CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

1

Outline

Separation Techniques‘Instrumentation for Gas Chromatography’

• Instrumental Configuration

• Carrier Gas

• Injection Port, Syringes andAutosamplers, Loops

• Packed and Capillary Columns

• Detectors

• Data Collection

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CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

2

Gas Chromatography Introduction

• Mobile Phase is a carrier gas.

• Stationary Phase is a solid orliquid (supported on a surface).

• Stationary phase is located in a(usually) long and narrow column .

• Sample introduced near beginningof column where it is vaporised.

• Detector senses compoundseluting from the column.

• GC is most important analyticalmethod for volatile compounds.

GC Measures Flavours Preservatives Additives Drugs Contaminants,e.g., from packaging, pesticides and herbicides

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

3

Instrument Layout

analystmobilephase supply separation in

heated column

sample loadingdetection

data analysis

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

4

Carrier Gas

• A pure gas usually suppliedfrom a pressurised cylinder

• Gas must be inert with respectto sample.

• Gas must not be retainedsignificantly by stationary phase.

• Prefer less expensive gases.

• Typical gases are He, H2 and N2

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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Carrier Gas

?• Which gas is best?

• Clue: analysis speed andresolution

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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Carrier Gas

• He and H2 give the bestresolution at higher flow rates.

• Higher flow rates mean fasteranalysis times.

• H2 is a fire/explosion risk

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

NS

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Injectors

• Syringes (liquids 1-5 mL; gases10-100 mL).

• Gas sampling valves

• Autosamplers

• Injection port has complex gasflows to make injection ‘sharp’and reproducible.

• The injection port (injector) isalways in a heated zoned of theGC and it is usually kept hotterthan the GC column

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

8

Syringes

• Syringes require good operatortechnique to be reproducible

• Autosamplers are roboticinjectors - very reproducible - upto 100 samples.

100 mL gas syringe

5 mL liquid syringe

1 mL liquid syringe

autosampler

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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Gas Sampling Loops

Step 1Flush loop withnew sample

Step 2Rotate valve and insertloop into mobile phase

For sampling gases

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

NS

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Columns

• Packed and Capillary columns are used

• Capillary columns most widely used

• Columns kept in a heated oven -temperature needed to keep compoundsin vapour form.

• Column temperature affects partitionequilibrium and diffusion - greatly affectsspeed of analysis.

• Temperature is very carefully controlled

• Isothermal and temperature programsare used

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

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11

Packed Columns

• Usually made from 3-6 mm diametertubing of about 1 to 3 m length

• Packed columns are inexpensive tomake, have high capacity butrelatively low resolution

• Require flow rates of 20-50 mL/min

Tube - metal, glassor teflon

Inert - finely dividedparticles act as support forliquid stationary phase orelse are porous and serve asadsorption stationary phases

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

NS

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Capillary (Open Tubular) Columns

• Long very narrow fused silica tubes typically0.3 mm diameter and 10-50 m long

• Stationary phase coated on the inside wall ofthe column in one of three ways

– Wall coated (WCOT)

– Support coated (SCOT)

– Porous layer (PLOT)

WCOT SCOT PLOT

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

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Capillary (Open Tubular) Columns

• Capillary columns requirelower flow rates (1-10mL/min)

• Low sample capacity usuallynecessitates a split injection(only small part of injectedliquid reaches column - rest isvented.

• Capillary columns are usuallypurchased ($500-$1000 ea)and are used for severalhundred analyses,

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

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Capillary Columns

• Higher resolution

• Shorter analysis times

• Greater sensitivity

• Less sample capacity

Compared to Packed Columns OT columns have:

Perfume Oil

Packed2mm x 1.5m

WCOT0.25 x 30m

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

15

Detectors

• Detectors sense the elution of compounds (other than thecarrier gas).

• They create an electrical signal that is recorded versus time tocreate the chromatogram.

• Usually the area under a peak increases in proportion to theamount of a particular substance eluting.

• Main detectors are:

– Flame Ionisation (FID)

– Thermal Conductivity (TCD)

– Electron Capture (ECD)

– Flame Photometric (FPD)

– Mass Spectrometer (MS)

GC Detectors arevery sensitive

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

NS

W 1999 - A

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16

FID

• Eluate mixed with air and H2

• Mixture ignites in flame

• Ions form in flame (about 1CHO+ for 105 reduced Catoms

• Flame becomes slightlyconductive

• Linear response to mass ofreduced carbon atoms ineluate (7-orders)

• Can detect 10-13 g/s

• Insensitive to inorganics

FID is an excellentgeneral detector fororganic compounds

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

17

TCD

• Less sensitive than FID

• Measures thermal conductivity ofcarrier stream (by resistance changeof heated wire).

• H2 and He have high TC and are usedas carrier gas

• Linear to concentration of analytesover 5 orders.

• Sensitivity about 10-8 g analyte / mL ofcarrier

• Can detect many inorganics

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

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18

Other Detectors

• Extremely sensitive to compoundscontaining F and Cl groups

• Highly selective for certain pesticideresidues found in food.

Electron Capture Detector:

• H2/O2 flame excites S and P to emitcharacteristic wavelengths.

• A selective detector for S and Pcontaining compounds

• Useful in food analysis

Flame Photometric Detector:

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

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19

GC-Mass Spectrometry

• The mass spectrometer obtains acharacteristic fingerprint (a massspectrum) of compounds as they elutefrom the column.

• This is an extremely powerfulcombination that is highly sensitive andselective.

• You will learn much more about thistechnique in CHEM3801.

The ultimate detector for a GC:

m/z

IonAbund.

CH3SCH2CH2CHNH2COOCH2CH3Methionine ethyl ester MW 177

GCMS

Micromass

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -3

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. Guilhaus U

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20

Data Collection

• Most GC systems are now sold with electronic integrators or PCs withsoftware to control the GC and analyse the data.

• Area of peaks for unknowns and standards are compared in thesoftware and concentrations are reported.

• Software can control the whole analysis including the autosampler,calibration, analysis and final reporting.

Shimadzu