Analyte Ionization

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Analyte Ionization. Lecture 3. Yuri Kazakevich Seton Hall University. Reversed-Phase HPLC Retention. The retention of ionizable analytes on the same bonded phase can be varied by: Type of organic modifier Concentration of organic modifier Temperature pH of the mobile phase - PowerPoint PPT Presentation

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<ul><li><p>Analyte IonizationLecture 3</p><p>Yuri KazakevichSeton Hall University</p></li><li><p>The retention of ionizable analytes on the same bonded phase can be varied by:</p><p>Type of organic modifierConcentration of organic modifierTemperaturepH of the mobile phase</p><p>However the pH of the eluent is dependent upon the type and concentration of the organic modifier and the temperature.Reversed-Phase HPLC Retention</p></li><li><p>IntroductionpH of the mobile phase affects:</p><p>analyte ionization and solvationinteractions of the analytes with the stationary phasestationary phase structure and propertiesbonded phase stabilitydissolution of silica matrix</p><p>pH and the type of pH modifier have a major impact on the selectivity alteration.</p><p>Recent developments in silica manufacturing and bonded phase chemistry significantly widened applicable pH range (1.5 - 10).</p></li><li><p>pH is the negative logarithm of the proton concentration in the solution</p><p>pH = -log[H+]</p><p>Equilibrium constants for acids are usually written in the following form:pH Definition</p></li><li><p>Ka=6.4 x10-5 , pKa=4.19Increase of the proton concentration in the HPLC mobile phase shifts equilibrium to the left.pKb = 9.4pKa= 4.6Ionization EquilibriapKa = 14 pKb</p></li><li><p>Dependencies of Analyte Ionization on the pH of the Mobile Phase</p></li><li><p>Basic compound: pKa=6Acidic compound: pKa=3pKa=Analyte is 50% ionizedDependencies of Analyte Retention on the pH of the Mobile Phase*Cs. Horvath, W.Melander, I.Molnar, Anal.Chem. 49 (1977) 142.*</p></li><li><p>Dependencies of Analyte Retention on the pH of the Mobile PhaseThe retention of ionizable compounds at a certain pH is dependent on their ionization state.</p><p>Acid and base are both ionized at pH=5Acid and base are both neutral at pH=5</p></li><li><p>Dependencies of Analyte Retention on the pH of the Mobile PhaseIonization in general decreases hydrophobicity causing a decrease of HPLC retention.</p></li><li><p>Effect of pH on Acidic Analyte RetentionHPLC Conditions:Mobile phase Column Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm 30% AcetonitrileFlow rate 1.0 mL/min 70% 20mM Na2HPO4 buffer; variable pHDetection UV 220nmacidic modifier = perchloric acidInjection volume 1L </p></li><li><p>Effect of pH on Retention Factor of Acidic AnalytesHPLC Conditions:Mobile phase Column Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm 30% AcetonitrileFlow rate 1.0 mL/min 70% 20mM Na2HPO4 buffer; variable pHDetection UV 220nm acidic modifier = perchloric acidInjection volume 1L </p></li><li><p>Effect of pH on Basic Analyte Retention2,4-lutidinepKa = 6.74Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18Eluent: 90% Aqueous / 10% MeCNBuffer: 10 mM Na2HPO47H2O + xH3PO4Flow rate: 1 ml/minTemp: 25oCTime (min.)</p></li><li><p>Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18Eluent: 90% Aqueous / 10% MeCNBuffer: 10 mM Na2HPO47H2O + xH3PO4Flow rate: 1 ml/minTemp: 25oCEffect of pH on Basic Analyte Retention</p></li><li><p>Aniline pKa=4.6Retention of Aniline as a Function of pHChromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18Eluent: 90% Aqueous / 10% MeCNAqueous: 10 mM Na2HPO47H2O + xHClO4Flow rate: 1 ml/minTime (min.)</p></li><li><p>Peak FrontingA condition where the rear of the peak is steeper than the front relative to the baseline. </p><p>Related to a secondary chemical equilibrium process-Analyte IonizationpH of analysis is close to analyte pKa-Reaction of analyte with mobile phase components(ex. Aldehydes in presence of water under acidic or basic conditions)</p></li><li><p>Peak TailingA condition where the front of the peak is steeper than the rear relative to the baseline.</p><p>Appears when the analyte concentration exceed the linear range of adsorption isotherm.</p><p>Tailing (Depends upon acidity of silanols, ionization state of basic analyte and mobile phase pH).</p></li><li><p>Concluding RemarkspH is an effective tool for adjustment of selectivity and retentionpH can be used to optimize the resolution Reversed phase packings are most stable between pHs 2 - 8. </p><p>Lecture 3Lecture 3ko= Retention factor of analyte in its neutral formk1 = Retention factor of analyte in its ionized form</p></li></ul>