GAS CHROMATOGRAPHY In gas chromatography (GC), the sample is injected onto the head of a...
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Transcript of GAS CHROMATOGRAPHY In gas chromatography (GC), the sample is injected onto the head of a...
GAS CHROMATOGRAPHY In gas chromatography (GC), the sample is
injected onto the head of a chromatographic column and immediately
vaporized. The components of vaporized sample are fractionated as a
consequence of partition between the mobile gaseous phase and the
stationary phase held in the column. The mobile phase is inert, it
does not inter act with the sample, it only carries the sample at
elevated temperature when it leaves the stationary phase thus. It
is also called the carrier gas. GC methods can either be:
a- Gas solid chromatographyGSC The stationary phase is a solid and
retention of substances occurs as a result of adsorption b-Gas
liquid chromatography GLC The stationary phase is a liquid
supported on an inert solid matrix. The components of the sample
have finite solubility in the stationary phase thus they distribute
themselves between this phase and the gas. Elution is accomplished
by forcing the inert carrier gas through the column. The rates of
which the various components move along the column depends on their
tendency to dissolve in the stationary phase. Types of samples for
GC: 1- Any compound that can be volatilized without decomposition,
such as low molecular weight hydrocarbons, aldehydes, ketones and
esters. 2- Samples that can be converted to volatile compounds,
such as fatty acids which can be converted to methyl esters which
is more volatile also amino acids can be converted to fluoroamide
ester derivative. 3-The sample may be organic or inorganic, but not
ionic. 4-The molecular weight ranges from 2 to 1000. Advantage of
GC High sensitivity ( gm.) High accuracy. High speed (short time of
analysis). Limitation of GC The sample must be volatile and
thermally stable below 4000C. Dirty and biological samples such as
blood, soil and tissues require clean up. GC cannot identify the
compounds surely ,it must be connected to another instrument such
as mass-spectrometer for accurate identification of compounds.
Instrument for gas chromatography
A gas chromatographic instrument is formed of FIVE components:
Carrier gas supply with its regulator, purifier, desiccant and flow
meter. Sample injection system. Column and thermostating system.
Detector. Integrator or computer data station. 1-Gas supply system:
This includes: a-carrier gas. b-gases used for detector. The most
commonly used carrier gases are nitrogen, helium, hydrogen and
argon. They are supplied in cylinders or produced by electrical
generators fitted with pressure regulator and manometer. The
carrier gas must be :
Pure (99.999% purity), N.B. impure gases will cause damage of the
stationary phase, noise and unidentified peaks or signals. To
ensure the purity of the gases, usually the gases are passed
through filters to remove impurities then moisture filter and
lastly oxygen filter since oxygen will destroy the stationary phase
and it gives signal in case of electron capture detector. Inert(
will not interact with the sample). Dry as traces of water at high
temperature will hydrolyze the sample and the stationary
phase.
The pressure of the gas is adjusted according to its viscosity and
the length of the column, to obtain the suitable flow rate. The
choice of the gas depends on the detector used: For flame
ionization detector, the carrier gas can be nitrogen ,helium, argon
or hydrogen. The detector gas is air and hydrogen. For electron
capture detector the carrier gas is nitrogen and no other gases are
needed for detector. For thermal conductivity detector low
viscosity, high thermal conductivity gas is needed as carrier gas
and which passes through the detector these are helium and
hydrogen. Column and Thermostating System:
The fundamental part of a gas chromatograph is the thermostating
oven. It is the place where the chromatographic column is put and
the sample is separated into its components. The optimum column
temperature depends upon the boiling point of the sample and the
degree of separation required. There are two types of columns
:Packed and Capillary. Packed columns: They are fabricated from
glass, metal or teflon and about two meter length and 0.5 cm
diameter. They are uniformly packed with packing material consists
of finely divided, uniform spherical solid inert support coated
uniformly with very thin layer of stationary liquid phase. The
carrier gas flow ranges from 3050 ml/min. The inner wall is coated
with thin film of liquid stationary phase.
Capillary or open tubular columns: These are capillary tubes made
of glass or stainless steel or fused silica. The inner wall is
coated with thin film of liquid stationary phase. Since there is no
resistance for the gas flow as the tube is open. The length of the
tube can be from meter. The flow rate of carrier gas is reduced to
1 ml/min. Stationary liquid phases:
Desirable properties for immobilized, liquid phase in GLC include:
1- Low volatility; ideally the boiling point of the liquid should
be at least 2000C higher than the maximum operating temperature for
the column. 2- Thermally stable 3-Chemically inert 4-To have a
reasonable retention time in the column, a species must show some
degree of solubility with the stationary phase. Generally, the
polarity of the stationary should match that of the sample
components. When the match is good, the order of elution is
determined by the boiling point of the eluate. Column temperature
and temperature program
The column(s) in a GC are contained in an oven, the temperature of
which is precisely controlled electronically. The temperature of
the column can be varied from about 50C to 250C. It is cooler than
the injector oven, so that some components of the mixture may
condense at the beginning of the column. The rate at which a sample
passes through the column is directly proportional to the
temperature of the column. A method which holds the column at the
same temperature for the entire analysis is called "isothermal."
Most methods, however, increase the column temperature during the
analysis, the initial temperature, rate of temperature increase
(the temperature "ramp"), and final temperature are called the
"temperature program." A temperature program allows analytes that
elute early in the analysis to separate adequately, while
shortening the time it takes for late-eluting analytes to pass
through the column. Detectors: The detector has the function to
detect the presence of chemical components in the gas flow. All
detectors measure a relative value; a sample component in the
carrier gas compared to the pure carrier gas. This change is
usually represented in the form of an electrical signal as a
function of time. A good detector should comply with the following
requirements:
1- Responds rapidly and reproducibly to low concentrations of
solutes emerged from the column. 2- Sensitive to very low
concentrations of sample, 10-9 to10-12 g of sample could be
detected. 3-Accurate and reliable. The following are some types of
GC detectors:
1-Thermal Conductivity Detector TCD The detector has the advantage
that it has no destructive effect on the sample. 2-Flame Ionization
Detector FID:
Most organic compounds, when pyrolyzed at the temperature of
hydrogen/air flame, produce ionic intermediates that provide a
mechanism by which electricity can be carried through the flame.
The FID is highly sensitive, has wide range of linear response but
it is destructive for the sample. 3-Electron Capture Detector
ECD
A -ray source (a radioactive substance that emit electrons) such as
Ni63. In the absence of the organic species, an amplifier electron
current is formed which runs in the direction of collector
electrode and is monitored as a continuous background current. The
moment there are electro-negative components present in the carrier
gas, the background current is reduced because these compounds
capture electrons. The change in the background current is
registered and that is the detector signal. ECD is a selective and
highly sensitive detector for molecules containing electronegative
functional groups such as halogens, peroxides, quinones, and nitro
groups. QUALITATIVE ANALYSIS Qualitative analysis by gas
chromatography is divided into two parts. The first is the
separation of component or components of interest from each others
in the mixture, and the second is the identification of the
separated components. Retention Data The retention volume, time or
distance of a peak is a qualitative property of the compound and is
constant for a given set of conditions (the same apparatus,
temperature and stationary phase). To identify a specific compound
in the mixture, a reference compound and the unknown sample are
co-chromatographed under identical conditions (spiking or
enrichment technique). The formation of one peak and the increase
of its height indicate that the unknown may be identical with the
reference compound, but it is not positive proof. To confirm the
identity of the unknown, other stationary phases of different
polarity could be also used and chromatography at variable
temperatures might be carried out. QUANTITATIVE ANALYSIS
A-Peak Height Measurement: The use of the peak height as the
quantitative measurement is to be preferred to the peak area
because of its simplicity. But this demands; The conditions of
chromatography be constant allover the operation and peak width
does not vary during the set of determinations, The response of the
detector must be carefully calibrated over the concentration range
of the components. This is done by allowing standard samples to be
chromatographed periodically. B-Peak Area Measurement:
In some cases the peak width is affected with certain factors, e.g.
adsorption, overloading of sample, long retention volumes and
variation in operating conditions like temperature. All these
factors cause broadening of the peak and measurement of the peak
height becomes of little importance. Therefore, it appears more
accurate to determine the peak area. This can be carried out as
follows: By means of a planimeter or counting square numbers on
group paper.
Weighting the cut-out peaks if paper thickness is uniform. By means
of some automatic integrating device. By a geometric method or
approximation such as multiplying the peak height by the width at
half height. The first three methods are useful in the
determination of asymmetrical peaks. Calibration: The calibration
involves the determination of chromatograms with pure components of
samples. A quantitative analysis through calibration can be
achieved by plotting chromatographed increasing amounts of pure
samples, against their peak areas. The value of the unknown
concentration can be deduced from the curve Quantitative
Determination of Atropine by Area Measurement