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 ( ).

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. Ionization Equilibria
Ka =
[C6H5COO-][H+]
[C6H5COOH]
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. Dependencies of Analyte Ionization on the pH of the Mobile Phase
[C6H5NH3+]
pKb=9.4
Aniline
% ionized pH
[C6H5COO-]
pKa=4.2
Benzoic acid
% ionized

7. Dependencies of Analyte Retention on the pH of the Mobile Phase
Lecture 3
Dependencies of Analyte Retention
on the pH of the Mobile Phase *
Basic compound: pKa=6
Acidic compound: pKa=3
ko= Retention factor of analyte in its neutral form
k1 = Retention factor of analyte in its ionized form
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. Dependencies of Analyte Retention on the pH of the Mobile Phase
RCOO-
RNH3+
RCOOH
RNH2 10 15 5
Retention Time (min.) A b s o r a n c e
Acid
pH=2.3
pH=6.0
Base
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. Effect of pH on Basic Analyte Retention
pyridine
pKa = 5.17 N H 3 C
2,4-lutidine
pKa = 6.74 N C H 2 3
4-ethylpyridine
pKa = 5.87 C H 3 N 2
2,3-dimethylaniline
pKa = 4.70 N C H 3
2-picoline
pKa=5.96
Chromatographic Conditions
Column: cm x 0.46 cm Zorbax Eclipse XDB-C18
Eluent: % Aqueous / 10% MeCN
Buffer: mM Na2HPO4•7H2O + xH3PO4
Flow rate: 1 ml/min
Temp: oC
phenylethylamine
pKa=9.83 C H 3 N 2
Time (min.)

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

14. Retention of Aniline as a Function of pH
pKa=4.6
Chromatographic Conditions
Column: cm x 0.46 cm Zorbax Eclipse XDB-C18
Eluent: % 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