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CHROMATOGRAPHY

CHROMATOGRAPHY. Analytical Chemistry II. Chromatography. Real samples are generally complex mixtures and substances interfere with each other in their analyses . Chromatography is the set of techniques used for the analyses of mixtures . Stationary phase and mobile phase

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CHROMATOGRAPHY

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  1. CHROMATOGRAPHY AnalyticalChemistry II

  2. Chromatography • Real samplesaregenerallycomplexmixturesandsubstancesinterferewitheachother in theiranalyses. • Chromatography is the set of techniquesusedfortheanalyses of mixtures. • Stationaryphaseand mobile phase • Beginning of 20th century, Tswett • Chroma:colorgraphein: writing • Columnchromatographyandplanarchromatography

  3. Intermolecularinteractions - + - + Ionicinteraction cation anion Hydrogenbonding water δ+ δ- δ+ δ- δ+ δ- δ+ δ- Dipole-dipoleinteraction δ+ δ- δ+ δ- δ+ δ- Dispersionforces

  4. POLARITY • Dipole moment, unitDebye Water: 1.85 D Benzene: 0 D • Octanol-waterpartitioncoefficientKow = CA, Octanol / CA, water • Likedissolveslike octanol water Soluteprefersoctanol High Kowvalue Nonpolarsolute Solutepreferswater LowKowvalue Polar solute

  5. Solvent carry solute molecules down the column in a continuousseries of transfers between the two phases. Because solute movement canoccur only in the mobile phase, the average rate at which a solute migrates dependson the fraction of time it spends in that phase. This fraction is small for solutes that arestrongly retained by the stationary phase (component B) and large where retention in the mobile phase is more likely (component A). Ideally,the resulting differences in rates cause the components in a mixture to separateinto bands, or zones, along the length of the column Detector • Elution is a process in which solutesare washed through a stationary phaseby the movement of a mobile phase. • The mobile phase that exits thecolumn is termed the eluate. • The eluent is the portion ofthe sample contained in the mobile phase moves down the column column sample Mobile phase A B B A B

  6. PaperChromatography • Abundant, cheapandporousmaterial • Inertsupport is cellulosewhich is polymer of glucose • Polaritydifferences Application: • Baselinemust be drawnbypencil. • Solution levelmust be underthelevel of baseline. • Never allow the running solvent to elute to the end of paper. This affects overall resolution by clustering components atthe top of the paper. • Whenthepaper is dried, analytesareseen as coloredplates. • Ifthecompunds do not have a colorthenthey can be monitoredunder UV lightorusingsublimation of iodine. • Moreintensecolormeanshigherconcentration.

  7. ThinLayerChromatography • Stationaryphase: An adsorbentmaterial is coated on thesurface of glassorplastic. • Adsorbentmaterials: alumina (Al2O3) andsilica (SiO2). • Alumina (more polar) forhydrocarbons, alkylhalogenides, ether, aldehydesandketones. • Silica (less polar) foralcohol, carboxylicacidsandamines. • TLC is usedmorethanpaperchromatography. • Quantitatively, • Spots can be scrapedoffanddissolved in a solventforanalysis. • The size of spots can be used • Optical densitometry • Twoway TLC

  8. ColumnChromatography • Adsorbents:Alumina, silicaorcalcinateddiatome • Supportmaterialglasscolumn • Belowadsorbentporousglassorcoton is placed. • Separatedcompounds can be observedwith a UV lampor a derivatizationwith a coloringagentiftheyarecolorless. Görsel: http://www.wikiwand.com/en/Column_chromatography Erişim Tarihi: 24.02.2019

  9. High Performance Liquid Chromatography (HPLC) • Automatizedcolumnchromatography • Sensitive, suitableforbothquantitativeandqualitativeanalyses, applicablefornon-volatilecompounds • Forpharmaceuticalsandfoodsamples, the golden standard • 10-30 cm length 4-10 mm diametersteelcolumnsfilledwith 2-25 µm diameterparticles • Stationaryphaseliquidor a thin film layer on solidparticles (adsorbingorchemicalbinding) • Theliquid in the mobile phaseandstationaryphasemust be immiscible. • Liquid-liquidchromatography is based on partition.

  10. High Performance Liquid Chromatography (HPLC) H2O Benzene n-HeptaneTolueneChlorobenzeneMetanolWaterAcetonitrile 0 D 0 D 0.43 D 1.58 D 1.70 D 1.85 D 3.44 D

  11. HPLC

  12. HPLC detector • A chromatogram is a plot of somefunction of solute concentration versuselution time. • Columnefficiency is stated as theoreticalplateheight (H) andnumber (N). The lower the theoretical plateheightand the greater the theoreticalplatenumber, the higher theefficienct . H = L / N L is thelength of column. sample Column mobile phase A B B A B

  13. Instrumentation in HPLC Main components of HPLC: • Mobile phasebottles • Pump: for mobile phaseflow • Injectionunit: toinjectsamplesto HPLC system • Column: stationaryphase • Detector: todetectseparatedsamples • Amplifier: toamplifythesignals of detector • Recorder: tovisualizeandrecordthesignals Görsel: https://laboratoryinfo.com/hplc/ adresinden alınarak düzenlenmiştir. Erişim tarihi: 24.02.2019

  14. Pump • Pressureupto 400 atm • Pressurewithoutpulse • 0.1 – 10 mL/minflow rate • ≤0.5 % repeatibility in flow • Resistancetocorrosion Pumptypes: • Reciprocatingpump • thescrew-drivensyringetype Nonlinearflow profile withpulse Linearflow profile withoutanypulse

  15. Sampleinjectionsystem • Manual injection • Automaticinjection • 1-100 µL injectionvolume (differentoptionsarealsoavailable)

  16. HPLC Columns Generally, • steelpipes 10 – 30 cm in length • 4 – 10 mm columninnerdiameter • 2 – 25 µm particle size Differentcolumns can be produceddepending on theneeds. «250 x 4.6 x 5µm» Packingmaterial: • Pellicular: spherical, nonporous, glass or polymer beads with typical diametersof 30 to 40 mm • Porousparticles: porousmicroparticles having diameters ranging from 3 to 10 mm. The particles are composed ofsilica, alumina, the synthetic resin polystyrene-divinyl benzene, or an ion-exchangeresin.

  17. HPLC columns • Toproduce a quantity of pureseparatedcompoundwidecolumns (preparativecolumns) areusedandthistechnique is called as preparative HPLC. • Therearespecificconditions (pH, solventtypes, temperatureetc) foreachcolumntoresist • Toincreasethelifetime of columnandtoprotect it fromcontamination, a guardcolumnwhich is a short column packed with a similarstationary phase as the analytical column can be used.

  18. HPLC Instrumentation - Detector • Detectors • Stack(based on comparison) detectors;respondstotheproperties of mobile phasethatarechangedbyanalytessuch as dielectricconstant, refractiveindexanddensity. • Analytespecificdetectors,respondstotheproperties of analytesthose mobile phasesdon’thavesuch as UV absorbance, fluorescenceintensityordiffusioncoefficient.

  19. Absorbace detectors: • Absorbancedetectorswithfilters • Provideshighsensitivity↑ • Allowsbroadrange of wavelengthpass↓ • Absorbancedetectorswithmonochromator • Diodearraydetectors (DAD) arethemostpowerfuldetectorsthatcollectfullspectrumaboutonesecond.

  20. Fluorescencedetectors • Lightsources: mercurylamp, xenonlamporlazers • Wavelengthselector: filtersormonochromators • Can be usedforthesubstances at verylowconcentrations • Onlyfluorescentsubstances can be analyzed (derivatization) Görsel: http://www.chromatographyonline.com/how-does-it-work-part-v-fluorescence-detectors Erişim tarihi:24.02.2019

  21. Refractiveindexdetector: Inthisdetector, solvent (reference) passthroughhalf of a cellwhileeluentpassthroughtheotherhalf of it. Thesetwocellsaredividedby a glassplatewhich is placed at an angleforrefraction of lightifrefractiveindex of twosolutionaredifferent. Whenthelightdeviatesfromitswaysthroughthesurface of photosensitivedetector, outputsignalchanges. Thechanges in theoutputsignalareamplifiedandrecorded as a chromatogram. Reference cell Computer– Monitoringdetectorresponse Photodiodedetector Lightsource Samplecell

  22. Electrochemicaldetectors Amperometry, polarography, coulometryandconductometry Advantages: High sensitivity, simplicityandselectivity Disadvantages: onlyelectroactivesubstances can be analyzed Görsel: https://community.asdlib.org/imageandvideoexchangeforum/2013/08/02/electrochemical-detector-for-hplc-separations/ Erişim tarihi: 24.02.2019

  23. Massspectrometricdetectors • Analysis using a massanalyzerafterionization • Broadranges of applications • Limit of detectionupto 1 – 10 pg • Duetohighsensitivity, LC-MS (Liquid chromatography – massspectrometry) is frequentlyusedforinvestigation of thepharmacokineticbehaviours of pharmaceuticalsandbiologicalsampleanalyses in bioanalytical. Forgettinghighersensitivtyandmorepreciseresultstwomassspectrometersareusedandthistechnique is called LC-MS/MS. LC-MS/MS is used in bioavailabilityandbioequivalancetests.

  24. Otherdetectors • Lightscatteringdetectors: Mobile phasepassingthroughthecolumns is convertedtosmallparticlesandsolvent is evaporatedandfinally hey aredetected in a lightscatteringcell. • Conductometricdetectorsmeasurestheconductivity of the mobile phasepassingthroughtwoelectrodes.

  25. Mobile phasebottlesandsolventtreatment in LC • Glass mobile phasebottles • Toremovedissolvedgasses • Degasser in theinstrument • Ultrasoundapplicationbeforeanalyses • Solventfilters • Isocraticelution: mobile phasecomposition is heldconstantduringseparation • Gradientelution: mobile phasecompositionchangesduringseparation degasser Görsel: https://www.schambeck-sfd.com/en/hplc-gpc-products/hplc-gpc-online-degasser.php Erişim tarihi: 24.02.2019

  26. Systemsuitabilityparameters Thevalidity of chromatographicseparation • Theoreticalplatenumber (N) • Selectivityfactor (α) • Resolution(Rs) • Capacityfactor(k) • Tailingfactor (T) • Assymetryfactor (As) System is suitable: at leasttwo of theparametersmeetrequirements.

  27. TheoreticalPlateNumber (N) • TheoreticalPlate is thesmalestplate at whichseparationtakesplace. • Sharpandnarrowpeaksshowshighernumber of platesandhighlyefficientcolumn. N = 16 []2 tRretention time of thepeak w: width of the peak at its base İdeally N > 2000 plate Column

  28. Selectivityfactor (α) Selectivityfactorshowslevel of separationbetweentwopeaks. It is alsocalled as relativeretention. α = İdeallyα > 1 . Althoughhigherαvaluesindicatesbetterseparationpower, it is not thedirectindicator of separation. Resolution is moreusefulforquality of separation. Görsel: Chromacademy, e-learningfortheanalyticalchemistrycommunity, The Theory of HPLCChromatographic Parameters

  29. Capacityfactor (k) Capacityfactor (k) is a measure of retention of an analyte in a column. It is the rate of retention of an analytetotheretention of andunretainedspecies. k = High k value Low k value Ideally 2 < k < 8 Capacityfactor can be usedforqualitativeanalysis of species in a sample.

  30. Rezolution (Rs) Resolution is themeasure of ability of a columntoseparatetwoanalytes. Themostimportantgoal of a HPLC method is getting optimum resolution at shortest time. Rs = Rs=1.6 Rs=0.4 IdeallyRs > 1.5

  31. Tailingfactor(T) andAsymmetryfactor (As) Bothareindicators of thepeaksymmetry. Intailingfactorpeakwidth is measured at 5% of thepeakheightwhile it is measured at 10% of thepeakheight in asymmetryfactor. • T = A+B / 2A (at w0.05) • As = B/A (at w0.10) Ideally 0.95 < As < 1.20 and T < 2

  32. Instrument, peakareaorpeakheightrepeatibility • Relativestandarddeviation of at least 6 timesrepeatedexperiments • Ideally, • General separations <% 1.5 • Bilogicalsamples < % 5.0 • Traceanalyses %5 – 15 • Forinstrumentrepeatibility, • retention time %relativestandarddeviation < 5% or <1% formoreexperiments.

  33. Systemsuitabilityparameters in EuropeanPharmacopoeia • Theoreticalplatenumber(N) • Seçicilik faktörü (α) • Resolution (Rs) • Capacityfactor(k) • Tailingfactor (T) • Asymmetryfactor(As) • Relativestandarddeviation of peakheightorarea • Instrumentrepeatibility

  34. Qualitativeandquantitativeanalyses in HPLC Qualitativeanalysis • Retention time, tR Quantitativeanalysis • Peakheight • Peakarea • Internalstandard • Regressionanalysis • Instrumentresponsefactor

  35. LC–MS combination Inthecombination of thesetwomethods, firstmixturesareseparated in LC andmorepurecompoundsare sent to MS fordetection. LC-MS also can alsoanalyzedifferent molecules having the same retention times because the mass spectra of themare different and selective analysis can be provided. Thereforecompoundsthatcannot be separatedbychromatography can be determined.Howeverforthissomething has to be takenintoconsideration: a.Interfacebetween LC and MS should not reducetheperformancebecausesamples. b. Analyteshould not undergoanyuncontrolledmodificationwhilepassingthroughtheinterface. c. Theyield of sample transfer must be high. If transfer occurs in theinterfacethenionizationefficienctmust be high. d. Thenoise in theinterfacemust be low. e. Interfacemust be reliable, repeatableanduserfriendly. f. Interfacemust be flexibletodifferentchromatographicconditions. Forexample, flow rate of mobile phase can varyfrom 20 nL/minto 2 mL/minorcomposition of mobile phasecomposition can varyfrom 100% percentorganicsolventto 100% aqueoussolution. g. Interfaceshould not changethevacuum of MS and it should not affecttheperformance of MS. h. Flow rate should be determinedby LC. i. Mobile phaseadditives in LC must be volatile (such as acetic acid, formic acid, trichloroacetic acid (TCA), trifloroacetic acid (TFA), trimethylamin, triethylamin, ammonium acetate, heptaflorobutiric acid (HFBA)). Alsoconcentraion of themmust be low ( upto 10 mM). Nonvolatilecompoundscontaminatetheinterfaceandthis can negativelyaffecytheanalysis. Metal phosphates, carbonates, mineral acidsand metal basesshouldnever be used.

  36. LC–MS combination Phases in LC-MS combination • Thesample in LC is liquid (sometimes it containshighamount of water) and it cannot be transferredto MS directly. The role of interface is toremovethe mobile phase. • In MS analysis is performed in gasphaseandsamplesmust be gasified. Somesamplesmay be nonvolatileandthey can be decomposedwhenthereareheated. Forthisreasondifferentionizationtechniquesaredevelopedratherthantheionizationtechniques in whichdecomposition can be observedsuch as chemicalionizationandelectronionization. Detectors in HPLC arepassive (sampledoes not change in thedetector). On theotherhand, MS is an activedetector in whichsamplesgasifiedandionized.

  37. REFERENCES: • Skoog DA, Holler FJ, Nieman TA, Principles of Instrumental Analysis, Harcourt&BraceCompany, USA, 1998. • Khan JI, Kennedy TJ, ChristianJr DR, Basic Principles of ForensicChemistry, Springer, New York, USA, 2012. • Hage DS, Carr JD, AnalyticalChemistryandquantitativeanalysis, PearsonPrenticeHall, New Jersey, USA,2011. • Onur F, Analitik Kimya II, Ankara Üniversitesi Eczacılık Fakültesi Yayın No:101

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