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Includes…… Introduction Theory Parameters Instrumentation Reference 6/18/2012 2PowerPoint Presentation: Introduction: Chromatography is a physical process of Chromatography means………. separation in which the components to be separated are distributed between 2 immiscible phases, a stationary phase of large surface area and a mobile phase which is in constant motion throughout the stationary phase. Based on the principle of separation, it is classified as: 6/18/2012 3PowerPoint Presentation: 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 mixture, but the drawback is that it is not an accurate method. e.g. TLC, GSC, and column chromatography. 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 former one. e.g. GLC, HPLC. 6/18/2012 4PowerPoint Presentation: 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, which is rare. Principle: The principle of separation in GLC is Partition . The components are separated according to their partition co-efficients. 6/18/2012 5 Gas chromatographyTheory:: Theory: There are 3 theories: The Plate theory. The Rate theory. The Random walk, non-equilibrium theory. 6 6/18/2012Plate theory: Plate theory 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. 7 6/18/2012PowerPoint Presentation: The plate model supposes 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. Hypothetical zone in which two phases establish an equilibrium with each other 6/18/2012 8Theoritical plate:: Theoritical plate: 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). 9 6/18/2012PowerPoint Presentation: 10 Plate 6/18/2012Plate theory: 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) 11 6/18/2012PowerPoint Presentation: 12 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 6/18/2012 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.Plate theory: 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. 13 6/18/2012Chromatogram: Chromatogram 14 w b t r Injection point Air peak t r W 0.5 Detector response time Wb-band breadth Tr-retention time T’r-adjudted retntion time 6/18/2012PowerPoint Presentation: Volatility of compound : Low boiling (volatile) components will travel faster through the column than will high boiling 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. 6/18/2012 15Column efficiency parameters: 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. 16 6/18/2012 Column efficiency parameters: : 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. 17 6/18/2012Number of theoritical plates (n): Number of theoritical plates (n) Each component separated on 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. 18 6/18/2012Column efficiency parameters: : 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. 19 6/18/2012 Column efficiency parameters: : 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 20 6/18/2012PowerPoint Presentation: 21 Plate HETP Smaller the h, the more efficient the column. 6/18/2012Column efficiency parameters: : 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. 22 6/18/2012Column performance parameters: 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. 23 6/18/2012Resolution : 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. 24 W bA W bC W bB t rA t rB t rC time 6/18/2012Column performance parameters: Column performance parameters Separation factor A related term used with resolution. Describe relative retention of two consecutive peaks. Disadvantages is that it does not indicates the “sharpness” or resolution of two peaks. 25 6/18/2012PowerPoint Presentation: 26 A B A B Two chromatograms with the same α A/B , but different resolution factors. α A/B = t rA / t rB 6/18/2012Peak Asymmetry : 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. 27 6/18/2012PowerPoint Presentation: 28 0.1 h 0.1 h a b a b Time (min) Time (min) h h Determination Of Peak Asymmetry 6/18/2012Peak asymmetry : 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. 29 6/18/2012Factors that decrease the column efficiency are: : 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 6/18/2012 30Limitation of plate theory: 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. 31 6/18/2012Rate theory: 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: 32 6/18/2012Van Deemter equation: Van Deemter equation h= 2 λ d p + 2 γ DG / u + 8kd 2 f u/ n 2 (k+1) 2 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 33 6/18/2012Van Deemter equation: 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 experimentally. To obtain minimum h (maximum efficiency), the constants A, B, and C must be minimized. 34 6/18/2012Van Deemter equation: 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. 35 6/18/2012A PLOT OF h VERSUS u: A PLOT OF h VERSUS u 36 MOLECULAR DIFFUSION FINITE RATE OF MASS TRANSFER MULTIPATH TERM A C B LINEAR GAS VELOCITY, u HETP FINITE RATE OF MASS TRANSFER 6/18/2012 Explanation First term, A (2 λ dp ) : Explanation First term, A (2 λ d p ) The A term accounts for the flow pattern effects. The first term, A (2 λ d p ), depends on the packing material’s size and shape. This is called “eddy diffusion” which takes into account the different path lengths traveled by the solute molecules. 37 6/18/2012PowerPoint Presentation: 38 Final band width Initial band width 6/18/2012First term, A: 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 λ . The plot for the term A shows straight line as particle size does not vary with the gas velocity. 39 6/18/2012Second term, B (2 γ DG) : 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. 40 6/18/2012Second term, B (2 γ DG): 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. 41 6/18/2012The final term, C : 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 . 42 6/18/2012The final term, C: 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. 43 6/18/2012The final term, C: 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. 44 6/18/2012Random walk and Nonequilibrium theory: 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. 45 6/18/2012INSTRUMENTATION Includes…..: INSTRUMENTATION Includes….. Carrier gas tank Pressure regulator Flow controller Injection port Column Detector Signal processor 6/18/2012 46PowerPoint Presentation: 6/18/2012 47 DIAGRAM:CARRIERGAS…: CARRIERGAS… Chemically inert – helium , nitrogen and hydrogen. HYDROGEN – good conductivity, low density, reacts with unsaturated compounds. HELIUM – excellent conductivity, but expensive. NITROGEN – inexpensive, low sensitivity. Choice of gas is depends on the type of detector used. Gas supply is associated with pressure regulators, gauges and flow meters. In addition carrier gas system often contain a molecular sieve to remove water and other impurities. 6/18/2012 48Flow regulators: Flow regulators Rota meter. Soap bubble meter 6/18/2012 49PowerPoint Presentation: Rotameter Placed 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 the carrier gas is pre- calibrated. Soap bubble meter Similar to rotameter, instead of float a soap bubble is formed and indicate the flow rate. It has a glass tube with inlet. A rubber is used to store the soap solution, when the bubble is gently pressed a drop of solution is formed which converted into bubble by the pressure of carrier gas to travel up . The distance travelled upward is a measure of the flow rate of carrier gas. 6/18/2012 50PowerPoint Presentation: Injection Port - Sample introduction Manual - Direct Injection Automated - Autosampler 6/18/2012 51Injection devices: Injection devices 6/18/2012 52PowerPoint Presentation: 6/18/2012 53PowerPoint Presentation: 6/18/2012 54PowerPoint Presentation: Criteria for sample Should be volatile Should be thermostable Type of injection system is depends on the physical state of the sample. Samples are analysed by GC are usually liquid at room temp.so 1.5-10µl sample is injected via gas syringe. Pyrolysis equipment 6/18/2012 55Columns: Columns Packed Capillary 6/18/2012 56PowerPoint Presentation: 6/18/2012 57PowerPoint Presentation: Packed - As suggested by the term, it is filled with a coated inert solid support such as fire brick, alumina, and graphite with a specific mesh size. The coatings are called phases and for best results are chemically bonded to the support. Chemical bonding provides for longer column life and less bleeding (major source of background noise) contributing to lower sensitivity. Column dimensions 1/8” - 1/4” ID x up to about 6’ 2-20mm diameter, 1-4m length using glass or stainless steel. Advantages - higher capacity (higher conc). Disadvantages: low resolution and low S/N. Capillary - Here the phase (film) is coated on the inside diameter of the capillary wall with film thickness range of 0.1 to 5μ where the ticker film provides for better resolution but also allows for more bleed. Typical dimensions .25mm - .53mm ID x up to 60m, 10-100m length made of fused silica coated with polyamide and glass. Advantages: high resolution and better S/N. Disadvantages: low capacity and cost. 6/18/2012 58Types of columns : Types of columns Support coated open tubular column Wall coated Irradiated open tubular column whisker open tubular column Whisker walled open tubular column Porous layer open tubular column 6/18/2012 59Advantages of capillary column: Advantages of capillary column Diameter of the column is less,the number of different path through the column that the sample component can flow is minimized Heat transfer to a column with a smaller diameter is superior to that of the larger packed column All of these factors combine to produce better resolution 6/18/2012 60Different stationary phase: Different stationary phase Liquid crystalline st.phase Separation is depends on the geometrical shape and dipole-dipole interaction between the component and st.phase. E,g;- methyl siloxane,Phenyl methylsilicone, pmvinylsilicone Solid stationary phase Retention time in gsc is longer than glc 6/18/2012 61Instrumentation - Detectors: Instrumentation - Detectors Ideal characters Good and high sensitivity Good stability A short response time i.e, independent of flow rate High reliability Non destructive of sample Simple and easy to maintaine In expensive linearity 6/18/2012 62Instrumentation - Detectors: Instrumentation - Detectors Destructive Mass Spectral (CI/EI) Flame Ionization (FID) Nitrogen-Phosphorus (NPD) Flame Photometric (FPD) Electrolytic Conductivity 6/18/2012 63PowerPoint Presentation: NON-DESTRUCTIVE Flame ionization Thermal ionization Argon ionization Electron capture 6/18/2012 64References : References 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 6/18/2012 65Cont…: Cont… 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. 6/18/2012 66PowerPoint Presentation: Thank you……. 6/18/2012 67 Thank you… You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.