gas chromatography (2)

GAS CHROMATOGRAPHY: 

GAS CHROMATOGRAPHY Presented by: Abdul Rasool. I.M .pharmacy Dept. of pharmaceutics

HISTORY: 

HISTORY Chromatography dates to 1903 in the work of the Russian scientist, Mikhail Semenovich Tswett . German graduate student Fritz Prior developed solid state gas chromatography in 1947. Archer John Porter Martin , who was awarded the Nobel Prize for his work in developing liquid-liquid (1941) and paper (1944) chromatography, laid the foundation for the development of gas chromatography and later produced liquid-gas chromatography (1950).

HISTORY: 

HISTORY The father of modern gas chromatography is Nobel Prize winner John Porter Martin , who also developed the first liquid-gas chromatograph. (1950)

CONTENTS : 

CONTENTS INTRODUCTION THEORY INSTRUMENTATION

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Introoduction: Chromatography means………. separation in which the components to be separated are distributed between 2 immiscible phases, a stationary phase and a mobile phase.

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Adsorption chromatography : In this the components of mixture are separated based on their relative affinities towards the stationary phase. This method is used for a larger quantity of mixtures Partition chromatography: In this the components of mixture are separated based on the relative solubilities or partition co-efficient. This method is used for a smaller quantity of mixture. This method is an accurate method than the GSC. Based on the principle of separation, it is classified as:

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It consists of gas solid chromatography (GSC) and gas liquid chromatography (GLC). In both types, gas is used as mobile phase and either solid or liquid is used as stationary phase. GSC is not widely used because of limited number of stationary phases available. In GSC, the principle of separation is adsorption. GSC is used only in case where there is less solubility of solutes in stationary phase . 7 Gas chromatography

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The chromatographic process Two different substances are partitioned between two phases. Depending on their affinity (toward the stationary phase) will spent different times adsorbed by the stationary phase.

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There are 3 theories: The Plate theory. The Rate theory. The Random walk, non-equilibrium theory. Theory :

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Developed by Martin and Synge Compared the GC separation to a fractional distillation. An analogy for this process would be multiple liquid-liquid extraction such as that which occurs in counter current distribution. The solute proceeds from one discrete extraction equilibrium to the next by the process of partitioning between two immiscible liquids. Plate theory

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The plate model supports that the chromatographic column contains a large number of separate layers, called theoretical plates . Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next plate. Hypothetical zone in which two phases establish an equilibrium with each other

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column in which the solute is complete equilibrium with the mobile and the stationary phases. This equilibrium is defined by the distribution coefficient, K D (partition coefficient). Theoritical plate: Plate

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Plate theory The distribution of a solute after ‘n’ equilibrations (plates) is defined by the expansion of the binomial in equation: ( a+b ) n-1 (n-1) = number of transfers between the plates. a = 1 / (K D +1) b = K D / (K D +1)

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14 A n = 10 n = 10 n = 20 n = 20 B A B FRACTION STAGE NUMBER Distribution curve for solutes A and B after 10 and 20 equilibrations 2/1/2012 Graph shows the distribution of 2 solutes, A and B, after 10-20 equilibrations where K D for A is 1 and K D for B is 0.5, and the volumes of both phases are equal.

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Plate theory As the number of plates increases (n > 100), the distribution becomes Gaussian. This illustrates two points: The elution peak is expected to be symmetrical. Band spreading will occur and will increase as the number of plates increases.

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Chromatogram w b t r Injection point Air peak t r W 0.5 Detector response time Wb -band width Tr -retention time

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Volatility of compound : Low boiling (volatile) components will travel faster through the column than high boiling point components. Polarity of compounds : Polar compounds will move more slowly, especially if the column is polar. Column temperature : Raising the column temperature speeds up all the compounds in a mixture. C olumn packing polarity : Usually, all compounds will move slower on polar columns, but polar compounds will show a larger effect. Flow rate of the gas through the column : Speeding up the carrier gas flow increases the speed with which all compounds move through the column. Length of the column : The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation. Factors which influence the GC separation.

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Column efficiency parameters Column efficiency parameters: The number of theoritical plates (n). The height equivalent to a theoritical plate, H (HETP). Column performance parameters: Resolution (R s ). Peak asymmetry. Separation factor.

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Column efficiency parameters: Number of theoritical plates (n): From the chromatogram, n is calculated by: n = 16 (t r / w b ) 2 = 5.54 (t r / w 1/2 ) 2 t r = retention time of the peak. w b = width of the peak at base line. The plate number is a measure of the extent of band broadening that a solute undergoes as it passes along the column.

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Number of theoritical plates (n) Each component separated in a column will have a different ‘n’ value for the column. This number is useful when comparing the efficiency of the same or similar columns for the same compound.

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Column efficiency parameters: The number of effective plates (N eff ) N eff = 5.54 (t r / w 1/2 ) 2 Where, t r = t r – t 0 This is used if the retention time of the analyte is short. This reflects the number of times, the analyte partitions between the two plates during its passage through the column.

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Column efficiency parameters: Height equivalent to a theoritical plate, h (HETP): When different columns of different composition and length are being compared, the H (HETP) is used. H = L / n H – height equalent to theoretical plate L- length of column required for one partition step to occur n- number of plates

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Plate HETP Smaller the H, the more efficient the column.

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Column efficiency parameters: If HETP is less, the number of theoritical plates in the column is more and so is its efficiency. If HETP is more, the number of theoritical plates in the column is less and its efficiency is also less.

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Column performance parameters Resolution Resolution R s defines the degree of separation of two adjacent bands The more efficient column has greater degree of resolution, it will produce between closely eluting peaks. The resolution between two peaks – A and B is expressed in the equation: R s = 2 (t rB - t rA ) / (W bB + W bA ) t rB and t rA are the retention times of peaks A and B W bB and W bA are the widths of peaks A and B at baseline.

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Resolution (R) : Resolution is the degree of separation between the two adjacent peaks i.e. the distance between the two adjacent peaks divided by the mean of the peak widths. where, d = distance between two peaks. tw1 = width of first peak. tw2 = width of second peak. If R = 1.5, baseline separation (99.7% resolution) is achieved.

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Resolution The larger the value of R s ,the greater the separation of the solute A and B. The resolution of 1.5 usually show baseline separation. 27 W bA W bC W bB t rA t rB t rC time 2/1/2012

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Column performance parameters A related term used with resolution . Column performance parameters Describe relative retention of two consecutive peaks Separation factor

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A B A B Two chromatograms with the same α A/B , but different resolution factors. α A/B = t rA / t rB

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Peak Asymmetry Peaks with tail have a high element of asymmetry. Asymmetry factor (AF): b / a a = the leading half of the peak measured at 10% of the peak height. b = the tailing half of the peak measured at 10% of the peak height.

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0.1 h 0.1 h a b a b Time (min) Time (min) h h Determination Of Peak Asymmetry

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Peak asymmetry The value should fall, ideally, in the range 0.95 – 1.15. Poor asymmetry may be caused through: Loading too much sample onto the column. Sample decomposition. The analyte adsorbing strongly onto active sites in the stationary phase. Poor trapping of the analyte when it is loaded onto the column or too much “dead volume” in the chromatographic system.

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Factors that decrease the column efficiency are: Very slow flow rate Large particle size of stationary phase Thick stationary phase coating Irregularly shaped particles of stationary phase Low temperature Uneven stationary phase coating Non-uniform stationary phase particle size Low diffusion coefficient in the mobile phase and stationary phase

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Limitation of plate theory Plate theory allowed for a comparison between different columns. But it did not suggest ways of improving the performance of the column.

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Rate theory This theory successfully describes the influence of variables which affects band separation (Retention time) and band broadening. This theory incorporate the facts that the mobile phase flows continuously and that solute molecules are constantly being transported and partitioned in a gas chromatographic column. Calculation of n, h, and R s from a chromatogram remains the same, except now h is described by equation:

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Van Deemter equation h= 2 λ d p + 2 γ DG / u + 8kdf 2 / Л 2 (k+1)D L u = average linear gas velocity λ = measure of the packing irregularities d p = particle diameter γ = tortuosity factor D G = coefficient of gaseous diffusion of the solute in the carrier gas k = ratio of the capacity of the liquid phase to that of the gas phase d f = film thickness D L = diffusion constant of solute in liquid phase 36

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Van Deemter equation This is frequently rewritten as: B h = A + + C u u A plot of h versus u, gives a hyperbola both in theory and practically. To obtain minimum h (maximum efficiency), the constants A, B, and C must be minimized.

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Van Deemter equation The equation states that the efficiency of column depends upon: A (eddy diffusion/multiple path diffusion) = length of the various paths along which the gaseous molecule move. B (longitudinal diffusion) = the diffusion of molecule within the gas phase. C (mass transfer) = the transfer of solute from the gas to the liquid phase and back again.

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A PLOT OF h VERSUS u MOLECULAR DIFFUSION FINITE RATE OF MASS TRANSFER MULTIPATH TERM A C B LINEAR GAS VELOCITY, u HETP FINITE RATE OF MASS TRANSFER

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Final band width Initial band width

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First term, A The particle diameter d p , when decreased, lowers the h but below a certain particle size, flow of carrier gas through the column is impeded and the pressure increases. The more uniform size and shape of the packing particles, the smaller the value of h . The plot for the term A shows straight line as particle size does not vary with the gas velocity.

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Second term, B (2 γ D G ) The B term accounts for the longitudinal diffusion. It relates to the molecular diffusion of the solute molecules in the vapour phase. γ measures the tortuosity of the carrier gas as it passes through the column. 42 2/1/2012

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Second term, B (2 γ D G ) γ is kept minimum by decreasing the particle size until there is a decrease in the performance due to an increase in pressure. D G is kept minimum by increasing the gas pressure and/or the molecular weight of carrier gas. 43 2/1/2012

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The final term, C It is a measure of mass transfer of the solute molecule from the stationary phase into the gas phase. D L is the measure of resistance to mass transfer of the solute into the stationary phase. D L value increases with increasing temperature, thereby increasing the efficiency. 44 2/1/2012

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The final term, C K, the capacity factor of the stationary phase, decreases with increasing temperature. The factor d f measures the thickness of the liquid phase on the stationary phase support. The thinner the liquid phase, the more efficient the column. But this also decrease the k. 45 2/1/2012

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The final term, C Therefore the overall implication of this term is to use the minimum amount of a liquid phase of low viscosity at the lowest possible temperature. 46 2/1/2012

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Random walk and Nonequilibrium theory This describes chromatographic separation in terms of a random walk using a statistical concept, the spreading of a solute band due to molecular diffusion, mass transfer, and flow pattern effects are equated to standard deviation σ or variance σ 2 terms. 47 2/1/2012

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INSTRUMENTATION Carrier gas Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Recorders & Integrators

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CARRIER GAS » Hydrogen better thermal conductivity disadvantage: it reacts with unsaturated compounds & inflammable » Helium excellent thermal conductivity it is expensive » Nitrogen reduced sensitivity it is inexpensive

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Requirements of a MOBILE PHASE Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance

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Flow regulatores & meteres Flow regulators are used to deliver the gas with uniform pressure or flow rate Flow rates of carrier gas: – Linear flow rate (cm/s): u = L/ tr – Volumetric flow rate (mL/min): u (π r2) L is length of column, tr is retention time, r is the internal radius of column Flow rate depends on type of column – Packed column: 25-100 mL/min – Capillary column: 1 to 25 mL/min Flow rate will decrease as column T increases 51

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Soap Bubble Meter ◊ Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate

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Soap bubble flow meter Aqueous solution of soap or detergent 54 soap bubbles formed indicates the flow rate. Glass tube with a inlet tube at the bottom. Rubber bulb-----store soap solution When the bulb is gently pressed of soap solution is converted into a bubble by the pressere of a carrier gas &travel up. Soap bubble meter

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55 inlet tube

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Injection Devices Gases can be introduced into the column by valve devices liquids can be injected through loop or septum devices

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Sample injection port Calibated Microsyringes are used to inject liquid sample Purge :volatile components are removed from sample by gentle heating Rubber or silicone diaphragm(septum) Sample port T: 50°C Packed C: sample sizes-1 to 20 μL Capillary C: 10 to 30 mL splitter is used to deliver a fraction of injection(1:50 to 1:500) Avaid over loading Slow injection & oversized samples cause band spreading & poor resolution 57

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58

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59 Micro syringe

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COLUMNS Important part of GC Made up of glass or stainless steel Glass column- inert , highly fragile COLUMNS can be classified Depending on its use 1. Analytical column 1-1.5 meters length & 3-6 mm d.m 2. Preparative column 3-6 meters length, 6-9mm d.m

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Depending on its nature 1.Packed column: columns are available in a packed manner S.P for GLC: polyethylene glycol, esters, amides, hydrocarbons, polysiloxanes … 2.Open tubular or Capillary column or Golay column Long capillary tubing 30-90 M in length Uniform & narrow d.m of 0.025 - 0.075 cm Made up of stainless steel & form of a coil Disadvantage: more sample cannot loaded

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3.SCOT columns (Support coated open tubular column Improved version of Golay / Capillary columns, have small sample capacity Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column Then coated with a thin film of liquid phase

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Columns Packed Capillary

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Some common liquid stationary phases used in GLC : STATIONARY PHASE TRADE NAME MAX. TEMP, o C APPLICATIONS Polydimethyl siloxane OV-1, SE-30 350 General purpose nonpolar phase, hydrocarbons, polynuclear aromatics, steroids. 5% Phenyl - Polydimethyl siloxane OV-3, Se-52 350 Fatty acid methyl esters, alkaloids, drugs, halogenated compounds. 50% phenyl - Polydimethyl siloxane OV-17 250 Drugs, steroids, pesticides, glycols. 50% Trifluoroproply - Polydimethyl siloxane OV-210 200 Chlorinated aromatics, nitro aromatics, alkyl substituted benzenes Polyethylene glycol Carbowax 20M 250 Free acids, alcohols, ethers, essential oils, glycols. 50% Cyanopropyl - Polydimethyl siloxane OV-275 240 Polyunsaturated fatty acids, rosin acids, free acids, alcohols. 66

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Equilibration of the column Before introduction of the sample Column is attached to instrument & desired flow rate by flow regulators Set desired temp. Conditioning is achieved by passing carrier gas for 24 hours

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Temperature Control Devices Preheaters : convert sample into its vapour form, present along with injecting devices Thermostatically controlled oven : temperature maintenance in a column is highly essential for efficient separation. Two types of operations Isothermal programming:- Linear programming:- this method is efficient for separation of complex mixtures Preheaters : convert sample into its vapour form, present along with injecting devices Thermostatically controlled oven : temperature maintenance in a column is highly essential for efficient separation. Two types of operations Isothermal programming:- Linear programming:- this method is efficient for separation of complex mixtures Preheaters : convert sample into its vapour form, present along with injecting devices Thermostatically controlled oven: temperature maintenance in a column is highly essential for efficient separation. Two types of operations Isothermal programming:- Linear programming:- this method is efficient for separation of complex mixtures

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Temperature Control Isothermal Gradient Instrumentation - Oven

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DETECTORS DETECTORS DETECTORS Characteristics of an Ideal detector : Adequate sensitivity : Generally a detector should detect the solute in picogram quantities. Good stability and reproducibility. A linear response to solutes that extends over several orders of magnitude. A temperature range from room temperature to atleast 400 o C. A short response time that is independent of flow rate. High reliability & ease of use. Similarity in response toward all solutes or, alternatively, a highly predictable & selective response toward one or more classes of solutes. Non destructive of sample.

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TYPES OF DETECTORS (1) THERMAL CONDUCTIVITY DETECTOR (2) FLAME IONISATION DETECTOR (3) ELECTRON CAPTURE DETECTOR (4) NITROGEN PHOSPHORUS DETECTOR

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Thermal Conductivity Detector (T.C.D) or Kathorometer: Principle: The rate of heat loss from a heated wire placed in a gas stream depend on thermal conductivity of the gas thus if the composition of gas stream changes,the rate of heat loss from the wire will change and so both the temperature &electrical resistance of the wire will be different.

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TCD filaments are made up of fine platinum, gold or tungsten wire. TCD thermistors are made of Oxides of manganese, Cobalt or nickel to which some trace elements are added. Advantages : Simple. Large linear dynamic range. It gives general response to both organic & inorganic species. Non destructive character, which permits collection of solutes after detection. Limitation : Low sensitivity. 73

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Relative Thermal Conductivity Compound Relative Thermal Conductivity Ohm -1 Carbon Tetrachloride 0.05 Benzene 0.11 Hexane 0.12 Argon 0.12 Methanol 0.13 Nitrogen 0.17 Helium 1.00 Hydrogen 1.28 75

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Flame Ionization Detector: Principle & working: When organic compounds introduced into a hydrogen –oxygen flame they are readily pyrolysed & ions produced which can be collected at a charged electrode & resulting current measured by an electrometer amplifier.

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Advantages: It is not sensitive for moisture &permanent gases like CO, CO 2 , SO 2 , NO 2 , NO, SiF 4. It gives high sensitivity. It has large linear range,. Lt produce low noise & easy to use. Disadvantages: It is destructive of sample. More complicated & more expensive.

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Electron Capture Detector: Construction : 1) Radio active material metal foil. 2) Anode and cathode electrode. 3)Potential difference of 20V to100V.

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Principle &working: The electron capture detector operates on the basis of differential electron absorption by molecules or functional groups within the molecules. One of the electrodes has on its surface a radioisotope that emits high energy electrons.(beta particles)

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These electrons bambard the carrier gas ,resulting in the formation of a plasma of positive ions,radicals &thermal electrons. N 2 ---------  N 2 + + e ¯ Electron absorbing compounds in the carrier gas stream react with the thermal electrons produce negative ions of larger mass. H + e ¯ ---------  H ¯ + Energy

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These electrons bambard the carrier gas ,resulting in the formation of a plasma of positive ions,radicals &thermal electrons. N 2 ---------  N 2 + + e ¯ Electron absorbing compounds in the carrier gas stream react with the thermal electrons produce negative ions of larger mass. H + e ¯ ---------  H ¯ + Energy

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Advantages: Highly sensitive for halogenated compounds,nitrogen compounds. Not altering sample significance. Detected compounds at 50 FG –1 PG. Level. Disadvantages: More contaminated detector than the other detectors It has high cost. Its linearity is poor.

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DETECTOR SELECTIVITY SENSITIVITY Gm solute/sec. LINEAR RANGE COMMENTS TYPE TCD NO 10 -8 10 4 Universal sensitivity, non destruction Conc. ECD HALOGEN 10 -13 10 2 Detect Halogen,oxygenated &highly conjugated compounds Conc. FID NO 10 -11 10 7 Detect all organic compound,most widely used G.C. detector destructive . Mass NPD NITROGEN, PHOSPHORUS, HALOGEN 10 -13 10 6 Mass Detects phosphorous ,Nitrogen,halogen

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REFERENCES: (1) Pharmaceutical Analysis By Munson J.W. Part A,Page No.16. (2) Instrumental methods of Analysis by Willard M. Page no.464 (3) www.pharmainfo.net

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The science and practice of pharmacy 21 st edition, -Remington , page no- 648-654 Pharmaceutical chemistry 4 th edition, -Bucket and Stenlake page no-129-149 The principles of instrumental analysis 5 th edition, - Douglas A Skoog p.no-701-722 Instrumental methods of chemical analysis, - Gurdeep r chatwal p.no-2.673-2.697 Internet sources: Morden pharmaceutical analysis aseminar report on gas chromatograph instrumentation by Pradeep H.K Theories of gas cg by Sumenth C.K An article of gc theory updated . An article from the hand book for organic chemistry lab. REFERENCES:

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Thank You