chromatography general principles and comparison - specially about gas chromatography)
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The document provides a comprehensive overview of chromatography, detailing its history, types, and applications, particularly focusing on gas chromatography. It outlines the principles of various chromatographic techniques, including their stationary and mobile phases, and compares normal and reverse phase chromatography. Additionally, it discusses the components and operational aspects of gas chromatography, including detectors and their advantages and disadvantages.
chromatography general principles and comparison - specially about gas chromatography)
- 2. Submitted to :- Dr. Ozair Alam Submitted by:- Shameer Course :- M .pharm (P.Analysis) Year & sem:- 1 yr. & 1 sem. Session :- 2020-2021
- 3. o Slight introduction to chromatography o Gas chromatography o Instrumentation o Applications o References
- 4. • Chromatography was first developed by the Russian botanist Michael Tswett in 1903 • In which he has separated colored pigments of plant (chlorophyll and xanthophyll) by percolating the petroleum ether extract through glass column packed with finely divided calcium carbonate • The separated species appeared as a color band on the column, which in turn accounts for the name
- 5. • The application of chromatography has grown explosively in the last half centaury, that just not only due to the development of several technique in this field but also due to the growing need by the scientist to study the more complex mixtures • Chromatography encompasses a diverse and important group of methods that allow the separation, identification and determination of closely related components of complex mixture many of these separation of mixtures are impossible by other method • Generally in all chromatographic method the sample is dissolve in a mobile phase, which can be a gas, liquid or a supercritical fluid. This mobile phase is then forced through an immiscible stationary phase, which is fixed in a column or on a solid surface
- 6. The two phases are chosen in such a way that the components of the sample distribute themselves between the two phases of varying degrees The components that are strongly retained by the stationary phase moves slowly with the flow direction of the mobile phase in comparison with the components that are not, (the weakly held components travels rapidly with the mobile phase) Flow of the mobile phase
- 7. There are different type for chromatography techniques on the basis of type of stationary phase and mobile phase used
- 8. • Based on the principle of selection Adsorption chromatography Partition chromatography • Separation on the basis of the affinity • Stationary phase – solid • GSC,TLC,CC & HPLC • Separation on the basis of partition coefficient • Stationary phase – liquid • GLC, Paper partition chromatography , column chromatography No two compounds have the same affinity for a combination of stationary phase , mobile phase and other condition No two components can have the same partition coefficient for a particular combination of stationary phase, mobile phase and other condition * The stationary phase as such can not be a liquid , hence a solid support is used over which a thin film or coating of liquid is made which acts as stationary phase
- 9. Based on the mode of chromatography Mode Normal phase chromatography Reverse phase chromatography Normal phase chromatography Reverse phase chromatography • Stationary phase is polar in nature (silica gel) & the mobile phase is non polar in nature Stationary phase is non polar in nature while the mobile phase is polar in nature • The normal phase chromatography is not much advantageous in the field of pharmaceutical as most of the drug are polar in nature • The reverse phase chromatography has advantage (as most of the pharmaceuticals preparation are polar in nature therefore they are not retained for a longer time and hence are eluted faster
- 10. Normal phase Reverse phase Stationary phase Polar Non-polar Mobile phase Non-polar Polar Compound eluted first and retained less Non-polar Polar Compound eluted last and retained more Polar Non-polar Example of stationary phase Silica gel ODS(C18),C8,C4 – bonded phases Comparison between normal phase and reverse phase chromatography
- 11. • Ion exchange chromatography :- an ion exchange resin is used reversible exchange of ions takes place between similar charged ions and with that of ion exchange resin • Gel permeation chromatography :- also known as gel filtration and size exclusion chromatography. A gel is used to separate the components of a mixture according to their molecular size • Chiral chromatography :-in this type of
- 13. • In gas chromatography the components of a polarized sample are separated as a consequence of being partitioned between a mobile phase and a liquid or a solid stationary phase held in a column • In performing gas chromatographic separation, the sample is vaporized and injected onto the head of a chromatographic column. • Elution is brought about by the flow of an inert gaseous mobile phase. • The separation of the component gets done on the basis partition coefficient • In comparison with most of the other type of chromatography, here the mobile phase doesn’t interact with the molecule of the analyte. • The only function which is to be performed by the mobile phase is just to act like the carrier for the analyte through the column
- 14. Types of gas chromatography Gas chromatography (GC) Gas-liquid chromatography (GLC) Gas-solid chromatography (GSC)
- 15. Gas liquid chromatography (GLC) Gas solid chromatography (GSC) • GLC finds widespread use in all field of science • In this the analyte is partitioned between a gaseous mobile phase and a liquid immobilized the surface of an inert solid packing or on the wall of a capillary tubing • GSC is based on a solid stationary phase in which retention of the analyte occur because of the physical adsorption • It has got limited application because of semi-permanent retention of active or polar molecule and also serve tailing of elution peaks. *Tailing is a result of the non-linear nature of the adsorption process *The concept of GLC was first suggested in 1914 by Martin & Synge (who were also responsible for he development of liquid-liquid partition chromatography
- 16. Carrier gas tank Flow regulator Sample injection chamber Column Detector Flow meter Data system Display Sampl e Oven Thermostat
- 17. • The mobile phase gas in GC is called as carrier gas • Most common mobile phase helium gas {although Argon, Hydrogen and Nitrogen} • These gases are available in pressurized tank and it’s requires pressure regulator, gauges, and flow meter to have a control over the flow rate {in addition the carrier gas system is also fitted with molecular sieve to remove impurities and water} • Flow rates are normally controlled by a two stage pressure regulator first at the gas cylinder and some sort of pressure regulator or flow regulator mounted in the chromatograph {inlet pressure 10-50
- 18. • Generally it is assume that the flow rate will be constant unless and until the inlet pressure remains constant • For the establishment of flow rate, rotometer at the head of column can be used however this device is not as accurate as simple soap-bubble meter • Usually the flow meter is located at the end of the column • Many modern computer-controlled gas chromatographs are equipped with electronic flow meters that can be regulated to maintain the flow rate at the desired level
- 19. • In order to achieve high column efficiency the sample must be of suitable size an introduces as a ‘plug’ of vapor {slow injection or oversize sample causes band spreading and poor resolution} • For this purpose micro-syringes are used to inject liquid samples through a rubber or silicone diaphragm or septum, into the heated sample port located at the head of the column • The sample port is kept at 50°C above the boiling point of the least volatile component of the sample • For ordinary packed analytical columns, sample size ranges from few tenths of a microliters to 20µL. • Capillary columns requires samples that are smaller by factor of 100 or more • Therefore a sample splitter is often needed to deliver a small known fraction (1:50 to 1:500) of the injected sample and rest of the sample will be wasted.
- 20. • Commercial gas chromatography intended for use with capillary columns incorporated with such splitters, they also allow for split-less injection to improve the sensitivity or for used with packed columns • Autoinjectors with automatic sampling trays are available for most of the higher gas chromatographs, that substantially improves the precision of the injected volume over the manual one
- 21. General type of column Open tubular (capillary) Packed • In earlier days vast majority od gas chromatographic analyses used packed columns • But as per for the current applications packed columns have been replaced by the more efficient Open tubular columns • Packed chromatographic columns vary in length from 1m to 5m , while the capillary Columns can have ranges from few meters to 100m • The material of construction for column are usually silica or stainless steel, although Teflon and glass can also be used for the purpose • In order to get fitted into the oven for thermostatting they are usually formed as coils having diameter ranges from 10 to 30 cm
- 22. • Column temperature is an important variable that must be controlled to a few tenths of a degree in order to get precise work, now that’s the reason for the column being ordinarily housed in a thermostatted oven • The optimal column temperature depends upon the boiling point of the sample and the degree of separation required roughly a temperature equal to or slightly above the average boiling point resulting in a reasonable elution time (for a samples with a broad boiling range it is often desirable to employ temperature programming in which the column temperature is increased either in a continuous or in steps as the separation proceeds)
- 23. • In market there are dozens of detector available which are incorporated with gas chromatography • In some cases, gas chromatographs are coupled with spectroscopic instruments like mass and infrared spectrometers. • Benefits of such system is that the spectral device not only detects the appearance of the analyte as they elute from the column but also helps to identify them • Adequate sensitivity (10-8 to 10-15 g solute/s) • Good solidity and reproducibility • A linear response to solutes that extends over several orders of magnitude. • A temperature to at least 400 °C • A short response time independent of flow rate • High reliability and ease of use • The detector should be nondestructive
- 24. Type Applicable sample Typical detection limit Flame ionization Hydrocarbon 1 pg/s Thermal conductivity Universal detector 500 pg/ml Electron capture Halogenated compounds 5 fg/s Mass spectrometer Tunable for any species 0.25 to 100 pg Thermionic Nitrogen and phosphorous compounds 0.1 pg/s (P), 1 pg/s (N) Electrolytic conductivity Compounds congaing halogens, sulphur, or nitrogen 0.5 pg cl/s, 2 pg S/s, 4 pg N/s Photoionization Compounds ionized by UV radiation 2 pg C/s Fourier transform IR Organic compounds 0.2 to 40 ng
- 25. Name of the detector Advantage Disadvantage Thermal conductivity detector • Applicable in most of the compounds • Good linearity • Sample is not destroyed & hence used at preparative scale • Simple, easy to handle and in- expansive • Low sensitivity affected by fluctuation in flow rate and temperature • Gives Relative response • Cannot analyze the biological sample • Flame ionization detector • The detector is extremely sensitive and background noise is low. Hence µg quantities of the solute can be detected • Stable and intensive to small changes in the flow rate of carrier gas and water vapor • Responds to most of the organic compounds • Linearity is excellent • Quite complex in handling • Require expertise guidance and supervision Argon ionization detector (AID) • Responds to most of organic compounds • Very much sensitive • Response is not absolute rather it is relative • Linearity is poor • Sensitivity is affected by water and is much reduced for halogenated compounds Electron capture detector (ECD) • high sensitivity • selectivity toward analyte with electronegative functional groups • nondestructive • nonlinear response under some circumstances • limited response range
- 27. Wall-coated open tubular (WCOT) Support-coated open tubular(SCOT). WCOT are simply capillary tubes coated with thin layer of the stationary phase. In SCOT the inner surface of the capillary is lined with a thin film (~30 µm) of a support material • Early WCOT column were constructed od stainless steel, aluminum, copper or plastic • Later glass column began to used • The most widely used capillary columns are fused silica wall coated (FSWC) open tubular columns
- 28. • The FSWC have much thinner wall then the glass columns. • Tubes have got extra strength by an outside protective polyimide coating. Which is applied when the capillary tubing is drawn. That results in columns having quite flexibility which in turn give them ease for being bent in coil having diameters of few inches. • Silica open tubular columns are available commercially and because they offer several important advantages (physical strength, much lower reactivity towards the sample components, and flexibility) for most of the applications, they have replaced the older type WCOT glass columns. • The most widely used silica open tubular columns have inside diameters of 0.32 and 0.25 mm. • Megabore columns are one of the new in the market. They can tolerate the sample sizes that are similar to those for packed columns they have significantly better performance characteristics then those of packed columns but not as much as that of smaller-diameter columns.
- 29. Types of solid phase Polarity Separation characteristi cs Application Operational temperature range (approx.) Methyl silicone Non-polar Boiling point order Petroleum, solvents, high boiling point compounds -60 to 360°C Phenylmethyl Slightly polar- moderately polar Phenyl group retain aromatic compounds. Perfumes, environmental compounds, aromatic compounds -60 to 340°C Cyanopropyl phenol Moderately polar-strongly polar Effective at separating oxygen- containing compounds, isomers, etc. Agriculture chemicals, oxygen containing compounds -20 to 280°C trifluropropyl Moderately polar-strong polar Specifically retains compounds that contains Halogen containing compounds, polar -20 to 340°C
- 30. • The modern glass columns are fabricated from glass or metal tubing • They are typically 2-3 m long and have inner diameter of 2-4 mm • These tubes are densely packed with uniform, finely divided packing material, or solid support, coated with thin layer (0.05-1 µm) of the stationary liquid phase. • The tubes are usually formed as coils with diameters of roughly 15 cm to permit convenient thermostatting in an oven
- 31. the packing or solid support in a packed column holds the liquid stationary phase in place so that as large a surface area as possible is exposed to the mobile phase. Ideal support • Small, uniform spherical particles • Good mechanical strength • A specific area of at least 1( m)2*/g • It should be inert (at elevated temperature) • And be uniformly wetted by the liquid phase (no material is yet available that meets all of these criteria perfectly) The earliest and the most widely used packing's for GC were prepared via naturally occurring diatomaceous earth
- 32. • The efficiency of a gas chromatographic column increases rapidly with decreasing particle diameter • The pressure difference required to maintain an acceptable flow rate of carrier gas varies inversely as the square of the particle diameter • The latter relationship has placed lower limits on the size of particles used in GC because it is not convenient to use pressure differences that are grater than about 50 psi • As a result the usual support particles are 60-80 mesh (250-170µm) or 80-100 mesh (170- 149µm)
- 33. Desirable properties of immobilized liquid phase in a gas liquid chromatographic column include • Low volatility (ideally the boiling point of the liquid should be at least 100°C higher than the maximum operating temperature fro the column) • Thermal stability • Chemical inertness • Solvent characteristics such that K & α {retention factor & selectivity factor} The retention time for an analyte on a column depends on its distribution constant, which in-turn is related to the chemical nature of the stationary phase
- 34. Stationary phase Common trade name Maximum temperature °C Common application Polydimethyl siloxane OV-1,SE-30 350 General-purpose non polar phase, hydrocarbons , poly- nuclear aromatics, steroids, PCBs 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% trifluropropyl- ploydimethyl siloxane OV-210 200 Chlorinated aromatics ,nitroaromatics, alkyl substituted benzenes Polyethylene glycol Carbowax 20M 250 Free acids, alcohols, ethers, essential
- 35. • Retention time • Retention volume • Separation factor • Resolution • Theoretical plate
- 36. G.C. Separation tool Detection tool In this role GC methods are unsurpassed when applied to complex organic, metal-organic And biochemical systems made up of volatile species or species that can be derivatised to Yield volatile substances In this GC plays role in the completion of an analysis, in this role retention time or volumes are used for qualitative identifications, and peak height or peak area provide quantitative information For qualitative purposes GC is much more limited than most of the spectroscopic methods
- 37. • Checking for the purity/impurity of the compound :- by comparing the chromatogram of the standard and that of the sample, the purity/impurity of the compound can be reported. If additional peaks are obtained then difference in purity/impurities are present and hence the compound is not pure/contain impurity . From the percentage area of the peaks obtained, the % purity/% impurity can also be reported. • Quantitative analysis It can be determined by different methods ▫ Direct comparison method ▫ calibration curve method ▫ Internal standard method • Isolation and identification of drugs or metabolites in urine, plasma, serum, etc. can be carried out. • Isolation and identification of
- 38. A textbook of principle of instrumental analysis A textbook of analysis by S. Ravi Sankar Wikipedia www.shimadzu.com Google for picture