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  1. 1. GOKARAJU RANGARAJU COLLEGE OF PHARMACY Presented by : Ch.Nikhila 170213884003 Dept. of Pharmaceutical chemistry. Under the guidance of M/s. CEEMA MATHEW, Asst.Prof. 1
  3. 3. INTRODUCTION:  CHROMATOGRAPHY: Chromatography is a separation of mixture into individual components by using a stationary phase and a mobile phase. There are various advanced chromatographic techniques, widely used for the estimation of multicomponent drugs in their formulations like:  High Performance Liquid Chromatography  High Performance Thin Layer Chromatography  Gas Chromatography 3
  4. 4. Gas chromatography In Gas chromatography, the components of a vapouraised sample are fractionated as a consequence of a partition between a mobile gaseous phase and a stationary phase held in a column. According to the nature of stationary phase, Gas chromatography may be (a). Gas solid chromatography [GSC] (b). Gas liquid chromatography [GLC] Carrier gas column detector Basic chromatographic arrangement 4
  5. 5. GSC not used because of limited no. of S.P GSC principle is ADSORPTION GLC principle is PARTITION Chromatographic Separation Mobile Phase – inert gas used as carrier. Stationary Phase– liquid coated on a solid or a solid within a column. The mixture of compounds to be separated is converted into vapor And mixed with gaseous M.P Component more soluble in the S.P → travels slower Component less soluble in the S.P → travels faster Components are separated according to their Partition Co-efficient Criteria for compounds to be analyzed by G.C 1.VOLATILITY: 2.THERMOSTABILITY: 5
  6. 6. HO-CH2-CH2-(O-CH2-CH2)n-OH polyethylene glycol 6
  7. 7. How a Gas Chromatography Machine Works  First, a vaporized sample is injected onto the chromatographic column.  Second, the sample moves through the column through the flow of inert gas.  Third, the components are recorded as a sequence of peaks as they leave the column. 7
  8. 8. PRINCIPLE: In GLC, the main principle 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 converted to vopour and mixed with gaseous mobile phase. The component which is more soluble in stationary phase travels slower and eluted later. The components which is less soluble in stationary phase travels faster and eluted out first. No two components has the same partition co-efficient for a fixed combination of stationary phase , mobile phase. Hence the components are separated according their partition coefficients. 8
  9. 9. THE CHROMATOGRAPHIC PROCESS - PARTITIONING (gas or liquid) MOBILE PHASE Sample in Sample out STATIONARY PHASE (solid or heavy liquid coated onto a solid or support system) 9
  10. 10. PRACTICAL REQUIREMENTS Carrier gas  Flow regulators & Flow meters  Injection devices  Columns  Temperature control devices  Detectors  Recorders & Integrators  10
  12. 12. Hardware and Columns 12
  13. 13. INSTRUMENTATION: o Carrier Gas: He, Ar, N2, H2 o Flow regulators & flow meters: o Injection Port: Rubber septum barrier (usually maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture) o Column: (fused silica with a thin coating of stationary phase on the inner surface) o Oven: Thermostat controlled forced air oven o Detector: o Data System: recorders & integrators 13
  14. 14. CARRIER GAS: Gas Supply Unit Carrier gas should be:  Inert, high purity, easily available.  Low cost, due to large quantities are used  Allow the detector or respond in an adequate manner  less risk of explosion or hazards.  Cheap Should not cause the risk of fire, Should give best column performance 14
  15. 15. CHOICE OF CARRIER GAS: Name of the carrier gas Hydrogen Advantages    Helium    Nitrogen    Disadvantages Cheap Gives the most time efficient separation Still very efficient at high gas velocities i.e.. 60 cm/ sec  Very inert, will not react with analytes Gives a very time efficient separation Non flammable  Cheap Very inert, will not react with analytes Non flammable     Can form an explosive mixture with air is a reductive gas Expensive A non-replenishable resource Very slow velocity to achieve good efficiency Narrow range for maximum efficiency 15
  16. 16. FLOW REFULATORS & METERS  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: 29-150 mL/min – Capillary column: 1 to 25 mL/min  16
  17. 17. FLOW REGULATORS: To regulate pressure& control the gas flow through the separation column. They are two types : 1. Rotometer 2. soap bubble flow rate ROTOMETER: It is like a burette with a float held on to a spring placed before column inlet it has a glass tube with a float held on a spring. the level of the float is determined by the flow rate of carrier gas 17
  18. 18. Soap bubble meter     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 pressure of a carrier gas &travel up. Aqueous solution of soap or detergent 18 Soap bubble flow meter
  19. 19. inlet tube 19
  20. 20. Injection Devices   Gases can be introduced into the column by valve devices liquids can be injected through loop or septum devices Micro syringes injection port Gas tight syringe microsyringe 20
  21. 21. 21
  22. 22. Micro syringe 22
  23. 23. COLUMNS Packed column 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 diameter 2. Preparative column 3-6 meters length, 6-9mm diameter  Columns in GC are two types based on its nature: 1) packed column 2) capillary column (open tubular column) Capillary column 23
  24. 24. Types of open tubular column: Solid support coated with liquid phase Liquid phase Wall-coated Open Tubular (WCOT) Support-coated Open Tubular(porous layer open tubular column) (SCOT) 24
  25. 25. 1.Packed column: 2.Open tubular column MADE UP OF Glass or metals columns are available in a packed manner Better resolution – efficient mass transfer between gas and SP Tubing – fused silica, glass, copper, stainless steel Long capillary tubing 30-90 M in length Uniform & narrow diameter of 0.025 - 0.075 cm Disadvantage: more sample cannot loaded 3.SCOT columns (Support coated open tubular column Improved version of 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 25
  26. 26. CHARACTERISTICS Type of Column FSOT WCOT SCOT Packed 10-100 10-100 10-100 1-6 0.1-0.3, 0.53* 0.25-0.75 0.5 2-4 2000-4000 1000-4000 600-1200 500-1000 Sample size (ng) 10-75 10-1000 10-1000 10-106 Relative pressure Low Low Low High Relative speed Fast Fast Fast Slow Flexible Yes No No No Chemical inertness Best Length (m) ID (mm) Efficiency (Plate/m) Poor 26
  27. 27. Equilibration of the column  Column is attached to instrument & desired flow rate by flow regulators  Set desired temperature.  Conditioning is achieved by passing carrier gas for 24 hours Temperature Control Devices Pre heaters: convert sample into its vapor 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 program:Linear programming:- this method is efficient for separation of complex mixtures. 27
  28. 28. The oven Inside here Column 28
  29. 29. Instrumentation - Oven Temperature Control • Isothermal • Gradient 240 Temp (deg C) 200 160 120 80 40 0 0 10 20 30 40 50 60 Time (min) 29
  30. 30. DETECTORS  Heart of the apparatus The requirements of an ideal detector are Applicability to wide range of samples.  Rapidity.  High sensitivity even small concentrations.  Linearity.    Response should be unaffected by temperature, flow rate… Non destructive. Simple & inexpensive. Instrumentation - Detectors • Thermal Conductivity (TCD) • Electron Capture (ECD) • Argon ionization detector ( AID) • Flame Ionization (FID) 30
  31. 31. Thermal Conductivity Detector (T.C.D) or Katharometer: • TCD is based upon changes in thermal conductivity of gas stream. • It consists of two cells i.e. reference cell and sample cell. • It is in the form of a wheat- stone bridge. • When carrier gas is passing there is no deflection in the galvanometer. • But when the column effluent is allowed to pass there is deflection in the galvanometer. • This deflection is recorded which is due to change in the thermal conductivity. 31
  32. 32. Advantages Linearity is good Applicable to most compounds Non destructive Simple & inexpensive Disadvantages Low sensitivity Affected by fluctuations in temperature and flow rate Biological samples cannot be analyzed 32
  33. 33. 33
  34. 34. Flame Ionization Detector    Organic compounds are readily pyrolysed when introduced into a hydrogen-oxygen flame and produce ions in the process. The ions can be collected at a charged electrode and the resulting current measured by electrometer amplifier. In FID the effluent gases are mixed with hydrogen and burned in presence of oxygen Advantages: •µg quantities of the solute can be detected •Stable •Responds to most of the organic compounds •Linearity is excellent Disadvantages: destroy the sample 34
  35. 35. Argon ionization detector Depends on the excitation of argon atoms to a metastable state, by using radioactive energy. Argon→ irradiation Argon + e- →collision MetastableArgon→ collision of sub. → Ionization →↑Current ADVANTAGES 1.Responds to organic compounds 2.High sensitivity DISADVANTAGES 1.Response is not absolute 2.Linearity is poor 3. Sensitivity is affected by water  35
  36. 36. Electron Capture Detector: ECD is based upon electron affinity of the molecule. It is composed of a radio active source which emits electrons. When the effluent from the column is allowed to flow through the chamber the electron are absorbed and current is observed. Construction : 1) Radio active material metal foil. 2) Anode and cathode electrode. 3)Potential difference of 20V to100V. 36
  37. 37.  If a compound is present that contains electronegative atoms, those electrons are captured and negative ions are formed, and rate of electron collection decreases  The detector selective for compounds with atoms of high electron affinity. This detector is frequently used in the analysis of chlorinated compounds   e.g. – pesticides, polychlorinated biphenyls ADVANTAGE: Highly sensitive DISADVANTAGE: Used only for compounds with electron affinity 37
  38. 38. RECORDERS & INTEGRATORS Record the baseline and all the peaks obtained. INTEGRATORS Record the individual peaks with Retention time, height. 38
  39. 39. Derivatisation of sample  Treat sample to improve the process of separation by column or detection by the detector  They are 2 types  Pre column derivatisation : this is done to improve some properties of the sample for separation by column. By this method Components are converted to volatile & thermo stable derivative. in following Conditions - Pre column derivatisation is done  if Component is less volatile  if Compounds are thermo labile( heat sensitive)  to decrease tailing & to improve separation  Ex: sugars, COOH, alcohols , phenols are converted to less polar by using( bis trymethyl silyl acetamide reagent )  They can also be converted to acetyl derivative or triflouro acetyl derivative. 39
  40. 40. Post column derivatisation  This is done to Improve response shown by detector  This is online technique where flow rate is neither stopped nor changed.  The components may not be detected by detector unless derivatisation is done.)  The components may be converted in such away that their ionization or affinity towards electrons is increased. Pretreatment of solid support  To overcome tailing .  Generally doing separation of non polar components like esters, ethers…. Techniques: 1. use more polar liquid S.P. 2. Increasing amount of liquid phase. 3.Pretreatment of solid support to remove active sites. 40
  41. 41. DERIVATISATION METHODS Derivatisation prior to GC is often desirable to 1.improve the thermal stability of compounds, particularly compounds that contain polar functional groups. 2.Change the separation properties of compounds by the purposeful adjustment of their volatility. TYPES OF DERIVATISATION METHODS: 1.Silylation 2.Alkylation 3.Acylation 41
  42. 42. SILYLATION OH O Cl O 2,4-dichlorophenoxyacetic acid (A cancer suspect agent). Cl (a) This is the most common type of derivation techniques used in GC. (b) It involves replacing an active hydrogen on the solute (i.e. R-OH, RCOOH, R-NH2, etc.) with an alkylsilyl group (usually –SiMe3). The result of this reaction is that the solute is converted into a less polar, more volatile and more thermally stable form. (c) The most common reagent used in silylation is trimethylchlorosilane (TMS). Examples of its use are shown below: R OH + Cl Si Si R O Me3 Me3 + HCl SiMe3 OH O Cl Cl O + Cl Si Me3 O Cl Cl O 42
  43. 43. (d) Besides trimethylchlorosilane, a number of other silylation reagents can also be used. These reagents have slightly different reactivity from trimethylchlorosilane. The byproduct of BSTFA is highly Volatile. N, O-Bis(trimethylsilyl)acetamide N,O-bis(Trimethylsilyl)trifluoroacetamide BSA and BSTFA are highly stable TMS derivatives, with most organic functional groups, under mild reaction conditions. Me3Si R OH + F3C Si O N SiMe3 R O O Me3 + F3C N SiMe3 43
  44. 44. (e) Alkylation I. Alkylation involves the addition of alkyl group to some active function group on the solute. A common example is esterification of a carboxylic acid, forming a volatile methyl ester. This is commonly done using borontrifluoride in methanol as the reagent. RCOOH + BF3/MeOH RCOOMe (f) Acylation I. Acylation involves the conversion of a solute into an acylate derivates. This is often used to improve the volatility of alcohols, phenols, thiols and amine (e.g., -OH, -SH and -NH) containing compounds. As is true for other GC derivations, acylation can also be used to increase the response of a solute to a given detector (e.g., allowing the use of electron capture in solute’s detection by including fluorine atoms in the derivitizing agent. 44
  45. 45. ii. Trifluoroacetic anhydride (TFAA) is one common reagent used for acylation. NH + O COCF3 N-CO-CF3 + HOCOCF 3 COCF3 methamphetamine Drug-of-abuse confirmation testing by GC iii. Another set of reagents used for solute with primary and secondary amines, as well as hydroxyl and thiol groups are N-Methylbis[trifluoroacetamide] (MBTFA). The reaction is under mild nonacidic conditions. H Me CF3 N Byproduct is volatile O 45
  46. 46. ADVANTAGES       Very high resolution power, complex mixtures can be resolved into its components by this method. Very high sensitivity with TCD, detect down to 100 ppm It is a micro method, small sample size is required Fast analysis is possible, gas as moving phase- rapid equilibrium Relatively good precision & accuracy Qualitative & quantitative analysis is possible 46
  47. 47. Applications : G.C is capable of separating, detecting & partially characterizing the organic compounds , particularly when present in small quantities. 1, Qualitative analysis Rt & RV are used for the identification & separation 2, Checking the purity of a compound Compare the chromatogram of the std. & that of the sample 3, Quantitative analysis It is necessary to measure the peak area or peak height of each component 4, used for analysis of drugs & their metabolites. 47
  48. 48. REFERENCES :      Instrumental Methods Of Chemical Analysis By Gurdeep R Chatwal &Sham K Anand pg no Principles Of Instrumental Analysis By Skoog Holler Niemen Page No.686 Willard merit, Dean settle, instrumental methods of analysis, 7th ed. 48
  49. 49. 49