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Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
Description of Module
Subject Name Biochemistry
Paper Name 12 Biochemical Techniques
Module Name/Title 12 Gas - liquid Chromatography
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
1. Objectives
1.1 To understand principle of Gas Liquid Chromatography
1.2 To explain the different components of GLC
2.0 Introduction and principle-
Introduction and principle-
Gas – Liquid chromatography (GLC) is one of the most useful techniques in analytical
chemistry. Claesson published one of the first important accounts of gas liquid
chromatography in 1946. Gas – liquid chromatography is a form of partition
chromatography in which the stationary phase is a film coated on a solid support and
the mobile phase is an inert gas like Nitrogen (N2) called as carrier gas flowing over the
surface of a liquid film in a controlled fashion. The sample under analysis is vaporized
under conditions of high temperature programming. The components of the vaporized
sample are fractionated as a result of partitioning between a mobile gaseous phase and
a liquid stationary phase held in a column.
Principle: When the vapours of sample mixture move between the stationary phase (liquid)
and mobile phase (gas) the different components of a sample mixture will separate
according to their partition coefficient between the gas and liquid stationary phase.
Concn. of solute in liquid (w/cc)
Partition coeff.(Kg) = --------------------------------------------
Concn of solute in gas (w/cc)
It is general assumption that if partition coefficient is low the emergence of the component
is fast and vice versa. The substances having low boiling point (B.P) i.e. more volatility
and higher vapour pressure will have more concentration in the mobile phase and thus
will elute or emerge first and so on. For example, lower carbon number compounds
have low B.P and higher volatility and vapour pressure will elute first than the higher
carbon number compounds e.g. lower chain fatty acids emerge first than long chain
ones. Therefore, less polar substances elute fast than polar substances. More polar
substances are more retained in the column and therefore move slowly as compared to
less polar substances which move at faster rate.
In chromatographic analysis there are two terms commonly used (i) Retention Time and (ii)
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
Retention volume.
Retention Time ( tR): It is the time required for the maximum for a solute peak (the peak
of that particular component) to reach the detector in a gas chromatographic column.
The retention time (tR) is characteristic of that component and the area under the peak
is proportional to its quantity. These parameters yield qualitative and quantitative data,
respectively.
The characterization of mixture in as unknown sample is done through retention time by
comparing with those of reference compounds. The relative proportion of varouis
components in a mixture is determined by calculating their peak areas and then
calculating the percentage of peaks are out of the total area of various peaks obtained.
Retention volume (VR) is defined as the volume of the gas required to carry a component
maximum through the column
VR = tR Fc
Where Fc is the volume flow rate of the gas at outlet.
3.0 Applications of GLC:
Gas liquid chromatography is generally used for
both qualitative and quantitative analysis of
organic compounds. This technique is much
sought technique in Agricultural Science,
Agriculture Industry, Food industry,
Environmental field, Forensic field,
Biotechnology field, Perfume and fragrance
industry i.e. cosmetic industry and chemical industry. This technique is very useful for
the estimation of (i) pesticide and insecticide residues in food and other consumables (ii)
estimation of pollutants in water and other food stuff (iii) Banned and controlled drugs
in urine, blood, tablets, energy drinks etc.
4. Apparatus:
The basic components of a typical gas chromatograph (GC) are as:
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
Carrier Gas Supply: The gaseous mobile phase must be inert. Helium is the most common
mobile phase, although argon, nitrogen, hydrogen are also used. Most of these gasses in highly
pure form – including mixtures such as nitrogen with hydrogen are available in cylinders.
Generally the gasses used in GC must be thoroughly dried because moisture entrapped in the
gasses leads to background noise. Now, a day’s GC suppliers are providing desiccant cartridges
and other filters along with the machines which take care of these problems as well as other
impurities. Otherwise, the best desiccant is a molecular sieve (Linde 5A) activated at 200 – 300
o C. the flow rate of these gases is controlled by the pressure gauges and flow meters.
Detector Carrier Gas
Thermal conductivity Helium
Flame ionization Helium or nitrogen
Electron capture Very dry nitrogen
4.1. Sample injection Systems:
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
Method of sample injection depends on the type of sample
i.e. gaseous, liquid or solid. In GLC the requirement is that
the suitable amount of sample should be injected as a “plug”
of vapors. It has been noticed that slow injection or oversized
samples cause band spreading and poor resolution. Sample
size depends upon the sensitivity of the detector; when an
ionization detector is used a liquid sample should not be greater than 0.5 l.
4.1.1. Liquid Samples: Liquids are injected by means of micro syringes through a silicon septum
into a heated sample port located at the head of the
column. The sample port is ordinarily above 50 o C above
the boiling point of the least volatile components of the
sample. For ordinary packed analytical columns, sample
sizes range from a few tenth of micro liter to 20 l .
Capillary columns require samples that are
smaller by a factor of 100 or more. Here a sample
splitter is often required to deliver only a small
known fraction (1:100 to 1:500) of the injected
sample, with the remainder going to waste.
4.1.2. Solid samples: Solid samples are generally
weighed into thin glass ampules which are placed
in the gas stream and then crushed.
4.2. Columns: Efficiency of any gas
chromatograph is very much dependent on the
columns used in GLC, which may be of glass or metal. These columns are mainly of two types –
packed columns and open – tubular (capillary) columns.
4.2.1. Packed columns: These columns can accommodate larger samples and are generally
more convenient to use. They are normally 2 –3 m long and have inside diameters of 2 –4 mm.
The tubes are ordinarily formed as coils with diameters of roughly 15cm to permit convenient
thermo stating in an oven. The packing or support, for a column hold the liquid stationary phase
in place, so that the surface area exposed to the mobile phase is as large as possible. The ideal
particle size of packing material for gas chromatography is in the range of 60 – 80 mesh (250 –
170 m) or 80 – 100 mesh (170 – 149 m).
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
4.2.2. Capillary columns: Capillary columns are generally made up of glass or fused silica. These
columns have inside diameters of 0.25 – 0.50 mm and lengths of 25 – 100 m. Silica capillaries
which have much thinner walls than their glass or metal counter parts, have outside diameters
of about 0.3 mm.
4.3. Column Oven: The oven used in GLC is usually having a high precision thermostat to
control the temperature of the column fitted inside the oven to get the reproducible retention
time. Range of temperature may vary from 0 - 400ᵒC
4.4. Detectors: Detectors are very sensitive and respond quickly to minute concentrations of
solutes exiting the columns. Detectors have the linear response stabile and uniform response
for a wide variety of chemical species. There are many
types of detectors are available. (a) Thermal conductivity
(b) Gas density (c) Flame ionization (d) ß – ray ionization (
Cross section, Argon, Helium, Electron capture, Electron
mobility) (e) Photo ionization. (f) Glow discharge (g) Flame
temperature (h) Dielectric constant. Out of the above-
mentioned detectors the two are
most commonly used.
4.4.1. Thermal Conductivity
Detector (TCD): this is also called
as Katharometer. This is madeup of
four filaments arranged in a
electrical bridge network. The
carrier gas flowing around these
filaments through cavities. The temperature of filament is determined by the rate of heat loss
by conduction through the carrier gas. As the components elute out from the column, the
composition of gas changes with the consequent changes in the thermal conductivity. This in
turn, produces change in temperature of filaments which generate electrical output from the
bridge circuit.
Merits: i) Simple ii) can be used in all applications iii) non destructive and thus suitable for
preparative fraction collection work.
Limitation: Low resistance ii) Low sensitivity ( 10 –9 g/ mL carrier ).
4.4.2. Flame Ionization Detector (FID): Most popular detector due to its high sensitivity, wide
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
range and greater reliability. It responds only to organic compounds. . It works on the principle
that most organic compounds, when pyrolize in a hot flame, produce ionic intermediates that
conduct electricity through the flame. It consists of a small hydrogen flame burning in an excess
of air and surrounded by an electrostatic field. Column effluent is mixed with hydrogen entering
the burner. Organic components eluted from the column are burnt producing CO2. During this
oxidation process, some ionizing particles and electron are formed as intermediate products of
oxidation. These ionizing particles are quantitatively proportional to the amount of carbon in
original compounds. These ionizing particles are collected and neutralized by the polarizing
electrodes generating an electric current which is picked up by electrometer and forms a peak
on the recording chart. The ionization detector exhibits a high sensitivity (10 – 13 g / mL ), a
large linear response and low noise. It is also rugged and easy to use. It responds only to
organic compounds.
Detection limit: 5 ppb for light hydrocarbon gases and 10 picograms for higher organic liquids
and gases. When column temperature of 200°C or higher is used, the practical detection limit is
about 1 nanogram (10-7 - 10-8g.).
Disadvantage: It destroys the sample.
4.4.3. Modified Flame ionization detector (FID): It is also called as thermo ionic alkali flame
detector (TID). In this case an alkali metal salt is added to the flame to enhance the ionization of
compounds containing P. Cl and N. The salts mainly PO4 and halides of Na, K, Rb or CS are fed
to the flame by (i) fixed wire (ii) heated capillary (iii) pallet fastened on jet. Salts should be
replaced when consumed.
4.4.4. Electron Capture Detector (ECD): It is based on the principal of electron capturing by
various substances being sensed which causes a reduction in the ion current. Chemicals
containing an electronegative element or group have a strong affinity for electrons. In the
gaseous state, they tend to capture electrons to form negative ions. Exposing these compounds
to a source of low energy electrons forms the basis of an extremely sensitive selective detector
for such compounds. The radioactive source employed for ECD include: Tritium (H3)and Ni63 .
Methane or argon are also used as a quench gas (mixed with carrier ga) to reduce the energy.
Non – radio active sources: Glow discharge in helium high sensitivity and high operating
temperature. (400°C). In all types of ECD, more important is specificity or selectivity than
sensitivity. Non capturing compounds (compounds which do not contain electronegative group)
should go undetected.
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
Advantages : Very high sensitivity and specificity for molecules such as oxygen, halogens
oxygen – containing and halogen containing compounds, which have high electron affinities.
4.4.5. The Nitrogen Phosphorus Detector (NPD): This detector is very-very sensitive and a
specific detector. It is highly responsive to organic compounds containing nitrogen and/or
phosphorus. Sensor in this detector is made up of a rubidium or cesium bead contained inside a
small heater coil. One side of the detector has the anode. The heated alkali Rubidium Sulphate
emits electrons by thermionic emission which are collected at the anode and thus produce ions.
When a solute containing nitrogen or phosphorus is eluted, the partially combusted nitrogen
and phosphorus materials are adsorbed on the surface of the bead. This adsorbed material
reduces the work function of the surface and, as consequence, the emission of electrons is
increased which raises the anode current. The sensitivity of the NPD is about 10-12 g/ml for
phosphorus and 10-11 g/ml for nitrogen).
4.5. Stationary Phases for GLC: Hundreds of liquids have been proposed as stationary phases in
the development of GLC. The important factors governing the selection of a liquid phase are its
polarity and operational temperature range. The later can be determined from tables or
supplier. From practical point of view, the greater the polarity of the liquid stationary phase,
the greater the retention of a polar solute relative to that of a non-polar solute with a similar
boiling point. Like is dissolved by like is the general rule. Thus if sample components are similar
in chemical structure but have different volatilities, a non polar liquid phase is generally better .
Conversely the components have different
functional groups, but are of similar boiling points
then polar phase is generally more suitable.
These stationary phases are mainly of two types –
polar and non-polar.
4.5.1. Polar (Selective) stationary phases: They
contain functional groups such as - CN, - CO, and
–OH. These phases include Polyethylene glycol
and polyester which retain polar solutes. Theses
phases separate solute molecules on the basis of
functional groups. Selective stationary phases
show selective retention for carbon – carbon double bonds. Such phases are used to separate
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
carbonyls, lactones and fatty acid esters.
4.5.2. Non-polar (non- selective) phases: They are generally hydrocarbon type (dialkyl siloxane)
such as methyl silicons, Apiezen greases and squalene. They tend to fractionate solutes by B.P.
Generally used for triglyceride.
All type of stationary phases has the following properties:
(1) Low volatility (ideally boiling point of the liquid should be at 100ᵒ C higher than the
maximum operating temperature of the column (2) Thermal stability (3) Chemical inertness (4)
Solvent characteristics.
4.5.3. Some Common Liquid Stationary phases: Polydimethyl siloxane, Phenyl – polydimethyl
siloxane, Polyethylene glycol, Cynopropyl – polydimethyl siloxane.
Selection of stationary phases depend upon the compounds, researches want to study.
Stationary phase Type Analyte type
100% Dimethyl polysiloxane Non- polar Solvents, Petroleum products, flavors, saturated hydrocarbons
5% : 95% Diphenyl : dimethyl polysiloxane
Non- Polar flavors, pesticides, aromatic hydrocarbons
35% : 65%; Diphenyl : dimethyl polysiloxane
Medium polarity Nitrogen containing pesticides
Polyethylene glycol Polar Fatty acid methyl esters, fatty acids, flavours, alcohols.
4.5.4.1. Conditioning of column: To remove low molecular weight residues from stationary
phase so as to maintain the minimum bleeds of liquid phase throughout the working range of
temp. This is normally accomplished by heating the column to a temperature about 20 – 30°C
above its proposed operating temperature with a stream of carrier gas passing through it. This
is essential to ensure that the complete removal of any solvent, water and volatile
contaminants that may have been retained during the preparation of the column material.
5.0 Derivatives for GLC: Most common derivatization is done prior to G.C. which makes the
sample more volatile; e.g.
1. Methyl esters: Both acidic and basic catalysts are used for esterification reactions of fatty
acids and fats.
Biochemistry Biochemical Techniques
12 Gas Liquid Chromatography
2. Silyl ethers: The silyl ethers are preferred for partial glycerides, sterol, carbohydrates etc.
3. 2,4 DNPH: Volatile carbonyl compounds are important flavour compounds in food stuffs,
accounting for flavour & off flavour. These are converted to their 2, 4 dinitrophenyl hydrzones.