GC PDF

1
Gas Chromatography (GC) and Food Analysis
Review Chapter 29
Introduction to GC
) Gas
chromatography is a separation technique used mainly for quantitative analysis mixtures such as food flavour extracts are injected into a gas chromatograph, the instrument used to perform the separation recorder prints out a chromatogram which is the record of the separation
@consists of a series of peaks, each one ideally representing a single component in the original sample
Schematic Diagram of a Gas Chromatograph
) Complex
)A
Gas Chromatographic Process

1. The sample solution is injected into the heated (250 C) injection port where it is rapidly volatilized 2. The volatilized sample is then swept via the carrier gas into the heated column (in an oven) where volatile compounds separate and are eluted separately

3. The eluted compounds are then detected in a heated detector to give an electrical signal which is amplified and recorded 4. The output is a plot of recorder response vs time called a chromatogram
Typical Chromatogram
) Gas
Introduction to GC
chromatography is a process by which a mixture is separated into its constituents by a gas phase moving over a stationary phase in a column Phase: inert carrier gas (eg. He or N2) Phase: high boiling liquid (or solid)
) Mobile
) Stationary
) Only
interested in gas-liquid chromatography (GC)
@ie. a liquid stationary phase
Basic Principles of GC Separation

) Sample

) Separation
molecules partition or equilibrate between the moving gas phase and the stationary liquid phase
occurs because components have different solubilities in the liquid stationary phase
@compounds with high solubility spend more time dissolved in the liquid stationary phase
thus, they move more slowly through the column
) When
in the gas phase, the sample molecules are carried through the column the sample molecules are dissolved in the liquid phase, they are stationary
) Separation
) When
occurs because components have different volatilities (ie. different boiling points)
@the lower the boiling point, the more time a compound will spend as a gas (vapourised) in the gas mobile phase
GC Partition Coefficient
) Solubility
GC Partition Coefficient
) Different
of the components in the liquid stationary phase and volatility of the components results in each compound having a partition coefficient between the gas mobile phase and the liquid stationary phase coefficient:
compounds coefficients, K
have
different
partition
) Example: ) Compounds
A and B KA= CmA / CsA KB= CmB / CsB
) Partition
@ratio of the concentration of a component in the mobile phase divided by the concentration of the component in the stationary phase
CmA = conc. of A in mobile phase CsA = conc. of A in stationary phase

) If
KA > KB then compound A spends more time on average than compound B in the mobile phase A migrates faster and separation
) The
) Compound
partition coefficient, K depends on the volatility (and bp) of the compounds being separated
occurs, and
@A is eluted first
Samples for GC Analysis

Gases
) Molecular ) Only
Liquids
Solids
) Samples
for gas chromatography cannot have a high molecular weight

@the upper limit of about 1000 excludes high molecular weight polymers, eg. wood and common plastics
Samples Not Suitable for GC Analysis
weight may range from 2 to about 1000
requirement is that the sample must be sufficiently volatile to be carried through the column samples remain condensed in the injector or on the front of the GC column
) Highly
polar and non-volatile samples such as sugars, amino acids or nucleotides cannot be analysed directly
@these materials decompose before they volatilise
) Non-volatile
) GC
cannot separate ionic compounds such as salts as they are non-volatile
Elution from a GC Column

1. Compounds are eluted in order of volatility, ie. the most volatile (generally lowest boiling) off column first 2. Compounds must be volatile to pass through the column
@if not, then they must be chemically modified to do so
Advantages of GC
) High
resolution speed
) High
) High
sensitivity accuracy
) High
High Resolution
) Ability
Compounds in Fermented Cabbage
to separate very closely eluting compounds shows remarkable separation capability for complex mixtures
) GC
Compounds in Orange Juice
High Speed with High Sensitivity
) Rapid
analysis can occur at 10-9 g or nanogram levels picogram, ie. 10-12 g levels can be determined
) Even
Other Advantages of GC
) Used ) Small
Sample Derivatisation
when the compounds to be analysed are:
amounts of sample are needed L
@thermally unstable @too low in volatility (eg. long chain fatty acids, sugars and amino acids) @poor chromatographic separation due to polarity (eg. phenols and acids)
) Good
detection system
) Quantitatively
analyzed
Examples of Derivatisation
) Silyl
Fatty Acid Methyl Esters (FAME)

) methanolic
reagents
@hydroxy, amino, carboxylic acid groups in sugars, sterols and amino acids
) Esterifying
HCl sodium methoxide ) boron trifluoride/methanol

Methylester
) methanolic
Fatty Acids
reagents
O R C OH + CH 3OH + H 2SO 4
@carboxylic acids such as fatty acids (use methyl esters), amines, amino acids, triglycerides, phospholipids
Reflux
O R C O CH 3 Volatile in Gas Chromatography
O CH 2 O C R CH O O C R CH 3ONa + CH 3OH O 3 R C O CH 3 Volatile in Gas Chromatography
Summarised in Chapter 29
O CH 2 O C R
Two Important GC Terms

) Time
Retention Time: tR
required for the sample to travel from the injection port through the column to the detector
@time from injection to the peak maximum @tR total time the component spends in the column
Response
D
) Retention ) Peak
Time tR
area / peak height

A
10
15
20
25
Retention Time
Retention Time: tR
) t0
Retention Time: tR
) Retention
dead time
@time from injection to the emergence of a sample gas that is unretained by the liquid stationary phase in the column, eg. butane or methane @time that the sample spends in the mobile gas phase
) t R'
time is used qualitatively to identify compounds in a chromatogram of a mixture samples of the compounds of interest are used as standards an unknown compound and the standard have the same retention times when run on the same instrument under identical GC conditions
@then the identification can proceed
) Authentic
adjusted retention time
@retention time (tR) - dead time (t0) @time that the sample spends in the liquid stationary phase
) If
) Area
under the peak is a measure of the quantity of the compounds present in a mixture
@peak area = peak height x width at half height @integrator or integration software used to determine the area under each peak
Response
Peak Area and Peak Height
GC Instrumentation
10
Retention Time
15
20
25
Operation of a GC Instrument Review Section 33.3 in Textbook (all examinable)
Gas Chromatography
Filters/Traps Data system
H
Schematic Diagram of Gas Chromatography
RESET
Regulators
Syringe/Sampler Inlets Detectors
) )
Column
) ) )
gas system inlet (injector) column detector data system
Gas Carrier
Hydrogen
Air
Requirements of GC Carrier Gas

) Ultra-pure
Carrier Gas
Mobile Phase
@free of oxygen
oxygen degrades column stationary phases
@free of organics
) Dry
@water can degrade some column stationary phases

) Compatible
with detectors
@helium used for most detectors @ultra-pure nitrogen used for electron capture detectors @helium for thermal conductivity detectors
Purification of Carrier Gas

) High
Gas Chromatography
H
quality gas in cylinders is needed

Regulators
@high purity grade @ultra high purity grade

) In
RESET
Syringe/Sampler Inlets
line filters to further purify the gas

Air
Detectors
Gas Carrier Hydrogen
@silica gel/molecular sieve filters remove water, carbon dioxide and light hydrocarbons @carbon filled Oxy traps remove oxygen after removal of water above @traps that remove organics
Column
GC Regulators and Flowmeters

) Flowmeter
is usually a needle valve, which opens and closes a small orifice to regulate the carrier gas pressure or column head pressure reproducible conditions of gas pressure and
GC Injector
) Want
flow
GC Injection Port
) Heated
Gas Chromatography
H
block surrounds the injector

Regulators Syringe/Sampler Inlets
@sample must be rapidly vapourised in the injector to ensure maximum column efficiency @heated to at least 50 C above the boiling point of the least volatile compound @not too high a temperature that decomposition occurs
RESET
Detectors
Gas Carrier Hydrogen Air
Column
Sample Introduction
) Liquid
Sample Introduction
) Manual
samples are normally the matrix analysed
by GC
) Liquid
sample injection
@largest source of poor precision in GC analysis
introduced into the injector using a syringe
@10 L syringes normally used

0.5 L to 3 L
) Sample
must be added a thin plug (small volume, eg. 1 L) through a correct and rapid injection technique
@slow injection of large samples broadening and loss of separation causes band
) Autosampler
@excellent precision @adjustable volume sizes
Sample Introduction
Injection volumes
) Packed
Functions of the Injector

) Standard
injectors produce flash volatilisation
@used for thermally stable compounds

) Temperature
injectors:
@1-20 L
) Split/splitless
programmed and on-column injectors use slower heating and volatilisation

@used for thermally labile compounds (ie. prone to decomposition by heat)
capillary injectors:
@0.1-1 L
)A
split injector splits the injection prior to entering the column from the injector
@used for capillary columns which have limited capacity and thus injection volume must be reduced
Components of an Injectors
) Septum
Packed GC Injector
) Use
@a soft silicone rubber septum seals the injection port (where the syringe needle is inserted) from the outside and thus maintains the pressurised conditions in the GC @penetrable self-sealing barrier
resealing capabilities depend on the temperature, flexibility of the silicone rubber (composition), sharpness of the syringe needle (pointed tip needle) many types of septa are available for use at different temperatures, including high temperatures such as >280 C
with packed columns is inserted well up into the injector do not have a glass liner
) Column
) Normally
) During
injection, the syringe needle is inserted into the top of the column
@on column packed injector
Packed GC Injector
heated block septum
Split/Splitless GC Injector
) Heated
chamber containing a glass liner into which the sample is injected through the septum with capillary columns where the small amount of liquid stationary phase demands sample sizes too small for direct injection (as for packed columns) rates in capillary columns are about 0.5-2 mL / min which is too small to rapidly sweep the sample from the injector onto the column in a very narrow band
... . ....... . .....

column needle heated block gas supply needle gas flowmeter syringe
) Used
) Flow
Packed GC Injector
)A
regular syringe is used to inject 0.2 to 2 L of sample into a heated glass liner is then rapidly vapourised and mixed with a fast flow of carrier gas, typically 100 L/min
) Sample
10
Glass injection Liner of Split/Splitless Injector

) Fits
neatly into the cylindrical heated block is injected into the middle of this liner
) Sample ) May
contain some glass wool to wipe the needle tip during injection
@any sample that decomposed is deposited on the glass wool or the inside of the glass liner
) After
Split Ratio
split vent flow rate + column flow rate split ratio = column flow rate
) The
thoroughly mixing with carrier gas in the glass liner, the sample stream is split
@a small % (say 1%) goes onto the column @remaining % (say 99%) exits as purge flow through the split outlet or vent needle valve @this would achieve a split ratio of 100:1
split vent flow rate (mL/min) is measured by placing a bubble flow metre on the outlet of the split vent and flow is adjusted using a needle valve on the split vent column flow rate (mL / min) is set by the dimensions of the column, the type of carrier gas and column head pressure
) Split
ratio is controlled by the flow rate through the column and out of the split vent
) The
Advantages of a Split Injector

) Only
Disadvantages of a Split Injector

) Thermally
a fraction of the sample enters the column its mass is very small and can be swept rapidly onto the column from within the glass liner narrow peaks result
sensitive compounds can thermally degrade in the hot syringe needle split injectors are non-linear
) Some ) Thus,
@a major limitation @high boiling components tend to be lost preferentially

called discrimination ) Not
) Very
suitable injector for trace analysis, since only a fraction of the sample enters the column
11
) In ) These
Grob Splitless Injector

the splitless mode, the split vent is closed during the injection, but opened at a later time
@0.5-3.0 L can be injected @95% of the sample enters the column @after 45-60 s, the split vent valve opens and the increased flow rate purges out any residual sample (and most of the solvent which is slower to enter the column)
injectors can operate in 2 modes
@split injection mode @splitless injection mode

developed by Dr Grob
Splitless Injector and the Solvent Effect

) Oven
Splitless Injector and the Solvent Effect

) After
is as cool as possible
some time the column temperature is raised
@most of the solvent then evaporates @the solvent effect leaves the sample molecules concentrated in a narrow band @as the column is further heated, the remaining solvent and sample molecules are rapidly vapourised
produces high column efficiencies and narrow peaks
) At
a time just after the injection
@solvent and sample are condensed in a long plug at the front of the column @the column temperature must be cold enough to condense the solvent
Advantages of Splitless Injection

) High
Cold On-Column Injectors

) In
sensitivity since 95% or more of the sample enters the column

@useful for trace analyses
the injector, the column is pushed tightly up against a tapered section of the glass liner syringe needle is inserted inside the column to inject the sample
@very thin syringe needles are used
) The
) Solvent
effect produces narrow bands leading to high column efficiencies serves a dual role
) Injector ) Hardware
is at the temperature of the column or at room temperature oven is then temperature programmed
@split injection @splitless injection
) The
12
Temperature Programmed Injector
) The
sample is introduced into a room temperature injection port injector is then temperature programmed to the desired temperature which vapourises the sample
GC Oven
) The
Importance of the GC Oven

) The
Gas Chromatography
H
temperature of the column is controlled by the insulated, fan forced, thermostatted oven
@controls the column temperature
Regulators
RESET
) Column
is coiled to fit into the oven

Gas Carrier Hydrogen
) Separation
in GC takes account of 2 factors:
Air
Column
@interaction of the volatiles with the liquid stationary phase @the boiling point of the volatiles
GC Oven Temperature
) The
Isothermal Operation of GC Oven

) Oven
GC analysis time is determined by the temperature of the oven (and thus the column)
@higher temperatures cause volatiles to elute at a faster rate with loss in resolution (peak separation)
retention times reduce as the oven temperature increases
temperature remains constant during the GC analysis for recording accurate retention times for compound identification purposes useful when the sample is a mixture of compounds that have a large range of boiling points
@ie. some low boiling point volatiles and some high boiling point volatiles (semivolatiles)
) Used
@low temperatures produce long analysis times

) Not ) Two
modes of operation
@isothermal @temperature programmed
13
Temperature Programmed Operation of GC Oven

) Temperature
of the oven is varied in a controlled linear mode throughout the GC analysis vary from 0.1 C/min to high heating rates
Application of Temperature Programmed GC

) Used
) Rates
@plus holding times at specific temperatures

) Normally ) GC
use rates of 2-10 C/min
for the analysis of mixtures consisting of > 15 components with different volatilities (boiling points) in a complex mixture
@eg. flavour extract from a food
analysis time can be sped up by using temperature program without compromising resolution (separating ability)
GC Column Differences
GC Columns
) Dimensions,
eg. internal diameter (i.d.)
Packed Megabore or Wide-bore Capillary
@packed columns have i.d. = or > 1 mm @megabore columns have i.d. = 0.53 mm @capillary columns have i.d. < 0.4 mm
Composition of Packed Columns Packed Column
) Glass
tubing of i.d. of 2 mm to 6 mm steel tubing of 3-10 mm o.d.
) Stainless
) Length
0.5-10 m long coiled to fit in the oven
14
Column Packing Material

) Columns
Column Packing Material

) Thin
packed with a granular material consisting of a liquid coated on an finely divided, inert solid support of uniform size
(for high performance) coating of a nonvolatile, inert liquid is called the liquid stationary phase
@liquid phase column loads of 5-20% by weight of the total packing material @support achieves the high surface area for contact between the carrier gas and the liquid stationary phase coating
) Inert
support is celite (diatomaceous earth) which is the skeletons of algae that have been purified, maybe chemically modified (eg. treated with silane) and sieved to a definite particle size
Effect of Stationary Phase Loadings in Packed Columns

) Retention
Packed Column Stationary Phases

) 200
time is proportional to the loading is improved by increasing the loading
available liquid coatings
) Resolution ) Bleed
) Silicone
(disadvantage)
based phases (methyl, phenyl, cyano substituted) are common (ester based) also used Se Chapter 29
@liquid coatings are somewhat volatile and can be lost from the packing material
bleed is observed as an increasing baseline during temperature programming
) Carbowax
@different stationary phases bleed at different rates

some have maximum temperatures of 320 C while other have a maximum of 220 C
) Normally
choose a liquid stationary phase of similar polarity to the compounds (analytes) to be separated
Other Packed Column Stationary Phases
Separation in a Packed Column

) Carrier
gas flows continuously through a packed
column
) Separation ) Porous
organic polymers that serve as stationary phases requiring no inert support
occurs because sample molecules distribute themselves between the flowing gas phase and the stationary liquid phase
@some molecules have a high solubility in the liquid phase, while others have a low solubility
) Distribution
between the two phases determines the speed of the column
15
Separation in a Packed Column

) Temperature
Disadvantage of Packed Columns

) Resolution
of the column and thus the liquid stationary phase affect the interaction of molecules with the stationary phase
@high temperatures favour little interaction with the liquid stationary phase
and thus more interaction with the mobile gas phase
or separating ability is much lower for packed columns than for capillary columns
) Due
mainly to the limitation on column length produced by the high pressure drop encountered along packed columns
@the wide bore of packed columns means there is a considerable pressure drop along the length of the column @if the column is too long then the head pressure required for gas to flow through the column would be too high and beyond the capabilities of the instrumentation
@low temperature permit molecules to dissolve more often in the stationary phase
and thus interact less with the mobile gas phase
Characteristics of Capillary Columns Capillary Columns

) Capillary
columns are chromatographic columns that are not filled with packing material tube made of fused silica
) Hollow
@thin walls of 25 m that are flexible
Characteristics of Capillary Columns

) Column
Capillary Columns
lengths of
5-100 m (low resistance to
flow)
) Column
) Thin
film of liquid phase or adsorbent coats the inside tube wall

@wall coated open tubular (WCOT) invented by Dr Marcel Golay
i.d. of 0.1 mm (microbore), 0.2-0.32 mm (normal capillary), or 0.53 mm (megabore) wall is coated with a polyimide to enhance strength and prevent breakage and oxidation
) Outer
16
Capillary Columns
) Liquid
Capillary Columns
) Now
phase is chemically bonded to the inner wall of the fused silica tube and internally crosslinked at phase thicknesses of 0.1 to 5 m
@thin films provide high resolution but very limited sample capacity @thick films have higher sample capacity but lower resolution
called fused silica open tubular (FSOT) columns

@most capillary columns now are FSOT
Fused Silica Capillary Columns

) Fused
silica is very inert (much more than glass)
@glass originally used but these column were too brittle and broke easily
) Flexible
so the columns can be wound into coils
@also easy to handle in and out of the GC oven

) Fused
silica is made by the reaction of SiCl4 and water vapour in a flame

@pure SiO2 results with about 0.1% hydroxyl or silanol groups on the surface and less than 1 ppm impurities

)A

) High
working temperature of 2000 C is required to soften and draw fused silica into capillary dimensions silica columns are drawn on expensive sophisticated machinery using advanced fibre optics technology
purity of fused silica produces a very inert chemical nature

@more uniform chemical surface than glass @absence of metallic cations makes it inert
) Fused
@inert surface and low surface area minimise sample adsorption leading to symmetrical peak shapes even for polar compounds
17
High Efficiency of Capillary Columns

) Superior
) Because
of fused silicas smooth, inert surface, capillary columns may be coated with a very thin, uniform liquid phase
@produces high efficiency
resolving (separating) power of capillary columns compared to packed columns
) However,
the separating ability (resolution) per unit length for the same liquid stationary phase is the same for both packed and capillary columns
@so why do capillary columns have superior efficiency? @purely a matter of the extra length of capillary columns
High Efficiency of Capillary Columns

) Capillary
Application of Capillary Columns

) Capillary
columns have a small pressure drop across their length due to their narrow bore size and lack of resistance to gas flow
@therefore, long columns are thus possible @ie. more unit lengths or theoretical plates
higher efficiency
columns are useful for separating mixtures that have a large range of boiling points such as food flavour volatiles
) Capillary
columns have total plate of 180,000 to 300,000 depending on their length columns generate only 4,000 plates
) Packed
Disadvantages of Capillary Columns

) The
liquid stationary is easily overloaded in capillary columns

@ie. it becomes saturated with sample resulting in changes in its separating characteristics
Overcoming Overloading in Capillary Columns

) Reduce
the amount of sample entering the
column
) Dilute
) During
overloading peaks broaden, lose their symmetrical shape and the peak height reduces
@integration of the area under the peak becomes difficult @closely eluting peak coalesce together and resolution reduces
considerably the sample, OR to split the sample using a split injector
) Easier
@eg. 100:1 or 300:1 @see the earlier section on this special injector
18
Parameters Used to Selecting Capillary Columns

Internal diameter (i.d.) Column length Film thickness Stationary phase composition Flow rate
Internal Diameter
) For
fused silica, internal diameters range from 0.1 mm to 0.53 mm mm columns have high resolution and high speed of analysis
@but, limited sample capacity making trace analysis difficult
) 0.1
) 0.25
mm or 0.32 mm represent the best compromise between resolution (separating ability), speed, sample capacity and ease of operation
Megabore Columns
) Capillary
Column Length
) Theoretical
columns with an i.d. of 0.53 mm
@liquid stationary phase still coated on the inside of the fused silica wall
) Less
plates are directly proportional to column length, L longer the column, the more theoretical plates and the better the separation resolution R is proportional to the L
) The
prone to overloading due to their larger amount of liquid stationary phase pressure drop than in normal capillary columns with i.d. < 0.53
@so column lengths must be less (maximum 30 m)
) However, ) Increased
) Larger
resolution is more easily obtained by choosing a small diameter column
) Efficiency
is between those of packed and normal capillary columns
) Retention
time tR is also proportional to column length, so long columns lead to slow analyses
Column Length
) When
high resolution (separating ability) is required

@use long column such as 50-60 m @used for separation of food flavour volatile, particularly when the mixture to be separated has 50 compounds or more @but analysis time will be long
) Standard
film thickness of 0.25 m is a good starting point for a GC analysis between:

@the high resolution with thin films, and @the high capacity with thick films
Film Thickness
) Compromise
)A )A
0.25 m film thickness has little column bleed
) For
fast analysis
@use short columns such as 10 m @only moderate resolution

) Compromise
column with a 0.25 m film thickness can be optimised for:

@high analysis speed using fast flow rates, or @for high resolution using slower flow rates
with a 25 m column
19
) Thick
films are 1.0 m or greater
Why Use Thick Films?

) Higher
Why Use Thick Films?

temperatures are required to elute strongly retained components from thick films temperatures produce higher bleed rates and thus more noise bleed is proportional to the amount of liquid phase in a column
@thus thick films bleed more
) Advantages: ) Increased
retention of sample components such as volatiles capacity permits injection of larger samples
) Higher
) High
) Disadvantages: ) Lower
) Column
efficiency, so greater lengths may be required to compensate for the lower number of theoretical plates per metre
Thin Films
) Films
less than 0.2 m
) Advantages: ) High
efficiency and thus high resolution operating temperatures can be used
@thus shorter column can be used

) Lower
Stationary Phases in Capillary Columns
@produces less column bleed

) Faster
analyses since retention time is proportional to the amount of liquid phase in the column of thinner films is the lower capacity
) Disadvantage
Required Characteristics of Stationary Phases in Capillary Columns

) High
Required Characteristics of Stationary Phases in Capillary Columns

) High
selectivity for the sample
chemical stability
) Fortunately,
the high number of theoretical plates in capillary columns makes this factor less important than in packed columns bleed rate
@so column can be used for many months with no variation in chromatographic properties
) Many
liquid phases meet these criteria
) Low
) 200
different liquid phases have been developed
@stationary phase must be stable at high temperatures
20
Stationary Phases in Capillary Columns

) As
Common Stationary Phases

1. Separation of mixtures of polar compounds
@Carbowax type stationary phases such as Carbowax 20M (polyethylene glycol) have limited lifetimes because of oxidation at high temperatures
maximum operating temperature is 250 C
capillary columns have replaced packed columns, fewer stationary phases are required since column efficiency has substituted for phase selectivity
@high efficiency has resulted in better separations even though the stationary phase is less suited for the separation
2. Separation of non-polar compounds

@polysiloxane liquid phases such as OV101 or SE-30 (polymer of methylsilicone) are heat stable and usable to 350 C
) Fewer
than 12 liquid phases in common use
@most durable and efficient phases are based on polysiloxane (-Si-O-Si-)
3. Methyl ester of fatty acids

@DEGS (diethylene glycol succinate)
Common Stationary Phases

) Crosslinked
Selection of Stationary Phases

) Intuition ) Knowledge ) Help
phases crosslinked bonds
contain
chemically
) Stable,
high-molecular-weight liquids or gums occurs between:
) Crosslinking
of chemistry from column manufacturers ) The literature

) General
@adjacent molecular chains; and @between the stationary phase and the wall
) Longer-life
Rules:
columns result
See Chapter 29
@polar phases to separate polar compounds @nonpolar phases to separate nonpolar compounds @phenyl-based phases to separate aromatic compounds
Selection of Stationary Phases

) However,
no hard and fast rule since a relatively non-polar phase such as 5% phenyl substituted methyl silicone will separate polar, in addition to non polar compounds
@commonly used liquid stationary phase
Carrier Gas Flow Rates
21
Flow Rates
)A
lower HETP values means less band broadening and greater column efficiency HETP and thus maximum efficiency occur at high linear flow velocities
GC Detectors
) Minimum
Gas Chromatography
H
What is a GC Detector?
)A
device situated at the exit of the column
RESET
Regulators
) Required
to measure the small amount of the separated components present in the carrier gas stream leaving the column entering the detector produce a response (different technology of response for each detector) that is outputted and amplified as an electrical signal
Gas Carrier
Hydrogen
) Different
selectivity
) Non-selective
detectors respond compounds except the carrier gas
) Selective
detectors respond to a single type of chemical compound

@eg. nitrogen containing or halogen containing
Air
Column
) Compounds
Functions of GC Detectors
detectors will give different types of
) Concentration
dependent detectors
to
all
@signal is related to the concentration of the solute in the detector @sample is not destroyed @dilution of the sample with make-up gas will lower the detectors response
22
) Mass
Types of GC Detectors
) Flame
flow dependent detectors
@sample is destroyed @signal is related to the rate at which solute molecules enter the detector @response is unaffected by make-up gas
Ionisation Detector (FID) ) Thermal Conductivity Detector (TCD) ) Electron Capture Detector (ECD) ) Flame Photometric Detector (FPD) ) Photoionisation Detector (PID) ) Electrolytic Conductivity Detector (ELCD) ) Thermionic Detector or Nitrogen Phosphorus Detector (NPD)
Types of Detectors
Detector Type Support gases Selectivity Detectability Dynamic range 107 107 105 106 103 Flame ionization (FID) Mass flow Hydrogen and Most organic cpds. air Universal Halides, nitrates, nitriles, peroxides, anhydrides, organometallics 100 pg 1 ng 50 fg 10 pg 100 pg
Thermal conductivity (TCD) Concentration Reference Electron capture (ECD) Concentration Make-up
Hydrogen and Nitrogen, phosphorus Nitrogen-phosphorus Mass flow air Hydrogen and Sulphur, phosphorus, tin, Flame photometric (FPD) Mass flow air possibly boron, arsenic, germanium, selenium, chromium oxygen Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, Photo-ionization (PID) Concentration Make-up organosulphurs, some organometallics Hall electrolytic conductivity Mass flow Hydrogen, oxygen Halide, nitrogen, nitrosamine, sulphur
Flame Ionisation Detector (FID)
2 pg
107
Flame Ionisation Detector

) Compounds
Schematic Diagram of Flame Ionization Detector
eluting from the column are burnt in a hydrogen flame (in an excess of air)
@H2 at 30 mL/min
@air at 300 mL/min through a porous frit for combustion of the hydrogen
23

) High

) An
temperature of hydrogen flame (H2 +O2 + N2) ionizes compounds eluted from column into flame ions collected on the collector electrode
electrical potential (+300 volts) is applied across the flame

@flame jet is grounded relative to the polarised collector electrode
) The
) Thus,
the flame carries a current across the potential that is proportional to the organic ions present in the flame from the burning of an organic compound current generated is amplified and recorded
@pure carrier gas produces a very small standing ion current of 10-14 amp
) The

) High
temperature of hydrogen flame (H2 +O2 + N2) ionizes compounds eluted from column into flame ions collected on the collector electrode
) The
) Thus,
the flame carries a current across the potential that is proportional to the organic ions present in the flame from the burning of an organic compound current generated is amplified and recorded
) The

Collector Detector electronics
- 220 volts
Flame
Chassis ground
Jet Column Signal output
24
Flame Ionisation Detector (FID)

) FID ) No

) Minimum
detectable quantity of 10-11 to 10-12 g/s is excellent, 1-106
responds to only organics on a weight basis response to H20, NO2, CO2, H2S or air
@50 ppb of a 1 mg sample

) Linearity ) FID
) Response
is best with compounds containing C-C and C-H bonds
is mass sensitive rather than concentration sensitive stability is excellent

@ie. it is relatively insensitive to minor drifts in temperature and flow rate
) Carrier
gas can be N2, He or H2 since these gases produce no FID response operation temperature is 400 C
) Thus,
) Maximum
) Destructive
detector
Food Analysis Applications of FID

) Organic
compounds are most common in food
analysis
) Wide
linear range in response, good sensitivity and dependability make this detector very useful for food analysis
Thermal Conductivity Detector
) Flavour ) Food
volatile analysis contaminants ) Sterol (eg. cholesterol) analysis

) Measures

) Principal:
The thermal balance of a heated
filament the changes of the thermal conductivity of a heated tungsten filament (in a sealed stainless steel block) due to the sample (g)
) Electrical
power is converted to heat in a resistant filament and the temperature will climb until heat power loss from the filament equals the electrical power input filament may loose heat by radiation to a cooler surface and by conduction to the molecules coming into contact with it
) Sample
can be recovered
) The
25
Thermal Conductivity Basics

) The
Thermal Conductivity Basics

The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas . When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal.
ability of a colliding molecule to carry off heat depends on its thermal conductivity and helium have high thermal conductivity and therefore will be more efficient at cooling a heated filament than will other gases
) Hydrogen
Flow

) As
the carrier gas passes over the hot filament, it cools the filament at a certain rate depending on carrier gas velocity and composition temperature of the filament determines its resistance to electrical current

) Newer
) The
TCDs employ a carrier gas switching valve to pass alternatively carrier gas or column effluent through the detector signal produced is a change in the cooling of the detector as a function of which gas is passing through the detector from the GC column or carrier gas supply carrier gas is normally used
) As
compounds elutes with the carrier gas, the cooling effect on the filament is less, resulting in an increase in resistance that is monitored by the GC electronics
@an imbalance in the Wheatstone bridge circuit generates an electrical signal
) The
) Helium

) The
Relative Thermal Conductivity

Compound Relative Thermal Conductivity
TCD will respond to any substance different from the carrier gas as long as its concentration is sufficiently high enough
@i.e. is a universal detector
Benzene
Carbon Tetrachloride Hexane Argon Methanol Nitrogen Helium Hydrogen
0.05 0.11 0.12 0.12 0.13 0.17 1.00 1.28
) Moderate
linear range of 1 to 104
) Good
stability
@controlled by the temperature stability of the detector block and the flow control of the carrier gas
Flow
26
Food Analysis Applications of TCD
) Used
where no other detector can be used used for fixed gas analysis
Electron Capture Detector
) Mainly
@eg. CO, CO2

) Detects
water

) For
pesticide analysis (picogram)
Electron Capture Detector Basics

) Electron
) Contains
a radioactive foil coating that emits electrons as it undergoes decay

@63Ni which emits particles
capture compound, X (highly electonegative element), tends to capture free electrons and increase the amount of ion recombination (F, Cl and Br) containing sample + (e) Xrecombination : X- + N2+ X + N2
) Ionization
@N2 (Carrier gas) + (e) N2+ + 2e) Electrons ) The
)X
are collected on an anode
) Ion
N2+ establish a base line or standing current
@the base line current due to the N2+ will decrease and this decrease constitutes the signal
27

) Very

) Conjugated
sensitive detector
) Requires
ultra-pure and very dry carrier gas (normally N2) since trace amounts of water and air degrade detector response as it only responds to certain compounds
@those with strong electron affinity
carbonyls, nitro organometallics (eg. lead)
compounds
and
) Phosphorous,
) Selective
silicone and polynuclear aromatics also can be detected
) Halogenated
compounds or those with conjugated double bonds give the greatest detector response
) Applied
to the analysis of insecticides, pesticides, vinyl chloride, and fluorocarbons

@particular trace analysis work
Olfactory Detector
Other GC Detectors
Flame Photometric Detector (FPD) Photoionisation Detector (PID) Electrolytic Conductivity Detector (ELCD) Thermionic Detector or Nitrogen Phosphorus Detector (NPD)
3 Types of Recording Devices
GC Recording Devices
Electrical output from the detector is further amplified and recorded Pen-trace recorder Integrator Computer workstation
28
Pen-Trace Recorder
) Converts
Integrator
) Records
electrical response of the detector into lines and peaks on chart paper producing a chromatogram times and areas under the peaks (for each exiting compound) must be manually calculated from the paper chromatogram is equal to the peak width at half height times the peak height
the electrical response of the detector
) Retention
) Prints
out the chromatogram
) Automatically
) Area
measures the retention times and area under each peak (for each exiting compound) using integration process
Computer Workstation
) Same
Gas Chromatography
H
as an integrator
Regulators
RESET
) Plus
a copy of the chromatogram and associated data can be stored as a file and later manipulated
Air
Gas Carrier
Hydrogen
Column
@eg. baseline corrections prior to reintegration

) Most
versatile GC data recording device
Other GC Terms
) Column
efficiency N or capacity factor k' (relative retention) R
Review Sections 33.4.2 (Examinable)
) Retention ) Selectivity
) Resolution
29
Column Efficiency - N
)A
Column Efficiency - N
) Column
good separation has narrow-based peaks, and ideally baseline separation

@baseline separation is where the valley between 2 neighbouring peaks is less than 10% of the peak height of the smallest of these 2 peaks
efficiency is measured by the number of theoretical plates N, which describes peak broadening as a function of retention or theoretical plates is a number that describes peak broadening as a function of retention
) Efficiency ) Peak
broaden as they pass through the column
@the more they broaden, the poorer is the separation of peaks and thus efficiency
Column Efficiency N
Column Efficiency: N
N = 5.54 ( tR / W1/2h )2 (best method)
W1/2h Wtan
[ W1/2h is the peak width at one half of the peak height ]

) The
retention times and width at base or halfheight must be measured in the same units
@N is thus unitless
Theoretical Plates
) Each
Column Efficiency: N
)A
theoretical plate represents one distribution equilibrium of sample molecules between the mobile phase and the stationary phase
@the more distributions, the more plates, the better the column
good packed column has N = 5000 plates
)A
good capillary column has 3000-4000 plates per metre for a total of 100,000 plates for say a 25 m column
30
Column Efficiency: HETP

) Length
of a column necessary for the attainment of compound distribution equilibrium (one theoretical plate) and is a measure of the efficiency of the column
@measure of peak broadening
Factors Affecting Column Efficiency (HETP)

) Factors
are related to the Van Deemter Equation: HETP = A1/3 + B/ + C
) Height
Equivalent to a Theoretical Plate HETP = L / N
)A )B
)N
is to be maximised so HETP is to be minimised capillary columns have HETP = 0.1-1 mm
= = )C = ) =
) Good
eddy diffusion band broadening due to diffusion resistance to mass transfer average linear flow velocity of the mobile phase
Van Deempter Equation
A - Eddy Diffusion Term

) Spreading
of the analytes in the column due to the carrier gas having various pathways or nonuniform flow
@A increases and thus efficiency decreases HETP increases and
) Packed
columns
@poor uniformity in solid support size or poor packing results in channelling and multiple pathways for the carrier gas
leads to spreading of the analyte in the column
@better to use commercially packed columns and high performance solid supports
) Capillary
A - Eddy Diffusion Term

Columns
@A term is very small

) As
B - Band Broadening Due to Diffusion

) Solutes
diameter of column increases
@flow properties deteriorate and band spreading results @smaller diameter columns have the best efficiency as the A term decreases as the diameter decreases
) Drawback
go from a high concentration to a low concentration slow flow rates () of the mobile phase result in large amounts of diffusion band broadening flow rates minimize the B term
) Very
of microbore (narrow bore) columns
@low capacity leads to overloading of the column which results in poorly shaped peaks and poor separation @normally, column diameters of 0.2-0.32 mm chosen to compromise efficiency with capacity
) Faster
31
C - Resistance to Mass Transfer

) If
@leading to poor efficiency

) Factors
affecting the C term
@thickness of the stationary phase

thick films lead to greater band spreading (larger C term) as they produce greater variation in diffusion properties in and out of the stationary phase, i.e. poor efficiency but good capacity ) Phase
HETP
the flow rate () is too fast, then the equilibrium between the mobile and stationary phases is not established and the C term increases
Relationship Between Carrier Gas Linear Flow Velocity () and Column Efficiency (HETP)
thickness of 0.25-1 m are common
Effect of Linear Flow Velocity

) Ideal
to operate GC at optimum linear flow velocity where HETP is minimised and thus efficiency is maximised
@linear flow velocity is like wind speed and is measured in cm/s
Effect of Carrier Gas Type and Linear Flow Velocity () on Column Efficiency (HETP)
N2 10 cm/s He 25-30 cm/s
HETP (mm)
) However,
if the analysis time needs to be shortened, then the linear flow velocity can be increased
@analysis time is directly proportional to the carrier gas velocity
H2 40-70 cm/s
10
20
30
40
50
60
70
80
90
Average Linear Flow Velocity (cm/s)
Effect of Gas Type on Efficiency

) The
Capacity Factor or Retention: k'

) k'
relationship between HETP and carrier gas linear flow velocity is influenced by the type of gas is the most efficient
) Nitrogen
@but has a low linear flow velocity leading to long analysis times
) Helium
is a direct measure of how strongly the sample components interact with the liquid stationary phase k' = ( tR - t0 ) / t0
is the most commonly used carrier gas

) k'
@high efficiency with small dependency on the linear flow velocity @can operate at high flow velocities without losing separation efficiency
= time spent in the liquid stationary phase time spent in the mobile gas phase
32
Retention or Capacity Factor - k'

k1' = ( t1 - t0 ) / t0 k2' = ( t2 - t0 ) / t0
Response
Capacity Factor or Retention: k'

) Capacity
factors range from 2 to 20 for most separations the temperature lowers the capacity factor or retention, k'
@a hotter column stationary phase means components spend less time in the stationary phase and more time in the mobile phase, ie. k' is smaller
t1 t0
A
) Raising
t2
10
Retention Time
15
20
25
Selectivity:
)
Selectivity -
= k2' / k1' = (t2 - t0) / (t1 - t0)
Response
is a number that describes how well a chromatographic system (GC) can separate two components
t1
) Selectivity
measures how well the liquid stationary phase exhibits selective solubilities (and thus retention) for the various components
t0
t2
10
Retention Time
15
20
25
Selectivity:
)
Resolution: R
) Measure
must be greater than 1 how well two peaks are separated
@if equal to 1 then the liquid stationary phase shows no selectivity for the 2 components since both compounds elute at the same time
) The
larger the value, the more selective is the liquid stationary phase and the easier the separation
) Resolution
is the retention time difference between peak divided by the average of the peak widths (at the base)
) Increase
by choosing a more polar liquid stationary phase
33
Resolution - R
R = (t2 - t1) / (W1 + W2)
Response
D
Alternate Resolution Equation
see HPLC notes for this equation

t2 t1
A B
5 W 1
10
W2
15
20
25
Retention Time
Effect of Resolution on Peak Separation

1. The sample solution is injected into the heated (250 C) injection port where it is rapidly volatilized 2. The volatilized sample is then swept via the carrier gas into the heated column (in an oven) 3. Sample components with high solubility in the liquid stationary phase move more slowly and thus spend a longer time in the column

4. The eluted compounds are then detected in a heated detector to give an electrical signal which is amplified and recorded 5. The output is a plot of recorder response vs time called a chromatogram

) Sample
components with a low solubility in the liquid stationary phase spend more time in the gas phase and are carried more rapidly through the column
@thus, volatile compounds eluted separately separate and are
34
Typical Chromatogram
Setting Up a GC Instrument
1. Turn the instrument on 2. Turn on the carrier gas (eg. helium), and FID gases (air and hydrogen) 3. Set up temperatures for injector (220 C) and detector (260 C) 4. Set up the oven temperature 5. Light the FID detector 6. Let column and detector equilibrate for at least 1 hr 7. Set up optimum linear flow velocity and split ratio
1. Setting Up Optimum Linear Flow Velocity ()

Why set up the carrier gas to the optimum linear flow velocity?
) The

) Determine
optimum linear flow velocity is where the carrier gas has a minimum HETP
@ie. maximum column efficiency or separating ability
the time (t0) for an unretained compound (eg. methane, butane) to flow through the column, knowing the optimum linear flow velocity () of the gas L x 100 cm
=
t0 x 60 sec L = length of the column in metres = cm/s; t0 = min
) This
ensures that the efficiency or column separating ability is not being affected by the carrier gas velocity

L (m) x 100 t0 = (cm/s) x 60
) Optimum

) Injections
min
after adjustment of the column head pressure are done until the required t0 is obtained the head pressure (using the valve)
) Increasing
linear flow velocity () varies with the carrier gas:

@nitrogen @helium @hydrogen 10 cm/s 25-30 cm/s 40-70 cm/s
@increases the column flow rate and decreases t0

) Decreasing
the head pressure (using the valve)
@decreases the column flow rate and increases t0
35

) Once
2. Calculation of GC Column Flow Rate (mL/min)

) Now
the required t0 is obtained, then the the carrier gas has been set to the optimum linear flow velocity
@minimising HETP and maximising column efficiency for the particular carrier gas
that the carrier gas has been set up to its optimum linear flow velocity or wind speed (cm/s),
@the actual column flow rate (mL/min) needs to be determined as we need this to set up the injector split ratio
volume of column (cm3) column flow rate = (mL/min) time for an unretained compound/carrier gas to flow through the column (t0 min)
2. Calculation of GC Column Flow Rate (mL/min)

Volume of a column (tube) = r2 L r = radius = i.d. / 2 i.d. = column internal diameter (mm)
3. Setting Up a Split Ratio for a Split GC Injection

split vent flow rate + column flow rate split ratio = column flow rate
) The
x i.d. (mm) x (L x 100)

column flow rate = 2 x 10
converts mm to cm
converts m to cm 2
split vent flow rate (mL/min) is measured by placing a bubble flow metre on the outlet of the split vent
t0 (min)
) The
column flow rate has been calculated for a particular carrier gas flowing at the optimum linear flow velocity (to maximise column efficiency)
Example of Setting Up a GC
Question
) Set
Answer ) choose = 25 cm/s for helium gas
) 1.
up a GC for optimum separation with a split ratio of 60:1. The following conditions apply:
@helium carrier gas @column i.d. of 0.2 mm @column length of 12 m
Calculate the dead time t0 L x 100 12 x 100 = x 60 25 x 60 = 0.8 min
t0 =
36
2. Calculate the column flow rate
2
split vent flow rate + column flow rate split ratio = column flow rate Split vent flow rate + 0.47
x i.d. (mm) x (L x 100)

column flow rate = 2 x 10 t0 (min) 60 =
0.47 = 3.14 x (0.2/20)2 x (12 x 100) / 0.8 = 0.47 mL/min Required split vent flow required = (60 x 0.47) - 0.47 = 27.7 mL/min
Quantification in GC
Use of Calibration Curve with the Internal Standard Method
Characteristics of an Effective Internal Standard

1. The internal standard must be volatile
3. The peak height or peak area for the internal standard peak in the chromatogram should be similar to those of the components to be measured 4. The internal standard should be chemically similar to the sample component(s) of interest
2. The internal standard must produce a completely resolved (separated) peak in the chromatogram, and be eluted close to the food components of interest
5. The compound to be used as the internal standard must not be naturally present in the original sample
37
Internal Standard Method

) Method
summary:
@a known amount of reference substance is added to the sample before injection onto the column
) Why
Use of Calibration Curve with the Internal Standard Method
use an internal standard?
@it eliminates any variations in those factors which influence the sensitivity and response of the detector
Internal Standard Methodology Involving a Calibration Curve

1. Add a fixed volume of an authentic standard (neat or as a solution) of the component of interest:
@a range of different amounts are added to separate volumetric flasks
3. The resulting mixtures (component and internal standard) are chromatographed:

@a calibration curve is constructed for the ratio of component peak area / internal standard peak area vs concentration of the component of interest
2. Add a constant volume of internal standard (neat or as a solution) to the above solutions, and dilute to the mark (fixed volume) with solvent (eg. water)
4. Prepare the sample:

@add a fixed volume (as per the standards) of the internal standard to a specified volume of the sample solution
Ratio Component Pk Area / ISTD Pk Area
5. Chromatograph the resulting mixture of sample and internal standard:

@then determine the peak area ratio from the sample chromatogram
Concentration (ppm)
38

6. The concentration (%) of the component of interest in the final sample solution is read off the calibration curve:
@based on the peak area ratio for the unknown sample
Ratio Component Pk Area / ISTD Pk Area For Sample
Ratio Component Pk Area / ISTD Pk Area
Concentration (ppm)
Unknown sample concentration read of graph
7. The concentration of the original sample solution (as opposed to the diluted sample solution just determined) is calculated using the dilution factor
Use of Relative Response Factors with the Internal Standard Method
What are Relative Response Factors (RRF)?

)A
Relative Response Factors (RRF)

) Prepare
one-point calibration curve using a single standard solution containing the compound of interest and an internal standard legitimacy of using a one-point curve for analysis of unknowns rests on method validation which is first performed
an ~ 1:1 standard solution of the compound of interest and the internal standard compound
) HPLC
analysis of the standard solution determines the peak areas of the compound of interest and the internal standard compound Conccompound of interest x Pk AreaISTD in standard
) The
RRF = Pk Areacompound of interest x ConcISTD in standard
39

) Prepare
the sample:
)
@add a fixed volume of the internal standard to a specified volume of the sample solution
) Chromatograph
Concentration of compound of interest in sample =
the resulting mixture of sample and internal standard:

@then determine the peak areas of the compound of interest and the internal standard from the sample chromatogram
RRF x ConcISTD in sample x Pk Areacompound of interest in sample Pk AreaISTD in sample
Advantages of the Internal Standard Method

) Results
HPLC Quantification: External or Internal Standard Method?

) Customary
are extremely accurate are independent of detector to use the Internal Standard Method in GC analysis since the response of GC detectors is less stable is the case for HPLC analysis where the External Standard Method is normally used
) Results
sensitivity
@ie. long-term variations
) Opposite ) Results
are independent of the sample size
injected
Applications of GC in Food Analysis
Tentative Identification of Unknown Compounds

Mixture of known compounds Response 1.6 min = RT Hexane Octane Decane
GC Retention Time on Carbowax-20 (min)
Response
Unknown compound may be Hexane 1.6 min = RT
Retention Time on Carbowax-20 (min)
40
Retention Times
Response
Semi-Quantitative Analysis of Fatty Acids

C 18
10
Peak Area
RT= 4.0 min on SE-30 Hexane
C16
8 6 4 2 0.5 1.0 1.5 2.0 2.5 3.0
Detector Response
GC Retention Time on SE-30
C 14
Response
RT= 4 min on SE-30 Unknown compound

Retention Time
C1 4 C +
14
Sample Concentration (mg/ml)
T h e c o n t e n t % o f C1 4 f a t t y a c i d s =
GC Retention Time on SE-30
C 16 + C
100
18
= t h e c o n t e n t % o f C1 4 f a t t y a c i d s
Gas Chromatogram of Methyl Esters of Fatty Acids
Effects of OH Groups of Carbohydrates

6 CH OH 2 O 5 4 HO 3 2 OH OH 1 OH 4 HO 3 2 OH 6 CH OH 2 O 5 OH 1 OH
Derivatisation of Glucose With Trimethylchlorosilane

6 CH 2 OH 5 4 HO OH 3 O 1 2 OH OH
Food Components Analysed by GC

) Triglycerides
CH 3 + 5Cl Si CH 3 Trimethylchlorosilane
6 CH 2 O-Si(CH3)3 5 O 1 O-Si(CH3)3
CH 3
) Cholesterol
Glucose
@Sugars as their silylated derivatives

trimethylsilyl (TMS) derivatives 5HCl ) Alcohol
4 (CH3)3-Si-O
O-Si(CH3)3 3
content is beverages and foods
2 O-Si(CH3)3
41
Food Components Analysed by GC

) Pesticides, ) Food
Other Examples
herbicides See Chapter 29 of Textbook
additives
@eg. organic acids (as TMS derivatives) used in foods

) Flavours ) Off
and odours
odours due to rancidity volatiles
) Packaging

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1

Gas Chromatography (GC) and Food Analysis

Schematic Diagram of a Gas Chromatograph

Gas Chromatographic Process

Gas Chromatographic Process

interested in gas-liquid chromatography (GC)

@ie. a liquid stationary phase

Basic Principles of GC Separation

Basic Principles of GC Separation

A and B KA= CmA / CsA KB= CmB / CsB

CmA = conc. of A in mobile phase CsA = conc. of A in stationary phase

Basic Principles of GC Separation

Basic Principles of GC Separation

Samples for GC Analysis

for gas chromatography cannot have a high molecular weight

Samples Not Suitable for GC Analysis

weight may range from 2 to about 1000

cannot separate ionic compounds such as salts as they are non-volatile

Elution from a GC Column

Compounds in Fermented Cabbage

Compounds in Orange Juice

High Speed with High Sensitivity

amounts of sample are needed L

Fatty Acid Methyl Esters (FAME)

HCl sodium methoxide ) boron trifluoride/methanol

O R C O CH 3 Volatile in Gas Chromatography

O CH 2 O C R CH O O C R CH 3ONa + CH 3OH O 3 R C O CH 3 Volatile in Gas Chromatography

Two Important GC Terms

area / peak height

adjusted retention time

Peak Area and Peak Height

Operation of a GC Instrument Review Section 33.3 in Textbook (all examinable)

Schematic Diagram of Gas Chromatography

Syringe/Sampler Inlets Detectors

gas system inlet (injector) column detector data system

Requirements of GC Carrier Gas

@water can degrade some column stationary phases

Purification of Carrier Gas

quality gas in cylinders is needed

@high purity grade @ultra high purity grade

line filters to further purify the gas

GC Regulators and Flowmeters

block surrounds the injector

samples are normally the matrix analysed

@largest source of poor precision in GC analysis

introduced into the injector using a syringe

@10 L syringes normally used

@excellent precision @adjustable volume sizes

Functions of the Injector

injectors produce flash volatilisation

@used for thermally stable compounds

programmed and on-column injectors use slower heating and volatilisation

... . ....... . .....

Glass injection Liner of Split/Splitless Injector

Advantages of a Split Injector

Disadvantages of a Split Injector

@a major limitation @high boiling components tend to be lost preferentially

Grob Splitless Injector

injectors can operate in 2 modes

@split injection mode @splitless injection mode

Splitless Injector and the Solvent Effect

Splitless Injector and the Solvent Effect

some time the column temperature is raised