The Science of Getting Rich PDF

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
) Complex
)A
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
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
Introduction to GC
) Gas
chromatography is mixture is separated into phase moving over a column
a process by which a its constituents by a gas stationary phase in a
) Mobile
Phase: inert carrier gas (eg. He or N2) ) Stationary Phase: high boiling liquid (or solid)
) Only
interested in gas-liquid chromatography (GC)
@ie. a liquid stationary phase
Basic Principles of GC Separation

) Sample
molecules partition or equilibrate between the moving gas phase and the stationary liquid phase in the gas phase, the sample molecules are carried through the column the sample molecules are dissolved in the liquid phase, they are stationary
) When
) When

) Separation
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
) Separation
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
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:
) Partition
@ratio of the concentration of a component in the mobile phase divided by the concentration of the component in the stationary phase
GC Partition Coefficient
) Different
compounds coefficients, K
have
different
partition
) Example: ) Compounds
A and B KA= CmA / CsA KB= CmB / CsB
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
) Compound
occurs, and
@A is eluted first
) The
partition coefficient, K depends on the volatility (and bp) of the compounds being separated
Samples for GC Analysis

Gases
) Molecular ) Only
Liquids
Solids
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
) Non-volatile
) 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
) Highly
polar and non-volatile samples such as sugars, amino acids or nucleotides cannot be analysed directly
@these materials decompose before they volatilise
) 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
to separate very closely eluting compounds shows remarkable separation capability for complex mixtures
) GC
Compounds in Fermented Cabbage
Compounds in Orange Juice
High Speed with High Sensitivity
) Rapid
analysis can occur at 10-9 g or nanogram levels
) Even
picogram, ie. 10-12 g levels can be determined
Other Advantages of GC
) Small
amounts of sample are needed L
) Good
detection system
) Quantitatively
analyzed
Sample Derivatisation
) Used
when the compounds to be analysed are:
@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)
Examples of Derivatisation
) Silyl
reagents
@hydroxy, amino, carboxylic acid groups in sugars, sterols and amino acids
) Esterifying
reagents
@carboxylic acids such as fatty acids (use methyl esters), amines, amino acids, triglycerides, phospholipids
Summarised in Chapter 29
Fatty Acid Methyl Esters (FAME)

HCl ) methanolic sodium methoxide ) boron trifluoride/methanol
Fatty Acids Methylester O R C OH + CH 3OH + H 2SO 4 Reflux O R C O CH 3 Volatile in Gas Chromatography
) methanolic
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
O CH 2 O C R
Two Important GC Terms
) Retention ) Peak
Time tR
area / peak height
Retention Time: tR
) Time
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
10 Retention Time
15
20
25
Retention Time: tR
) t0
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
) tR'
adjusted retention time
@retention time (tR) - dead time (t0) @time that the sample spends in the liquid stationary phase
Retention Time: tR
) Retention
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
) 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 D
Peak Area and Peak Height
10 Retention Time
15
20
25
GC Instrumentation
Review Section 33.3 in Textbook (all examinable)
Operation of a GC Instrument
Gas Chromatography
Filters/Traps Data system
H
RESET
Regulators
Syringe/Sampler Inlets Detectors
) )
Column
) ) )
gas system inlet (injector) column detector data system
Gas Carrier
Hydrogen
Air
Schematic Diagram of Gas Chromatography
Carrier Gas
Mobile Phase
Requirements of GC Carrier Gas

) Ultra-pure
@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
quality gas in cylinders is needed
@high purity grade @ultra high purity grade

) In
line filters to further purify the gas
@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
Gas Chromatography
H
RESET
Regulators
Gas Carrier
Hydrogen
Air
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
) Want
flow
GC Injector
GC Injection Port
) Heated
block surrounds the injector
@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
Gas Chromatography
H
RESET
Regulators
Gas Carrier
Hydrogen
Air
Column
Sample Introduction
) Liquid
samples are normally the matrix analysed
by GC
) Liquid
introduced into the injector using a syringe
) Sample
must be added a thin plug (small volume, eg. 1 L) through a correct and rapid injection technique
@slow injection of large samples causes band broadening and loss of separation
Sample Introduction
) Manual
sample injection
@largest source of poor precision in GC analysis @10 L syringes normally used

0.5 L to 3 L
) Autosampler
@excellent precision @adjustable volume sizes
Sample Introduction
Injection volumes
) Packed
injectors:
@1-20 L
) Split/splitless
capillary injectors:
@0.1-1 L
Functions of the Injector

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

) Temperature
programmed and on-column injectors use slower heating and volatilisation

@used for thermally labile compounds (ie. prone to decomposition by heat)
)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
@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
Packed GC Injector
) Use
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
... . ..... . .......

column needle heated block gas supply needle gas flowmeter syringe
Packed GC Injector
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
) Used
) Flow
)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
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
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
ratio is controlled by the flow rate through the column and out of the split vent
Split Ratio
split vent flow rate + column flow rate split ratio = column flow rate
) The
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
) The
Advantages of a Split Injector

) Only
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
) Thus,
) Very
Disadvantages of a Split Injector

) Thermally
sensitive compounds can thermally degrade in the hot syringe needle split injectors are non-linear
) Some
@a major limitation @high boiling components tend to be lost preferentially

called discrimination ) Not
suitable injector for trace analysis, since only a fraction of the sample enters the column
) These
injectors can operate in 2 modes
@split injection mode @splitless injection mode

developed by Dr Grob
Grob Splitless Injector

) In
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)
Splitless Injector and the Solvent Effect

) Oven
is as cool as possible
) 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
Splitless Injector and the Solvent Effect

) After
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
Advantages of Splitless Injection

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

@useful for trace analyses
) Solvent
effect produces narrow bands leading to high column efficiencies serves a dual role
) Hardware
@split injection @splitless injection
Cold On-Column Injectors

) In
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
) Injector
is at the temperature of the column or at room temperature oven is then temperature programmed
) The
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
) The
GC Oven
Importance of the GC Oven

) The
temperature of the column is controlled by the insulated, fan forced, thermostatted oven
@controls the column temperature
) Column
is coiled to fit into the oven in GC takes account of 2 factors:
) Separation
@interaction of the volatiles with the liquid stationary phase @the boiling point of the volatiles
Gas Chromatography
H
RESET
Regulators
Gas Carrier
Hydrogen
Air
Column
GC Oven Temperature
) The
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
@low temperatures produce long analysis times

) Two
modes of operation
@isothermal @temperature programmed
Isothermal Operation of GC Oven

) Oven
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
) Not
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
) Rates
@plus holding times at specific temperatures

) Normally ) GC
use rates of 2-10 C/min
analysis time can be sped up by using temperature program without compromising resolution (separating ability)
Application of Temperature Programmed GC

) Used
for the analysis of mixtures consisting of > 15 components with different volatilities (boiling points) in a complex mixture
@eg. flavour extract from a food
GC Columns
Packed Megabore or Wide-bore Capillary
GC Column Differences
) Dimensions,
eg. internal diameter (i.d.)
@packed columns have i.d. = or > 1 mm @megabore columns have i.d. = 0.53 mm @capillary columns have i.d. < 0.4 mm
Packed Column
Composition of Packed Columns
) 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
Column Packing Material

) Columns
packed with a granular material consisting of a liquid coated on an finely divided, inert solid support of uniform size 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
) Inert
Column Packing Material

) Thin
(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
Effect of Stationary Phase Loadings in Packed Columns

) Retention
time is proportional to the loading is improved by increasing the loading
) Resolution ) Bleed
(disadvantage)
@liquid coatings are somewhat volatile and can be lost from the packing material
bleed is observed as an increasing baseline during temperature programming
@different stationary phases bleed at different rates

some have maximum temperatures of 320 C while other have a maximum of 220 C
Packed Column Stationary Phases

) 200
available liquid coatings
) Silicone
based phases (methyl, phenyl, cyano substituted) are common (ester based) also used Se Chapter 29
) Carbowax
) Normally
choose a liquid stationary phase of similar polarity to the compounds (analytes) to be separated
Other Packed Column Stationary Phases
) Porous
organic polymers that serve as stationary phases requiring no inert support
Separation in a Packed Column

) Carrier
gas flows continuously through a packed
column
) Separation
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
Separation in a Packed Column

) Temperature
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
@low temperature permit molecules to dissolve more often in the stationary phase
and thus interact less with the mobile gas phase
Disadvantage of Packed Columns

) Resolution
or separating ability is much lower for packed columns than for capillary columns 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
) Due
Capillary Columns
Characteristics of 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
lengths of 5-100 m (low resistance to
flow)
) Column
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
Capillary Columns
) Thin
film of liquid phase or adsorbent coats the inside tube wall

@wall coated open tubular (WCOT) invented by Dr Marcel Golay
Capillary Columns
) Liquid
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
Capillary Columns
) Now
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
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
) Fused

) High
purity of fused silica produces a very inert chemical nature

@more uniform chemical surface than glass @absence of metallic cations makes it inert @inert surface and low surface area minimise sample adsorption leading to symmetrical peak shapes even for polar compounds
) Because
of fused silicas smooth, inert surface, capillary columns may be coated with a very thin, uniform liquid phase
@produces high efficiency
High Efficiency of Capillary Columns

) Superior
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
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
) Capillary
columns have total plate of 180,000 to 300,000 depending on their length columns generate only 4,000 plates
) Packed
Application of Capillary Columns

) Capillary
columns are useful for separating mixtures that have a large range of boiling points such as food flavour volatiles
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
) 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
Overcoming Overloading in Capillary Columns

) Reduce
the amount of sample entering the
column
) Dilute
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
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
) 0.1
mm columns have high resolution and high speed of analysis

@but, limited sample capacity making trace analysis difficult
) 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
columns with an i.d. of 0.53 mm
@liquid stationary phase still coated on the inside of the fused silica wall
) Less
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)
) Larger
) Efficiency
is between those of packed and normal capillary columns
Column Length
) Theoretical
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
) However, ) Increased
resolution is more easily obtained by choosing a small diameter column
) 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
) For
fast analysis
@use short columns such as 10 m @only moderate resolution

) Compromise
with a 25 m column
) 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
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
) Thick
films are 1.0 m or greater
Why Use Thick Films?
) Advantages: ) Increased
retention of sample components such as volatiles capacity permits injection of larger samples
) High
) Disadvantages: ) Lower
efficiency, so greater lengths may be required to compensate for the lower number of theoretical plates per metre
Why Use Thick Films?

) Higher
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
) Higher
) Column
) Films
less than 0.2 m
Thin Films
) Advantages: ) High
efficiency and thus high resolution operating temperatures can be used
@thus shorter column can be used

) Lower
@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
Stationary Phases in Capillary Columns
Required Characteristics of Stationary Phases in Capillary Columns

) High
selectivity for the sample
) Fortunately,
the high number of theoretical plates in capillary columns makes this factor less important than in packed columns bleed rate
) Low
@stationary phase must be stable at high temperatures
Required Characteristics of Stationary Phases in Capillary Columns

) High
chemical stability
@so column can be used for many months with no variation in chromatographic properties
) Many
liquid phases meet these criteria
) 200
different liquid phases have been developed
Stationary Phases in Capillary Columns

) As
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
) Fewer
than 12 liquid phases in common use
@most durable and efficient phases are based on polysiloxane (-Si-O-Si-)
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
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
3. Methyl ester of fatty acids

@DEGS (diethylene glycol succinate)
Common Stationary Phases

) Crosslinked
phases crosslinked bonds
contain
chemically
) Stable,
high-molecular-weight liquids or gums occurs between:
) Crosslinking
@adjacent molecular chains; and @between the stationary phase and the wall
) Longer-life
columns result
See Chapter 29
Selection of Stationary Phases

) Intuition ) Knowledge
of chemistry ) Help from column manufacturers ) The literature

) General
Rules:
@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
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
) Minimum
GC Detectors
Gas Chromatography
H
RESET
Regulators
Gas Carrier
Hydrogen
Air
Column
What is a GC Detector?
)A
device situated at the exit of the column
) 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
) Compounds
Functions of GC Detectors
) Different
detectors will give different types of
selectivity
) Non-selective
detectors respond compounds except the carrier gas
to
all
) Selective
detectors respond to a single type of chemical compound

@eg. nitrogen containing or halogen containing
) Concentration
dependent detectors
@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
) Mass
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
Types of GC Detectors
) Flame
Ionisation Detector (FID) ) Thermal Conductivity Detector (TCD) ) Electron Capture Detector (ECD) ) Flame Photometric Detector (FPD) ) Photoionisation Detector (PID) ) Electrolytic Conductivity Detector (ELCD) Detector or Nitrogen ) Thermionic 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 Nitrogen-phosphorus Mass flow
Hydrogen and Nitrogen, phosphorus air Hydrogen and Sulphur, phosphorus, tin, Flame photometric (FPD) Mass flow air possibly boron, arsenic, germanium, oxygen selenium, chromium Aliphatics, aromatics, ketones, esters, aldehydes, Photo-ionization (PID) Concentration Make-up amines, heterocyclics, organosulphurs, some organometallics Hall electrolytic conductivity Mass flow Hydrogen, oxygen Halide, nitrogen, nitrosamine, sulphur
2 pg
107
Flame Ionisation Detector (FID)
Flame Ionisation Detector

) Compounds
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
Schematic Diagram of Flame Ionization Detector

) 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

) An
electrical potential (+300 volts) is applied across the flame

@flame jet is grounded relative to the polarised collector electrode
@pure carrier gas produces a very small standing ion current of 10-14 amp

) 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
Flame Ionisation Detector (FID)

) FID ) No
responds to only organics on a weight basis
response to H20, NO2, CO2, H2S or air
) Response
is best with compounds containing C-C and C-H bonds gas can be N2, He or H2 since these gases produce no FID response operation temperature is 400 C
) Carrier
) Maximum

) Minimum
detectable quantity of 10-11 to 10-12 g/s
@50 ppb of a 1 mg sample

) Linearity ) FID
is excellent, 1-106
is mass sensitive rather than concentration sensitive stability is excellent

@ie. it is relatively insensitive to minor drifts in temperature and flow rate
) Thus,
) 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
) Flavour
volatile analysis ) Food contaminants ) Sterol (eg. cholesterol) analysis
Thermal Conductivity Detector

) Measures
the changes of the thermal conductivity of a heated tungsten filament (in a sealed stainless steel block) due to the sample (g)
) Sample
can be recovered

) Principal:
The thermal balance of a heated
filament
) 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
) The
Thermal Conductivity Basics

) The
ability of a colliding molecule to carry off heat depends on its thermal conductivity
) Hydrogen
and helium have high thermal conductivity and therefore will be more efficient at cooling a heated filament than will other gases
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.
Flow
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
) The
) 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

) Newer
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
) The
) Helium

) The
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
) 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
Relative Thermal Conductivity

Compound Relative Thermal Conductivity
Carbon Tetrachloride Benzene Hexane Argon Methanol Nitrogen Helium Hydrogen
0.05 0.11 0.12 0.12 0.13 0.17 1.00 1.28
Food Analysis Applications of TCD
) Used
where no other detector can be used used for fixed gas analysis
) Mainly
@eg. CO, CO2

) Detects
water
Electron Capture Detector

) For
pesticide analysis (picogram)
) Contains
a radioactive foil coating that emits electrons as it undergoes decay

@63Ni which emits particles
) Ionization
@N2 (Carrier gas) + (e) N2+ + 2e) Electrons ) The
are collected on an anode
N2+ establish a base line or standing current
Electron Capture Detector Basics

) Electron
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
)X
) Ion
@the base line current due to the N2+ will decrease and this decrease constitutes the signal

) Very
sensitive detector
) Requires
ultra-pure and very dry carrier gas (normally N2) since trace amounts of water and air degrade detector response as compounds it only responds to certain
) Selective
@those with strong electron affinity

) Halogenated
compounds or those with conjugated double bonds give the greatest detector response

) Conjugated
carbonyls, nitro organometallics (eg. lead)
compounds
and
) Phosphorous,
silicone and polynuclear aromatics also can be detected
) 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)
GC Recording Devices
3 Types of Recording Devices

Electrical output from the detector is further amplified and recorded Pen-trace recorder Integrator Computer workstation
Pen-Trace Recorder
) Converts
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
) Retention
) Area
Integrator
) Records
the electrical response of the detector
) Prints
out the chromatogram
) Automatically
measures the retention times and area under each peak (for each exiting compound) using integration process
Computer Workstation
) Same
as an integrator
) Plus
a copy of the chromatogram and associated data can be stored as a file and later manipulated
@eg. baseline corrections prior to reintegration
) Most
versatile GC data recording device
Gas Chromatography
H
RESET
Regulators
Gas Carrier
Hydrogen
Air
Column
Other GC Terms
) Column
efficiency N or capacity factor k' (relative retention) R
) Retention ) Selectivity
) Resolution
Review Sections 33.4.2 (Examinable)
Column Efficiency - N
)A
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
) 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 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
Column Efficiency N
W1/2h Wtan
Column Efficiency: N
N = 5.54 ( tR / W1/2h )2 (best method) [ 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
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
Column Efficiency: N
)A
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
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
) Height
Equivalent to a Theoretical Plate HETP = L / N
)N
is to be maximised so HETP is to be minimised capillary columns have HETP = 0.1-1 mm
) Good
Factors Affecting Column Efficiency (HETP)

) Factors
are related to the Van Deemter Equation: HETP = A1/3 + B/ + C
)A
= )B = )C = ) =
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
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
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
B - Band Broadening Due to Diffusion

) Solutes
go from a high concentration to a low concentration
) Very
slow flow rates () of the mobile phase result in large amounts of diffusion band broadening flow rates minimize the B term
) Faster
C - Resistance to Mass Transfer

) If
the flow rate () is too fast, then the equilibrium between the mobile and stationary phases is not established and the C term increases
@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
thickness of 0.25-1 m are common
Relationship Between Carrier Gas Linear Flow Velocity () and Column Efficiency (HETP)
HETP
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
) 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
Effect of Carrier Gas Type and Linear Flow Velocity () on Column Efficiency (HETP)
N2 10 cm/s He 25-30 cm/s
HETP (mm)
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
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 the most commonly used carrier gas
@high efficiency with small dependency on the linear flow velocity @can operate at high flow velocities without losing separation efficiency
Capacity Factor or Retention: k'

) k'
is a direct measure of how strongly the sample components interact with the liquid stationary phase k' = ( tR - t0 ) / t0
) k'
= time spent in the liquid stationary phase time spent in the mobile gas phase
Retention or Capacity Factor - k'

k1' = ( t1 - t0 ) / t0 k2' = ( t2 - t0 ) / t0
Response D
t1 t0
A
t2
10 Retention Time
15
20
25
Capacity Factor or Retention: k'

) Capacity
factors range from 2 to 20 for most separations
) Raising
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
Selectivity:
)
is a number that describes how well a chromatographic system (GC) can separate two components
) Selectivity
measures how well the liquid stationary phase exhibits selective solubilities (and thus retention) for the various components
Selectivity -
= k2' / k1' = (t2 - t0) / (t1 - t0)
Response D
t1 t0
A
t2
10 Retention Time
15
20
25
Selectivity:
)
must be greater than 1
@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 by choosing a more polar liquid stationary phase
) Increase
Resolution: R
) Measure
how well two peaks are separated
) Resolution
is the retention time difference between peak divided by the average of the peak widths (at the base)
Resolution - R
R = (t2 - t1) / (W1 + W2)
Response D
t2 t1
A
5 W 1
10
W2
15
20
25
Retention Time
Alternate Resolution Equation
see HPLC notes for this equation
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
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
optimum linear flow velocity is where the carrier gas has a minimum HETP
@ie. maximum column efficiency or separating ability
) This
ensures that the efficiency or column separating ability is not being affected by the carrier gas velocity

) Determine
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

L (m) x 100 t0 = (cm/s) x 60
) Optimum
min
linear flow velocity () varies with the carrier gas:

@nitrogen @helium @hydrogen 10 cm/s 25-30 cm/s 40-70 cm/s

) Injections
after adjustment of the column head pressure are done until the required t0 is obtained the head pressure (using the valve)
) Increasing
@increases the column flow rate and decreases t0

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

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

) Now
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)
x i.d. (mm) x (L x 100)

column flow rate = 2 x 10
converts mm to cm
converts m to cm 2
t0 (min)
3. Setting Up a Split Ratio for a Split GC Injection

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

column flow rate = 2 x 10 t0 (min)
= 3.14 x (0.2/20)2 x (12 x 100) / 0.8 = 0.47 mL/min
split vent flow rate + column flow rate split ratio = column flow rate Split vent flow rate + 0.47 60 = 0.47 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 2. The internal standard must produce a completely resolved (separated) peak in the chromatogram, and be eluted close to the food components of interest
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 5. The compound to be used as the internal standard must not be naturally present in the original sample
Internal Standard Method

) Method
summary:
@a known amount of reference substance is added to the sample before injection onto the column
) Why
use an internal standard?
@it eliminates any variations in those factors which influence the sensitivity and response of the detector
Use of Calibration Curve with the Internal Standard Method
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
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)
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
Ratio Component Pk Area / ISTD Pk Area
Concentration (ppm)
4. Prepare the sample:

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

@then determine the peak area ratio from the sample chromatogram
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
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
) The
Relative Response Factors (RRF)

) Prepare
an ~ 1:1 standard solution of the compound of interest and the internal standard compound 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
) HPLC
RRF = Pk Areacompound of interest x ConcISTD in standard

) Prepare
the sample:
@add a fixed volume of the internal standard to a specified volume of the sample solution
) Chromatograph
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
Concentration of compound of interest in sample =
RRF x ConcISTD in sample x Pk Areacompound of interest in sample Pk AreaISTD in sample
Advantages of the Internal Standard Method

) Results
are extremely accurate are independent of detector
) Results
sensitivity
@ie. long-term variations
) Results
are independent of the sample size
injected
HPLC Quantification: External or Internal Standard Method?

) Customary
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
) Opposite
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)
Retention Times
Response
RT= 4.0 min on SE-30 Hexane
GC Retention Time on SE-30
Response
RT= 4 min on SE-30 Unknown compound
GC Retention Time on SE-30
Semi-Quantitative Analysis of Fatty Acids

C 18
10
Peak Area
C16
8 6 4 2 0.5 1.0 1.5 2.0 2.5 3.0
Detector Response
C 14
Sample Concentration (mg/ml)
Retention Time
C1 4 C +
14
T h e c o n t e n t % o f C1 4 f a t t y a c i d s =
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 2 OH O 1 OH
CH 3 + 5Cl Si CH 3 Trimethylchlorosilane
6 CH 2 O-Si(CH3)3 O 5 1 O-Si(CH )
3 3
CH 3
Glucose
4 (CH3)3-Si-O
5HCl
O-Si(CH3)3 2 O-Si(CH3)3
Food Components Analysed by GC

) Triglycerides ) Cholesterol
@Sugars as their silylated derivatives

trimethylsilyl (TMS) derivatives ) Alcohol
content is beverages and foods
Food Components Analysed by GC

) Pesticides, ) Food
herbicides
additives
@eg. organic acids (as TMS derivatives) used in foods

) Flavours ) Off
and odours
odours due to rancidity volatiles
) Packaging
Other Examples
See Chapter 29 of Textbook

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Gas Chromatography (GC) and Food Analysis

recorder prints out a chromatogram which is the record of the separation

Schematic Diagram of a Gas Chromatograph

Gas Chromatographic Process

Gas Chromatographic Process

chromatography is mixture is separated into phase moving over a column

a process by which a its constituents by a gas stationary phase in a

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

weight may range from 2 to about 1000

for gas chromatography cannot have a high molecular weight

Samples Not Suitable for GC Analysis

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

analysis can occur at 10-9 g or nanogram levels

picogram, ie. 10-12 g levels can be determined

amounts of sample are needed L

when the compounds to be analysed are:

Fatty Acid Methyl Esters (FAME)

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

Review Section 33.3 in Textbook (all examinable)

Syringe/Sampler Inlets Detectors

gas system inlet (injector) column detector data system

Schematic Diagram of Gas Chromatography

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

Syringe/Sampler Inlets Detectors

GC Regulators and Flowmeters

block surrounds the injector

Syringe/Sampler Inlets Detectors

samples are normally the matrix analysed

introduced into the injector using a syringe

@largest source of poor precision in GC analysis @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

injectors can operate in 2 modes

@split injection mode @splitless injection mode