GAS CHROMATOGRAPHY FOR B.PHARM AND M.PHARM STUDENTS BY P.RAVISANKAR.

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WHAT IS GAS CHROMATOGRAPHY?, INTRODUCTION,COLUMN OPERATION, INSTRUMENTATION AND DETECTION,APPLICATIONS. …

WHAT IS GAS CHROMATOGRAPHY?, INTRODUCTION,COLUMN OPERATION, INSTRUMENTATION AND DETECTION,APPLICATIONS.
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  • 1. Gas-liquid chromatography (often just called gaschromatography) is a powerful tool in analysis.Prof. p. RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.banuman35@gmail.com00919059994000GAS CHROMATOGRAPHY
  • 2. Chromatograph(or)ChromatogramColumn( A separation Technique)Injection portGas separator (or) Aerograph.Gas chromatography (GC) is most widely used analytical method for the separation ofvolatile and semi-volatile organic compounds without decompositionSmall amounts of sample For ex.1mlof air 1µl (microlitre) uL; 1/1000 of amL)the solutions either Liquids insolids in solutionThe compounds are separated primarily based on the relative(differences in their) volatilitiesWhat is gas chromatography?Genarates a written record of analysis
  • 3. All forms of chromatography involve a stationary phase and a mobile phase. In all the otherforms of chromatography you will meet at this level, the mobile phase is a liquid. In gas-liquid chromatography, the mobile phase is a gas such as helium and the stationary phase isa high boiling point liquid absorbed onto a solid.Introduction
  • 4. From the column, the separated solutes passthrough a detector where they are sensedgenerating an electronic signal. The signal isthen amplified and normally displayed on astrip chart recorder. The trace plotted on therecorder is called a "Chromatogram".It is a plot of the detector response in millivoltsas a function of time.Time is the Abscissa(horizontal or X-axis)Andmillivolts the Ordinate.(y- coordinate(verticalaxis)
  • 5. Gas Chromatograph ComponentsFlameIonizationDetectorColumnOvenInjection Portfront view
  • 6. One milligram in a kg is 1 ppm (by mass). Oneliter . so 1 mg/L is 1 ppmOne ppb represents one microgram ofsomething per liter of water (ug/l), or onemicrogram of something per kilogram of soil(ug/kg).GCwith a TCD the components can continue on to another detector afterpassing through the TCD; thus it is considered a non-destructive detector(this can be useful for further analysis.
  • 7. What are advantages of GC?The GC is one of popular instrument used in the worldSeveral advantages includeHigh resolution.High speed.High sensitivityThe GC is robust, flexible, and user-friendly.Most importantly, it has the best value in the market..High resolution :Many compounds can be resolved nicelyfor ex: Gasoline has been resolved in to over 300 different peaks complexsample of Petroleum. (complex mixture can be resolved in constituents)The term resolution refers to well separated two peaksare from each other.If two peaks are so close together that you can’t tell when one peakbegins and the other ends the resolution is poor; you can also say thatthe separation is poor.If you can clearly identify two different peaks- the resolution is good.The longer and more narrow the column, the better the resolution.Increasing the carrier gas flow rate and/or the temperature will sendthe vapours through the column faster, which will lower the retentiontime and worsen the resolution. Lowering the temperatureand/or flow rate increases retention times and broadens the peaks.
  • 8. Analyze in a matter of minutes and few compounds cananalysis in a matter of few seconds also possible.we see both rapid analysis as well as high sensitivity.(detectability) evenProviding selective(identification)For ex a 2 1/2 min of separation 3 common pesticidesMethyl parathion, Malathion, Ethion at pg levels. Pg 10-12 grams means these areparts per billion. (One trillionth of a gram )This is a very good ex of both the high speed aswell as a very sensitive detection..Methyl parathionMalathionEthion
  • 9. : It gives good precision and accuracy. (free from errors)Accuracy just means we do quantitative analysis we get a very good results writeanswer, good quantitative results. (possible or actual deviation from the exactanswer(exactness).GC is also a very easy technique compare to some very well known .most widely used instruments in the world todayGC and liquid chromatography together just been the premier techniques for Trace analysisof organic and inorganic compounds.All the work which has been done byAir pollutionand water pollutionand food safetywe hv. to analyze for pesticides toxic chemicals founds in Food and food products all ofthese things are done by routinely daily and rapidly by gc and or liquid chromatography.In essential role of chromatography is the QC and foods, and drugs control in raw materialand finished products ensuring the safety of the people. we are so dependent on the worldtoday on chemicals synthetic chemicals made by chemist.Primarily pesticides very good for agriculture and very harmful for humans. SoChromatography is the best separation technique for quantitative trace analysis of toxicchemicals.
  • 10. 1. GC the samples must be volatile.Allow to heat the samples up to 3000 and 3500C at that point we must generateVapors that can be easily carried by the carrier gas.2. Dirty samples require cleanup. dirty samples Duran,waste water Extracts of many things.and these samples normally contaminate the system andmay even Plug up the column and destroy the column and these case often timeswe will extracts with the Solvents to take off the imp components.3. Another limitation is we must use another instrument for ex a Mass spectrometerfor conformationTypically we use the retention time of standards of un knows to decide what thepeak can be.but Legally in the us the retention times are not considered a conformation. A MSis need for conformation if necessary.,and finally of course some training/ and some experience is necessary in order toget good results.Samples for GC:They can be gasesAlmost every gas has been analyzed liquids and some solidssolids are Usually dissolved in a low boiling solvents and analyzed.Molecular wt. has been done easily from Molecular weight 2 hydrogen, up to over 800(2 to~800).But we must be honest also limitations….
  • 11. In exceptional cases simple hydrocarbons MW up to 1200 have been separated.Samples can be organic and inorganicBut most of the things in GC are organic compounds.Inorganic would be water or gases.The samples always must be volatile but you cant do rocks , sticks or stones andYou can’t also do proteins, peptides and biological molecules those are better doneby liquid chromatography.For Quantitative analysis peak area is proportional to the concentrationI would like to show u some data herethis is a very simple sample of hydrocarbonsDecane,(C10H22)undecane(C11H24) ,dodecane(C12H26), tridecane(C13H28)which would blended up volumetrically andUsing density is calculated mass in grams this is determined by GC plus 1 SDand results and relative error is less than 1% in this case. This is a simple sampleDone by flame ionization to gather with aid of computer typical very high accuracyOne can obtain.Quantitative analysis is the major advantages of GC.
  • 12. ChromatographyIntroduction:The term chromatography is derived from a Greek wordsChromatos= meaning colour.Graphos = written.Initially used for analysis of coloured compounds now due to vastDevelopments it is also applied to colourless compounts.Chromatography is the separation technique of a mixture of compounds(solutes) into separate components By using a stationary phase and amobile phase so it is easier to identify (qualitative) and measure theamount (quantitate) of the various sample components.Chromatography makes use of 2 phases. The mobile phase and the stationary phase.Mobile phase refers to the mixture of analyte while stationary phase consists of fixed solid orLiquid medium. The mobile phase run over the stationary phase and the mixture gets distributedb/n these 2 phases resulting in the separation of analyte/solute. Finally separated analyte isIdentified qualitatively or quantitatively by Massspectrometry,IR,NMR.The technique for GC is similar to that of column Chromatography exceptthat the liquid mobile phasein the column chromatography is replaced bya moving gas.
  • 13. History– Russian botanist and physicalchemist Scientist MikhailSemenovich Tswett is creditedfor the discovery of chromatographyin the early 1900’s (1903).– Germangraduate student Fritz Prior iscredited for developingsolid state gas chromatography(1947).– However the foundation of the GSCwas laid down by Damkohler andThiele in 1943.
  • 14. Mikhail Tswett, Erika Cremer, A.J.P. Martin og Richard L. M. Synge
  • 15. • Modern GC was invented in 1952 by Martinand James.• The father of modern gas chromatography isNobel Prize winner(1952) John PorterMartin,Synge who also developed the firstliquid-gas chromatograph. (1950).• The gas chromatography technique was firstcarried out in Austria in 1944 by thechemist Erika Cremer, who used a solidstationary phase.• Griffin and George (London, UK) probablymanufactured the first commercial GCsystem in 1954, and severalcompanies, including PerkinElmer, Fisher/Gulf, BarberColeman, Podbelniak (all U.S.-based) andPye Unicam (UK), followed shortly in 1955and 1956.• Harold McNair was lucky to be around in thebeginning of gas chromatography (GC).Harold heard about it in 1956, made his firstinjections in 1957, and he is still workingwith it today.• Dr. R. Gohlke had introduced the first GC–mass spectrometry (MS) experiment in 1959using a packed column Harold McNair
  • 16. H C HHHH HO3.Bonding electrons are not shared evenly.The end of the bond with electrons becomes partially negative.The end of the bondwithout electrons becomes partially positive.PolarPolar compoundNon-polar compoundPolar compounds are soluble in polar solvents.Non-polar compounds are soluble in non-polar solvents.Basic rule in organic chemistry is that “like dissolves like rule .” Thus the polarsolvent water dissolves the polar solute ethanol but not the hydrocarbon octane. Thenonpolar solvent benzene will dissolve octane but not ethanol. Polar stationary phases willretain polar solutes and pass those that are nonpolar. The order of emergence is reversedwith nonpolar stationary phases.
  • 17. Principle:5 Principle 1.Adsorption in GSC 2.Partition of molecules between gas (mobile phase)and liquid (stationary phase) in GLC (gas) MOBILE PHASE Sample in Sample outSTATIONARY PHASE (solid or heavy liquid coated onto a solid or support system)Packing materialThe most popular packing material is silica gel.It is believed that silanol radicals ( -Si-OH ) on the surface of silica gelact as the active site and the sample is separated.SiSiSithe surface of silica gel
  • 18. Gas-liquid chromatography (often just called gas chromatography)Depending upon the nature of stationary phase used gas chromatographyis divided in to 2 types.1.Gas liquid chromatography (GLC) or (VPC) or (GC): Principle of separation is PARTITION.In this technique an inert porous solid is coated by a high boiling viscus liquid orNon volatile liquid(carbowax 200m(1500C)Applications –CHO,>C=0,PEG(2000C)Alcohols,pestisides, poly siloxane(2500C)steroids,glycols,pestisides, Silicon rubber gum(300-3500C maximum temp.)Alkaloids,vitamines,gums,fattyacids.,or polymer .The inert solidsupport which acts as a stationary phase ,then this chromatography Is termed as GLC (or)(VPC) vapour-phase chromatography .Separation is based on the relative volatilities.Commonly used support for liquid phase is Diatomaceous earth (or) Kieselguhr.supports maybe either firebrick materials such as (chromosorb-p ,Anakrom- ABS)Generally this technique is most widely used.Gas-solid chromatography the solid adsorbent is used as a stationary phase , it is termed asGSC.In this technique the components of the mixture get distributed between thegas and the solid phase due to differences in their adsorptive behavior.This technique is mainly used for the separation of gases and it is rarely used.The common adsorbants are Zeolite, activated alumina, carbon, Granular silica gel etc.It has few applications because-The active gases get retained on the solid surfaces.-Reproduction of surface area is difficult.2. Gas solidchromatography (GSC): In GSC the principle of separation is ADSORPTION
  • 19. In gas chromatography, the basis for separation is the distribution of solutesbetween two phases. One of these phases is a stationary bed of large surface area(stationary phase) and the other is a gas (mobile phase) which percolates throughthe stationary phase. If the stationary phase is a solid adsorbent, then it is known asgas- solid chromatography (GSC).Here, the adsorptive properties of the stationary phase areresponsible for separating solutes, primarily gases. Common solid stationary phasesare silica gel, molecular sieves, porous polymers, alumina and charcoal. In case, thestationary phase is liquid, it is called gas- liquid chromatography(GLC). The liquid isspread (coated) as thin film over an inert support. The basis of separation is thepartitioning of the solutes in and out of the liquid film. There is a wide range of theliquid phases with usuable temperatures up to 400ºC. This makes GLC the mostversatile and the selective form of chromatography. It is used for analysis of gases,liquids and solids.
  • 20. X = Amount of gasm = Amount of liquidC = Concentration of liquid gasK = Constant.The principle of GC is similar to the Column chromatography,HPLC as well as TLCwith the following differences.1. In GC the separation of mixture of components occurs b/n gas mobile phaseand liquid stationary phase where as in other chromatographic techniquesit occurs b/n liquid mobile phase and a solid stationary phase.2. The concentration of solute(sample) of components in the carrier gas is entirelya function of vapour pressure of the carrier gas.3. In GC the column is placed in an oven whose temperature can be controlledwhere as in other chromatographic techniques temperature programming is notessential.
  • 21. Mover ever principle of GC is also similar to the principle of fractional distillation.Where in the components of the mixture are separated depending up on the differencesIn their boiling points. The GC is used on micro scale and the fractional distillationon the large scale basis.After the known volume of sample to be analyzed has been injected in to the injectionPort which is maintained at temp. higher than the boiling point of the sample.In the column the partitioning of the sample components occurs in between the carrier gasAnd the high boiling liquid in accordance with the equilibrium law.The partitioning properties of sample components differs from one another.The components that have greater tendency to get dissolved in the liquid stationary phaseMove slowly through the column while negligible solubility move rapidly. Hence theComponents are carried along the column at different rates and at different retention times.
  • 22. Basic Chromatography TheoryA GC separation, like extraction, involves a partitioning of solutes between phases. In the caseof GC, one phase in stationary and the other is mobile. The more a solute is partitioned in themobile phase, the more it moves; in other words, the partitioning between the stationary andmobile phases affects the time required for a solute to travel through the instrument.A solute that interacts very little with the stationary phase (via van der Waals forces such asdispersion, dipole-dipole interactions, etc.) moves relatively quickly through the column. Such asolute is not retained by the stationary phase material.A solute with strong interactions with the stationary phase is retained by that phase; such asolute will take longer to travel through the column.This is essentially the same for all types of chromatography (thin layer, paper, liquid-liquid, etc). Gas chromatography is more precisely described as gas-liquid chromatography.
  • 23. Distribution of analytes between phasesThe distribution of analytes between phases can often be described quite simply. An analyte isin equilibrium between the two phases;Amobile A stationaryThe equilibrium constant, K, is termed the partition coefficient; defined as the molarconcentration of analyte in the stationary phase divided by the molar concentration of theDiatomaceous earth which is a very porous rock)
  • 24. The process where a substance divides itself between two immiscible solventsbecause it is more soluble in one than the other is known as partition. Now, youmight reasonably argue that a gas such as helium cant really be described as a"solvent". But the term partition is still used in gas-liquid chromatography.You can say that a substance partitions itselfbetween the liquid stationary phase and thegas. Any molecule in the substance spendssome of its time dissolved in the liquid andsome of its time carried along with the gas
  • 25. when a mixture is introduced into the hot column, a component that does not dissolve in theliquid would be vaporized by the heat and carried straight though the capillary column at thesame speed as the helium gas which is called the carrier gas. A compound of the mixturethat dissolves in the liquid called the stationary phase and has less interactions with the gasphase would remain near the start of the column and move through it with difficulty.Consequently, different compounds are separated within the column because they movethrough it at different rates, depending of the partition between the stationary phase andthe mobile carrier gas.
  • 26. Principle of searation in Gas liquid chromatography: The principle of separation in GLC 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 vapor and mixed withGaseous mobile phase. The component which is more soluble in the stationary phase travels slower and eluted later. The component which is less soluble in the stationary phase travels faster and eluted out first. No two components has the same partition co-efficient for a fixed combination ofStationary phase, mobile phase and other conditions. Hence the components are separated according to their partition co-efficients. Partition co-efficient is the ratio of solubility of a substance distance between two immiscibleliquids at a constant temperature.2 important criteria for compoundsTo be analysed by GC areVolatility: unless a compound is volatile,It cannot be mixed with mobile phase.Hence volatility is important.Thermostability: All the compounds willNot be in the form of vapour. The solidAnd liquid samples convert them to a vapourForm higher temparature is required. That’sWhy compounds have to be thermostable.4mmm
  • 27. Theory of gas chromatography:The column efficiency (or) performance is usually measured in terms of No. of TheoreticalPlates as well as HETP.( Hight equivalent to theoretical plates).Plate theory was initially developedAccording to plate theory The vapors come in ,they partition in to the stationary phase andred Stationary phase ,Vapours come to equilibrium with moving gas stage and come back againin the series of steps adsorbing- disrobing ,adsorbing disorbing each of the steps is calleda theoretical plate . (Parallel layers of discrete and Continuous horizontal plates)Theoritical plate is an imaginary or hypothetical unit of a column and they do not reallyExist.They help to understand the functioning of the column and serve to measure theColumn efficiency.A theoretical plate can also be called as a functional unit of the column.The efficiency of the column can be increased by increasing the no. of theoretical plates.The no of theoretical plates can be calculated (determined) by using the formula:the chromatographic column is contains a large number of separate layers, called theoreticalplatesNo of theoretical plates also known as column efficiency.HETP
  • 28. It is important to remember that the plates do not really existMeasuring column efficiency, either by stating the number of theoreticalplates in a column, N (the more plates the better), or by stating the plate height;the Height Equivalent to a Theoretical Plate (the smaller the better).2WhereN = Number of theoretical platestR = Retention time.W = Peak width obtained upon drawing tangents from 2/3rd height of the peakup to the base line.W
  • 29. The main reason why different compounds can be separated this way is theinteraction of the compound with the stationary phase“(like-dissolves-like”-rule). Thestronger the interaction is the longer the compound remains attached to thestationary phase, and the more time it takes to go through the column (=longerretention time).
  • 30. Theory of Operation• Velocity of a compound through the columndepends upon affinity for the stationary phaseArea under curve is______ of compoundadsorbed to stationaryphaseGas phase concentrationCarrier gasmass
  • 31. How does peak look ..lets look at the peak.you see here Selectivity and efficiency is imp. In the case of the peakWhat are the desired characteristics of how peak look like…If the response is distinguished from all other responses the method is said to be selective.selectivity obtained by choosing optimal columns and setting chromatographicConditions such as mobile phase composition, column temparatureand detector wavelength.Who will decide this one efficiency of the detector selectivity of the detector andAlso the partition ratio.the sharpness of the peak is anindication of how good, orefficient a column is. The platenumber depends on column length: the longer the column,the larger the plate number. Therefore,the plate height term has been introducedto measure how efficiently column hasbeen packed, h = L/N .The lower the plate height and thehigher the plate number, the moreefficient the chromatographic column.
  • 32. Scheme of a chromatogramtR Total retention time of the compound (in the wholechromatographic system)t’R Adjusted retention time of the compound (retention time in thestationary phase)tM "Dead time" (retention time in the mobile phase)W0,5 Peak width at half heighth Height of a signalThis yields in following equation: tR = t’R + tM
  • 33. What influences the separation?1. Polarity of the stationary phasePolar compounds interact strongly with a polar stationary phase, hence have a longer retentiontime than non-polar columns. Chiral stationary phases based on amino acidderivatives, cyclodextrins, chiral silanes, etc are capable to separate enantiomers, because oneform is slightly stronger bonded than the other one, often due to steric effects.2. TemperatureThe higher the temperature, the more of the compound is in the gas phase. It does interactless with the stationary phase, hence the retention time is shorter, but the quality ofseparation deteriorates.3. Carrier gas flowIf the carrier gas flow is high, the molecules do not have a chance to interact with thestationary phase. The result is the same as above.4. Column lengthThe longer the column is the better the separation usually is. The trade-off is that the retentiontime increases proportionally to the column length. There is also a significant broadening ofpeaks observed, because of increased back diffusion inside the column.5. Amount of material injectedIf too much of the sample is injected, the peaks show a significant tailing, which causes apoorer separation. Most detectors are relatively sensitive and do not need a lot of material(see below).6. ConclusionHigh temperatures and high flow rates decrease the retention time, but also deteriorate thequality of the separation.
  • 34. (average velocity of the mobile phase)
  • 35. it is ideal to have the bands of the individual components as narrow as possible. This is to saythat it is best to have each component occupying as little space as possible within thecolumn:From this figure it can be seen that a better separation between narrow bands ofcomponents is ideal for easier collection of the individual samples.H is the height equivalent of a theoretical plate4 (HETP) and u is the velocity (flowrate) of the mobile phase.The lower the resulting value of H is, the greater the efficiency of the procedure.So, ideally, a scientist will want to minimize all three terms in order to minimize H.
  • 36. The following figure helps in visualizingEddy diffusion:The A factor is determined by a phenomenon called EddyDiffusion. This is also called the multi-path term. Solute molecules will take different paths through thestationary phase at random. This will cause broadening of thesolute band, because different paths are of different lengthsIn the figure, particle B will be eluted before particle C, and bothwill be eluted before particle A. Since it is improbable for allparticles of one compound to find the shortest path, there will befractions of the component that will behave like particles A, B, andC. This leads to the broadening of the band. There is little ascientist can do to minimize the Eddy Diffusion factor, can bedecreased by using smaller particle size(particle size should beoptimum) Shape and manner of packing,Column diameter.very smaller size particles also increase the pressure drop leading to disturbance in lineargas velocity that’s why decrease the column efficiency.Generally Eddy diffusion can be minimized by using small particles of uniform sizeand smaller diameter columns.columns should be 1/8inch inner diameter and particle size up to 100-200 mesh rangeare used for good resolution. asgranular bed of thepacking particles
  • 37. The A term is loosely affected by the flow rate of the mobile phase, and sometimes theaffect of the flow rate is negligible.B/u is called the longitudinal diffusion term, and is caused by the components naturalmigration from a place of high concentration (the center of the band) to a place of lowerconcentration (either side of the band) within the column. Diffusion occurs because moleculesin a place of high concentration will tend to spread out to areas of lower concentration toachieve equilibrium.B - Molecular diffusion( Longitudinal diffusion)
  • 38. . Longitudinal diffusion is a chief cause of band broadening in Gas Chromatography, asthe diffusion rates of gaseous species are much higher than those of liquids.The magnitude of the term B/u can be minimized by increasing the flow rate of themobile phase. Increasing the velocity of the mobile phase does not allow thecomponents in the column to reach equilibrium, and so will hamper(restrict theactivity or free movement) of longitudinal diffusion.Hence the molecular diffusion can be decreased by using the optimum linear gasvelocity (flow rate) And using high molecular weight carrier gas eg: Nitrogen, argonthan hydrogen or helium.At time zero in the figure above, the particles of a compound are generally localized in anarrow band within the separating column. If the mobile phase flow rate is too small or if thesystem is left at rest, the particles begin to separate from one another. This causes a spread inthe concentration distribution of that compound within the column, thus bringing about bandbroadening for the band of that particular compound. As the time that the system is left stillapproaches infinity, the compound reaches complete concentration equilibrium throughoutthe entire column.B - Longitudinal diffusionThe concentration of analyte is less at the edges of the band than at the center. Analytediffuses out from the center to the edges. This causes band broadening. If the velocity of themobile phase is high then the analyte spends less time on the column, which decreases theeffects of longitudinal diffusion.
  • 39. . Nonequilibrium or mass transfer: The analyte takes a certain amount of time to equilibratebetween the stationary and mobile phase. If the velocity of the mobile phase is high, and theanalyte has a strong affinity for the stationary phase, then the analyte in the mobile phase willmove ahead of the analyte in the stationary phase. The band of analyte is broadened. The higherthe velocity of mobile phase, the worse the broadening becomes.The flow rate of the mobile phase should not be increased in excess, however, as the term Cu ismaximized when u is increased. Cu is referred to as the mass transfer term. Mass transfer refersto when particles are so strongly adhered to the stationary phase that the mobile phase passesover them without carrying them along. This results is particles of a component being leftbehind. Since it is likely that more than a single particle of any given compound will undergothis occurrence, band broadening results. This results in a phenomenon called tailing, in whicha fraction a component lags behind a more concentrated frontal band. Non-equilibrium effectscan be caused by two phenomena: laminar flow and turbulent flow. Laminar flow occurs intubular capillaries, and so is most prominent in Capillary Electrophoresis. Turbulent flow occursas a result of particles becoming overwhelmed by the stationary phase and is more commonin column chromatography.
  • 40. In the above figure, particles of the adsorbent solid become occupied by particles of thesample. If too many particles of the adsorbent are occupied, particle A will have nothinghindering it from flowing through the column. So, the particles of a single compoundseparate from one another. Also, as the mobile phase moves through the column, particlesof the sample leave the stationary phase and migrate with the mobile phase. However, if theflow rate of the mobile phase is too high, many of the sample particles are unable to leavethe stationary phase and so get left behind. These occurrences result in band broadening, asthe individual particles of a single compound become less closely packed. The high flow rateof the mobile phase makes it more difficult for the components within the column to reachequilibrium between the stationary and mobile phase. It is for this reason that the Cu termis also called the non-equilibrium factor. Minimization of this factor can be achieved bydecreasing the flow rate of the mobile phase. Decreasing the flow rate of the mobile phasegives sample components more time to leave the stationary phase and move with themobile phase, thus reaching equilibrium.By observing the Van Deemter equation, it can be deduced that an ideal mobile phase flowrate must be determined to yield the best (lowest) value of H. Decreasing the flow rate toomuch will result in an increase of the longitudinal diffusion factor B/u, while exceedinglyincreasing the flow rate will increase the significance of the mass transfer term Cu. So, H canbe minimized to a finite limit depending on the various parameters involved in thechromatography being performed.
  • 41. van Deemter profile for hydrogen, helium, and nitrogen carrier gases.The curves were generated by plotting the Height Equivalent to a Theoretical Plate(H.E.T.P., the length of the column divided by the total number of theoretical plates)against the columns average linear velocity. The lowest point on the curve indicatesthe carrier gas velocity at which the highest column efficiency is reached.Hydrogen is the fastest carrier gas (upto : 40cm/sec.) and exhibits the flattest van Deemterprofile. Helium is the next best choice (uopt: 20cm/sec.). The head pressures at optimumflow rates are similar for hydrogen and helium because hydrogen has half the viscosity anddouble the linear velocity of helium. Nitrogens performance is inferior for capillarycolumns and is usually not recommended because of the slow optimum linear velocity(uopt: 12cm/sec.) and steep van Deemter profile.
  • 42. Van Deemter plotsA plot of plate height vs. average linearvelocity of mobile phase.Such plots are of considerable use indetermining the optimum mobile phase flowrate.The van-Deemter-equation demonstrates thedependence of getting sharp peaks (low HETP)from the following terms:small particle’s diameter (particle size),small column’s diameter,small film thickness of the stationary phase.
  • 43. When HETP is plotted against u we getHyperbola with minimum HETP.This minimum is the optimum flow rate (u)At which column efficiency is maximum.This graph represents the van deemter curveWhich is a hyperbola with minimum HETP.It shows that the affects ofA,B,C on the relation ship b/n HETPAnd the gas flow rate.Minimum HETP indicates max. efficiency ofThe column.Hence the ideal flow rateCorresponding to the minimum value ofHETP is used.Such plots are of considerable use in determining the optimum mobile phase flow rate.A- Eddy diffusionC-Resistance to mass transferB- Molecular diffusion
  • 44. 2.Symmetry Factor:Apart form the the N and HETP another term used to measure the column efficiency orColumn performance is symmetry factor(S) of a peak.H1/20It is given by the following equation S = --------------2AS = Symmetry factorH = Peak width at 1/20th level of its height.A = Distance between the perpendicular dropped from the maximum peak heightupto 1/20th peak height.When the symmetry factory of a peak is equal to 1 it implies that the peak is reasonablySymmetrical.Hence peak height may be used for the calculation of chromatogram.However, when the symmetry factor of peak is less or greater than 1, thenFronting( is due to saturation of stationary phase and can be avoid by using lessQuantity of sample)or tailing of the peak( is due to more active adsorption sites and can beeliminated by support pretreatment,more polar mobile phased increasingthe amount of liquid phase) may be seen.
  • 45. Asymmetry factor:A chromatograhic peak should be symmetrical about centre and peak should be like anIsosceles triange.But in practice due to some factors peak is not symmetrical and shows tailing and fronting .Asymmetry factor (0. 95 to 1.05) can be calculated by using the formulabAF = ---------- b and a calculated at 5% or 10% of the peak height.a
  • 46. D = Distance b/n the 2 consecutive peak maximaW1 +W2 = peak widths of peak 1 and peak 2.When R = 1. 98% resolution is achieve.R= ≥ 99.7% resolution is achieved.
  • 47. Where N is no. of theoretical plates.α=Solvent efficiencyR =ResolutionK’2 =partition ration of second peak.=Adjusted retention time(T’R)-----------------------------------------Retention time in Air
  • 48. Interaction forces:Forces that help to carry out the separation process in GCa)Debye forces: Debye or induced dipole forces are the minor forces that result from theinteraction of 2 molecules i.e. Induced dipole in in one molecule and dipole in anothermolecule.b) Orientation forces: These forces are the result of interaction between 2 permanentdipoles.c)Specific interaction forces: These forces are the resultant of the variations that are madeto occur in the immediate dipoles of any two interacting species.d)Non Polar forces: These forces are the resultant of the variations that are made to occurin the immediate dipoles of any two interacting species.4.Partition Ratio (K)™Partition ratio is the ratio of total concentration or amount of solute in the columnor stationary phase(t’R) to the total concentration or amount of solute in the gas ormobile phaseAmount of solute in column t’RK = ---------------------------------------- K = -----------Amount of solute in gas tMPartition ratio is the resultant effect of combination forces i.e., Debyforces ,orientation forces, specific interaction forces and non polarForces.It depends upon the temp.of column ,nature of components of solute and liquid phase as well asOn the quantity of liquid phase in the column.
  • 49. Retention Time (tR)The retention time is the total time that a compound spends in both the mobilephase and stationary phase. Retention time is generally reported in minutes.Dead Time (tm)The dead time is the time a non-retained compound spends in the mobile phasewhich is also the amount of time the non-retained compound spends in the column.Dead time is generally reported in minutes.Adjusted Retention Time (tR)The adjusted retention time is the time a compound spends in the stationary phase.The adjusted retention time is the difference between the dead time and theretention time for a compound.
  • 50. Capacity Factor (or Partition Ratio) (k)The capacity factor is the ratio of the mass of the compound in the stationary phaserelative to the mass of the compound in the mobile phase. The capacity factor is aunit less measure of the columns retention of a compound.Phase Ratio (ß)The phase ratio relates the column diameter and film thickness of the stationaryphase. The phase ratio is unitless and constant for a particular column andrepresent the volume ratioß.Distribution Constant (KD)The distribution constant is a ratio of the concentration of a compound in thestationary phase relative to the concentration of the compound in the mobile phase.The distribution constant is constant for a certain compound, stationary phase, andcolumn temperature.
  • 51. Selectivity (or Separation Factor) (alpha)The selectivity is a ratio of the capacity factors of two peaks. The selectivity isalways equal to or greater than one. If the selectivity equals one the twocompounds cannot be separated. The higher the selectivity, the more separationbetween two compounds or peaks.Linear Velocity (u)The linear velocity is the speed at which the carrier gas or mobile phase travelsthrough the column. The linear velocity is generally expressed in centimeters persecond.
  • 52. EfficiencyThe efficiency is related to the number of compounds that can separated by thecolumn. The efficiency is expressed as the number of theoretical plates (N,unitless) or as the height equivalent to a theoretical plate (HETP, generally inmillimeters). The efficiency increases as the height equivalent to a theoretical platedecreases, thus more compounds can be separated by the column. The efficiencyincreases as the number of theoretical plates increases, thus the columns ability toseparate two closely eluting peaks increases.
  • 53. The oven is used for maintaining the precise temparature control around theColumn. Hence the oven should be free from the influence of changingAmbient temparature and should have adequate air flow system.To guard against sample deposition After long use,narrow glass or metal inserts areprovided in the injection port. The sample deposition on inserts can be taken outperiodically for cleaning purpose
  • 54. A typical GC system used is shown below(a gas chromatograph)Carrier gas: He (common), N2, H2Pinlet 10-50 psigFlow = 25-150 mL/min packed columnFlow = 1-25 mL/min open tubular columnColumn: 2-50 m coiled stainless steel/glass/TeflonOven: 0-400 °C ~ average boiling point of sampleAccurate to <1 °CDetectors: FID, TCD, ECD, (MS)
  • 55. How separation works on the columnOne of three things might happen to a particular molecule in the mixture injected into thecolumn:It may condense on the stationary phase.It may dissolve in the liquid on the surface of the stationary phase.It may remain in the gas phase.None of these things is necessarily permanent.A compound with a boiling point higher than the temperature of the column will obviously tendto condense at the start of the column. However, some of it will evaporate again in the sameway that water evaporates on a warm day - even though the temperature is well below 100°C.The chances are that it will then condense again a little further along the column.Similarly, some molecules may dissolve in the liquid stationary phase Some compounds will bemore soluble in the liquid than others. The more soluble ones will spend more of their timeabsorbed into the stationary phase; the less soluble ones will spend more of their time in thegas.The process where a substance divides itself between two immiscible solvents because it ismore soluble in one than the other is known as partition. Now, you might reasonably argue thata gas such as helium cant really be described as a "solvent". But the term partition is still usedin gas-liquid chromatography.You can say that a substance partitions itself between the liquid stationary phase and the gas.Any molecule in the substance spends some of its time dissolved in the liquid and some of itstime carried along with the gas.
  • 56. A compound with a boiling point higher than the temperature of the column will obviouslytend to condense at the start of the column. However, some of it will evaporate again in thesame way that water evaporates on a warm day - even though the temperature is well below100°C. The chances are that it will then condense again a little further along the column.Similarly, some molecules may dissolve in the liquid stationary phase Some compounds willbe more soluble in the liquid than others. The more soluble ones will spend more of theirtime absorbed into the stationary phase; the less soluble ones will spend more of their timein the gas.The process where a substance divides itself between two immiscible solvents because it ismore soluble in one than the other is known as partition. Now, you might reasonably arguethat a gas such as helium cant really be described as a "solvent". But the term partition isstill used in gas-liquid chromatography.You can say that a substance partitions itself between the liquid stationary phase and the gas.Any molecule in the substance spends some of its time dissolved in the liquid and some of itstime carried along with the gas.
  • 57. Retention timeThe time taken for a particular compound to travel through the column to the detectoris known as its retention time. This time is measured from the time at which the sampleis injected to the point at which the display shows a maximum peak height for thatcompound.Different compounds have different retention times. For a particular compound, theretention time will vary depending on:the boiling point of the compound. A compound which boils at a temperature higherthan the column temperature is going to spend nearly all of its time condensed as aliquid at the beginning of the column. So high boiling point means a long retention time.the solubility in the liquid phase. The more soluble a compound is in the liquidphase, the less time it will spend being carried along by the gas. High solubility in theliquid phase means a high retention time.
  • 58. the temperature of the column. A higher temperature will tend to excite molecules into the gasphase - either because they evaporate more readily, or because they are so energetic that theattractions of the liquid no longer hold them. A high column temperature shortens retentiontimes for everything in the column.For a given sample and column, there isnt much you can do about the boiling points of thecompounds or their solubility in the liquid phase - but you do have control over thetemperature.The lower the temperature of the column, the better the separation you will get - but it couldtake a very long time to get the compounds through which are condensing at the beginning ofthe column!On the other hand, using a high temperature, everything will pass through the column muchmore quickly - but less well separated out. If everything passed through in a very shorttime, there isnt going to be much space between their peaks on the chromatogram.The answer is to start with the column relatively cool, and then gradually and very regularlyincrease the temperature.At the beginning, compounds which spend most of their time in the gas phase will pass quicklythrough the column and be detected. Increasing the temperature a bit will encourage theslightly "stickier" compounds through. Increasing the temperature still more will force the very"sticky" molecules off the stationary phase and through the column.
  • 59. It should be inert and available at low costHigh purityEasily availableLess risk of explosion or fire hazardsPressure:-Inlet  10 to 50 psi-packed column  25 to 150 mL/min.- capillary column  1 to 25 mL/min
  • 60. CARRIER GAS:The choice of carrier gas determines the efficiency of chromatographic separation.The main purpose of the carrier gas is to transport sample components through the column.Most commonly used carrier gases are Hydrogen, Helium, Nitrogen and Argon.1. It should be chemically inert and should not interact with sample and stationary phase.2. It should be suitable for the detector to be utilized and the type of sample analyzed.3. Easily available.4. It should be readily available, cheap, and of high purity.5. It should not cause the risk of fire or explosion hazard.6. It should give best column performance consistent with the required speed of analysis.Hydrogen: It has a better thermal conductivity, low density.It is useful in TCD,FID. The disadvantage is that it reacts with unsaturated compounds andIt is inflammable.Helium: It is also has excellent thermal conductivity, but it is expensive. It is very goodCarrier gas when used with TCD.Nitrogen: It is inexpensive but has reduced sensitivity.Argon: For electron capture detector argon is used as carrier gas. However, argon is notReadily available in India.
  • 61. • Impurities in the carrier gas such as air water vapor and tracegaseous hydrocarbons can cause sample reaction, columncharacter and affect the detector performance.• The carrier gas system should contains a molecular sieve andfilter, drier and absorbing tubes to remove water(moisture) andother gases impurities.• These gases are available in pressurized tanks. pressure regulatorsand flow meters are required to control the flow rate of the gas.• The gases are supplied from the high pressure gas cylinder , beingstored at pressure up to 300 psi (pounds per sq. inch).• carrier gas should be better then 99.99% moles % is desirable and99.999% is often used.
  • 62. Soap bubble flow meterAqueoussolution ofsoap ordetergent 68A soap bubble meter is an accuratedevice for reproducing the rate of thecarrier gas. formed indicates the flowrate.Glass tube with a inlet tube at the bottom.Rubber bulb-----store soap solutionWhen the bulb is gently pressed of soapsolution is converted into a bubble by thepressure of a carrier gas &travel.The time required for the soap film to move betweenTwo graduations on the burette is then measured andConverted to flow rate.Soap bubble meter and flow metersAs carrier gases are stored under high pressureflow regulators are used to deliver gasWith uniform pressure or flow rate.Flow meters are used to measure the flow rate of carrier gas.They are soap bubble meter and Rotameter.BuretteGas fromcolumnGas exit
  • 63. 69inlet tube
  • 64. Rotameter:
  • 65. Sample injection port Calibrated Micro syringes are used to inject liquidsample Sample must be introduced as a vaporin the smallest possible volume and minimum oftime with out decomposition. Liquid samples, 1-10 microliters in the volumeare usually injected by a micro syringe through aself sealing silicone rubber septum. The most accurate and precise method forgas samples used a calibrated sample loop(0.5-10 ml) and a multiport rotary valve. Smaller the sample better the peak shape.
  • 66. Sample injection portFor optimum column efficiency, the sample should not be toolarge, and should be introduced onto the column as a "plug" ofvapour - slow injection of large samples causes bandbroadening and loss of resolution. The most common injectionmethod is where a micro syringe is used to inject samplethrough a rubber septum into a flash vaporiser port at thehead of the column. The temperature of the sample port isusually about 50°C higher than the boiling point of the leastvolatile component of the sample. For packedcolumns, sample size ranges from tenths of a microliter up to20 microliters. Capillary columns, on the other hand, needmuch less sample, typically around 10-3 mL. For capillaryGC, split/splitless injection is used. Have a look at this diagramof a split/splitless injector; The injector can be used in one oftwo modes; split or splitless. The injector contains a heatedchamber containing a glass liner into which the sample isinjected through the septum. The carrier gas enters thechamber and can leave by three routes (when the injector is insplit mode). The sample vapourises to form a mixture ofcarrier gas, vapourised solvent and vapourised solutes. Aproportion of this mixture passes onto the column, but mostexits through the split outlet. The septum purge outletprevents septum bleed components from entering the column.
  • 67. Split: The inlet is continuously purged with vent gas as some flow ratio to the column flow (atthis lab, we use 60:1). This means the flow through the column is of the total flow. This resultsin most of the injected solution being vented rather than deposited on the column, which inturn gives a tight, spatially limited band of analyte on the column.Split injection typically gives the best chromatography (highest theoretical plates).Most of the sample is lost, so split injection is not used when absolute sensitivity is required.However, this does give 1-2 orders of magnitude of dynamic range in instrument responsewithout changing any actual detector parameters (simply by splitting or not).Split less: The split vent is closed during the actual injection. Some time after the injection (forexample, one minute), the split vent is opened to purge excess solvent. This technique allows agreater amount of the injected sample to be deposited on the column.Split less injection gives a greater response for a given solution than split since most of thesample is actually deposited (rather than vented).Split less injection gives the most precise quantitative results, but the chromatographicresolution may be less than with split injection.The instrument parameters needed to produce that maximum resolution are compoundspecific. This means that each analysis must be optimized to achieve maximum resolution.Pulsed Splitless: Pulsed splitless is similar to splitless injection, but during the vent closedportion of the timing cycle, the column flow is pulsed to a relatively high rate. This a relativelynew technique that combines the advantages of split (better chromatographic resolution) andsplitless (better quantitative results and greater response).
  • 68. a. Gas samples: The sample gas can also be injected at the top of the column by meansof a hypodermic syringe. The Hamilton Teflon coated gas syringe is particularlysuitable.Generally gases are introduced by typical hex port gas sampling valve which is alsoInstalled on the gas chromatograph.b. Liquid sample:Liquid samples are most conveniently introduced by means of micro syringe whichare different sizes.Liquids can be injected through loop or septum devices. Generally high quality siliconeRubber septum through which sample solution is injected.The rubber is made up of good quality silicone rubber which can with stand high temp.c. Solid samples:Solid samples are dissolved in a suitable volatile solvent and inject like a liquid sampleand they are injected through a septum.
  • 69. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters.Capillary columns, on the other hand, need much less sample, typically around 10-3 mL. Forcapillary GC, split/splitless injection is used.
  • 70. Ovens:The oven is maintaining the precisetemperature around the column.Hence the column oven should be free frominfluence of changing ambientTemperature(temp. of the surroundings ) andfree and should have well designedand adequate air flow system.The column temperatureThe temperature of the column can be variedfrom about 50°C to 250°C. It is cooler than theinjector oven, so that some components of themixture may condense at the beginning of thecolumn.In some cases, as you will see below, thecolumn starts off at a low temperature andthen is made steadily hotter under computercontrol as the analysis proceeds.
  • 71. Column temperature and temperature programColumn selectionA gas chromatography oven, open to show a capillary columnThe column(s) in a GC are contained in an oven, the temperature of which is preciselycontrolled electronically. (When discussing the "temperature of the column," an analyst istechnically referring to the temperature of the column oven. The distinction, however, is notimportant and will not subsequently be made in this article.)The rate at which a sample passes through the column is directly proportional to thetemperature of the column. The higher the column temperature, the faster the sample movesthrough the column. However, the faster a sample moves through the column, the less itinteracts with the stationary phase, and the less the analytes are separated.In general, the column temperature is selected to compromise between the length of theanalysis and the level of separation.A method which holds the column at the same temperature for the entire analysis is called"isothermal." Most methods, however, increase the column temperature during theanalysis, the initial temperature, rate of temperature increase (the temperature "ramp") andfinal temperature is called the "temperature program."A temperature program allows analytes that elute early in the analysis to separate
  • 72. Column temperatureFor precise work, column temperature must be controlled to within tenths of adegree. The optimum column temperature is dependant upon the boiling point ofthe sample. As a rule of thumb, a temperature slightly above the average boilingpoint of the sample results in an elution time of 2 - 30 minutes. Minimaltemperatures give good resolution, but increase elution times. If a sample has awide boiling range, then temperature programming can be useful. The columntemperature is increased (either continuously or in steps) as separation proceeds.The rate at which a sample passes through the column is directly proportional to thetemperature of the column. The higher the column temperature, the faster the sample movesthrough the column. However, the faster a sample moves through the column, the less itinteracts with the stationary phase, and the less the analytes are separated.In general, the column temperature is selected to compromise between the length of theanalysis and the level of separation.A method which holds the column at the same temperature for the entire analysis is called"isothermal." Most methods, however, increase the column temperature during theanalysis, the initial temperature, rate of temperature increase (the temperature "ramp") andfinal temperature is called the "temperature program
  • 73. How a Gas Chromatography Machine Works:• How does this column and system separate things.(How does GC work?• How does the column work? What happens inside the column? How dothe compounds move through the column? Why do some compoundsstay in the column longer than others? How does the sample get into thecolumn? These are some of the most basic questions asked about gaschromatography.• This shows us a schematic of a GC packed column.• There are 2 columns:• Packed columns and capillary.• In this packed column we have are 2 phases.• mobile phase which is moving stationary phase which is not moving.• Mobile phase also called a carrier gas typically it is He some times H2 andsome times Nitrogen.• It is now possible to separate hundreds of components of a mixture in asingle chromatographic experiment.
  • 74. How does this column and system separate things.(How does GC work?)This shows us a schematic of a GC packed column.There are 2 types of columns.Packed columns large sample capacitypreparative workGood packed column will have 1000 to3000 plates/m.capillary columns.(opentubular column)Packed column-3m in length.Capillary column- 50-150 METER LENGTHliquid stationary 1 Micron thickness.higher efficiency smaller sample sizeanalytical applications .Good capilary column range from 1000 to 4000plates/m. More no of plates better separation.diameter
  • 75. What are guard columns?Guard columns are short lengths of deactivated, uncoated fused silica or metal tubingplaced between the injection port and the analytical column. They protect andprolong the lifetime of an analytical column .Why use a guard column?Capillary gas chromatography (GC) guard columns protect analytical columns inseveral ways. Guard columns trap non-volatile residues, preventing them fromcollecting at the head of the analytical column. These non-volatile residues may bevery high molecular weight organic compounds, inorganic salts, or particulates.If these contaminants enter the analytical column, they can cause adsorption ofactive compounds, loss of resolution, and poor peak symmetryA guard column canprotect your analytical column andensure reproducible analyses.
  • 76. A guard column or retention gap are the samething, but they serve different purposes. Bothare 1-10 meters of deactivated fused silicatubing attached to the front of the column .Deactivated fused silica tubing does notcontain any stationary phase; however, thesurface is deactivated to minimize soluteinteractions. A suitable union is used to attachthe tubing to the column. In most cases, thediameter of the retention gap or guardcolumn should be the same as the column.Guard columns are used when samples contain non-volatile residues that maycontaminate a column. The non-volatile residues deposit in the guard column and not inthe column. This greatly reduces the interaction between the residues and the samplesince the guard column does not retain the solutes (because guard column contains nostationary phase). Also, the residues do not coat the stationary phase which often resultsin poor peak shapes. Periodic cutting or trimming of the guard column is usually requiredupon a build-up of residues. Guard columns are often 5-10 meters in length to allowsubstantial trimming before the entire guard column has to replaced. The onset of peakshape problems is the usual indicator that the guard column needs trimming or changing.
  • 77. UnionsThere are a variety of unions that can be usedto connect fused silica tubing. Stainlesssteel, stainless steel-glass combinations, glasspress-fit and quick connectors are some of themore common types.All mobile phases, samples and additives should be filtered through a .45µm syringe filter.It is recommended that guard columns are packed with the same stationary phase as theanalytical column to be protected. (Eliminate the possibility of any loss of performance orselectivity.)Pump seals and rotor seals should be replaced on a routine basis.A guard column provides saturation of your mobile phase with silica by “bleeding” silicainto the mobile phase instead of from the analytical column. This can be achieved withoutthe loss of resolution or performance by using a Guard Column. When the guard column isdestroyed, replace it with another cartridge in minutes.According to most experts, a guard column can increase the usable life of your columns bya factor of four. You will save money and valuable time. Another source of problems arecompounds that irreversibly bond to the stationary phase and are often injected intoanalytical columns. These compounds cause permanent damage to columns that are notprotected by a guard column. Shifting of retention time and loss of resolution often results.
  • 78. 85Coated with 30 micro metersThick adsorbant such as diatomaceous earth.Which consists of singled -celled sea -plant skeletons.Then this adsorbant is treated with liquid stationary phase.SCOT columnsare capable ofholding agreater volumeof stationaryphase than aWCOT columndue to itsgreater samplecapacity, WCOTcolumns stillhave greatercolumnefficiencies.modern WCOTcolumns aremade ofglass, but T316stainlesssteel, aluminum, copper(capillary tube whose wallsare coated with liquidstationary phase)(the inner wall of the capillary is lined with a thin layer of supportmaterial such as diatomaceous earth, onto which the stationaryphase has been adsorbed).More efficientThan scot columns
  • 79. Columns:There are two general types of column, packed and capillary (also known as open tubular).Packed columns contain a finely divided, inert, solid support material (commonly basedon diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5- 10m in length and have an internal diameter of 2 – 4 mm.Capillary columns have an internal diameter of a few tenths of a millimeter. They can beone of two types; wall-coated open tubular (WCOT) or support-coated opentubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coatedwith liquid stationary phase. In support-coated columns, the inner wall of the capillary islined with a thin layer of support material such as diatomaceous earth, onto which thestationary phase has been adsorbed. SCOT columns are generally less efficient than WCOTcolumns. Both types of capillary column are more efficient than packed columns
  • 80. In 1979, a new type of WCOT column was devised - the Fused Silica OpenTubular (FSOT) columnThese have much thinner walls than the glass capillary columns, and are givenstrength by the polyimide coating. These columns are flexible and can be wound intocoils. They have the advantages of physical strength, flexibility and low reactivity.
  • 81. One of the most popular types of capillarycolumns is a special WCOT column called thefused-silica wall-coated (FSWC) open tubularcolumn.The walls of the fused-silica columns aredrawn from purified silica containing minimalmetal oxides. These columns are muchthinner than glass columns, with diameters assmall as 0.1 mm and lengths as long as 100m. To protect the column, a polyimidecoating is applied to the outside of the tubingand bent into coils to fit inside the thermo-statted oven of the gas chromatographyunit. The FSWC columns are commerciallyavailable and currently replacing oldercolumns due to increased chemicalinertness, greater column efficiency andsmaller sampling size requirements. It ispossible to achieve up to 400,000 theoreticalplates with a 100 m WCOT column, yet theworld record for the largest number oftheoretical plates is over 2 million plates for1.3 km section of column.Computer Generated Image of a FSWC column (specialized to withstand extreme heat)
  • 82. For example, the FSWC column is designed specially for blood alcohol analysis. It producesfast run times with baseline resolution of key components in under 3 minutes. Moreover, itdisplays enhanced resolutions of ethanol and acetone peaks, which helps with determiningthe BAC levels. This particular column is known as Zebron-BAC and it made with polyimidecoating on the outside and the inner layer is made of fused silica and the inner diameterranges from .18 mm to .25 mm. There are also many other Zebron brand columns designedfor other purposes.Another example of a Zebron GC column is known as the Zebron-inferno. Its outer layer iscoated with a special type of polyimide that is designed to withstand high temperatures. Itcontains an extra layer inside. It can withstand up to 430 °C to be exact and it is designed toprovide true boiling point separation of hydrocarbons distillation methods. Moreover, it isalso used for acidic and basic samples.
  • 83. The common liquid phases for Gas chromatography:Liquid stationary phase Maxium temerature ApplicationsSqualane(C30H62).High molecular wt.hydrocarbon used for nonpolar hydrocarbons)140-150 Hydrocarbons.Corbowax 200Carbowax 20M(PEG)150oC200-250Aldehydes, ketones.Alcohols,aromatics,pesticides,ketonesPoly siloxane 250oC Steroids,pesticides,Glycols.Polyethylene glycol(PEG)(more effective for polarcomounds)200-250oC Alcohols,pesticides etc.Silicon rubber gum(SE-30) 300-350oC. Alkaloids,alcohols, gasesfatty acids,gums,bile andurinary compounds,vitamins,Sugars,pesticides,
  • 84. Stationary Phases:Stationary phase in GC is the main factor determining the selectivity and retentionof solutes.There are three types of stationary phases used in GC:Solid adsorbentsLiquids coated on solid supportsBonded-phase supports1.) Gas-solid chromatography (GSC)- same material is used as both the stationary phase and support material- common adsorbents include: aluminamolecular sieve (crystalline aluminosilicates [zeolites] and clay) silica active carbonMagnified Pores in activated carbon
  • 85. Gas-solid chromatography (GSC):advantages:- long column lifetimes- ability to retain and separate some compounds not easily resolved by other GCmethods geometrical isomers permanent gasesdisadvantage:- very strong retention of low volatility or polar solutes- catalytic changes that can occur on GSC supports- GSC supports have a range of chemical and physical environments different strength retention sites non-symmetrical peaks variable retention times
  • 86. 2.) Gas-liquid chromatography (GLC)- stationary phase is some liquid coated on a solid support- over 400 liquid stationary phases available for GLC many stationary phases are very similar in terms of their retention properties- material range from polymers (polysiloxanes, polyesters, polyethylene glycols) tofluorocarbons, molten salts and liquid crystalsBased on polarity, of the 400 phases available only 6-12 are needed for mostseparations. The routinely recommended phases are listed below:NameChemical nature ofpolysiloxaneMax.temp.McReynolds’ constantsx’ y’ z’ m’ s’SE-30 Dimethyl 350 14 53 44 64 41Dexsil300 Carborane-dimethyl 450 43 64 111 151 101OV-17 50% Phenyl methyl 375 119 158 162 243 202OV-210 50% Trifluoropropyl 270 146 238 358 468 310OV-225 25% Cyanopropyl-25% phenyl250 238 369 338 492 386Silar-SCP 50% Cyanopropyl-50% phenyl275 319 495 446 637 531SP-2340 75% Cyanopropyl 275 520 757 659 942 804OV-275 Dicyanoallyl 250 629 872 763 1106 849McReynolds’ constants based on retention of 5 standard “probe” analytes– Benzene, n-butanol, 2-pentanone, nitropropanone, pyridineHigher the number thehigher the absorption.
  • 87. Preparing a stationary phase for GLC:- slurry of the desired liquid phase and solvent is made with a solid supportsolid support is usually diatomaceous earth (fossilized shells ofancient aquatic algae (diatoms), silica-based material)- solvent is evaporated off, coating the liquid stationary phase on the support- the resulting material is then packed into the columndisadvantage:- liquid may slowly bleed off with time especially if high temperatures are used contribute to background change characteristics of the column with time
  • 88. 3.) Bonded-Phase Gas chromatography- covalently attach stationary phase to the solid support material- avoids column bleeding in GLC- bonded phases are prepared by reacting the desired phase with the surface of a silica-based supportreactions form an Si-O-Si bond between the stationary phase and supportorreactions form an Si-C-C-Si bond between the stationary phase and support- many bonded phases exist, but most separations can be formed with the followingcommonly recommended bonded-phases: Dimethylpolysiloxane Methyl(phenyl)polysiloxane Polyethylene glycol (Carbowax 20M) Trifluoropropylpolysiloxane Cyanopropylpolysiloxaneadvantages:- more stable than coated liquid phases- can be placed on support with thinner and more uniform thickness thanliquid phasesSiCH3CH3OnSiCH3CH3OnSiC6H5C6H5OmC CHO OHHHHHn
  • 89. HO-CH2-CH2-(O-CH2-CH2)n-OHpolyethylene glycol(Polar)(intermediate polarity)(non-polar)Polydiphyl siloxanePolyalkyline glycolPoly bis cyano propyl siloxane (very polar non-bonded phase upto 250oC.
  • 90. Phases
  • 91. Solid Phase:The main function of the solid phase is to provide mechanical support to the liquid phase.The commonly used solid phases include: Diatomaceous earth or Kieselguhrcommonly abailable as dicalite,calite,sterchamol etc.Firebrick coated with metalic silver or gold commercially available asChromosorb P ,, chromosorb W, Kieselguhr ,Anakrom ABS.Others include glass beads,unglazed tiles, porous polymers, sand etc.The criteria for an ideal support include:1. It should have large surface area2. It should be chemically inert i.e., it should not react with the liquid phase as well as withthe body of the column.3. It should be thermostable.4. It should be a poor adsorbent.5. It should get uniformly wet with the liquid phase.6. It should be strong enough to prevent the fractionating of the column.
  • 92. Liquid phase:Many liquid phases are available of gas chromatography columns. Choice depends uponthe trial/error basis.The requirements for a good liquid phases are1. It should be non-volatile2. It should have low volatility i.e., should be stable at the operating temarature.3. It should be chemically inert.4. It should possess low vapour pressure at column temperature.5. I t should have high decomposition temperatures.Liquid phases are classified as:Very polar liquids: Glycerols,glycols, Polyphenols.Polar liquids: Alcohols, ketones, esters.Intermediate polar liquids: Aldehydes, ketons, esters.Low polar liquids: Aromatic hydrocarbons, chloroform, dicloromethane.For getting very good results…………..The liquid phase should be chemically and structurally similar to the slolute(sample)i.e.,The polar liquid phase for polar soluteThe non-polar liquid phase for non-polar solute.
  • 93. 3,6,12 foot lengthpacked with a solidsupport likeDiatomaceous earthSupports may beeither fire brickderived materials likeChromosorb –p ;Anakrom ABS etc.packed columns can only achieve about 50% of the efficiency of a comparable WCOTcolumn.Due to the difficulty of packing the tubing uniformly, these types of columns have alarger diameter than open tubular columns and have a limited range of length.The diatomaceous earth packing is deactivated over time due to the semi-permanentadsorption of impurities within the column.In contrast, FSWC open tubular columns are manufactured to be virtually free of theseadsorption problems.1.6 to 9.5 mm inDiameter.Capilary columns haveTubing coiled in to anOpen spiral ,A basket-coil orFlat pancake shape.
  • 94. Column temperatureFor precise work, column temperature must be controlled to within tenths of a degree.The optimum column temperature is dependant upon the boiling point of the sample. Asa rule of thumb, a temperature slightly above the average boiling point of the sampleresults in an elution time of 2 - 30 minutes. Minimal temperatures give goodresolution, but increase elution times. If a sample has a wide boiling range, thentemperature programming can be useful. The column temperature is increased (eithercontinuously or in steps) as separation proceeds.The column efficiency can be improved by variation in temperature, low liquidphase, loading, narrow particle size distribution and tight packing. The length of thecolumn can be, as deemed necessary, from a fraction of an inch (capillary column) toseveral hundreds of feet.For a given species, the ratio of the times spent in the moving and stationary regions isequal to the ratio of its concentrations in these regions, known as the partitioncoefficient.
  • 95. Packed columns are made of a glass or a metal tubing which is densely packed with a solidsupport like diatomaceous earth. Due to the difficulty of packing the tubinguniformly, these types of columns have a larger diameter than open tubular columns andhave a limited range of length. As a result, packed columns can only achieve about 50% ofthe efficiency of a comparable WCOT column. Furthermore, the diatomaceous earthpacking is deactivated over time due to the semi-permanent adsorption of impurities withinthe column. In contrast, FSWC open tubular columns are manufactured to be virtually freeof these adsorption problems.
  • 96. Temperature Control• Isothermal • Gradient040801201602002400 10 20 30 40 50 60Time (min)Temp(degC)Instrumentation - Oven
  • 97. The best detector must have a high sensitivityto traces, good stability, and satisfactoryresponse to a wide variety of substances.Detectors are classified as integral ordifferential, and destructive or non-destructive.The integral detector measures the totalamount of the component .The differentialdetector measures some property related tothe concentration of the resolved componentsIn case of the destructive detector, the sampleis destroyed in the process of detection, suchas the case of the flame ionization detector(FID). The thermal conductivity differentialdetector (TCD) is the most widely utilized non-destructive detector. Fast scanning mass
  • 98. Detectors:The detector senses the presence of the individual components as theyLeave(elute) the column. The detector out put after amplification is tracedon a recorder.As peaks at intervals on the chromatograph.The duration of the intervals is usually a single second or even less than that.Hence the detector is considered to be the brain of the gas chromatograph.The most desirable criteria for a gas chromatographic detectors are:1.It should be highly sensitive towards wide range of compounds.2. It should produce uniform and linear responses towards wide range of vaporizedsolute particles.3. It should be stable during operation conditions.4. It should have concentration reproducibility.5.It should be easy to operate.Generally gas chromatography detectors are about 4-5 orders of magnitude moresensitive than the liquid chromatography detectors.
  • 99. generally different detectors gives different types of selectivity.Non-selective detector- It responds to a wide range of compounds except the carrier gas.Selective detector - it responds to a group of compounds with similar physical orchemical properties.Specific detector -specific detector responds to a single chemical compound. .In general detectors may be divided in to 2types.(detectors in chromatography operated in2 ways. Respond either to the concentration of solute or the mass flow rate.1. Concentration dependent detectors:The signal from a concentration dependant detector is related to theconcentration of solute reaching detector, and usually these detectors do not destroythe sample.Dilution of response with make-up gas lowers the response.Ex: Thermal conductivity detector (TCD)Electron capture detector (ECD)Argon ionization detector (AID)Helium ionization detector (HID)
  • 100. 2. Mass flow dependant detectors usually destroy the sample, and the signal is related to therate of solute particles enter the detector. The response of a mass flow dependant detector isun affected by make-up gas.In differential detectors that responds to the mass flow rate, the peak area is directlyproportional to the total mass and there is no dependency on the flow rate of the mobilephase.. These detectors are suitable for quantitative analysis.EX:- Flame ionization detector.(FID)Flame Photometric detector (FPD)Nitrogen phosphorous detector(NPD)Depending upon the reason for operation chromatography may bePreparative chromatography = separation of components of a givenmixture for future use. This chromatography is also known aspurification process.Analytical chromatography = It can work even with minuteconcentrations of the sample mixture and measures the componentsof given mixture. Therefore it is used for quantitative estimation ofanalytes.
  • 101. Detectors may be non-destructive, whereby sensing does not alter the nature of thesolutes, as in the case of light absorption, so they may be collected for further use.Destructive detectors, on the other hand, destroy the solutes. Detectors include notonly the component that senses the solutes but also those that perform the associatedtransduction, electronic amplification, and final readout.
  • 102. To obtain optimal separations, sharp, symmetrical chromatographic peaks must beobtained. This means that band broadening must be limited. It is also beneficial tomeasure the efficiency of the column.
  • 103. Column efficiencyThe efficiency of a column is reported as the number of theoretical plates (platenumber), N, a concept Martin borrowed from his experience with fractional distillation:where tr is the retention time measured from the instant of injection and w is the peakwidth obtained by drawing tangents to the sides of the Gaussian curve at the inflectionpoints and extrapolating the tangents to intercept the baseline.The plate number depends on the length of the column. The extreme value of106 plates was obtained with an open tubular gas chromatographic column 1.6kilometres (1 mile) long. A more appropriate parameter for measuring efficiencyis the height equivalent to a theoretical plate (or plate height), HETP(or h), which is L/N, L being the length of the column. Efficient columns havesmall h values (see below Theoretical considerations: Plate height).HETP = L/N ( Efficiencyresolution: Column efficiency).
  • 104. There are many detectors which can be used in gas chromatography. Differentdetectors will give different types of selectivity. A non-selective detector responds to allcompounds except the carrier gas, a selective detector responds to a range ofcompounds with a common physical or chemical property and aspecificdetector responds to a single chemical compound. Detectors can also be groupedinto concentration dependant detectors and mass flow dependant detectors. The signalfrom a concentration dependant detector is related to the concentration of solute inthe detector, and does not usually destroy the sample Dilution of with make-up gas willlower the detectors response. Mass flow dependant detectors usually destroy thesample, and the signal is related to the rate at which solute molecules enter thedetector. The response of a mass flow dependant detector is unaffected by make-upgas. Have a look at this tabular summary of common GC detectors:
  • 105. Type of Detector Applicable Samples Detection LimitMass Spectrometer(MS)Tunable for any sample .25 to 100 pgFlame Ionization (FID) Hydrocarbons 1 pg/sThermal Conductivity(TCD)Universal 500 pg/mlElectron-Capture(ECD)Halogenatedhydrocarbons5 fg/sAtomic Emission(AED)Element-selective 1 pgChemiluminescence(CS)Oxidizing reagent Dark current ofPMTPhotoionization (PID) Vapor and gaseousCompounds.002 to .02 µg/LTypical gas chromatography detectors and their detection limits
  • 106. Detector Type Support gases Selectivity Detectability Dynamic rangeFlame ionization (FID) Mass flow Hydrogen and air Most organic cpds. 100 pg 107Thermal conductivity(TCD)Concentration Reference Universal 1 ng 107Electron capture (ECD) Concentration Make-upHalides, nitrates,nitriles, peroxides,anhydrides,organometallics50 fg 105Nitrogen-phosphorus Mass flow Hydrogen and air Nitrogen, phosphorus 10 pg 106Flame photometric (FPD) Mass flowHydrogen and airpossibly oxygenSulphur, phosphorus,tin, boron, arsenic,germanium, selenium,chromium100 pg 103Photo-ionization (PID) Concentration Make-upAliphatics, aromatics,ketones, esters,aldehydes, amines,heterocyclics,organosulphurs, someorganometallics2 pg 107Hall electrolyticconductivityMass flow Hydrogen, oxygenHalide, nitrogen,nitrosamine, sulphur
  • 107. Thermal conductivity detector (TCD),KATHAROMETER (OR)Hot wire detector:Thermal conductivity detector is the one of the oldest detector still using because ofSimplicity of system and it is widely used. Data’s are available over the years.It is simple inexpensive, non-selective, accurate, non destructive of the sample.The TCD is based on the changes in thermal conductivity of the gas stream..Thermal conductivity of the most of the compounds is lesser than the commonly used carriergases (He, H) because the thermal conductivity of He is about 6 to 10 times greater than mostorganic compounds.When He is used as a carrier gas the presence of small amounts of organic materials causesA relatively large decrease in thermal conductivity of the column effects. As a result of thedecrease in conductivity.The detector undergoes a marked rise in temperature.Since detector response depends upon the difference in thermal conduction of the carrier gasand sample, a large difference is essential. An increase in temp. of the detector causes achange In the resistance of wire or thermistor and this resistance gives a measure of thethermal conductivity of the gas.
  • 108. TCD consists of a temperature controlled metal block into which 2 cylindrical chambersOr cavities are present.Both chambers consists of filaments(thermistors,resistance wires) made up of platinum,tungsten or alloys.The filaments constitute of the reference(R) and the sensing (S) elements.Both these filaments are connected to the arms of the Wheatstone bridge arrangement.At the given temp. when carrier gas alone is flow through the both cylindrical chambers theResistance of the both the filaments are thermal equilibrium (they are constant).As long as the composition of the gas does not change the rate of heat loss from the filamentsWill not alter.Once the effluent passes through (s) the temp. of the filament (s) changes .Because of thermal conductivities of sample and the carrier gas are different.The differences result in heat loss form the filament eventually changes in the resistanceOf (s).This resistance changes are send by the Wheatstone bridge arrangement.The bridge then produces a measurable voltage change that is amplified and signaled to theRecorder which is recorded on the chromatogram.
  • 109. Widge balanceameterExcitation voltageUnbalancedVoltage.Same carrierGas is passingThrough theDetectors.Initially whatTo do is widgeBalance with theBy varying thesePotentiomenrI will balance theBridge.When ever the elution peak comes out side gases comes detector outPut change there is a thermal conductivity is changed I will get aUnbalanced voltage recorded on the recorder.We can have 4 detectors in one block itsef.Amlifier andrecorderBattery(direct current source)
  • 110. ) Thermal Conductivity Detector (TCD)- katherometer or hot-wire detector- first universal detector developed for GCProcess- measures a bulk property of the mobile phase leaving the column.- measures ability to conduct heat away from a hot-wire (i.e., thermal conductivity)- thermal conductivity changes with presence of other components in the mobile phase
  • 111. Disadvantages:An oven is essential for the working of detector to attain column temp.The detectors are relatively insensitive i.e., sensitivity is only about 10-6 to 10-8.Advantages:The detectors are simple in construction having no moving parts and are inexensive.They give accurate results.They are durable and possess long life.They are non- selective hence known as universal detectors.Use of wheatstone bridge increases the sensitivity of the detector.It does not result in the destruction of the sample.Linear response range is about 3 orders of magnitude.The filament resistances supplies a measure of the thermal conductivity of the gas. HenceBy measuring the filament resistance, the changes in the effluent stream can be monitored.
  • 112. As the name suggests, analysis involves the detection ofions. The source of these ions is a (tiny)small hydrogen-air flame. Sometimes hydrogen-oxygen flames are useddue to an ability to increase detectionsensitivity, however for most analysis, the use ofcompressed breathable air is sufficient. Columneffluents are led into the flame wherein ionisation ofcompounds may takes place.In order to detect these ions, two electrodes are used toprovide a potential difference. The positive electrodedoubles as the nozzle head where the flame isproduced. The other, negative electrode is positionedabove the flame. The ions thus are attracted to thecollector plate and upon hitting the plate, induce acurrent.When only carrier gas passes through the flame, itsmolecules are ionised and the resulting ionisationcurrent after amplification is fed to the sutable recorder.This current is measured with a high-impedancepicoammeter and fed into an integrator.A FID is sensitive to almost all the organic compoundsBut insensitive to noblegases,oxygen,N,CO,C02,water,H2S,S02,CS2 and Nitrogen
  • 113. The eluent exits the GC column (A) and enters the FID detector’s oven (B). The oven isneeded to make sure that as soon as the eluent exits the column, it does not come out ofthe gaseous phase and deposit on the interface between the column and FID. Thisdeposition would result in loss of effluent and errors in detection. As the eluent travels upthe FID, it is first mixed with the hydrogen fuel (C) and then with the oxidant (D). Theeluent/fuel/oxidant mixture continues to travel up to the nozzle head where a positive biasvoltage exists (E). This positive bias helps to repel the reduced carbon ions created by theflame (F) pyrolyzing the eluent. The ions are repelled up toward the collector plates (G)which are connected to a very sensitive ammeter, which detects the ions hitting the plates,then feeds that signal (H) to an amplifier, integrator, and display system.FIDs are best for detecting hydrocarbons and other easily flammable components.n FID essentially can only detect components which can be burned. Other components maybe ionized by simply passing through the FIDs flame, but they tend not to create enoughsignal to rise above the noise of the detector.
  • 114. The effluent from the column is mixed with hydrogen andair, and ignited. Organic compounds burning in the flameproduce ions and electrons which can conduct electricitythrough the flame. A large electrical potential is appliedat the burner tip, and a collector electrode is locatedabove the flame. (mixture burns at the tipIons and the free electrons are in the formed ) Thecurrent resulting from the pyrolysis of any organiccompounds is measured. FIDs are mass sensitive ratherthan concentration sensitive; this gives the advantagethat changes in mobile phase flow rate do not affect thedetectors response. The FID is a useful general detectorfor the analysis of organic compounds; it has highsensitivity, a large linear response range, and low noise. Itis also robust and easy to use, but unfortunately, itdestroys the. Positive ions and electrons are produced inthe flame when organic substances are present. The ionsare collected at electrodes and produce a small,measurable current. The flame-ionization detector ishighly sensitive to hydrocarbons, but it will not detectcarrier gases, such as nitrogen, or highly oxidizedmaterials, such as carbon dioxide, carbonmonoxide, sulfur dioxide, and waterParrlar orcylendrical½ cm 1cm above the tipHydrogenTip.This was the restinceAcross the gap andCauses a current to flow.mountedThis ensures thatMake current flow onlyIonized materials entersThe external resisteris sensed voltage droppedAmplified and displayed onThe detector.Platinum jet-ve electrode.
  • 115. advantages:- universal detector for organics-doesn’t respond to common inorganic compounds.- mobile phase impurities not detected.- carrier gases not detected.- limit of detection: FID is 1000x better than TCD.- linear and dynamic range better than TCDdisadvantage:- destructive detector.Flame ionization detectorHydrocarbon groups are enter the flame and a complex process takes place which+ve ly charged carbon species and electrons are formed. Now the current is greatlyIncreased .This FID responds only to the substances only for ionizedTo the substances that produced charged the ions that is burned in H2 flame.organic compounds the response is proportional to the no. of oxidisable carbonatoms. This is basic principle of falme ionization detector.
  • 116. The electron-capture detector, a stream of electrons from a radioactive source is produced ina potential field. Materials in the gas stream containing atoms of certain types captureelectrons from the stream and measurably reduce the current. The most important of thecapturing atoms are the halogens—fluorine, chlorine, bromine, and iodine. This type ofdetector, therefore, is particularly useful with chlorinated pesticides. Certain elements willemit light of distinctive wavelength when excited in a flame.
  • 117. The Flame Thermocouple DetectorThe "flame thermocouple detector" was thenext detector to be reported which wasdeveloped by Scott and was, in fact, theforerunner to the flame ionization detectorFID. A diagram of the flame thermocouple isshown.bellow
  • 118. 3.) Nitrogen-Phosphorus Detector (NPD)- used for detecting nitrogen- or phosphorus containing compounds- also known as alkali flame ionization detector or thermionic detectorProcess- same basic principal as FID- measures production of ions when a soluteis burned in a flame- ions are collected at an electrode tocreate a current- contains a small amount of alkali metalvapor in the flame- enhances the formation of ions fromnitrogen- and phosphorus- containing compoundsAlkali Bead
  • 119. Flame photometric detector:The flame photometric detector measures the intensity of light with a photometric o circuit.Solute species containing halogens, sulphur, or phosphorus can be burned to produce ionicspecies containing these elements and the ions sensed by electrochemical means.It is a selective detector. It uses the luminous emissions of S or P from the effluent in a lowtemp. hydrogen flame. These emissions are Serves as a analytical information i.e.,s pecificFor compounds containing these atoms. The intensity for band is recorded photometrically.The detector consists of combustion chamber that houses flame. H2 as fuel, air or oxygento Support combustion. A thermal filter for the separation of UV and visible rays emitted byThe flame.An interchangeable optical filter for the selection of S,P an insulated Photomultiplier tube(PMT) and chimney as exhaust for combustion products.Applications:For the detection of heavy metals like Iron, chromium, selenium, strontium, tin etc. inOrganometallic compounds.For the analysis of pesticides, coal, hydrogenated products as well as air and waterPollutions.
  • 120. Electron capture detector:Invented by Dr.J.E.Lovelock in the late 1950s.It is one of the most sensitive detectors used in GC.It is specifically used for trace amounts of halaogen containingCompounds like pesticides,polychlorinated biphenyls.ECD contains a radioactive β-emitter for the generation of electrons.This detector functions in 2 modes DC and pulsed mode.DC mode: constant DC potential is applied b/n 2 electrodes.
  • 121. The separation of the sample air is accomplished using gas chromatography. Tiny porousbeads, sometimes coated with a liquid, interact with the mixture of molecules impedingthere movement either because of their size or their solubility. In our case, this separationcolumn is divided into two parts. When the chemicals of interest move onto the secondcolumn, the first columns flow is reversed to clean heavier compounds off before thenext injection. This is called "backflushing".Detection of the halocompounds is by an electron capture detector. A radioactive foil ofnickel-63 is inside the pin-in-cup detector. The beta decay (an electron) ionizes the carriergas forming an electron cloud. Periodically a large positive pulse is applied to the centerelectrode. This causes the free electrons to move to the electrode where they aremeasured as a tiny current. When a molecule containing halogen atoms, which has theproperty of enhanced affinity for electrons, enters the detector, it readily attracts andholds a free electron. The background current is thus reduced. In due time the electroncapture molecules are flushed out of the detector and the current returns to its previouslevel.The measure of the dip in the current curve is a measure of the amount of chemicalpresent. By periodically injecting gas mixtures containing known quantities of thechemical of interest, calibration of the detector is accomplished.Recording of the inverted current produces a curve called a "chromatogram".
  • 122. Advantages: It is highly sensitive in its response.Sensitivity is 10-13 g.It is 1 million times more sensitive than TCD and about 10,000times more sensitiveThan FID.It is non-desturctive. i.e., lt does not alter the sample significantly.It is highly sensitive for the detection of compounds like Halogens(Cl,Br,I), Quinones,Peroxides,nitrites,nitro and phosphorous compounds, organometallic compounds.Disadvantages:It is least sensitive for compounds whose molecules have negligible affinity for electrons.It is insensitive to functional groups like alcohols, amines and hydrocarbons.Linear response is limited to 2 orders of magnitude.The carrier gas used should be in absolutely pure form like pure nitrogen or mixture ofPure methane-argon.The temperature limitations of the detector is 2200C.Applications:Analysis of halogenated compounds.Analysis of environmental pollutants.Detection and quantitative determination of chlorinated insecticides.
  • 123. Electron-capture DetectorsElectron-capture detectors (ECD) are highly selective detectors commonly used fordetecting environmental samples as the device selectively detects organiccompounds with moieties such as halogens, peroxides, quinones and nitro groupsand gives little to no response for all other compounds. Therefore, this method isbest suited in applications where traces quantities of chemicals such as pesticidesare to be detected and other chromatographic methods are unfeasible.The simplest form of ECD involves gaseous electrons from a radioactive ? emitter inan electric field. As the analyte leaves the GC column, it is passed over this ?emitter, which typically consists of nickle-63 or tritium. The electrons from the ?emitter ionize the nitrogen carrier gas and cause it to release a burst ofelectrons. In the absence of organic compounds, a constant standing current ismaintained between two electrodes. With the addition of organic compounds withelectronegative functional groups, the current decreases significantly as thefunctional groups capture the electrons.The advantages of ECDs are the high selectivity and sensitivity towards certainorganic species with electronegative functional groups. However, the detector hasa limited signal range and is potentially dangerous owing to its radioactivity. Inaddition, the signal-to-noise ratio is limited by radioactive decay and the presenceof O2 within the detector.
  • 124. Electron Capture Detector (ECD)- radioactive decay-based detector- selective for compounds containing electronegative atoms, such as halogensPrinciple of Operation- based on the capture of electrons byelectronegative atoms in a molecule- electrons are produced by ionization of thecarrier gas with a radioactive source3H or 63Ni- in absence of solute, steady stream ofthese electrons is produced- electrons go to collector electrode wherethey produce a current- compounds with electronegative atomscapture electrons, reducing currentAdvantages:- useful for environmental testingdetection of chlorinated pesticides or herbicidesdetection of polynuclear aromatic carcinogensdetection of organometallic compounds- selective for halogen- (I, Br, Cl, F), nitro-, and sulfur-containing compounds- detects polynuclear aromatic compounds, anhydrides and conjugatedcarbonyl compounds.Disadvantages: Could be affected by the flow rate.e-e-e-e-e-HeHeHeHeHe+He+He+He+He+e-e-HeHeHeHeHe+He+He+He+He+
  • 125. Atomic Emission DetectorsAtomic emission detectors (AED), one of the newest addition to the gaschromatographers arsenal, are element-selective detectors that utilize plasma, which isa partially ionized gas, to atomize all of the elements of a sample and excite theircharacteristic atomic emission spectra. AED is an extremely powerful alternative thathas a wider applicability due to its based on the detection of atomic emissions.There arethree ways of generating plasma: microwave-induced plasma (MIP), inductively coupledplasma (ICP) or direct current plasma (DCP). MIP is the most commonly employed formand is used with a positionable diode array to simultaneously monitor the atomicemission spectra of several elements.InstrumentationThe components of the Atomic emission detectors include 1) an interface for theincoming capillary GC column to induce plasma chamber,2) a microwave chamber, 3) acooling system, 4) a diffration grating that associated optics, and 5) a position adjustablephotodiode array interfaced to a computer
  • 126. Schematic of atomic emission detector
  • 127. 1. Mass Spectrometer (GC/MS)Many GC instruments are coupled with a mass spectrometer, which is a very goodcombination. The GC separates the compounds from each other, while the mass spectrometerhelps to identify them based on their fragmentation pattern.2. Flame Ionization Detector (FID)The detector is very sensitive towards organic molecules (10-12 g/s, linear range: 106 –107), but relative insensitive to a few small molecules e.g. N2, NOx, H2S, CO, CO2, H2O. Ifproper amounts of hydrogen/air are mixed, the combustion does not afford any ions. If othercomponents are introduced that contain carbon atoms cations are produced in the effluentstream. The more carbon atoms are in the molecule, the more fragments are formed and themore sensitive the detector is for this compound (-- > response factor). However, due to thefact that the sample is burnt (pyrolysis), this technique is not suitable for preparative GC. Inaddition, several gases are usually required to operate a FID: hydrogen, oxygen (compressedair), and carrier gas
  • 128. 3. Thermal Conductivity Detector (TCD)This detector is less sensitive than the FID (10-5-10-6g/s, linear range: 103-104), but is wellsuited for preparative applications, because the sample is not destroyed. It is based on thecomparison of two gas streams, one containing only the carrier gas, the other one the carriergas and the compound. Naturally, a carrier gas with a high thermal conductivity e.g. heliumor hydrogen is used in order to maximize the temperature difference (and therefore thedifference in resistance) between two thin tungsten wires. The large surface-to-mass ratiopermits a fast equilibration to a steady state. The temperature difference between thereference cell and the sample cell filaments is monitored by a Wheatstone bridge circuit.4. Electron Capture Detector (ECD)The detector consists of a cavity that contains two electrodes and a radiation source thatemits b-radiation (e.g. 63Ni, 3H). The collision between electrons and the carrier gas(methane plus an inert gas) produces a plasma containing electrons and positive ions. If acompound is present that contains electronegative atoms, those electrons are “captured”and negative ions are formed, and the rate of electron collection decreases. The detector isextremely selective for compounds with atoms of high electron affinity (10-14 g/s), but has arelatively small linear range (~102-103). This detector is frequently used in the analysis ofchlorinated compounds e.g. pesticides, polychlorinated biphenyls, which show are very highsensitivity.
  • 129. Submultiples MultiplesValue Symbol Name Value Symbol Name10−1 g dg decigram 101 g dag decagram10−2 g cg centigram 102 g hghectogram10−3 g mg milligram 103 g kg kilogram10−6 g µgmicrogram (mcg)106 g Mgmegagram (tonne)10−9 g ng nanogram 109 g Gg gigagram10−12 g pg picogram 1012 g Tg teragram10−15 g fgfemtogram1015 g Pg petagram10−18 g ag attogram 1018 g Eg exagram10−21 g zgzeptogram1021 g Zg zettagram10−24 g ygyoctogram1024 g Yg yottagram