(d) Chromatography - Chromatography is a valuable technique for the separation, purification and identification of the constituents of a mixture.

This technique was originally confined to the separation of coloured substances such as plant pigments and dyestuffs. But the technique is now well applied to colourless substances also. Now chromatography has become an advanced separation as well as purification technique that finds application in almost all branches of science The chromatographic technique is different from other traditional purification and separation methods such as distillation extraction, crystallisation and sublimation etc. The traditional methods of purification of a mixture or crude product can be applied only in those cases where large amounts of the material are available. Moreover, they give satisfactory results when the crude mixture contains two or three components. Separation by using these traditional methods does not give satisfactory results in the case of materials which are available in small quantities containing three or more components with very similar solubilities in a range of solvents or whose boiling points are very close. In these cases, a perfect separation can be achieved by making use of the chromatographic techniques even when the material is present in minute quantity. be chromatography technique was first invented by M. Tswett in 1906. He used the technique to separate various plant pigments such as chlorophyll and xanthophylls by passing a solution of these compounds through a glass column packed with finely divided calcium carbonate. The separated species appeared as coloured bands on the column. Since the discovery, this technique has undergone tremendous modifications and now a days various types of chromatographic techniques have been developed for separating almost any kind of given mixture, whether coloured or colourless into its constituents and to test the purity of these constituent The applications of hromatography have grown explosively in the last four decades, owing not only to the development of several new types of chromatographic techniques, but also due to the growing needs of scientists for better methods of separating the complex mixtures. The term Chromatography (Greek : Khromatos - colour and graphos- written) and its

principles were first -d-is covered by Mikhail Tswett. In his experiment, a leaf extract sample in petroleum ether was allowed to pass through"a" column of CaC03. Pure'ether was continued to flow through the column, as a result of which various chlorophyll pigments, etc., were separated into a series of differently coloured and easily distinguished zones. In the first monograph he writes : TYPES OF CHROMATOGRAPHY I) 1. 2. 3. 4. Based upon the nature of stationary and mobile phase There are different types of chromotagraphy based on the type of stationary and mobile phase used they are, Gas Gas Solid Liquid Solid Chromatography Liquid Chromatography Liquid Chromatography {Column Chromatography, thin layer chromatography, HPLC (High performance liquid chromatography)} Liquid chromatography (Paper partition chromatography) Column Partition chromatography)

II)

Based on the principal of separation The principal of separation can be either adsorption or partition. Hence they can be called as adsorption chromatography or partition chromatograph.

1.

Adsorption chromatography When a mixture of compounds (adsorbate) dissolved in the mobile phase (eluent) moves through a column of stationary phase (adsorbent) they travel according to the relative affinities towards stationary phase. The compound which has more affinity towards stationary phase travels slower and the compound which has lesser affinity towards stationary phase travels faster. Hence the compounds are separated. No two compounds have the same affinity for a combination of stationary phase, mobile phase and other conditions. Examples : Gas-solid chromatography, thin layer chromatography, Column

chromatography and HPLC (High Performance liquid chromatography) 2. Partition chromatography When two immiscible liquids are present a mixture of solute will be distributed according to their partition coefficient. The component which is more soluble in the stationary phase, that travels slower. The component which is more soluble in mobile phase travels slower. The component which are separated because of the differences in their partition co-efficients. No two components have the same partition co-efficient for a particular combination of stationary phase, mobile phase and other conditions. Examples : Gas Liquid chromatography, Paper Partition chromatography III) Based on the modes of chromatography There are two types. They are based upon the polarity of te stationary phae and mobile phase used. 1. Normal Phase chromatography : In this the stationary phase is Polar and mobile phase ois Non-Polar. 2. Reverse phase chromatography : In this stationary phase is Non-Polar and mobile phase is polar. This is most widely used in pharmaceutical analysis. A comparison of Normal Phase and Reverse phase mode Normal Phase Polar Non- Polar Non- Polar Polar Silica Gel Reverse Phase Non- Polar Polar Polar Non-Polar ODS (C18), C8, C4 Bounded phases

Stationary Phase Mobile Phase Compound eluted first and retained less Compound eluted last and retained less Example of stationary phase

INTRODUCTION OF GAS CHROMATOGRAPHY

Gas chromatography, GC, is probably the most utilized of all the chromatographic techniques. Since the theory was developed and amino acids were separated in the early 1950's, literally thousands of applications have been reported in organic, general, and biochemistry. It has been applied to a wide variety of theoretical and practical problems in the separation and identification of components of the atmosphere, gases liquids, drugs, and commercial products. The primary limitation is that the sample must be capable of being volatilized without undergoing decomposition, Because of this limitation it is now being replaced, to a large extent, by high performance liquid chromatography. In gas-liquid chromatography, GLC, the mobile phase is a gas and the stationary phase is a thin layer of a non-volatile liquid bound to a solid support. A partition process occurs. On the other hand, gas-solid chromatography, GSC, utilizes a solid adsorbent as the stationary phase and an adsorption process takes place. Gas chromatography is quite similar to column chromatography, except that a gas is used as the mnhile phase instead of a liquid. Gas-solid chromatography (GSC) is based upon selective adsorption on a solid, whereas gas-liquid chromatography is base ,:poi, th partit.or, bct:.ceo c gee "'t an liquid phase. Gas solid chromatography was developed by G.D. Kohler and K. Thieie in 1943 -followed by E. Cremer, T. Janak, H.W. Patton, J.S. Lewise and W.I. Kaye, C.S.G. Phillips contributed to frontal analysis whereas P. Schuftan, N.C. Turner, S. Claesson and N.M. Turkel gave displacement development of the elution technique. The gasliquid chromatography is more popular than gas-solid chromatography and has many more applications. Gas-liquid chromatography was originated by A.J.P. Martin in 1951 together with A.T. James. It is noteworthy that Martin had theoretically predicted the feasibility of GLC about a decade earlier while presenting his work on liquid -liquid chromatography (LLC). In 1952 Martin and Synge were awarded the Nobel Prize in chemistry for their work on the development of partition chromatography. The main advantages of gas chromatography are given below

(i) (ii) (iii) (iv) (v) (vi)

The technique has strong separation power and even complex mixture can be resolved into constituents. The sensitivity of the method is quite high. It is a micromethod and only a few mg of the sample is sufficient for analysis. It gives good precision and accuracy. The analysis is completed in a short time. The cost of instrument is relatively low and its life is generally long. The technique is suitable for routine analysis because the operation of a gas chromatograph and related calculations do not require highly skilled persons. Gas chromatography is at present one of the most widely used and powerful tools

available for separations. The major factors for this are the speed, resolving power and extreme sensitivity of the Techinique. It is possible to separate the 10 isomerse of Heptane in less than 10 Seconds PRINCIPLE OF SEPARATION The principle of separation in GLC is partition. Gas is used as mobile phase. Liquid which is coated on to a solid support is used as stationary phase. The mixture of components to be separated is converter to vapour and mixed with gaseous mobile phase. The component which is more soluble in the stationary phase travels slower and eluted later The component which is less soluble in the stationary phase, travels faster and eluted out first. No two components has the same partition co-efficier.: for a fixed combination of stationary phase, mobile phase and other conditions. Hence the components are separated according to their partition co-efficients. (Partition co-efficient is the ratio of solubility ze a substance distributed between two immiscible liquids at a constarr temperature)

CRITERIA FOR COMPOUNDS TO BE ANALYSED BY GAS CHROMATOGRAPHY

Two important criteria are 1. Volatility: Unless a compound is volatile, it cannot be mixed mobile phase. Hence the compound must have volatile nature. 2. Thermostability: All the compounds will not be in the form of vapour. There will be solid as well as liquid samples. Hence is convert them to a vapour form, they have to be heated to a high temperature. At that temperature, the compounds have to be thermostable. If they are not thermostable, the compounds car-rim be analyzed by Gas chromatography, since they will be decomposed. INSTRUMENTATION OF GAS CHROMATOGRAPHY

1. 2. 3. 4. 5. 6. 7.

Carrier gas Flow regulators and flow meters Injection devices Columns Temperature control devices Detectors Recorders and Integrators

The gas Chromatographic Separation is carried out in a tubular column made of glass, metal or Teflon. In hti column a sorbent is filled as stationary phase the absorbents are packed in

the form of fine science graded powder, where as the liquids are either coated as fine film and the column or is coated over an inert size graded porous support such as firebrick powder followed by packing in the column. A gas serving as mobile phase, flows continuously through the column. It is known as the carrier gas and serves to transport sample components in the column. The sample is introduced is the vapour form at the carrier gas entrance end of the column. Different components of the sample are sorbed on the stationary phase to different extents depending upon their distribution coefficients. The portion of each component in the gas phase is swept further immediately by the carrier gas. As a res.ilt a fraction of the sorbed amount also desorbs out to maintain the K-value. At the same time, out of swept amount some amount will go into sorbent at the next point in the column again to maintain the K-value. This goes on successively. and continuously and as a whole the band for each component moves further in'the column and having the shape of, more or less, Guassis,. J ;tribut:on. The separ_sson_ of a binary mixtures. It should be kept in mind that the gas is the driving force for the movement of zones through 6e column and the solid (for GSC or liquid for GLC) provides the selective retarding force. The detector can be regarded as the brain of the chromatograph and the column as its heart. CARRIER GAS Requirements of a carrier gas: 1. 2. 3. 4. 5. 6. 7. Inertness Suitable to the detector used High purity Easily available Cheap Less risk explosion or fire hazards Should give best column performance consistent with the required speed of analysis. Considering these requirements, and a compromise among inertness, efficiency and

operating cost, etc make Nitrogen and Helium as the most common carrier gas. *As carrier gas is compressible, gases are stored under high pressure in cylinders and used when required. The choice of carrier gas determines the efficiency of chromatographic separation. Most widely used carrier gases are Hydrogen, Helium, Nitrogen and Argon. The most widely used carrier gases are Hydrogen, Helium and Nitrogen and Organ Hydrogen: 1) 2) 3) 4) Helium: 1) 2)

It has better thermal conductivity, low density. It is useful in case of thermal conductivity detector and flame ionisation detector. It is more advantageous as compare to other gases but is dangerous to use. The disadvantage is that it reacts with unsaturated compounds and create fire and explosive hazards. It is 2nd best gas but it is expensive. It is generally used because of its inertness, low density and it allows greater flow rates.

Nitrogen: It is inexpensive but has reduced sensitivity. 2. FLOW REGULATORS AND FLOW METERS As carrier gases are stored under high pressure, flow regulators are used to deliver the gas with uniform pressure or flow rate. Flow meters are used to measure the flow rate of carrier gas. They are Rotameter and Soap bubble flow meter. Rotameter: It is placed conveniently before the column inlet. It has an ordinary glass tube (like burette) with a float held on to a spring.

The level of the float is determined by the flow rate of carrier gas and is precalibrated. Soap bubble meter: It is similar to rotameter and instead of a float, soap bubble formed indicates the flow rate. It has a glass tube with a inlet tube at the bottom through which gas comes in. A rubber bulb is used to store soap solution.

When the bulb is gently pressed, a drop of soap solution is converted into a bubble by the pressure of carrier gas and travels up. The distance travelled upwards is a measure of flow rate of carrier gas. The graduations are also precalibrated.

INJECTION DEVICES Samples for introducing in to the column can be any of tyre either gas, liquid or solid in nature. Gases can be introduced in to the Column by valve devices. Liquids can be injected thorough septum devices. Solid samples are dissolved in a suitable solvent and then they are injected through a septum. SAMPLE INTRODUCTION SYSTEM The real chromatographic analysis starts with the introduction of the sample onto the column. The development of capillary gas chromatography resulted in many practical problems with the injection technique. The technique of on-column injection, often used with packed columns, is usually not possible with capillary columns. The injection system in the capillary gas chromatograph should fulfill the following two requirements: Some general requirements which a good injection technique should fulfill are:

It should be possible to obtain the columns optimum separation efficiency. It should allow accurate and reproducible injections of small amounts of representative samples.

It should induce no change in sample composition. It should not exhibit discrimination based on differences in boiling point, polarity, concentration or thermal/catalytic stability.

Injection Devices are of Two Types SPLIT INJECTION ON-COLUMN INJECYION

The Split Injection System

The basic difference between the two types of injection systems is that the capillary column now projects into the glass liner and a portion of the carrier gas sweeps past the column inlet to waste. As the sample passes the column opening, a small fraction is split off and flows directly into the capillary column, ipso facto this device is called a split injector. The split ratio is changed by regulating the portion of the carrier gas that flows to waste which is achieved by an adjustable flow resistance in the waste flow line. This device is only used for small diameter capillary columns where the charge size is DISADVANTAGE The device has certain disadvantages due to component differentiation and the sample placed on the column may not be truly representative diffusivities (higher molecular weights).

Consequently, quantitative analyses carried out using the high efficiency small diameter capillary columns may have limited accuracy and precision, depending on the nature of the sample. This problem was partially solved by using larger diameter columns that would permit on-column injection. The columns are constructed to have an I.D. of about 0.056 in; which is slightly greater than the diameter of a certain hypodermic needles. This injection system is depicted following figure ON-COLUMN INJECTION SYSTEM

On-Column Injector On injection, the sample breaks up into separate portions, and bubbles form at the beginning of the column causing the sample to be deposited at different positions along the open tube as the solvent evaporates On starting to develop the separation, each local concentration of sample acts as a separate injection.

As a consequence, a chromatogram containing very wide or multiple peaks is produced. Procedures have been introduced in an attempt to eliminate sample splitting in this manner.

COLUMNS
Column Is One Of The important part of Gas Chromatography which decides the separation efficiency. Columns are made up of glass or stainless steel. Stainless steel columns have the advantage of long life and can be easily handled with out fear fragility. But some samples react with them hence in such cases, glass columns are used ex: Steriods. Glass Columns have a advantage they are inert and do not react with the any kind of sample. The great disadvantage is that they are highly fragile and are difficult to handle.

COLUMN SELECTION The choice of column depends on the sample and the active measured. The main chemical attribute regarded when choosing a column is the polarity of the mixture, but functional groups can play a large part in column selection. The polarity of the sample must closely match the polarity of the column stationary phase to increase resolution and separation while reducing run time. The separation and run time also depends on the film thickness (of the stationary phase), the column diameter and the column length

Columns are classifieds as follows : DEENDING ON ITS USE 1) Preperative 2) Analytical 1) PREPERATIVE These are larger when compared to analytigal columns since larger amount of sample has to be loaded .they have a lenth of 3-6meters and outside diameter of 6-9mm ANALYTICAL : Analytical Columns Have A Length Of 1-15 Meters And diameter of 3-6mm. They arepacked columns made up of glass or stainless steel. Only small quantity of samples can be loaded

DEPENDING ON ITS NATURE 1) Packed column 2) Open tubular column. 3) SCOT columns support coated open tubular columns

The Packed GC Column

Packed columns are usually constructed from stainless steel or Pyrex glass. Pyrex glass is favored when thermally labile materials are being separated such as essential oils and flavor components. However, glass has pressure limitations and for long packed columns, stainless steel columns are used as they can easily tolerate the necessary elevated pressures. The sample must, of course, be amenable to contact with hot metal surfaces. Stainless steel columns are usually washed with dilute hydrochloric acid, then extensively with water followed by methanol, acetone, methylene dichloride and n-hexane. This washing procedure removes any corrosion products and traces of lubricating agents used in the tube drawing process. Adsorbents There are two types of packing employed in GC, the adsorbents and the supports, on which the stationary phase is coated. There are both inorganic and organic types of GSC adsorbents, each of which have specific areas of application. All are ground and screened to provide a range of particle sizes that extend from about 30/40 mesh to 100/120 mesh. In general, the smaller the particle size the higher the column efficiency, but the packing procedure is more difficult. It is also essential that the particle size range should be as narrow as possible. Packing materials that have a wide size range not only produce columns with poor efficiencies, but again, are also far more difficult to pack.

THE CAPILLARY OR OPEN TUBULAR COLUMN OR GOLY COLUMNS

They are made up of long capillary tubing of 30 - 90 meters in length and have uniform and narrow internal diameter of 0.025 - 0.075 cm. These are made up of stainless steel and are in the form of a coil. The inner wall of the capillary is coated with the stationary phase liquid in the form of a thin film (0.5 to l). These columns offer least resistance to the flow of carrier gas and hence they are more efficient than packed columns which offer more resistance to the flow of carrier gas. But the disadvantage is that more sample cannot be loaded.

Capillary columns are fabricated from stainless steel or quartz. Metal capillary columns must be carefully cleaned to remove traces of extrusion lubricants before they can be coated, usually by washing with methylene dichloride, methanol and then water. After removing oil and grease, the columns are washed with dilute acid to remove metal oxides or other corrosion products that may remain adhering to the walls, washed with water and the again washed with methanol and methylene dichloride.
SCOT COLUMNS (SUPPORT COATED OPEN TUBULAR COLUMN):

This is an improved version of Golay or capillary columns. As Golay or capillary columns have small sample capacity, they can be modified into SCOT columns. These columns are made by depositing a micron size porous layer of support material on the inner wall of the capillary column and then coated with a thin film of liquid phase. These columns also have low resistance to the flow of carrier gas but offers the advantage of more sample load or capacity. STATINOARY PHASES FOR GLC : The no. of liquid stationary phases available for GLC is almost unlimited. In general the most important requirement of a liquid phase for a satisfactory separation. 1) It should be a good solvent for the component of sample. 2) It should be thermally stable. 3) The Solvent power of liquid phase should be different for each component of the sample. 4) It should be chemically inert towards the sample. 5) It should be of low volatility.

TEMPERATURE CONTROL DEVICES PREHEATERS: Preheaters are used in Gas chromatography to convert the sample into its vapour form and mix them with the mobile phase or carrier gas. The preheaters are present along with injecting devices. As soon as liquid samples are injected, they are converted into vapour form. THERMOSTATICALLY CONTROLLED OVEN: The principle of separation in Gas chromatography is partition. Partition co-efficient is the ratio of concentration of a solute distributed between two immiscible liquids. Since partition coefficient as well as solubility of a solute depends upon temperature, temperature maintenance in a column is highly essential for efficient separation. Hence the column as well as injecting devices should be maintained at a particular temperature. As columns are long, they cannot be enclosed in oven easily. Hence the columns are in a coiled form and enclosed in thermostatically controlled oven. These ovens are highly accurate and can maintain temperature nearest to 0.10C. Two types of operations are available. They are Isothermal programming: (Iso means same) in which the same temperature is maintained throughout the process of separation. Linear programming: in which the oven is heated linearly over a period of time. eg.

150C initially to 200C at the end of separation with an increase in temperature at the rate of 5C/minute. This type of linear programming is required when a sample has a mixture of low boiling and high boiling point compounds. This method is efficient for separation of such complex mixtures.

Detector are the most important part of gas chromatographic instruments they are considered

as heart of the apparatus a detector uses uses some property by which it can detect the difference between a pure carrier gas and a eluted component. The requirements of an ideal detector are: i. ii. iii. iv. v. vi. vii. Applicability to wide range of samples. High Sensitivity to even small concentrations. Rapidity of response. Linearity: i.e. less response to low concentration and proportional response to high concentration. Response should be unaffected by temperature, flow rate or characteristics of carrier gases. Non destructive to the sample in case of preparative work. vii. Simple and easy to maintain. Inexpensive.

The different detectors used commonly are a) Katharometer or Thermal Conductivity Detector (TCD) b) Flame Ionisation Detector (FID) c) Argon Ionisation Detector (AID) d) Electron Capture Detector (ECD) The Katherometer Detector The katherometer detector (sometimes spelt catherometer and often referred to as the thermal conductivity detector or hot wire detector) is relatively insensitive but has survived largely as a result of its catholic response and, in particular, its response to the permanent gases. Consequently, it is often the detector of choice for gas analysis and environmental testing. Its frequent use in these special types of application, somewhat surprisingly, has made it the fourth most commonly used GC detector. A filament carrying a current is situated in the column eluent and, under equilibrium conditions, the heat generated in the filament is equal to the heat lost by conduction and convection and consequently the filament assumes a

constant temperature. At the equilibrium temperature, the resistance of the filament and thus the potential across it is also constant.

The Katherometer Detector ("In-Line Cell") The heat lost from the filament will depend on the thermal conductivity of the gas and its specific heat and both these parameters will change in the presence of a foreign gas or solute vapor. The presence of a different gas entering the detector causes the equilibrium temperature to change, producing a change in potential across the filament. This potential change is amplified and fed to a suitable recorder. A diagram of the katherometer is shown in figure

The katherometer may have an "in-line" sensor where the column eluent passes directly over the filament or an "off-line" sensor where the filaments are situated out of the main carrier gas stream and the gases or vapors reach the sensing element by diffusion. Due

to the high diffusivity of vapors in gases, diffusion can be considered as almost instantaneous. The katherometer detector is very flow and pressure sensitive and the sensor must be carefully thermostatted and fitted with reference cells to compensate for changes in pressure or flow rate. The filaments of the reference and measuring cell are made to form two of the arms of a Wheatstone bridge and the out-of-balance signal amplified and fed to a recorder or computer data acquisition system. The Flame Ionization Detector The FID, invented by Harley and Pretorious (7), and separately by McWilliams and Dewer (8), evolved from the Heat of Combustion Detector developed by Scott (9). The FID detector employs hydrogen as the combustion gas which is mixed with the column eluent (helium, nitrogen or other appropriate gas) and burnt at a small jet situated inside a cylindrical electrode. A potential of a few hundred volts is applied between the jet and the electrode and when a carbon containing solute is burnt in the jet, the electron/ion pairs that are formed are collected at the jet and cylindrical electrode. The current is amplified and fed to a recorder or to the A/D converter of a computer data acquisition system. A diagram of the basic FID is shown in figure 22. During the process of oxidation, oxidized or partially oxidized fragments of the solute are formed in the flame which are thought to generate electrons by thermionic emission. The background current (ions and electrons from the hydrogen flame alone) is very small (1-2 x 10-12 amperes) and consequently, the noise level is also commensurably small (about 10-14 amperes).

Figure 22. The Flame Ionization Detector The ionization process is not very efficient, only 0.0018% of the solute molecules produce ions, (about two ions or electrons per 10 5 molecules). Nevertheless, because the noise level is very small, the minimum detectable mass of n-heptane is only 2 x 10-12 g/sec. At a column flow rate of 20 ml/min. this is equivalent to a minimum detectable concentration of about 3 x 10-12 g/ml. The detector responds to mass per unit time entering the detector, not mass per unit volume consequently the response is almost independent of flow rate. This is particularly advantageous and allows it to be used very effectively with capillary columns. Although the column eluent is mixed with the hydrogen prior to entering the detector, as it is mass sensitive and not concentration sensitive, the diluting effect has no impact on the sensitivity. The FID detects virtually all carbon containing solutes, with the exception of a small number of small molecular compounds such as carbon disulfide, carbon monoxide, etc. In fact, due to its diverse and comprehensive response, it is considered a universal detector.

An example of the use of the FID in a paraffin, isoparaffin, aromatic, naphthene and olefin analysis of a hydrocarbon mixture (frequently called the PIANO analysis) is shown in figure 23. The column was the Petrocol DH 50.2, 50 m long and 0.5 mm I.D. and made from fused silica. The column temperature was held a 35oC for 5 minutes and then programmed up to 200C at 2/min. The carrier gas was helium and the mobile phase velocity of 20 cm/sec. Many standard tests carried out in the hydrocarbon and pharmaceutical industries and for environmental testing have been designed to utilize the FID as the detector

The Nitrogen Phosphorus Detector (NPD) The nitrogen phosphorus detector (NPD), is a highly sensitive but specific detector and evolved directly from the FID. It gives a strong response to organic compounds containing nitrogen and/or phosphorus. Although it appears to function in a very similar manner to the FID, in fact, it operates on an entirely different principle. A diagram of an NP detector is shown in figure 24.

Figure 24. The Nitrogen Phosphorus Detector

The actual NPD sensor is a rubidium or cesium bead contained inside a small heater coil. The helium carrier gas is mixed with hydrogen and passes into the detector through a small jet. The bead is heated by a current passing through the coil which is situated above the jet, and the helium-hydrogen mixture passes over it. If the detector is to respond to both nitrogen and phosphorus, then a minimum hydrogen flow is employed to ensure that the gas does not ignite at the jet. In contrast, if the detector is to respond to phosphorus only, a large flow of hydrogen can be used and the mixture burned at the jet. A potential is applied between the bead and the anode. The heated alkali bead emits electrons by thermionic emission which are collected at the anode and thus produce an ion current. 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). Unfortunately, the performance deteriorates with time. Reese (10) examined the function of the NPD in great detail. The alkali salt employed as the bead is usually a silicate and Reese showed that the reduced response was due to water vapor from the burning hydrogen, converting the alkali silicate to the hydroxide. At the operating temperature of the bead, the alkali hydroxide has a significant vapor pressure and consequently, the rubidium or cesium is continually lost during the operation of the detector. Eventually all the alkali is evaporated, leaving a bead of inactive silica.

The Electron Capture Detector The electron capture detector contains a low energy b-ray source which is used to produce electrons for capturing by appropriate atoms. Although tritium adsorbed into a silver foil has been used as the b particle source, it is relatively unstable at high temperatures, the Ni63 source was found to be preferable. The detector can be used in two modes, either with a constant potential applied across the cell (the DC mode) or with a pulsed potential across the cell (the pulsed mode). In the DC mode, hydrogen or nitrogen can be used as the carrier gas and a small potential (usually only a few volts) is applied across the cell that is just sufficient to collect all the electrons available and provide a small standing current. If an electron

capturing molecule (for example a molecule containing an halogen atom which has only seven electrons in its outer shell) enters the cell, the electrons are captured by the molecule and the molecules become charged. The mobility of the captured electrons is much smaller than the free electrons and the electrode current falls dramatically. The DC mode of detection, however, has some distinct disadvantages. The most serious objection is that the electron energy varies with the applied potential. The electron capturing properties of a molecule varies with the electron energy, so the specific response of the detector will depend on the applied potential Operating in the pulsed mode, a mixture of 10% methane in argon is employed which changes the nature of the electron capturing environment. The electrons generated by the radioactive source rapidly assume only thermal energy and, in the absence of a collecting potential, exist at the source surface in an annular region about 2 mm deep at room temperature and about 4 mm deep at 400C. A short period square wave pulse is applied to the electrode collecting the electrons and producing a base current. The standing current, using 10% methane in argon is about 10-8 amp with a noise level of about 5 x 10-12 amp. The pulse wave form is shown in figure 26.

Figure 26. Wave form of Electron Capture Detector Pulses In the inactive period of the wave form, electrons having thermal energy only will attached themselves readily to any electron capturing molecules present in the cell with the consequent production of negatively charged ions. The negative ions quickly recombine with the positive ions (produced simultaneously with the electrons by the b particles) and thus

become unavailable for collection. Consequently the standing current measured during the potential pulse will be reduced. The period of the pulsed potential is adjusted such that relatively few of the slow negatively charged molecules (molecules having captured electrons and not neutralized by collision with positive ions) have time to reach the anode, but the faster moving electrons are all collected. During the "off period" the electrons re-establish equilibrium with the gas. The three operating variables are the pulse duration, pulse frequency and pulse amplitude. By appropriate adjustment of these parameters the current can be made to reflect the relative mobilities of the different charged species in the cell and thus exercise some discrimination between different electron capturing materials. A diagram of an electron capture detector is shown in figure 27.

Figure 27 The Electron Capture Detector

The basic electron capture detector consists of a small chamber one or two ml in volume enclosing two metal electrodes. The electrodes may be concentric cylinders or metal discs separated by an insulator. The cell contains the radioactive source, electrically connected to the entrance conduit and to the negative side of the power supply. A gauze "diffuser" is connected to the cell exit and to the positive side of the power supply. The output from the sensor is processed by suitable electronics and the output passed to either a potentiometric recorder of a computer data acquisition system. The electron capture detector is very sensitive, probably the most sensitive GC detector available (ca. 10-13 g/ml) and is widely used in the analysis of halogenated compounds, in particular, pesticides. An example of a pesticide analysis employing an electron capture detector is shown in figure 28. Recorders and Integrators Recorders are used to record the responses obtained from detectors after amplification, if necessary. They record the baseline and all the peaks obtained, with respect to time. Retention time for all the peaks can be found out from such recordings, but the area of individual peaks cannot be known. Integrators : Integrators are improved version of recorders with some data processing capabilities. They can record the individual peaks with retention time, height and width of peaks, peak area, percentage of area, etc. Integrators provide more information on peaks than recorders. DERIVATISATION OF SAMPLE Derivatisation is a technique of treatment of the sample to improve the process of separation by column or detection by detector. There are two types based upon its need. They are Precolumn derivatisation: This is done to improve some properties of the sample for separation by column. By this derivatisation technique, the components are converted to more volatile and thermostable derivatives. Moreover improved separation and less tailing will be seen after such treatment. In the following conditions, pre column derivatisation is done. 1. The component is less volatile.

2. 3. 4.

The compounds are thermolabile, i.e. heat sensitive. To reduce tailing. To improve separation factor. Example: Carboxylic acids, sugars, phenols, alcohols, etc can be converted to less polar compounds by using reagents like BSA reagent (Bis trimethyl Silyl Acetamide reagent).

They can also be converted to acetyl derivative or triflouro acetyl derivative. Post column derivatisation: Post column derivatisation is done to improve the response shown by detector. The components may not be detected by detector unless derivatisation is done. The components may be converted in such away that their ionisation or affinity towards electrons is increased. Normally this is 'On-Line' detection technique where the f l owrate is neither stopped nor altered. PRETREATMENT OF SOLID SUPPORT
Solid support is used to hold the stationary phase liquid as a thin film. But sometimes due to some defects, uniformity and stability of the film of liquid stationary phase may not exist. In such cases, tailing of peaks and low separation efficiency can be observed. Therefore to overcome such demerits, it is best to do pretreatment of the stationary phase.

Generally while doing separation of non-polar components like esters, ethers, etc tailing of peaks are observed. These problems can be overcome by the following techniques: 1. 2. 3. 4. By using more polar liquid stationary phase. Increasing the amount of liquid phase on the support. By selecting a less active support. Pretreatment of the solid support to remove active sites.

Example: By using hexamethyl disilazone or dimethyl silyl dichloride. This converts the hydroxy groups (polar) to dimethyl group (non polar) and hence the active adsorption sites are deactivated.

PARAMETERS USED IN GAS CHROMATOGRAPHY Retention time (Rt) Retention time is the difference in time between the point of

injection and appearance of peak maxima. Retention time is the time required for 50% of a component to be eluted from a column. Retention time is measured in minutes or seconds. Retention time is also proportional to the distance measured in cm or mm. moved on a chart paper, which can be

Retention volume (Vr) Retention volume is the volume of carrier gas required to elute 509 of the component from the column. It is the product of retention time and flow rate. Retention volume = Retention time x flow rate Separation factor (S) Separation factor is the ratio of partition co-efficient of the tv, components to be separated. It can be expressed and determined by using the following equation:

Where

to Kb, Ka tb, ta S

= Retention time of unretained substance = Partition coefficients of b and a S = Retention time of substance b and a = depends on liquid phase & column temperature

If there is more difference in partition coefficient between two compounds, the peaks are far apart and the separation factor is more. If the partition coefficients of two compounds are similar, then the peaks are closer and the separation factor is less.

RESOLUTION

Resolution is a measure of the extent of separation of two components and the baseline separation achieved. It can be determined by using the following formula:

Theoretical Plate (Plate theory) A theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. A theoretical plate can also be called as a functional unit of the column.
HETP - Height Equivalent to a Theoretical Plate

A theoretical plate can be ofany height, which decides the efficiency of separation. If HETP is less, the column is more efficient. If HETP is more, the column is less efficient. HETP can be calculated by using the following formula:

HETP is given by the Van Deemeter equation

Where A =

Eddy diffusion term or multiple path diffusion which arises due to packing of the column. This is unaffected by carrier gas velocity or flow rate. This can be minimised by uniformity in packing. B= C= u= Longitudinal diffusion term or molecular diffusion which depends on flow rate. Effect of mass transfer which depends on flow rate Flow rate or velocity of the mobile phase.

The following figure is the effect of flow rate on HETP. A column is efficient only when IIETP is minimum. Hence an ideal flow rate corresponding to the minimum value of HETP is used. Efficiency (No. of theoretical plates) Efficiency of a column is expressed by he number of theoretical plates. It can be etermined by using the formula:

Where n Rt w

= No. of throatily plates ] = rentition time = peak width at base

Rt and w are measured in common units (mm or cm or minutes or seconds) and are proportional to the distances marked on chart paper. If the number of theoretical plates is high, the column is said to 'be highly efficient. If the number of theoretical plates is low, the column is said to be less efficient. For gas chromatographic columns, a value of 600/metre is sufficient. But in HPLC, high values like 40,000 to 70,000/metre are recommended.

Asymmetry factor A chromatographic peak should be symmetrical about its centre and said to follow Gaussian distribution. In such cases, the peak will be like an isosceles triangle. But in practice, due to some factors, the peak is not symmetrical and shows tailing or fronting as shown in the following figures. Fronting is due to saturation of stationary phase and can be avoided by using less quantity of sample. Tailing is due to more active adsorption

sites and can be eliminated by support pretreatment, more polar mobile phased

increasing the amount of liquid phase. Asymmetry factor formula: AF = b/a (0.95 to 1.05) can be calculated by using the (b and a calculated at 5% or 10% of the peak height)
APPLICATIONS OF GAS CHROMATOGRAPHY

1. Qualitative analysis: It is nothing but identification of a compound. This is done by comparing the retention time of the sample as well as the standard. Under identical conditions, the retention time of the standard and the sample are same. If there is a deviation, then they are not the same compound. 2. Checking the purity of a compound: By comparing the chromatogram of the standard and that of the sample, the purity of the compound can be reported. If additional peaks are obtained, impurities are present and hence the compound is not pure. From the percentage area of the peaks obtained, the percentage purity can also be reported. 3. Presence of Impurities: This can be seen by the presence of additional peaks when compared with a standard or reference material. The percentage of impurities may also be calculated from peak areas. 4. Quantitative analysis: The quantity of a component can be determined by several methods like a. Direct comparison method By injecting a sample and standard separately and comparing their peak areas, the quantity of the sample can be determined. Area of the peak = peak height x width of peak at the half height

where Al and A2 are peak area of sample and standard Wl and W2 are weight or concentration of sample and standard is the response factor b. Calibration curve method

Calibration curve method In calibration curve method, standards of varying concentrations are used to determine their peak areas. A graph of peak area Vs concentration of the drug is plotted. From the peak area of the unknown sample, by intrapolation, the concentration of the sample can be determined. This method has the advantage that errors, if any are minimised. Internal standard method In this method, a compound with similar retention characteristics is used. A known concentration of the internal standard is added separately to the standard solution and sample solution whose concentration is not known. The chromatogram is recorded and the peak area ratio of standard and internal standard is determined. By using the peak area ratio of sample and internal standard, the concentration of the unknown solution is determined. This method is useful when more extraction steps are involved in sample preparation and the sample matrix is complex. 5.Multicomponent analysis or Determination of mixture of drugs: Similar to the quantification of a single drug, multicomponent analysis can also be done easily. The quantity of each component is determined by using any one of the above methods. Marketed formulations are available which contain several drugs and each component can be determined quantitatively. 6. Isolation and identification of drugs or metabolites In urine, plasma, serum etc can be carried out. 7. Isolation and identification of mixture of components like amino acids, plant extracts, volatile oils, etc.