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CHROMATOGRAPHY - 2

Gas chromatography is a technique for separating vaporized samples by distributing them between a mobile gaseous phase and a liquid or solid stationary phase held in a column. This separation is achieved by the flow of an inert gaseous mobile phase. Various stationary phases, such as liquid and solid, are used in gas chromatography. This technique is commonly used in analytical chemistry for qualitative and quantitative analyses.

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CHROMATOGRAPHY - 2

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  1. CHROMATOGRAPHY - 2 AnalyticalChemistry II

  2. GasChromatography • Mobile phase: inertgases (helium, nitrogen, argon etc) • Stationaryphases: in capillarycolumn • Liquid (siloxanepolymers, polyethyleneglycol) • Solid (alumina, silicagelorcarbon) • Solid – liquid Image: James W Zubrick, GasChromatography (Lab Manual)

  3. GasChromatography • In gas chromatography, the components of a vaporized sample are separated by beingdistributed between a mobile gaseous phase and a liquid or a solid stationary phase heldin a column. • In performing a gas chromatographic separation, the sample is vaporized andinjected onto the head of a chromatographic column. • Elution is brought about by the flow ofan inert gaseous mobile phase. • In contrast to most other types ofchromatography, the mobilephase does not interact with molecules of the analyte. The only function of the mobile phaseis to transport the analyte through the column.

  4. GasChromatography • Ingas-liquidchromatography, while mobile phase (gas) washawaytheanalytes, theyareseparatedowingtothedifferentpolaritiesthatresults in differences in interactionwithstationaryphase. • Gasmovingwiththepressure (unitmL/min) • Injectionunit, gasification at veryhightemperature • Derivatizationfornonvolatilecompounds Görsel:https://www.chromacademy.com/Headspace-GC.html Erişim tarihi: 28.02.2019

  5. Derivatization 1- trimethylsilylation: Alkoller, fenoller, steroidler, karbohidratlarve amino asitlerinbutürevlerihazırlanır. Bu işlemiçin, trimetilklorsilan (TMCS) vehekzametildisilazan (HMDS) karışımıkullanılır. RXH + (CH3)3SiCl → (CH3)3SiXR + HCl 2 RXH + (CH3)3SiNHSi(CH3)3 → 2 (CH3)3SiXR + NH3 2- Esterification: 3- Acylation:

  6. Stationaryphases in gaschromatography Packedcolumns • Glass or metal tubing. • Typically2 to 3 m long and have inside diameters of 2 to 4 mm. • Packingmaterial: siloxane polymers (methyl,phenylandcyanosiloxanemixtures),polyethyleneglycol Capillarycolumns Fusedsilicacolumns • offer several important advantages over glass columns, such as physicalstrength, much lower reactivity toward sample components Wall coatedopentubularcolumns (WCOT) • Wall-coated columns are capillary tubescoated with a thin layer of the liquid stationary phase. Supportcoatedopentubularcolumns (SCOT) the inner surface of the capillary is lined with a thin film (~30 µm) ofa solid support material, such as diatomaceous earth, on which the liquid stationaryphase is adsorbed. This type of column holds several times as much stationary phaseas does a wall-coated column and thus has a greater sample capacity Fusedsilica Liquid film WCOT Fusedsilica Liquid film SCOT

  7. Detectors in gaschromatography

  8. ColumnTemperature • Chooseconsideringtheboilingpoint of thesample • Reasonableelutiontimes: 2–30min • Temperature of injection port must be higherthancolumntemperature. • GC-MS

  9. GasChromatography Qualitativeandquantitativeanalyses can be performedusingretentiontimes (tR) andpeakarea/heightrespectively. Comparison of GC and HPLC

  10. Size-exclusion chromatography (SEC) • Molecularweightdetermination • Columnpackingmaterials: polyacrylamide, dextran, agarose, silica orcrosslinkedpolystyrenes • Viscositymust be low at workingtemperature. Themostusedsolventsaretetrahydrofuran (THF) andtoluen. • Pumps: Pistonorperistaltic pumps • Concentrationresponsivedetectors: UV absoption (themostsensitiveone), refractiveindex (RI) anddifferentialrefractiveindex (DRI , themostused), IR absorpt • Molecularweightresponsivedetectors: lightscatteringdetectors (Low-anglelaserlightscattering; LALLS andmultianglelaserlightscattering; MALLS)

  11. Size-exclusion chromatography (SEC) Chromatogramsshowthedecomposition of a mixturecontainsthreecompounds in time

  12. Affinitychromatography • Affinitychromatography is based on thebiologicalinteractionbetweenantigenandanitbody, enzymeorsubstrateandligand.A receptor is immobilized on a agarose gel. • A moleculefrom a sample can be purifiedorconcetrated. • Amount of somecomponenets can be reduced in a mixture. • Biologicalmoleculesthatbindstospecificcompounds can be determined (e.g. Pharmaceuticals). • Enzymes can be purifiedorconcentrated in theirsolutions. Image: https://www.creative-biostructure.com/custom-affinity-chromatography-service-257.htm Access date: 02.05.2019

  13. Ion-Exchange Chromatography • Ion Exchange resins • Cationexchangechromatography (separation of alkali compoundslikeamines) • Anionexchangechromatography (compoundsthatcontainsnegativeionslikephosphate,sulfonateandcarboxylate) • Usedforsubstanceswithmolecularweightlowerthan 1500 g/mol, watersolubleandionizable. • Parameters: pH, temperatureandiontype of mobile phase • Suitableforpharmaceuticalsandtheirmetabolites, serums, foodadditives,vitaminmixtures, sugars. Görsel: https://www.lcgclabs.com/ion-exchange-chromatography/ Erişim tarihi: 28.02.2019

  14. Ion-pairchromatography Ioniccompoundsformsneutralion-pairwithion-pairingreactive (counterion) in the mobile phase.Hydrophobicity of formedion-pairincreasesandresults in increase in theaffinitytowardstationaryphase (non-polar). Separationoccursduetothedifferentaffinities of differention-pairs. AcidicsampleR–COO– + R’4N+ R–COO– + NR’4 Basicsample R–+NH3 + R’–SO3– R–+NH3–O3S –R’ https://www.youtube.com/watch?v=TSWP4igRwU0

  15. Ion-pairchromatography • Generally UV andfluorescencedetectorsareused. Forhighsensitivitymassspectrometrydetectorsareused. • İon-pairreactives: Alkanesulfonates andquaternaryammoniumsalts • Forseparation of smallinorganicandorganiccompounds • Columns can only be usedforonceduetothecontaminations • Trifluoroaceticacid can be usedforbiologicalmoleculessuch as proteinsandpeptides.

  16. Monolithiccolumns in HPLC • Columnswith porouschannels • There is almostnodeadvolume in thecolumnandthecolumn is durableto 9 mL/minflow rate • Betterresolutionandquickseparation • High backpressure– UHPLC - 1000  bar • High speed- especiallykineticstudies, analysis of highlydecomposedsubstancesandstabilitystudies • Lowsolventconsumption • Columnlength < 15 cm • BroadpHrange • Effective in separation of bigmoleculesduetothebigchannelsizes

  17. 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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