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Quantitative Analysis

by: Gavin Harvey

Quantitative Analysis CHEM 253

Marketplace > University of Idaho > Chemistry > CHEM 253 > Quantitative Analysis
Gavin Harvey
GPA 3.91


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This 32 page Class Notes was uploaded by Gavin Harvey on Friday October 23, 2015. The Class Notes belongs to CHEM 253 at University of Idaho taught by Staff in Fall. Since its upload, it has received 64 views. For similar materials see /class/227961/chem-253-university-of-idaho in Chemistry at University of Idaho.


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Date Created: 10/23/15
High Performance Liquid Chromatography HPLC Harris Chater 25 1212005 Chem 253 7 Chapter 25 1 o Separation of nonvolatile orthermally unstable compounds lfthe analytesample can be found to be sufficiently soluble in a solvent system then that system can usually to used as the mp in an HPLC separation 0 Common method used for analysis of 0 Biological compounds Pharmaceuticals 0 or Nonvolatile environmental cpds eg PCB 1212005 Chem 253 7 Chapter 25 2 Michael Tswett 1906 separation of plant pigments by organic solvent mobile phase amp chalk stationary phase Martin and Synge 1941 liquidliquid partition chromatography 1952 Nobel Prize in chemistry Other variants Paper chromatography Thinlayer chromatography TLC Preparative column chromatography Medium pressure chromatography Ionexchange chromatography Sizeexclusion chromatography 1212005 Chem 253 7 Chapter 25 3 HPLC components Liquid Mobile gt Pump gt Injection gt Separation gt Detector Phase Valve Column 0 Also an integrator usually records the detector response a We will discuss each component but let s first discuss the band broadening aspects of LC This discussion tells us why high pressures are required for analytical separations 1212005 Chem 253 7 Chapter 25 4 wo pum s for Inlection port gradiem elullon Column I Uliraviolel Compuler for cnnirol and display detector lzlllz Chem 25370hapi2125 s Band Broadening in LC 0 Back to the van Deemter Equation H A Bu Cu 0 Which of the three components is the largest contribution to H Consider the following o Bu effects Diffusion is usually 100x less in liquids than in the gas phase 0 Cu effects By process of elimination we will assume that mass transport effects are the largest contribution to H 39n LC lzlznna Chem 25370hapi2125 s Cu Mass Transfer MT Effects I This is the effect of the kinetics of mass transfer to 39om the mobile phase tofrom the stationary phase M bquot9 DiVeCilon m Ell eitltiDn Stationary phase I Bandwidth Slow equiiibration Cux P l Bandwidth after some travel 1 illiZEIEIS Review of MT effects C a fltk gtd MTinthemp m Dm Where dP is the diameter ofthe packing particle in Le Stationary as ph Packing particle silica Smaller d increases the surface areavolume ratio and thus increases MT indie mp imam ii Smaller clp increases the surface areavolume ratio and thus increases MT in the mp I Volume 43 7 r3 Sur ce Area 4 1 r2 Sur ce areavolume 1l3xr The effect is dramatic in gures 252 amp 253 e cost of small packing particles is that the pressure required to force liquid through the column follows as AP and The typical particle sizes in HPLC is 310 pm In order to achieve ow ra es of 0 0 mLmin for a 1030 cm column pressures of 70 to 400 am 1000 to 6000 psi are required izmznna Chem 25370hapt2125 s E 3 E E7 a C E L n Flow rate mLmin izwznna Chem 25370hapt2125 1o Figure 252 Chmmlugllm arm mm samplv my on Mum Wm will at law um rm 51m pnnlcle mmmm PmmR E mm chmm u mg n as 12mm Chem 2537 Chapter 25 11 0 Requirements for HPLC pressures to 6000 psi pulse free prevents remixing of solutes control flow rate from 01 to 10 mLmin 0 Types of HPLC pumps Reciprocating pumps most commercial systems are based on this design e Syringe pumps manna Chem 2537 Chapter 25 12 Reciprocating pumps o Disadvantages pulses from single piston See dual piston design in figure 2514 of your text Outlet Intake Stroke 121 2005 Chem 253 Chapter 25 13 Springloaded mullet valve M I z 2 7 Damper V Cooling water 7 i To column l l intake filter I 50u Lvolume So Ivenl 10011 L volu me mam reservoir piston 1212005 Chem 253 Chapter 25 1 4 Syringe Pumps 25 Mmlumammnchmmmgmyn m o Pulsefree output limited mobile phase capacity rag zu Cm mm on mam pimp m pislnn am Meier ram m yislun rimm mm m usenmrcyllmrby mum m anew m Thwmubilz pm Kul mu mung mm quotpm 02mm of m w M llIDES Chem 25370hapl2125 1s Pulse Dampers o Diaphragm n Slainless o Coil 3 to 20 meters in length lZlZEIEIS Chem 25370hapl2125 1e httpwwwchromtechcom2001catalogSeparatePgs303pdf 1212005 Chem 253 Chapter 25 1 7 Introduces sample to the column Mobile gt Pump gt Injection gt Column Phase Valve Valve consists of a rotor and stator stationary backplane See schematics below and gure 2515 of your text Two oumus 10 Median pm gladmm mmron Commquot Caqmmm to camel and display quot 1212005 Flash Animation of Injection Valve ohtt wwwrestekcominfo six ortas Y sunpl a Lawn smplc Wmquot quot In From Ta Km 13 Pump Commquot Pump Column FILL Fulllan INJECT Posllloll Fig mm A sample injection vulva The sv mn valve shown allows Lhrec pairs of polls to be I m uquot r on pair of pom w t r r m vave I he quotmacr pnsiunn dlmds ma mobilrpllas ow mm mm sample lonp leadmg m nJacucn of nmplc omu me column mm 2 n mm wth Jawmam Mum um m mm Mm m m 01mm 1quot Coma u mm mm 1212nn5 Chem 25370ham2125 20 To o Solvent in column Load position Inject position a WWWZEUS Chem 2537 ChapVLErZE 0 HPLC Syringes unbeveled tips 1212EIEI5 Chem 2537 ChaMErQS Precolumn filters 2 types porous stainless frit 05 to 2 pm or a little piece of sacri cial column Injection gt Precolumn gt Column gt Detector Valve Prevents the contamination of the expensive analytical columns with ne particles that can eventually clog the mobile phase ow 1212005 Chem 2537Chapler25 23 Analytical Columns 0 Common configuration to the right 0 Generally stainless steel and teflon components 0 The stationary phase packings are microporous silica 210 pm in diameter o Unmodified silica is very polar J 1212005 Chem 253 7 Chapter 25 24 i um 2574 uwc wlumnwlllv Mimi mm mmquot moum Imnnhlyulwngtcd mpumz numm ms mnmm m We evanlymumdlmwruldxcmlum Counzxy umm scum Oak HKMJVAJ manna Chem 2537 Chap ev 25 Hydrogen bonded wcinal silanol groups IsuIaled silanal groups manna Chem 2537 Chaptev 25 29 Fig 255 silica particles 1212nn5 Chem 25370ham2125 0 Where R can vary typically 018 Cs CHZCBH5 39CH2339NH21 39CH2539CN 1212nn5 Chem 25370ham2125 Fig 258 protection from hydrolysis llIDES Chem 25370hapl2125 Reversed amp Normal Phase Se arations 0 Normal Phase Polar sp amp Nonpolar mp Early HPLC work was conducted on unmodified silica highly polar this required the use of nonpolar mobile phases in order to get adequate separations o Reversed Phase Nonpolar sp amp Polar mp 4 Later HPLC research led to silica 018 modified surfaces which required the use of polar mobile izwznna Chem 2537 Chapter 25 Partition vs Adsorption Chromatorah o nu WM u D sed 39 quot surface which is very polar Solute analyte species is adsorbed to this surface 0 Partition Chromafo raphy Based on modi ed silica surfaces The 018 bonded phases dissolve rather than adsorb the analyte solute species thus partitioning ofthe so ute between two essentially liquid phases A A m m manna When should we use Partition nonpolar sp vs Adsorption polar sp phases in chormatographic separations mu maniac mm zu Anllnllum a uun hmmun lIphy mum mmmmm lmkhumm bxme nu mm manna Molem ar mass lt2 mm Soluble m urgamc salvems Soluble m wanar Scdubve In Nomamc m modsmiely pom summed palm salvems w paler 5mm hexane m mews cm a canon Bonded Soluble m a cnho Banded revevsedphass Somme in CHCI acelommle or nnvmalrphase Onvexchange or you chmmamgrapny emyl aceme cnmmzmgvaphy chmWme om cm 7 oz m phenyl nyanu mum Bands nuvmaL chiomakogmvhy P 355 Cyana ammu chmmamglaphy cyano ammo may 1212nn5 Chem 25370ham2125 3 Sulubl n urga c schems Somb e m wam Malecular Molecular we Nomomc or I size lt so nrn ao aua nm Dnvpalred W7 Bonded musedphase WSW mm Motecular Mmecular sue Vonrexchange chromamgvapw chmmamgvaphy svze lt so nm ao 4uo nm chmmamgvaphy E Morecmav uc uslun chmmamgraphy cnmmamgyapny c c 0 Dr phenyl or pa yemer resn 1212nn5 Chem 25370ham2125 4 HPLC solvents o Operator experience plays a large role in the design and selection of an HPLC solvent system 0 Generally we want a significant difference between the polarities of the sp and the mp the reason being is that separation is based on solu iity differences between the mp and 5p partitioning K CSICm 0 Almost all reversed phase separations polar mp amp nonpolar sp can be carried out with combination of acetonitrile CchN andor methanol and water as a mp iZiZEIEIS Chem 25370hapl2125 Water is the most polar of all possible solvents Tab225v1 mm m and ultraviolet mm wimp w mm in mm mmmwymm s Mummy Ulnmivlurulo inm Mm 000 um Wm 0g m cutoffis Inc ef s rkg important to keep in 1 01th 3 mindwhen wegetto Ethyl acme Melhyl min emu detectors ulinvlniri mm m it w m mm L 1 3mm ii Mm Llumdrhnmummphy in quotmm m m a mswn AWN quotmamammmmm mm Mu nuui l mm uka mum IM iZiZEIEIS Chem 25370hapl2125 Practical notes Separation of most organic compounds can be handled by 018 stationary phases Most mobile compositions can be handled by either CH30NHZO or CH30HHZO Solvents must be miscible eg waterethanol An immiscible solvent system such as watertoluene would create a mess in the column tillZEUS Chem 25370hapt2125 l lls 254 glariiuu rundilinm fur mused phase du39nmamgraplly Stationary phase CIx of G8 on Seplnrdialneter spherical silica particles Less acidic Type B silica Figure 254 is preferred For upemiiou above 50 C sterically protected silica Figure 253 is preferred Column 0 46 gtlt 5 cm column torsum particlesquot 046 x 75 em column for 35w panicles shoner run same resolution Flow rate 20 mLmin Mobile phase CHSCNHZO for neutral aaalyies CHJCNnqueous bulTe rai ionic analyies 5 vol 0 CHJCN in 120 to 100 CHJCN for gradient elution Temperature 35 4o c if iempemiure control is available Sample size 25 50 uL conlaining 25 50 pg ofelich analer d A Lilli x 0467cm meler reducing me ow to LHJKZJl mLmiul 086 mUmilL la suite 4will Koll Kquot will n0 be used quickly tillZEUS Chem 25370hapt2125 Mobile Phase Compositions I Isocratic Elutions Constant solvent composition mobile phase polarity stays constant throughout elution process This is equivalent to isothermal separations 39n GC Gradient Elutions Mobile phase com position and thus polarity varies throughout elution This is equivalent to temperature programming in Consider the series ofisocratic elutions on the next page We can see that an ef cient separation is never achieved A H20 B CHGCN manna Chem 2537 Chapter 25 15 1 3 mmrm39w 12mnu5 cum m7 Wm 1 39 80 CHacN 80 i z o f 60 v C 0 45 CH N s 4 3 a 0 30 CHGCN gt 20 t llllllllit39lllIIlllllllllltllt 0 15 20 25 30 35 Time min 8 3 E Tu 8 4 g I 7 g l E 6 llmIllIlHllllltlutlllllllllldnnl 5 0 15 20 25 30 35 Time min a Figure 251 1 Gradient elution of the same mixture of aromatic compounds from Figure 250 with the same column ow rate and solvents The upper trace is the segmented gradient pro le so named because it is divided into several different segments 1212005 Chem 253 Chapter 25 41 The pump system for gradient elution is more expensive than for isocratic systems The metering valves require electronic control o Solute elution times under gradient programs are not as reproducible as isocratic elutions Fig 168 A system for low pitssur gradient generation for a ternary solvent system The mobile phase composition is controlled by metering valves located in the lines feeding the mixing chamber The mixed solvent amen the highpressure pump to be pumped Lhmugh the column Changes in mo bile phase composition are accomplished by changing the scltings for the metering valves 1211 42 21 Column Heaters in HPLC Healing the column lrl HPLC will improve masstranspurt decreasestne Cu term in the van Dreamer equatan Cunsidertne following Example w H mm lm changes We should Expectvvltn T manna Chem 2537 Chapter 25 Detectors in HPLC 0 Ideal Characteristics 0 Universal 0 Small volume prevents remixing amp band broadening 0 Fast response to owing system manna Chem 2537 Chapter 25 Refractive Index RI detector Nearly universal but poor detection limit Passes visible light through 2 compartments sample amp ference I When the solvent composition are the same the light passed through the compartments the light beam that passes through is recorded as zero en a solute is in the sample compartment refractive index changes will shi the light beam 39om the detector Limit of detection LCD 10 ng of solute tillZEUS Chem 25370hapt2125 45 Path taken when Reference out refractive index of eluate changes Path taken with pure solvent in Sample out Sample in l tillZEUS Chem 25370hapt2125 as UV vis absorbance detector I Based on electronic transitions within molecules Chapter 19 Most common type of detector for LC Fixed wavelength Hg lamp 254 nm 7 gt n Tunable wavelength selectable for speci c wavelengths monochromators or lters Still limited to single wavelegths I 1ngOD I Solvent limitations with UVvis abs Detectors iznznna Chem 25370napt2125 41 Eluate out conmin only 3 L nf liquid are mum Eluate in nwzuna Chem my 0mm 25 4 Table 2 Eluotmpi series and ultraviolet cutoff wavelengths of solvents for adsorption chromatography on silica Solvent Eluenl strength 2 Ultraviolet cutDFfnm Theulmviolclcumn mxwatu is 190 um 5mm L x Snyder in H mm mm Chmmalugmphy 1c wa lh ed y Vol 2 mm York Audum mu 8 5mm 5 lnduml 32mm Gmdr am am Muxkcgnn Mt Bummk ammu Luhnnmnet 199m quotv 1212nn5 Chem 25370hapt2125 49 Diode array detector 0 See lecture notes on the diode array spectrometer Chapter 21 Allows for the m 1 m OMWWMMWW H mm rm passed through the Mwwwwm diode array detector 1212nn5 Chem 25370hapt2125 so Pholodiade manna Deuterium lamp 5 Chem 2537 Ghana 25 Sample flow cell Reference aperlure i wanna Chem 2537 Ghana 25 Fluorescence Detectors 0 Review based on emission of excited state molecules 0 Detector 900 from excitation axis oLOD10fg 12mm Chem 2537 Chapter 25 From Chapter 18 Emission Instrumentation I Note that the signal is measured at 90 relative to the light source ummesmnca n L x many wavelength manna Chem 2537 Chapter 25 54 IR Detectors o FTIR allows for spectrum records of flowing systems analgous to the diode array system 0 Wateralcohols can be major interferences to solute detection oLOD 100 ng 1212005 Chem 2537Chapter25 55 Evaporative Light Scattering Detector o Responds to any analyte that is significantly less volatile than the mobile phase a Eluate is mixed with N2g and forms a fine mist o Solvent mp evaporates leaving fine particles of analyte The particles themselves are detected by light scattering 0 Response is proportional to analyte mass J 1212005 Chem 253 7 Chapter 25 56 25 HighPerformance Liquid Chromatography rlgm 2549 Optmlumnlnn vpunliv lightmum emu lCmmuy Allwch Anaemia imam n l nwznna Chem 25370hapl2125 Electrochemical Detectors 0 Based on amperometric response of analyte to electrode usually held at constant potential 0 lfthe analyte is electroactive can be highly sensitive since response is based on a surface phenomenon ratherthan a solution bulk property eg UVvis absorbance tillZEUS Chem 25370hapl2125 Dxxdllwns Hydmcamons Anncsi Mum Aminn Pncnnlx Ammmc hydmxylx 0mm A 2 n I o s v mm 23 memmu m hoxlznnlal llne Show Ln 0 mmquot 25c Imm mmu for 1un chwmmxmphy lt R ducliom gt Ole n Eum Kclnncx Aldzhydu 7 Cunjugllzd mm am i Dim campmmds i Mum mmwundx Mam 1 o 72 Clzv n Poxznnally ammable olgznlc mnmunal group by ampemmeni mu xa momm on m ledumun pomntiais s when comp undscon 5 me intimated functinnal gmups m dxlmanlvev 737 1212nn5 Chem 25370ham2125 To Infcrencc nnd cuumer cectmdes and men to waste Machinzd Kel F blocks From column Te nn or Mylar spnccr kaing clactmde Figure 2x13 Ampemmet c thinlayel detector cell for 5ch 1212nn5 Chem 25370ham2125 oLOD1 pg 12mm Selected ionmonitoring focuses the mass spec onto one particular me ratio SIN enhancement occurs when scanning mode is off I I VMJVKLJLL Jr m 1 mmmnmkmmwmwu mummmwmmem m rmmm mummwmwmmr wornmm Wmquot mva RWMMMW rum I pm w m n quotonquot ml 1212nn5 Summary of LC Detector mu um mmmzrclz y Mm Lnu mum nmmuzn dmeams muofmum w a mmrmmummym mm Mumquot m m m WWW m wn hmmtmm w W yum new quotmurennth mt n v murmur 1mm mummyumy mwmrwn znw mummm Mr3 YMu mnLsynva L 67mm may rm m We 12mm Chem 2537 Chapter 25 a Table 2 39nlupnrisun of minimrural HPI39 detecturs Approximate limit a of detection Useful with Detector Hg gradient Ultraviolet 014 Yes Refractive index 00 000 No Evaporative liglltscattering 01 Yes Electrochemica 001 1 N0 Fluorescence 00010l Yes Nitrogen NMNOLNOQ Hw 03 Yes Conductivily 0571 No ass spectrometry 0 I I Yes Fourier transform infrared l 000 Yes 1 Must detection limits from E W Yeung and R E Synuvec Detectors for Liquid ChromatographyquotAnal Chem 1936 58 1237A 1212nn5 Chem 2537 Chapter 25 u


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