Gas Chromatography

OUTLINES

7.2 Gas chromatography

7.2.1 Sample preparation
7.2.2 Chromatographic theory
7.2.3 Application of GC
 Gas Chromatography and Food
Analysis
• Applications:
• fatty acids, cholesterol, gases, solvent purity, water,
alcohol, sugars, amino acids, vitamins, pesticides,
food additives, antioxidants, nitrosoamines, drugs,
flavor compounds etc.

• Often a derivative is made to make it volatile.

 SAMPLE PREPARATION FOR GAS
CHROMATOGRAPHY
• One cannot generally directly inject a food product into a
GC without some sample preparation.
• Sample preparation often involves grinding,
homogenization, or otherwise reducing particle size.
• Sample is vaporized (if it is not already a vapor)
• Passes through a column where interaction occurs - does
analyte move with gas phase or stay with stationary
phase (column coating)
• Separation occurs in column
• Detection with several types of detectors depending on
type of component involved.
GC System and components

Schematic Diagram of GC
Basic Instrumentation of Gas Chromatography

• Gas System
• Injection System
• Column
• Column Oven
• Detector
• Data System
1. Gas System
• Carrier gas (Inert gas) → Helium / Argon / Nitrogen -
Chromatographic grade gases (high purity)

• O2 is usually avoided since it will oxidize the stationary

phase.

• Choice dictated by type of detector, cost, availability.

• Need - Pressure regulator for constant inlet pressure &

flow controller for constant flow rate.
 Regulators
• Pressure regulators – to regulate the pressure of
chromatography system

• Flow regulators - Determine gas flow rates through

system (sensitive precision instruments)

Two Stage Tank Regulator

 2. Injection system

(a) Injector port (sample introduction)

(b) Syringe Needle / Auto sampler
 Injection port
• Introduce sample
• Vaporize sample
• Dilute and Split sample
Schematic of a GC injection port.
 Injection system
• Split Injection- only a portion of injection goes on column.
• Splitless Injection- “all” material injected goes on column.
• On-Column Injection- cold injection (sensitive materials)
• Temperature Programmed Injection - sensitive material

Enter from Injector Exit to Detector

Packed Column
installed in
Oven Compartment
 Columns: Packed/Capillary/PLOT

• Packed column
• Capillary (generally open tubular
but can be a wall coated PLOT
type)
• Porous Layer Open Tubular
(PLOT) GC columns are Packed
commonly used for separations
of small molecules, such as
permanent gases, light
hydrocarbons, and volatile sulfur
compounds.
Capillary
 Columns
• Generally fused silica - strong and inert
• Inner diameters - 0.10 - 0.53 mm
• Length - 1 - 60 m
• Coatings - several - range in thickness from 0.1 - 5
um
Common Stationary Phase Coatings
 Column coatings
(stationary phases)

• Polar to nonpolar
• Polar - Carbowax
• Non Polar - silicone based phases
 Column ovens
• Usually oven is heated to help in separations by
heating up the column.

• Ovens can be controlled from about -60 to 400C

 Detectors
• Many types varying in sensitivity and selectivity

• The most common detectors are the flame

ionization (FID), thermal conductivity (TCD),
electron capture (ECD), flame photometric(FPD),
and photo ionization(PID) detectors.
 Flame Ionization Detector (FID)

• Specificity - most organics.

• Sensitivity - 10-12 g/sec for most organics -- this is
quite good.
• Linear range 106 - 107 - this is good.
Flame ionization detector
 FID Operating Principles
• As compounds elute from the analytical column, they are
burned in a hydrogen flame.

• A potential (often 300 volts) is applied across the flame.

• The flame will carry an electric current across the potential

which is proportional to the organic ions present in the flame
from the burning of an organic compound.

• The current flowing across the flame is amplified and recorded.

• Give no response to H2O, NO2. SO2, H2S.

 Electron Capture Detector

• Specificity - sensitive to halogens, conjugated

carbonyls, nitriles, and a few others - no response
with ordinary organics or H2O.
• Sensitivity 5 x 10-14 g/sec - excellent
• Linear range 104
• The radioactive detectors have definite
temperature limits.
Electron Capture Detector
 ECD Operating Principles
• The electron capture detector contains a radioactive foil
coating that emits electrons as it undergoes decay.
• The electrons are collected on an anode, and the standing
current is monitored by instrument electronics,
• As an analyte elutes from the GC column, it passes between
the radioactive foil and the anode.
• Compounds that capture electrons reduce the standing current
and thereby give a measurable response. Halogenated
compounds or those with conjugated double bonds give the
greatest detector response.
• Unfortunately this detector becomes saturated quit easily and
thus has a very limited linear response range
Thermal conductivity detector
 TCD Operating Principles
• As the carrier gas passes over a hot filament (tungsten), it cools
the filament at a certain rate depending on carrier gas velocity
and composition.
• The temperature of the filament determines its resistance to
electrical current.
• As 2 compound elutes with the carrier gas, the cooling effect on
the filament is typically less, resulting in a temperature increase
in the filament and an increase in resistance that is monitored by
the GC electronics.
GC separation
 Separation theory
The principles of chromatographic separations
and chromatographic theory are discussed as
follows:

1. Adsorption
2. Molecular exclusion
3. Partition
 Adsorption chromatography
Adsorption chromatography is used to separate very
volatile polar compounds (e.g., alcohols, water, and aldehydes)
on porous polymer columns (e.g., Tenax phase).

Interaction with a granular support e.g. Tenax, charcoal, silica gel.

 Molecular exclusion
For example, size-exclusion chromatography is used in
the separation of permanent gases such as N2, 02 and H2.

A variation of size exclusion is used to separate chiral

compounds on cyclodextrin- based columns; one
enantiomorphic form will fit better into the cavity of the
cyclodextrin than will the other form, resulting in
separation.
 Partition chromatography
• Partition chromatography is the
workhorse for gas chromatographic
separations.
• Partitioning between mobile phase
and carrier gas vapor pressure.
• Separation is based on the boiling
point of the materials.
Applications of Gas Chromatography
in
the Food Research
Aroma research
• Biotechnology in flavor production – natural flavor.
• Fermentation of oleic acid by Sporobolomycetes
odorous to produce delta decalactone – key
coconut aroma compound
 Sample preparation

• Surface residue so simply wash surface with a solvent

and concentrate for analysis.
• Three min soak in hexane, concentration followed by a
GC/MS determination.
 Sample preparation via derivatization
• The compound must be thermally stable under GC
condition employed
• Eg. Pesticides, aroma compound and volatile
contaminants

• Compound that thermally unstable, too low in volatility

(sugar and amino acids), or yield poor chromatographic
separation due to polarity ( acid or phenol ) a derivatives
step need to be done.
Reagent to make volatile derivative in
GC analysis
 Fatty acid profile
• Interested because the only way to determine if fat
in food is saturated, unsaturated or poly
unsaturated – needed to meet label requirements.
 Method for fatty acid analysis
1. Add 3 ml of boron trifluoride and about 2 drops of fat
into a 20 ml test tube.
2. Cap with Teflon lined cap
3. Put tube in boiling water bath for 1 hr.
4. Cool and add 3 ml of distilled water
5. Add 10 ml of hexane and shake well
6. Decant top layer and concentrate under a stream of N2 to
about.1 ml
7. Inject about 2 ml into gas chromatograph.
Typical FA chromatogram of vegetable oil