UNIT - II Methods of Separat On-ry cally aim at separati cals with the h as Gas chromatograp) (Van d Gel chromatography ys pte! Fes 2.1 GAS CHROMATOGRAPHY shy is undoubtedly the most important Hy of complex mixture of subsiqhn romatography was originally deyere yee (awarded Nobel pr 2 iograpl) 88. purely anatase nd AT. James (1952) were the first to separate fay Ud pinent of continuo Acids Processes base phy isa physical method of separation, accom ple solution in the vapor nd a fixed phase. When the fixed phase ig ique is called Gas solid chromatog jon-volatile liquid held in a co moving gas pl adsorbent, the te (GSC).When fixed phast technique is called Gas liquid chromatography (GLC). Gas liquid chromatography is based upon the partition of the between a gaseous mobile phase and a liquid phase immobilised on the surface of an inert solid. ase raphy mn, analyte ‘The sampl ed in Gas chromatographic separation must either te s or be capable of being converted to a gas at the temparature of column.Gas chromatography provides both qualitative and quantitative formation. Gas liquid chromatogra in \ds widespread use in all fields of science, ‘where its name is usually shortened to Gas chromatography (GC). Generally, gas chromatography cannot be used for analysis of ‘organic salts, because inorganic salts can not be converted to gases.al 178 prof Separation-t1 oe orature at which, i Is between y Portant ph jon of solute between the wo nj ono - column, its molecules are die RE Stationa peter meals of each type is a constanrate Concentration of solute in the st Ke ing two compounds and B. “A? any distribute equally Between the two phases qu and gaseous) +B’ on the other hand may be highly solubleint se veut relatively few ofits molecules will be femt the vape ame wile molecules of “A’ will distribute equally between the wo fneee equilibrium is reached. Under this condition, the carrier gos wil drag the molecules of compound A along the column leaving tehia molecules of compound B. As soon as the molecules of Areach a. region ining fresh liquid, some of them will redissolve and a new «quilibrium is established, During this time when fresh mobile gas (carrier gas) passes over the liquid containing molecules of B, some of again enter the gas phase in order to establish the «quilibrium. Thus, fhe more volatile molecules are swiftly swept along and the Tess volatile component move along The equilibrium ratio (partition ratio) depends upon (the nature of the solute ( the nature of the solvent i (ii) the concentration of the I (iv) the temperature the stationary liquid phase ands of Analytical Che, sty 182 rn ‘< =, from e% L Ms TK" an. ©) =a) -@) Je phase me taken by an unretaineg aig a ahe cotum THUS yy repre hb ~@) rue E from the equation (9) i), we get tp =tull +) (10) , *y ) Retention Volume, Vp. can also be ORE an Nea a) nical species. Retention volume, yt analyse pile phase required £0 elute the sy defined 28 volume omn ahe column. The retention volume, v component con to each other as = fy are related retention time, fy ar Ie phase. where F is the flow rate of mol be a isan slue of fy from equation (10) in equation (11), weg, Substituting ¥ fm cota Va= uF + ®) le phase ‘Substituting val nis imple Rand = () = retention time of the mol But, 4 F = Viv ; where Vyy = retention volume of mobile phase or void volume interstitial volume or dead volume of mobile phase (3) Relative Retention (a) : A change in any of several experiment conditions like stationary phase, mobile phase, flow rate, temparatue etc. can alter Vp and fg, hence a third parameter called relative retention, a is defined for identification and characterisation of component. It eliminates the effect of experimental variables. where fy pelative retaintion, ‘@” can ‘pution coefficient of the su : aiference CompOUNA. (Kr) K Kree itati (14) 4) HETP - Quantitative Deseri affewency of a chromatographic column 9g ¢ uum” Efficiency : The mn Suma to separate components of a mixy me eee Of ability of a itati fa mixtures, E; 3s ly by the num ‘ures. Efficienc fxpressed quantitatively by the number of thee -y can be al plate (n) or the oxint equivalent to a theoretical plate (He, ‘ETPis the length of column corresponding toa. = 2h A single theoretical HETP is mathametically defined as, Het n (15) where L=Length of the column n= Number of theoretical plates H=HETP An efficient column is the one for which ‘n? is large or 4 small. th ‘n” is large or *H? is A theoretical plate can be thought of as a single equilibration of sample component between the stationary and mobile phase. Within a column many equilibrations occur. As the number of theoretical plates (a) in the column increases, the efficiency of the column increases. ‘ete number of plates (n) can be computed by using the following ton. also be stance (kK) to a=Y pantwans of snalytical Ch, hod f Separary, ve nt 134 M tne Rate Theo, , ~ (6) Tooreadths of ene F nei6| sorts ot brag ; ave (in the units of ime) {SiR mechan ara ocrtes ws ac! here we width of peak A component. Substitituting they, st ty Eady Diffusion {2 dlentyardor Se jon time of 8 we get eddy Diffusion , ° fq = Retention tt ation (15) NE ie pomMosraply aris, fey digg er cof sn from Squation (16) tow VAPIOUS pathy Pom Ying die ttl broadenins factor in pets (7) behavion, © Tange solutes travel, as they Vote the separatic ve.a measure of the Separation bg, Ge Resolution : Resolution 59 09.2) Wee, Fi .jacent peaks in 2 chromatogram. ( i en majority of the time, Momorated in the statio fime. Thus, two molecules oj ye time. Eon res jonsible fori ‘The magnitude of contribu depends on size of packing particles hej ttsion of band broadening sneir distribution in the column, "©" shape and the uniformity of @) Longitudinal Diffusion : Longiendingy diffusi nal diffusion is a band broadening factor in chromatograpiyi ofthe solute molecules. ‘Ply caused by the random motion average length of get eluted at the Detector Responise —r cents used to calculate the resolution, R of two peaks Fig.2.2 Me " aks is a function of ty Solute molecules tend to diff aration between two peaks is a. n iffuse froma more concer The amou's the diference between the retention time ofthe ply | yoltion to a more dilute par the tie pe eueemEaE Ft of a parameters. As the differs nereases. i.e. resolution is direciy | soncentration difference. Inthe center of @ chromatographic band the increases, the sep mntion times of the peaks. Goncentration of a species is high, eae the difference in retei - While at the two e also dependent upon that portion ofthe | concentration approaches zero. Th eatxoled| mal to (propria) lerefore, molecules tend to The amount of sere nearest to the adjacent peak. As half widis | either edge of the band. This results in bard bani with ofeach peak which is nearest to ie paration between the peas | ia HETP) and factor responsible fori ems Toagi of the peak intre#scelution decreases. ) Non-equilibrium in mass transfer : Nonequilibrium in mass decreases, i. the resolution transfer is a band-broadening factor in chromatography coured br the Resolution R’ is given by ite time required by a solute to equilibrate benveen vo phases, This pw 2a =ed For (ty )2 > (lg )s arises from the fact that equilibrium cannot be attained Ter the mame distribution of solute betwean Stationary and mobile phases: TeTack of continuous flow of the mobile phase in the column, This resus i where), and (), re retention times of peaks of the two compere +», and w, are width of peaks of the two components at the base.ea" eT plate is refe which Jocity (u), referred to as he dashed lines on the g, Cuterms to HETP at va, va raph Tio Heates an overall effect yey! icity where the best balan” ete Carrier Gas Veleoity, v (em 5") ig23 Schemati depiction of the van Deemter equation {tography = Instrumentation of Gas Chromat eat “The important components involved and their functions are as follows 1, Agas cylinder containing a carrier gas, 2. Pressure regulator and flow control, 3. Sample injection system, 18 6 “the recorder Yt Prossure Regulator & Flow Control -— | Amplifier Recorder gram of. Carrier Gas Supply : Th pelium, hydrogen, nitrogen, Krier gas depends upon (i) a © Most comm ae a it loyed the type of being employed (iii) co = a cost suitable fOr Use With athe mand helium fe iney have high thermal conductivity and lew dere ae ical purposes hydrogen, however ie oe of its fire explosion hazard and ts prunsaturated sample components, Th pressurised tanks. The mobile phase does not react with molecules ct bralyte. Its only function isto transport the analyte through the eelarmn 2, Pressure Regulator and Flow Control : The carrier gas eylinder isattached to pressure regulator and flow control valve whose fenetion isto regulate the pressure and flow rate respectively to desired level ‘ypically pressure of 10 to 50 psi and flow rate of 25 to 30 em maintained. 3.Sample injection system : The function of sample injection system is 10 vaporise the sample instantaneously so that sample is introduced assaplug of vapour into the column. The type of injection system depends onthe physical state of the sample. Liquid samples are usually injected by a graduated micro syringe through a self sealing rubber septum into a preheated flash evaporation injector located at the head of the column. Here the sample is rapidly not used as a carrier gas teactivity towards reducible ese gases are available in marepariovars of Analvtical Che, 18S geature is maintained gy 1% ate ng compo yagerised pote boiling point famaple from ingen in yt 323 K above the bor ashes the ¥ th packed wector sample. The cares £3 Te volumes used WiHN P2cKed Colum, column. Typica (My Tro about Sem Te GC vary from about OT to aoM | CY by GC if they can be Melteg scteg pment les whit Sag carpe ca as Bee ed an then by dissolved in gitable solve! The ress caseS, porelyss at or eraphand ‘ised to vaporize solid sam fon ith eat B85, into the column duced ii a cas sampling valves. c lamas "The columns used in Gas chromatograph ol : ‘bular (or capillary), cked column (ii) open tl ce vo pes. (D Packs Seas reked columns are made from glas G@ Packed columt Bf) 3 m long and have inside diame subing. Then of gre normally formed as coils to permit o mm. The ormostating in an oven, squid « —_ vnents of an ideal support to be used for liquid : ear packing or support, for a column holds the Viguid sta na jnase in place, so that the surface area exposed to the mobile phase ad phase in place, large as possible. (2) The ideal so particles with good at least | m? P 4 tetperan 3 terial should be inert at elevate: peratures and be fl 3) Te ened by the liquid phase. No substance that meets perfectly Il of these criteria is yet available. ° ‘The earliest packings for gas chromatography were prepared from naturally occuring diatomaceous earth. These packings are i wanely wsed support materials and are often treated chemically with ‘ethylchlorosilane, which gives a surface layer of methyl groups. This {reatment reduces the tendency of the packing to adsorb polar molecules, The particle size of packings for gas chromatography typically falls in the range of 60 to 80 mesh (250 to 170 j1m) or 80 to 100 mesh (17010 149 pm). Desirable properties of immobilised liquid in GC : (1) Low volatility (ideally, the boiling point of the liquid should beat 373 K higher than the maximum operating temparature for the column) (2) Thermal stability at the operating temparature Y are olumy S orm a torts ONVenien i i: Il, uniform, tid packing consists of small, uniform, spheri vid trenieal strength and with a specific surface st ‘ane (623K), 50 Filuoropropy! poly, eter or po sige a2 be bent ase of fi molecules *¢ d strength by an exter gpite flexible and strong. fehes. An important ady, Morb minimum analyte 5 Thermal Compartm, e ina rea For samples with a broad boil temperature programming, whe either continuously or in steps 6.Detector = ‘The function of detector in GC components as they leave the col electrical signal. The temperature sufficiently high to prevent condensat decomposition. Ideally a detector sho (i It must respond rapidly to mi they exit the column, (ii) The time during which a peak passes the detector i typically one second or less, which requires that the device should be capable of exhibiting its full response during this brief period, Other desirable properties for a detector include linear response, and uniform response for a wide variety of chemical species. No single detector fulfills all of these requirements. sony hts above the average Soli ion period (2 to 30 min). INS range, it may 30 min), sreby the colina) DE necessary to employ a the sep lumn temperature is increased aration proceeds, i 10 sense the arrival of separated mn and provide a corresponding OF detector compartment must be ion of sample vapours, yet not ours ld have the following propersce, inute concentrations of solutes asnalytical Che ys of Mistry ‘ofa substance to cong, y hermal conductivitigg ing sample. TCD jg ‘ifferential detector jg q” 1954. 1 prass cells fitted wig 0 ibe es constitute reference ®) statemn two arms Of Wheatson) i Flowed to flow through one "her cell. Each of these wires : fom an accumulator ecu emparature. An near path wires and hence a change ls, the temperay hn both the cells, the temperature thee remo carter B88 MIO ment in wheatstone bridge i sang ristance of B mene in composition in gas flowin, Cre ‘alaneed circuit. Achange ord therefore a change jg which shows 2 heat los through the sensing ¢ se 2 fh the aid of wheatstone bridge. rou istance wires, (MGaance which can Be reco" ster instead of resista 5,35 "a modern tren eration. mm rensitive in low temperature oP- they are more tance gas flows throug! Lead to Wheatstone Bridge Column Cantar Effluent In Gas in s R = Column carder —= Effluent Out Gas Out Electricity Heated Wire Fig2.5 Diagram of a two-filament thermal conductivity detector YY yothod OF Separaygy, ghe Advantages 7 191 rs ac Are (a) its simplicity, (b) its large linear destruct °SPOnse to both organic and ion, Ve character which permits collection of ‘tion co er)" °F TCD is its relatively low sensitivity ‘ation Dete, ‘etame ionisation detean st" (FID) junds, when pyrolysey's © based on the fact that most organic eotgonduct electricity thes? ho flame, produee ionie intermediates that ith this detector, ‘8h the flame. Hydrogen is used as carrier detect hydactor (FID), the eluate coming from the e. Thekt (the fuel) and air (the oxidant) to ignited mixture forms a flame which RETRY {0 ionia yple components in the eluate “© St organie and a few inorganic omides sufficient energy ® sao Chimney Electrical igniter ‘Connection (+) Hy Column Etfivent Fig.2.6 Diagram of a flame-ionization detector The gaseous cations formed during ioniza attracted to a negative collector electrode and re oran electrode placed near the base of the flame. Upon si collector electrode, the positive ions cause a current to flow in anles, Th ugh he = ¢ the collector the concentration of ionizable Sam le probes can bi . center th es that ci Zed j, a sae rier only 10 SUStaMeES ean} compa sctparesen f fof response 10 STEARIC Compa, air bedroees gen, nitrogen, SUIPRUT and jad clu . ‘ces on the compour ces on “although the FID is about 2 seed times MOTE Sensitive th, thousane’s of sample Componeme” the jeiect ot ents, a it cannot be used to deteh a's diagram of an FID jg shoe destroys sample entering it destroys in Fig 2.6. tor (ECD) * j (© Electron Capture ee capture detector (ECD) is based on the The principle of IS mpounds bing negative element ors electron absorp cone paving an ele a = har erapot commons OE pe colin eae pasta elas TES aca parle, The ener Gt th col element, whicl and the carrier gas molecule: S between the bets PAT rons emitted during the ionization are capyeg carrier gas ions. the Stetjectrode, and cause an nt to fy by a positive collector 7 rent is amplified and recorded. An eas an external circuit, The FUTOR: “ hosen for use with the detec jonized gas, such as Melt ure of methane with helium, nj gen, affinity for electrons entey sé le component that has an 7 When 2 sample he electrons emitted by the carrer gas are cpt the detector, some OFT re net result is the removal of electron from the by the component. The Me standing current. The decrease is recordaj 5 adecrease il iz Seem She peak on the recorder. or argon. Fig.2.7 Diagram of en electron capture detector. wipe advantage of the q, _siructiVe NALUTE as fay rar “almost all the detec jenal- It is therefore, neo? sig letecto, aS samp n Guta series of Peaks on the pap, er. This gechnique = The carrier gas, obtained from a stg a flow regulator for the adjustmen, & i gas y Sample injector. A small amon, low rat jajector is mai ie highest boilin, ization of the liquid s, vaporization of the liquid samples. Ty Se injector sweeps off the vaporized samnie Syemeril 3s at different rates. separation of the components o f the sample. The separated components now enters the detects change in composition or con r concentration of the c: through it. This change is amplified before carrier gas with the Which measure the arrier gas as it passes fed into a recorder which drives the recordin; tive and Quantitative Analysis : Qualitative analysis using gas chromatography is done by comparing retention times or retention volumes of sampie components with retention times or retention volumes of reference compoundssof Analytical Chem, ions. That is often q, "sample Component ine gparately injected kn, hy lone ang vy, corded at different carries 8 rantageous 10 USE reten itis aeyse retention volumes nes jon volume 8 found to dopant wer nd to depg er ey of clu, stationary pa nm wn the other hand, depeng 4, "smpound etc. " to eliminate the effect gp tg? is used dj is es then provides a better ingar on of unknown compoung j, F e and then to record the le of si np pentic sample of suspects pound, and enhancement of rovides an excellent means of this Peompound in the mixture, The a suspen if effect can be duplicated op c a Ce. corent temperature: : umn and at titative chromatography iS based upg, Analysis # Qepror the area of the analytical peak wig ihe standards. then this P! ifferent co Quantitative a comparison of that of one or more (a) Analysis Bas! js obtained by connect i Sain ing BPP ro pn eh oe to peak, Peat gare obiained with peak heights only if variation in acco onditions do not alter the peak widths during the period ‘equired to obtain chromatogram for sample and standards. {) Analysis Based on Peak Areas : Peak areas are independent of brodening effects.Hence areas are more satisfactory analytical parameter than peak heights. Peak area is found out by multiplying half the peak height. Most modem peak height by its width at one Instruments are equipped with digital electronic integrators, which permit precise estimation of peak areas. (©) The Internal Standard Method : This method gives highest 5 lines on ing the base Uwndicular distance from this line | shods of €paration-ty Mer jon because the unce; 195 ijection are ternal standard I = aed ‘on this plot. GAS SOLID CHROMATOGRAPHY solid chromatography (GSC) is hase Gas Gus substances on a soli al 1 8 Solid station, of Srficients are generally much ee cotsequently, GSC is useful for the sepatat ined by GLC columns, such as tompor nge, carbon disulphide, nitregees 9° le, and the rare gases." O*ide, “he GSC has the same basic components is in column packing. GS giference is in col '8. GSC is performed with both ij open tubular columns. Packed columns Cae Ruth uniform, finely divided adsorbent like silica wel. ata Packed {harcoal oF various grades of diatomaceous earth tm activated ‘Nowadays molecular sieves and s gsed for packing the column, in open tubular column, a thin layer is jonen ey orm ter fot i aad the open tubular column ie. PLOT columns. Two types of adsordente sre inuse, (1) Molecular Sieves. (2) Porous Polymers. mens ore (1) Molecular Sieves : Molecular sieves are aluminium silicate ion exchangers, whose pore size depends upon the kind of cation present. Commercial molecular sieves come in pore sizes of 4, 5, l0and 13 A®, Molecules smaller than these dimensions penetrate into the interior of particles where adsorption take place. Molecular sieves can be used to separate small molecules from large molecules. @) Porous Polymers : Porous polymer beads of uniform size are manufactured from styrene cross linked with divinyl benzene. It has found considerable use in the separation of gaseous polar species as mentioned above and methanol, vinyl chloride etc. . In GSC distribution than those for GLC. n of species that are not nents of air, hydrogen carbon monoxide, carbon as that of GLC. The only Porous polymers are also commonlyWANS € r winlytical Op, ving to semipermanent y ations On Mare taHiNyE OF | Mee at adsorption process). jon except for separati Chas lin fr moles active oF polar mole Sonsequence of not aque has not fou molecular weight £2 year charact inet wide applica Gas sald commbanenhy (SO 1. | station Monte pare . the soli 2 | Matos Adsorption on th | of separation surface. sorents are poked, in seam fines raded powder. Liquids are either co Fine fi on the cco oron the inet seo Packing of the| coleman ag, ’ al 4. | Thermal stability} Operation can be at higher Fs decided by tate than in GLC. ae of the stationary) (rperayimit decided by | Below the boiling yo 20 pe eee ability of samples | the liquid int Vary rarely with facking may catalyse to 's, | Reactions on the| Packing mey fay sacly with Column. produce some chemical he Fxg, due liability. | (a) Limited app! @ Plicabitty, due specific conditions and surface catalysis (b) Useful for low boiling liquids and in the sepa- ration of permanent gases. separations. (b) All volatile materiay| except the more permansy gases. Applications of GC : GC is useful in the analysis of natural gases, gaoline plant samples, refinery gases, synthetic rubber and plastic, intermediates, steroidal hormones and in the analysis of trace and atmospheric constituents GC finds its major applications in the detection, identification and determination of organic substances in complex mixtures, that have Sy An impo: Oa indus peaiytical dist Rona hur and DitrOgen compere S tO! been separated by ce ms: hy eetometric techniques, entific GLC is successfiy Prelohexane (BP 333 gS rs ant (5) The GLC is a versatile BENS, ete, have jnorganics (6) Purity of metals can yolatile complexes. Thus Yetected. (1A large number of other industri pesticides, pharmaceuticals, cosmetics plastic-materials, fertilizers, alcohotig heeemneS: POtective coatings, products, soap and synthetic detergents how yest ane Fubber feparated by Gas chromatography, ave been analysed and (8) GC is used in the field of centered around the trace com flavour of foods. (9) The technique i: EO) ae iq is useful in analysis of fuel gases, auto exhaust (10) The technique is a boon in biomedical applications. Analysis of body fluid within a few minutes may result ie the conte diaganosis of the state of the health of individuals, (11) GLC has been used in the separation of radioactive products. (12) GC can be used in analysis of natural products and help in synthesizing such materials. tool for the Separation of volatile be tested b; Y converting traces of Ay neti the metals to their uranium ean be food products complete