Gas chromatography
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The document provides a comprehensive overview of gas chromatography, detailing its introduction, principles, instrumentation, and various applications in analytical chemistry. It discusses the types of chromatography, the importance of carrier gases, sample injection methods, and the function and characteristics of detectors. Various applications in pharmaceuticals, environmental analysis, and food quality control are highlighted, emphasizing its significance in research and industry.
Gas chromatography
- 1. GAS CHROMATOGRAPHY Mr. SHINDE G.S PRAVARA RURAL COLLEGE OF PHARMACY, PRAVARANAGAR DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
- 2. Contents :- 2 Introduction Principle Instrumentation Working Evaluation Applications Reference
- 3. Ganesh Shashikant Shinde Pravara Rural college of pharmacy, pravaranagar
- 4. The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950) 4
- 5. INTRODUCTION:- 5 Gas chromatography – “It is a process of separating component(s) from the givencrude drug by using a gaseous mobilephase.” It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the columnby the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.
- 6. Twomajor types: • Gas-solid chromatography: Here, the mobile phase is a gas while the stationary phase is asolid. Used for separation of low molecular gases, e.g., air components, H2 S, CS2 ,CO2 ,rare gases, CO and oxides of nitrogen . • Gas-liquid chromatography: The mobile phase is a gas while the stationary phase is a liquid retained on the surface as an inert solid by adsorption or chemicalbonding. 6
- 7. Principles: 7 The principle of separation in GC is“partition.” The mixtureof component to be separated is converted to vapourand mixed with gaseous mobilephase. The component which is more soluble in stationary phase travel slower and eluted later. The component which is less soluble in stationary phase travels faster and eluted outfirst. No two components has same partition coefficient conditions. So thecomponentsare separated according to their partitioncoefficient. Partition coefficient is “the ratio of solubility of a substancedistributed between two immiscible liquidsat a constanttemperature.’
- 8. Instrumentation:- 8 Carriergas - He (common), N2, H2, Argon Sample injectionport - micro syringe Columns Detectors Thermal conductivity (TCD) Electron capture detector(ECD) Flame Ionization detector(FID) Flame photometric(FPD)
- 9. Gas chromatography Video Demonstration
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- 11. Carrier gas:- 11 The cylinder/ gas tank is fitted with a pressure controller to control the pressure of gas, a pressure gauge that indicates the pressure, a molecular sieve to transfer filtered drygas and a flow regulatortoensurea constantrateof flow of mobile phase to thecolumn. It should meet the followingcriteria: Should be chemicallyinert Should be cheap and readilyavailable Should beof high qualityand notcauseany fire accidents Should give best possibleresults Should be suitable for the sample to beanalyzed and for thedetector
- 12. Hydrogen, helium, nitrogen and carbon dioxide are commonlyused. Hydrogen has low density andbetter thermal conductivity. However, it reacts with unsaturated compounds and is inflammable and explosive in nature. Nitrogen is inexpensive but itgives reduced sensitivity. He is the most preferredgas. Inlet pressure ranges from: 10-50psi -Flow rate : 25-150 mL/minfor packed columns -Flow rate: 2-25 mL/min foropen tubularcolumn 12
- 13. Sampling unit:- 13 Sampling unitor injection port is attached to the columnhead. Since the sample should be in vapourized state, the injectionport is provided with an oven that helps to maintain its temperature at about 20-500 C above the boiling point of thesample. Gaseous samples may be introduced by useof agas tight hypodermic needle of 0.5-10 ml capacity. For Liquid samples , microsyringes of 0.1-100µL capacity may beused. Microsyringe
- 14. Injections of samplesinto capillarycolumns:- a. Split injections- it splits the volume of sample stream into twounequal flows by means of a needle valve , andallow thesmallerflow to passon to the columns and the bigger part is allowed to be vented to the atmosphere. This technique is not suitable when highest sensitivity is required. 14
- 15. b. Splitless injectors- They allow all of the sample to pass through the column for loading. Sample should beverydilute toavoid overloading of thecolumn and a high capacity column such as SCOT or heavily coated WCOT columns should beused. c. On column injectors: A syringe with a very fine quartz needle is used. Aircooled to -200c below the b.p. of the sample. After then the warmer air is circulated to vaporize the sample. 15
- 16. d. Automatic injectors: For improving the reproducibility and if a large number of samples areto be analyzed or operation is required without an attendant, automatic injectors areused. • The solid samples are introduced as a solution or in a sealed glass ampoule, crushed in the gas stream with the helpof a gas tight plunger, and the sample gets vapourized and flows intocolumn under the influence of carriergas. 16
- 17. Column unit:- Columnsare of different shapes and sizes that includes: “U” tube typeorcoiled helix type. They are mainly made of copper, stainless steel, aluminium, Glass, nylonand othersynthetic plastics. Support material:- it’s main function is to provide mechanical supportto the liquid phase. An ideal supportshould havea large surface area, chemically inert, should get uniformly wet with liquid phase, should be thermostable. Commonly used solid phases are: diatomaceous earth or kieselguhr, glass beads, porous polymers,sand,etc. 17
- 18. Liquid phase:- It should have the followingrequirements: It should be non-volatile Should have high decomposition temperature Should be chemicallyinert Should posses lowvapourpressure atcolumn temperature Should be chemically and structurally similarto thatof the solute i.e., polar for polarsolute. 18
- 19. Examples of different liquid phases: 19 CATEGORY EXAMPLES Non-polar hydrocarbon phases Paraffin oil (nujol), siliconoil, silicon rubber gum (used for high temp of about4000 Polar compounds (having polar groups like -CN, -CO and –OH) Polyglycols (carbowaxes) Liquids having hydrogen bonding Glycol, glycerol, hydroxy acids
- 20. Types of columns:- • There are two general types ofcolumns: 1. Packed columns:- In GLC, they are denselypacked with finely divided, inert, solid supportmaterial ( diatomaceous earth) coated with liquidstationary phase. In GSC, thecolumnsare packed with adsorbents or porouspolymers. Length- 1.5 - 10m internal diameter- 2 - 4mm. 1. Capillary columns- length ranges from 10-100m inner diameter is usually0.1-0.5mm. 20
- 21. • It is mainly of twotypes: Wall-coated columns - consist of a capillary tube whosewallsarecoated with liquid stationary phase. Support-coated columns- the inner wall of the capillary is lined with a thin layer of support material such as diatomaceousearth, ontowhich the stationary phase has been adsorbed. It is also known as PLOT (porous-layer open tubularcolumns). SCOTcolumnsaregenerally lessefficient than WCOT columns. Both types of capillary column are more efficient than packedcolumns. 21
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- 23. Equilibrium of the column:- • The packed columns areequilibrated before introduction of the sample. This is done by allowing continuous flow of heated carrier gas through the columns for a specific duration of time (24hrs) at prescribedtemperature. • Ideally prepared and conditioned columns show a zero base lineon the recorder upon passage of carriergas alone. Column temperature:- • This can be controlled by jackets equipped with vapours of a boiling liquid, electrically heated metal blocks or circulating air baths. • Compounds of low B.P- eluted at lowertemperature • Compounds of high B.P- boils at highertemperature resulting in broader and shallower peaks, require temperature programming. 23
- 24. Detectors:- 24 The eluted solute particles along with the carrier gas exit from the column and enter thedetector. Thedetectorthen produces electrical signals proportional to the concentration of the components of solute. The signals areamplified and recorded as peaks at intervals on thechromatograph.
- 25. Properties of an ideal detector: 25 Sensitive Operate at high T (0-400 °C) Stable and reproducible Linearresponse Wide dynamicrange Fast response Simple (reliable) Nondestructive Uniform response to allanalytes
- 26. I. Thermal conductivitydetector:- 26 “TCD is based upon the fact that the heat lost from a filament depends upon the thermal conductivity of the stream of surrounding gasas well as its specificheat.”
- 27. When only carrier gas flows heat loss to metal block is constant, filament Tremains constant. When an analyte species flows past the filament generally thermal conductivity changes, thus resistance changes which is sensed by Wheatstone bridgearrangement. The imbalance between control andsample filament temperature is measured and a signal is recorded. 27
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- 29. • Advantages :- Simple and inexpensive Durable and posses long life Accurate results Non-selective, hence known as universaldetectors • Disadvantages:- Low sensitivity Affected by fluctuations in temperature andflow rate. 29
- 30. II. Electron capturedetector:- 30 Molecules of compounds, which posses affinity for electrons, differ in their electron absorbing capacities. This difference is utilized in this detectorfor identificationof the compounds. Working- A foil made up of a radioactive metal like Ni63 (β- emitter) is placed inside a Teflon coated cell which also containsacathodeand an anode. In the absence of organic species, the produced electrons migrate towards positive electrode and produce a certain constant standingcurrent. When a sample/eluent is present itcaptures theelectrons, elutes from column, there is adrop in this constantcurrent. The potential across twoelectrodes is adjusted tocollectall the ions and a steady saturation current, is therefore, recorded.
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- 32. Advantages:- Highly selective Highly sensitive for thedetectionof compounds like halogens, quinones, peroxides, nitrites,etc. It is non-destructive More sensitive than TCD andFID. Disadvantages:- Least sensitive tocompoundswhose molecules have negligible affinity forelectrons. Carriergas used should beof pure form like pure nitrogen. 32
- 33. III.Flame ionizationdetector:- 33 This employs hydrogen flame that is maintained in a small cylindrical jet made up of platinum or quartz. Effluent from the column with helium or nitrogen as carrier gas are fed into the hydrogen flame, gets ignited and undergoes pyrolysis to produceions. Fordetection of these ions, twoelectrodes are used that provide a potentialdifference. The ions produced are repelled by the positive electrode which hit the collector plate. The current produced indoing so is amplified and fed to an appropriaterecorder.
- 35. IV.Flame photometricdetector:- 35 It is a selective detector that is responsive to compounds containing sulphur orphosphorous The detection principle is the formation of excited sulphur (S2*) and excited hydrogen phosphorous oxide species (HPO*) in areducing flame. A photomultiplier tube measures the characteristic chemiluminescent emission from these species. The optical filter can be changed toallow the photomultiplier toview light of 394 nm for sulphur measurement or 526 nm for phosphorus.
- 36. Flame photometric detector 36 Applications: -For detectionof heavy metals like chromium, selenium, tin, etc, in organometallic compounds. -Also for analysis of pesticides, coal, hydrogenated products as wellas air andwater pollutants.
- 37. Working:- 37 • Fill the syringe withsample. • Record the setting i.e., column temperature, detector temperature and injection port temperature. • Introduce sample into the injection port by completely inserting the needle into the rubber septum. Note down the injection time. • The samplegetsvapourized due to highertemperatureof injection port and is swept intocolumn bycarriergas. • This samplecomponents now get distributed between the gas and stationary liquid phase depending upon their solubilizing tendencies. • Thecomponents with minimal solubility move faster and those with maximum solubility travelslowly. • Thecomponents leaving thecolumn activatedetector and recorder to give aplot.
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- 39. The components that slowly leave the column give a bell shaped curveof shorterpeak while theonewhich travels faster gives a bluntly pointed curve of larger peak. In abovegraph, thecomponent that first emergesout of thecolumn is component 4 followed by 2,5,3 and 1. Thearea under thecurve is determined in order to obtain the percentage compositionof the mixture. 39
- 40. Evaluation :- 40 HETP (height equivalent to theoretical plate)- It is the distance on the column in which equilibrium is attained between the solute in thegas phase and thesolute in liquid phase. Larger the numberof theoretical plates/ smaller the HETP, the moreefficient thecolumn is forseparation. HETP = Length of column/n ; Where n = number oftheoretical plates Retention Time: defined as theabsolute time taken bya sample toshow maximum peak after injecting. Retention Volume: defined as thevolume of gas required to eluteabout half of thesolute through thecolumn. VR = tR ×F F =average volumetric flow rate (mL/min) ,estimated by using soap bubble meter (some gases dissolving in soap solution)
- 41. Applications:- 41 Qualitative Analysis – by comparing the retention time or volume of the sample to the standard / by collecting the individual components as they emerge from the chromatograph and identifying these compounds byother methods like UV, IR, NMR. Quantitative Analysis- area under a single component elution peak is proportional to thequantityof thedetected component/response factor of the detectors. It is doneby: I. Direct comparison method- A(sample) / A(std) = α C(sample)/C(std) Where, α is the response factordetermined forevery pure compound under givenconditions.
- 42. II. Calibration curve- a graph is plotted by taking peak areas on Y axis and concentration of standard compound on X axis. Concentrationof unknown sample is then determined byplotting its peak area on samegraph. III. internal standard method- A known concentration of internal standard, which has similar retention characteristics as that of sample is added to both reference standardand testsample. 42
- 43. Pharmaceutical applications- Qualitycontrol and analysis of drug products like antibiotics (penicillin), antivirals (amantidine), general anesthetics (chloroform, ether), sedatives/hypnotics (barbiturates), etc. Assayof drugs – purityof a compound can be determined for drugs like: Atropine sulphate Cloveoil Stearicacid In determining the levels of metabolites in bodyfluids like plasma, serum, urine,etc. 43
- 44. Miscellaneous: analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticides residues,trace elements. Pollutants like formaldehyde, carbonmonoxide, benzene, DDT etc. Dairy productanalysis like milk, butter–fordetection of aldehydes, milk sugars, ketones and fattyacids. Separation and identification of volatile materials, plastics, natural and synthetic polymers, paints,and microbiological samples. 44