Analyte Ionization
Lecture 3
Yuri Kazakevich
Seton Hall University
1
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.
2
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).
3
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:
[ A ]  [ H  ]
Ka 
[ AH ]
 [ AH ] 
pK a  pH  log   
 [A ] 
4
Ionization Equilibria
[C6H5COO-][H+]
Ka = [C H COOH]
6 5
-
COOH
COO
+
+
Ka=6.4 x10-5 , pKa=4.19
H
 [ AH ] 
pK a  pH  log   
 [A ] 
Increase of the proton concentration in the HPLC mobile phase shifts
equilibrium to the left.
NH2
NH3
+
H2O
pKb = 9.4
pKa= 4.6
+
+
-
OH
pKa = 14 – pKb
5
Dependencies of Analyte Ionization
on the pH of the Mobile Phase
[C6H5NH3+]
100
90
80
70
60
50
40
30
20
10
0
pKb=9.4
pH
0
2
4
6
8
Aniline
10
12
14
[C6H5COO-]
100
90
80
70
60
50
40
30
20
10
0
pKa=4.2
pH
0
2
4
6
8
10
Benzoic acid
12
14
6
Dependencies of Analyte Retention
on the pH of the Mobile Phase
* Basic compound: pKa=6
A
B
Acidic compound: pKa=3
C
ko
B
C
ko
k
k
pKa
pKa
k1
k1
pH
ko  k1  e[ 2.3( pKa  pH )]
k
1  e[ 2.3( pKa  pH )]
0
1
2
4
3
5
6
7
pH
pKa=Analyte is 50% ionized
7
*Cs. Horvath, W.Melander, I.Molnar, Anal.Chem. 49 (1977) 142.
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.
8
Dependencies of Analyte Retention
on the pH of the Mobile Phase
RNH3+
RNH2
Base
RCOO- pH=2.3
Acid
Base
pH=6.0
Acid
pH=2.3
Absorbance
pH=6.0
RCOOH
5
10
15
Retention Time (min.)
Ionization in general decreases hydrophobicity causing a decrease of HPLC
retention.
9
Effect of pH on Acidic
Analyte Retention
HPLC Conditions:
Column – Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm
Flow rate – 1.0 mL/min
Detection – UV 220nm
Injection volume – 1L
Mobile phase
30% Acetonitrile
70% 20mM Na2HPO4 buffer; variable pH
acidic modifier = perchloric acid
10
Effect of pH on Retention Factor of
Acidic Analytes
8
7
P-toluic acid
6
Phenylacetic acid
m-toluic acid
5
k
o-toluic acid
4-aminobenzoic acid
4
Lithium lactate
3
2
1
0
2.02
2.48
3.01
3.52
4.00
4.96
5.98
7.02
8.02
8.86
pH
HPLC Conditions:
Column – Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm
Flow rate – 1.0 mL/min
Detection – UV 220nm
Injection volume – 1L
Mobile phase
30% Acetonitrile
70% 20mM Na2HPO4 buffer; variable pH
acidic modifier = perchloric acid
11
Effect of pH on Basic Analyte Retention
A : P y rid in e ,
pK
B : 2 -P ic o lin e ,
p K a= 5 .9 6
C : 2 , 4 L u t id in e ,
p K a= 6 .7 4
a
= 5.17
D : 4 - e t h y lp yr id in e
p K a= 5 .8 7
E : P h e n y l e th y l a m i n e
p K
F : 2 , 3 d im e t h y l a n i l i n e
p K a= 4 .7
a
C
A
pH = 8.04
CH3
N
=9 .83
B
D
pyridine
pKa = 5.17
F
2,4-lutidine
pKa = 6.74
H3C
E
CH2CH3
C
B
A
F
D
E
pH =7 .04
B
N
C
4-ethylpyridine
pKa = 5.87
A
D
F
E
pH =6 .03
B+ C
C
A
D
E
F
p H = 5 .0 8
CH3
2,3-dimethylaniline
pKa = 4.70
CH3
N
B+ C
NH2
A
D
E
F
pH = 4.04
A+ B
C
D
E
p H = 3.0 5
B
A
pH = 2.04
F
C
D
E
F
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
2-picoline
pKa=5.96
phenylethylamine
pKa=9.83
N
CH3
NH2
12 CH3
Time (min.)
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
13
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.)
14
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)
15
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).
16
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.
17