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Biochemical Techniques 12 Gas Liquid

Description of Module

Subject Name Biochemistry

Paper Name 12 Biochemical Techniques

Module Name/Title 12 Gas - liquid Chromatography

Biochemical Techniques Biochemistry 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 . 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 like (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)

Biochemical Techniques Biochemistry 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 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, 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:

Biochemical Techniques Biochemistry 12 Gas Liquid Chromatography

Carrier Gas Supply: The gaseous mobile phase must be inert. is the most common mobile phase, although , nitrogen, 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:

Biochemical Techniques Biochemistry 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).

Biochemical Techniques Biochemistry 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

Biochemical Techniques Biochemistry 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. : 5 ppb for light 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 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 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 . 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.

Biochemical Techniques Biochemistry 12 Gas Liquid Chromatography

Advantages : Very high sensitivity and specificity for 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 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

Biochemical Techniques Biochemistry 12 Gas Liquid Chromatography

carbonyls, lactones and . 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, products, , saturated 5% : 95% Diphenyl : dimethyl Non- Polar flavors, pesticides, polysiloxane aromatic hydrocarbons 35% : 65%; Diphenyl : dimethyl Medium polarity Nitrogen containing polysiloxane 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 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.

Biochemical Techniques Biochemistry 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.

Biochemical Techniques Biochemistry 12 Gas Liquid Chromatography