electronegative compounds
(e.g. halogenated compounds)
Nitrogen-phosphorus
detector (NPD) Compounds containing nitrogen or phosphorus
Flame-photometric
detector (FPD) Compounds containing sulphur or phosphorus
Conventional detectors in GC
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fg pg ng ug mg
TCD
NCD (N)
NCD (P)
ECD
FID
FPD (S)
FPD (P)
Conventional detector in GC
SENSITIVITY
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Detector Selectivity Linearity
Flame-ionization (FID) No 107
Thermal-conductivity (TCD) No 104–106
Electron-capture (ECD) Halogens 104
Nitrogen phosphorus (NPD) N, P 104–107
Flame-photometric (FPD) S, P S 103; P 105
SELECTIVITY & LINEARITY
Conventional detector in GC
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Food applications
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Matrix: juice
soya sauce
Target analytes: preservatives
Ethyl 4-hydroxybenzoate
Propyl 4-hydroxybenzoate
Butyl 4-hydroxybenzoate
Sorbic acid Dehydroacetic acid
Benzoic acid
Methyl 4-hydroxybenzoate
GC system: GC-FID (Agilent 7820A)
Column: HP-Innowax (30m × 0.25mm × 0.25µm)
Inlet: 1 µl, split (10:1), 240°C
Oven temp.: 160°C → 15°C/min to 240°C (10 min)
Carrier gas: Helium (2.5 mL/min)
FID: Temperature 250°C
H2 flow 40 mL/min
Air flow 400 mL/min
Make up (N2) 45 mL/min
Data acquisition: 20 Hz
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GC-FID chromatogram of preservatives (100 µg/mL) spiked in juice sample
Repeated analysis of soyabean sauce
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Matrix: infant formula
Target analytes: fatty acids
Fat is major component of infant formula
Long chain polyunsaturated FA:
docosahexaenoic acid (DHA)
arachidonic acid (ARA)
eicosapentaenoic acid (EPA)
Trans-fatty acids: < 3% of total FA
→ Fatty acid methyl esters (FAMEs)
GC system: GC-FID (Agilent 7890A)
Column: HP-88 (100m × 0.25mm × 0.2µm)
Inlet: 1 µl, split (30:1), 260°C
Oven temp.: 140°C (5 min) → 4°C/min to 240°C (15 min)
Carrier gas: Nitrogen (1 mL/min)
FID: 280°C
Data acquisition: 20 Hz
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GC-FID chromatogram of 37 FAMEs (standard mixture).
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Infant formula (milk powder)
Standard of 37 FAMEs
GC-FID chromatogram of FAMEs
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Target analytes:
Volatile organic acids (C1-C10)
GC system: GC-FID (Agilent 7890A)
Column: DB-624UI (30m × 0.25mm ×1.4µm)
Inlet: 1 µl, split (116:1)
Oven temp.: 70°C (isothermal)
Carrier gas: Hydrogen (42 gm/s))
FID: 300°C
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GC-FID chromatogram of VOC at ng levels
Calibration curve for n-butyric acid
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Column: DB-624UI (30m × 0.25mm ×1.4µm)
Inlet: 1 µl, split (200:1), 250°C
Oven temp.: 70°C (1 min), 20°C/min to 160°C
Carrier gas: Hydrogen (3 mL/min)
FID: 260°C
Column: DB-624UI (30m × 0.25mm ×1.4µm)
Inlet: 1 µl, split (200:1), 250°C
Oven temp.: 70°C (1 min), 20°C/min to 260°C
Carrier gas: Hydrogen (3 mL/min)
FID: 260°C
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Matrix: Essential oils
Target analytes:
Monoterpens
Monoterpenoids
Sesuiterpenes
GC system: GC-FID (Agilent 6980)
Column: HP-5 (50m × 0.32mm ×1.05µm)
Inlet: 1 µl, split (25:1), 250°C
Oven temp.: 50°C → 2°C/min to 280°C
Carrier gas: Helium (constant pressure, 12.33 psi)
FID: 300°C
Retention time database (400 compounds)
R.T. locking: n-pentadecane (70.0 min)
Sesuqiterpenoids
Diterpenes
Diterpenoids
GC-FID chromatogram of Spanish orange oil
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Matrix: Blood
Target analytes: Alcohols
Methanol
Ethanol
2-propanol
Acetone
N-propanol
GC system: GC-FID (Agilent 7890B) + HS Autosampler 7697A + GC-MS 5977A
Column: DB-ALC-1 (30m × 0.32mm ×1.8µm)
Oven temp.: 55°C (isothermal)
HS GC-FID MS
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Chromatographic separation of multicomponent mix at 0.4% g/dL
Calibration curve for ethanol (0.02–0.4 g/dL)
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THANK YOU FOR YOUR ATTENTION...
KAMILA KALACHOVÁ
Application specialist
GC–MS, GC–MS/MS
tel. 244 001 248
mobile 702 281 172
IVO NOVOTNÝ
Product specialist
GC/MS, ICP/MS, NMR
tel. 244 001 240
mobile 724 309 027