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Analyte Ionization

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

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Analyte Ionization

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  1. Analyte Ionization Lecture 3 Yuri Kazakevich Seton Hall University

  2. 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 • However the pH of the eluent is dependent upon the type and concentration of the organic modifier and the temperature.

  3. Introduction • pH of the mobile phase affects: • analyte ionization and solvation • interactions of the analytes with the stationary phase • stationary phase structure and properties • bonded phase stability • dissolution of silica matrix • pH and the type of pH modifier have a major impact on the selectivity alteration. • Recent developments in silica manufacturing and bonded phase chemistry significantly widened applicable pH range (1.5 - 10).

  4. pH Definition pH is the negative logarithm of the proton concentration in the solution pH = -log[H+] Equilibrium constants for acids are usually written in the following form:

  5. [C6H5COO-][H+] Ka = [C6H5COOH] Ionization Equilibria Ka=6.4 x10-5 , pKa=4.19 Increase of the proton concentration in the HPLC mobile phase shifts equilibrium to the left. pKb = 9.4 pKa= 4.6 pKa = 14 – pKb

  6. [C6H5NH3+] [C6H5COO-] pKb=9.4 % ionized pKa=4.2 % ionized pH pH Aniline Benzoic acid Dependencies of Analyte Ionization on the pH of the Mobile Phase

  7. Dependencies of Analyte Retention on the pH of the Mobile Phase * Basic compound: pKa=6 Acidic compound: pKa=3 pKa=Analyte is 50% ionized *Cs. Horvath, W.Melander, I.Molnar, Anal.Chem. 49 (1977) 142.

  8. Dependencies of Analyte Retention on the pH of the Mobile Phase Acid and base are both ionized at pH=5 Acid and base are both neutral at pH=5 • The retention of ionizable compounds at a certain pH is dependent on their ionization state.

  9. RNH3+ RCOOH RNH2 Base RCOO- Acid pH=2.3 Base pH=2.3 Acid pH=6.0 pH=6.0 e c n a b r o s b A 5 10 15 Retention Time (min.) Dependencies of Analyte Retention on the pH of the Mobile Phase Ionization in general decreases hydrophobicity causing a decrease of HPLC retention.

  10. Effect of pH on Acidic Analyte Retention HPLC Conditions: Mobile phase Column – Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm 30% Acetonitrile Flow rate – 1.0 mL/min 70% 20mM Na2HPO4 buffer; variable pH Detection – UV 220nm acidic modifier = perchloric acid Injection volume – 1L

  11. Effect of pH on Retention Factor of Acidic Analytes HPLC Conditions: Mobile phase Column – Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm 30% Acetonitrile Flow rate – 1.0 mL/min 70% 20mM Na2HPO4 buffer; variable pH Detection – UV 220nm acidic modifier = perchloric acid Injection volume – 1L

  12. C H C H C H C H N H 2 3 3 3 2 C H 3 C H 3 N C H N N H H C N N 3 2 3 2,3-dimethylaniline phenylethylamine 2-picoline pyridine pKa=9.83 pKa=5.96 pKa = 5.17 pKa = 4.70 4-ethylpyridine pKa = 5.87 Effect of pH on Basic Analyte Retention 2,4-lutidine pKa = 6.74 Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 Eluent: 90% Aqueous / 10% MeCN Buffer: 10 mM Na2HPO4•7H2O + xH3PO4 Flow rate: 1 ml/min Temp: 25oC Time (min.)

  13. Effect of pH on Basic Analyte Retention Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 Eluent: 90% Aqueous / 10% MeCN Buffer: 10 mM Na2HPO4•7H2O + xH3PO4 Flow rate: 1 ml/min Temp: 25oC

  14. Retention of Aniline as a Function of pH Aniline pKa=4.6 Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 Eluent: 90% Aqueous / 10% MeCN Aqueous: 10 mM Na2HPO4•7H2O + xHClO4 Flow rate: 1 ml/min Time (min.)

  15. Peak Fronting • A condition where the rear of the peak is steeper than the front relative to the baseline. • Related to a secondary chemical equilibrium process • -Analyte Ionization • pH 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)

  16. Peak Tailing • A condition where the front of the peak is steeper than the rear relative to the baseline. • Appears when the analyte concentration exceed the linear range of adsorption isotherm. • Tailing (Depends upon acidity of silanols, ionization state of basic analyte and mobile phase pH).

  17. Concluding Remarks • pH is an effective tool for adjustment of selectivity and retention • pH can be used to optimize the resolution • Reversed phase packings are most stable between pH’s 2 - 8.

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