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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

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analyte ionization

Analyte Ionization

Lecture 3

Yuri Kazakevich

Seton Hall University

slide2

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.
introduction
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).
slide4

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:

slide5

[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

slide6

[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

slide7

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.

slide8

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.
slide9

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.

slide10

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

slide11

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

slide12

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.)

slide13

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

slide14

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.)

slide15

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)
slide16

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).
slide17

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.