INTRODUCTION TO GAS CHROMATOGRAPHY IN FOOD...

Autorizovaný distributor Agilent Technologies HPST, s.r.o. HPST, s.r.o. HPST, s.r.o.

INTRODUCTION TO GAS

CHROMATOGRAPHY IN

FOOD ANALYSIS

“Food Safety, Quality and Nutrition Course”

July 14th, 2014

KAMILA KALACHOVÁ

Application specialist GC–MS

Autorizovaný distributor Agilent Technologies HPST, s.r.o. HPST, s.r.o. HPST, s.r.o. 2

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)


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


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


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.


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


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


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


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


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


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


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


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

×


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


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


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


–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)



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



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



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


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

Autorizovaný distributor Agilent Technologies HPST, s.r.o. HPST, s.r.o. HPST, s.r.o.

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


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


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


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


Stationary phases – siloxane chemistry

MEDIUM POLAR NON-POLAR MEDIUM POLAR


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


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


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


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



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)



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



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)


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


fg pg ng ug mg

TCD

NCD (N)

NCD (P)

ECD

FID

FPD (S)

FPD (P)

Conventional detector in GC

SENSITIVITY


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


Food applications


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


GC-FID chromatogram of preservatives (100 µg/mL) spiked in juice sample

Repeated analysis of soyabean sauce


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)


Column: HP-88 (100m × 0.25mm × 0.2µm)


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


GC-FID chromatogram of 37 FAMEs (standard mixture).


Infant formula (milk powder)

Standard of 37 FAMEs

GC-FID chromatogram of FAMEs


Target analytes:

Volatile organic acids (C1-C10)


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


GC-FID chromatogram of VOC at ng levels

Calibration curve for n-butyric acid



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


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


Matrix: Essential oils

Target analytes:

Monoterpens

Monoterpenoids

Sesuiterpenes

GC system: GC-FID (Agilent 6980)

Column: HP-5 (50m × 0.32mm ×1.05µm)


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


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


Chromatographic separation of multicomponent mix at 0.4% g/dL

Calibration curve for ethanol (0.02–0.4 g/dL)


THANK YOU FOR YOUR ATTENTION...

KAMILA KALACHOVÁ

Application specialist

GC–MS, GC–MS/MS

[email protected]

tel. 244 001 248

mobile 702 281 172

IVO NOVOTNÝ

Product specialist

GC/MS, ICP/MS, NMR

[email protected]

tel. 244 001 240

mobile 724 309 027

INTRODUCTION TO GAS CHROMATOGRAPHY IN FOOD...

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