Analyte Ionization

1

Analyte Ionization

Lecture 3

Yuri KazakevichSeton Hall University

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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.

Reversed-Phase HPLC Retention

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).

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pH is the negative logarithm of the proton concentration in the solution

pH = -log[H+]

][

][][

AH

HAKa

Equilibrium constants for acids are usually written in the following form:

][

][log

A

AHpHpKa

pH Definition

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COOH COO-

+ H+Ka=6.4 x10-5 , pKa=4.19

Ka =[C6H5COO-][H+]

[C6H5COOH]

Increase of the proton concentration in the HPLC mobile phase shifts equilibrium to the left.

NH2 NH3+

+ OH-+ H2O

pKb = 9.4pKa= 4.6

Ionization Equilibria

pKa = 14 – pKb

][

][log

A

AHpHpKa

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Dependencies of Analyte Ionization on the pH of the Mobile Phase

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

pH

[C6H5COO-]

pKa=4.2

Benzoic acid

% io

nize

d0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

pH

[C6H5NH3+]

pKb=9.4

Aniline

% io

nize

d

7

A B C

k1

ko

pH

k

pKa

)](3.2[

)](3.2[1

1 pHpK

pHpKo

a

a

e

ekkk

B C

ko

k1

pH

k

pKa

1 2 3 4 5 6 70

Basic compound: pKa=6 Acidic compound: pKa=3

pKa=Analyte is 50% ionized

Dependencies of Analyte Retention on the pH of the Mobile Phase

*Cs. Horvath, W.Melander, I.Molnar, Anal.Chem. 49 (1977) 142.

*

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Dependencies of Analyte Retention on the pH of the Mobile Phase

• The retention of ionizable compounds at a certain pH is dependent on their ionization state.

Acid and base are both ionized at pH=5 Acid and base are both neutral at pH=5

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Dependencies of Analyte Retention on the pH of the Mobile Phase

Ionization in general decreases hydrophobicity causing a decrease of HPLC retention.

RCOO-

RNH3+ RCOOH

RNH2

10 155

Retention Time (min.)

Abs

orba

nce

AcidpH=2.3Acid

pH=6.0

BasepH=6.0

BasepH=2.3

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Effect of pH on Acidic Analyte Retention

HPLC 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

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Effect of pH on Retention Factor of Acidic Analytes

0

1

2

3

4

5

6

7

8

2.02 2.48 3.01 3.52 4.00 4.96 5.98 7.02 8.02 8.86

pH

k

P-toluic acid

Phenylacetic acid

m-toluic acid

o-toluic acid

4-aminobenzoic acid

Lithium lactate

HPLC 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

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E

A

A

pH = 2.04

BC

D

E F

p H= 3.0 5

A+BC

D

E F

pH = 4.04

B+C

A

D

EF

p H= 5 .0 8

B+C C

D

F

pH =6 .03

E

A

B

C

D

F

pH =7 .04

A

E

B

C

D

F

pH = 8.04

A

E

B

C

D

F

C

A : P y rid ine , pK = 5.17

B : 2 -P icolin e, p K = 5 .9 6

C : 2,4 Lutid ine, p K = 6 .7 4

D : 4-ethy lp yr idine p K = 5 .8 7

E : P h eny le th yla m ine p K =9 .83

F: 2 ,3d im ethy lan iline p K = 4 .7

a

a

a

a

a

a

Effect of pH on Basic Analyte Retention

N

pyridinepKa = 5.17

N

CH2CH3

4-ethylpyridinepKa = 5.87

NH3C

CH3

2,4-lutidinepKa = 6.74

N CH3

CH3

CH3

NH2

2,3-dimethylanilinepKa = 4.70

CH3

NH2

phenylethylaminepKa=9.83

2-picolinepKa=5.96

Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18Eluent: 90% Aqueous / 10% MeCNBuffer: 10 mM Na2HPO4•7H2O + xH3PO4

Flow rate: 1 ml/minTemp: 25oC

Time (min.)

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Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18Eluent: 90% Aqueous / 10% MeCNBuffer: 10 mM Na2HPO4•7H2O + xH3PO4

Flow rate: 1 ml/minTemp: 25oC

Effect of pH on Basic Analyte Retention

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Aniline pKa=4.6

Retention of Aniline as a Function of pH

Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18Eluent: 90% Aqueous / 10% MeCNAqueous: 10 mM Na2HPO4•7H2O + xHClO4

Flow rate: 1 ml/min

Time (min.)

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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)

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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).

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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.