Salt Effects
Lecture 5
Yuri Kazakevich
Seton Hall University
1
Effect of Modifier Type and
Concentration of Salt on HPLC
Separations
• Analyte Solvation
• Retention of Basic compounds in low pH region
• Increase in retention
• Concentration vs. pH?
• Chaotropic effect
• Disruption of solvation
• Effect of counteranion concentration
• Type of counteranion
2
Solvation
Solvation is the association of the analyte with the solvent molecules
primarily by the formation of hydrogen bonds.
H
O
H
H
H
O
O
O
H
H
O
C
d-
d-
O
O
H
O
C
H
Acid in its neutral form
is more hydrophobic
H
H
H
O
Acid in its ionized form
is less hydrophobic
H
H
O
3
Solvation with Eluent Components
CH3CN/H2O
CH3OH/H2O
H
H
O
H
d-
d-
O
O
H
H
O
H
O
C
H
O
H
H
H
O
CH3
d-
OH
HO
O
O
CH3
d- H
• Acetonitrile is not able to solvate analyte since it cannot form
hydrogen bonds.
• Solvation with methanol forms a partially hydrophilic shell that
could be retained on the RP adsorbent.
H
O
4
Solvation with Eluent Components
Benzoic acid in MeOH/H2O
Benzoic acid in MeOH/H2O
no buffer
pH= 2.5
5
Solvation with Eluent Components
Salicylic acid in MeOH/H2O
Salicylic acid in MeOH/H2O
no buffer
pH= 2.5
6
Note 20 times difference in signal intensity
Solvation with Eluent Components
Benzoic acid in MeCN/H2O
Benzoic acid in MeCN/H2O
no buffer
pH= 2.5
7
Eluent Additives
Buffer components: Salt, Acid
Retention of Basic Compounds in a Low pH Region is
Affected by Salt Concentration and Type of Acid
•
•
•
Basic compounds that are fully ionized have a low
retention.
Goal is to increase basic analyte retention in a low pH
region.
The addition of various acids and salts to the mobile
phase may effect the retention of protonated basic
analytes.
8
Effect of pH on Retention Factor
of Bases
Low Retention: Fully ionized
Chromatographic Conditions
Column: 15 cm x 0.46 cm Zorbax XDB-C18
Eluent:
90% Aqueous / 10% MeCN
Aqueous:
10 mM Na2HPO4•7H2O + xH3PO4
Flow rate: 1 ml/min
Temp:
25oC
9
Retention of Aniline as a
Function of pH
Aniline
pKa=4.6
VR
10
Retention increase of 4-Ethylpyridine
with TFA as Acidic Modifier
VR=3.5
VR=2.6
VR=2.8
VR=3.6
pH=4.1
pH=3.1
pH=2.6
pH=1.3
2
3
Time (min.)
4
11
Retention Increase of a Basic Compound
Using HClO4 as Acidic Modifier
0
10
Time (min.)
Total ClO4[mM]
pH
47
41
20
14
7
1.4
1.5
1.8
2.0
2.2
12
A
B
Concentration Versus pH ?
0
Total ClO4[mM]
pH
47
41
20
14
7
1.4
1.5
1.8
2.0
2.2
Rt.
(min.)
7.5
5.2
10
Total ClO4[mM]
pH
Rt.
(min.)
100
89
79
70
55
2.0
2.0
2.0
2.0
2.0
9.9
7.7
13
Schematic of Chaotropic Process
14
Chaotropic Counteranions
•Characteristics of a chaotropic counteranion
-Anion of less localized charge
- High Polarizability
-Low degree of hydration
-Greater disorder
•Type of chaotropic counteranions
-Inorganic and organic ions
-Phosphate, Perchlorate, Trifluoroacetate
- PF6, BF4, CCl3CO2-, CF3CO2-
15
Anionic Chaotropes in Reversed
Phase HPLC
•
Basic analyte must be fully ionized in order to ensure electrostatic
interaction with anionic chaotrope.
•
Effects retention of Basic Analytes.
•
Hydrogen bonding between water molecules disrupted.
•
Decrease in solvation of protonated basic analyte since hydration shield
around protonated analyte becomes less structured.
•
Facilitate the approach and increased interaction of the analyte to the
stationary phase.
•
Retention generally increases with increase in counteranion concentration.
•
Changes in selectivity may be observed.
16
Effect of Counteranion Concentration
on Retention
2.8
2.6
pH=2.0 pH=1.8
2.4
2.2
pH=2.2
pH=2.0
k
2
1.8
1.6
pH=1.8
pH=2.0
pH=2.2
pH=2.0
pH=2.0
1.4
1.2
1
0
20
40
60
80
100
Conc. ClO4- (mM)
Variable pH
Variable pH
pH=2.0
HClO4
KH2PO4 adj. w/ HClO4
KH2PO4 adj. w/ HClO4 and NaClO4
17
Effect of Counteranion Concentration
on Retention
4-ethylpyridine
pH = 1.91
pH = 1.91
1.2
Variable pH
pH = 1.91
k
0.9
Constant pH
pH = 1.73
2-ethylpyridine
pH =1.91
pH = 2.10
0.6
Chromatographic Conditions
Column: 15 cm x 0.46cm Zorbax XDB-C18
Eluent: 90% Aqueous / 10% MeCN
Aqueous: Water + xHClO4 and HClO4 +xNaClO4
Flow rate: 1 ml/min
0.3
0
0
0.03
0.06
ClO4- [M]
0.09
0.12
• The increase in retention is independent of the pH if the analytes are fully ionized18
• The increase in retention is attributed to an increase of the perchlorate concentration
Buffer (Salt) Concentration
• Ionic compounds are solvated
Desolvated
Solvated
k s - k us
k
 k us
K [A ]  1
Solvation-desolvation equilibria is dependent on buffer (counteranion) concentration
19
Neutral, Acidic and Basic Compounds
O
H3C
CH3
CH3
O
Metoprolol pKa 9.7
(base)
NH
CH3
OH
SO3 H
p-toluenesulfonic acid pKa<2.5
(acid)
O
OH
OH
H2N
HN
HO
CH3
Labetalol pKa 8.7
(base)
SO3 H
Phenol
(neutral)
Benzene sulfonic acid pK<2.5
a
(acid)
How will the retention change for neutral and acidic analytes
with an increase of perchlorate concentration?
20
Effect of Salt Concentration on
Retention of Neutral, Acidic and Basic
Compounds
5
phenol
labetolol (base)
4
p-toluenesulfonic acid
3
benzene sulfonic acid
k
phenol (neutral)
labetolol
2
metoprolol
metoprolol (base)
1
p-toluene sulfonic acid (acid)
0
benzene sulfonic acid (acid)
-1
0
10
20
30
40
ClO 4-
50
[mM]
60
70
Chromatographic Conditions
Column: 15 cm x 0.46cm Zorbax
Eclipse XDB-C18
Eluent: 70% Aqueous / 30% MeCN
Aqueous: Water + xHClO4 + yNaClO4
pH= 3.0
Flow rate: 1 ml/min
• The retention factor of the acidic and neutral compounds do not increase as a
result of increasing perchlorate anion concentration.
• Changes in selectivity can be observed as a result of the retention increase of
21 the
basic compounds.
Chaotropic Approach for Basic
Compounds of Different pKa
•
•
Retention of o-chloroaniline governed by ionization.
Retention of phenylethylamine governed by chaotropicity.
22
Separation of Basic Compounds
Using Chaotropic Approach
A
B
C
eCN
+ 50 mM NaClO4
C
A+C
D
B
+ 10 mM NaClO4
70 H
2O
(ad (pH =
j. H
ClO 3.0 )
4)
D
A+B
: 3
0M
Compounds
pKa
A: Theophylline
>9
B: 2,4 Lutidine
6.7
C: Benzylamine
9.3
D:Phenylethylamine 9.8
A+B
D
+ 5 mM NaClO4
D
C
23
no salt added
1
2
3
4
Structures of Beta Blockers
CH3
OH
OH
NH
H3C
H3C
CH2
O
CH3
O
O
HN
CH3
O
NH
O
CH3
CH3
OH
Propranolol
pKa = 9.45
H3C
NH
Alprenolol
pKa = 9.70
H
N
OH
Acebutolol
pKa = 9.67
H3C
CH3
HN
OH
H3C
H3C
NH
O
H3C
CH3
OH
OH
Pindolol
pKa = 8.8
NH
O
H2N
O
O
OH
CH3
Atenolol
pKa = 9.55
Nadolol
pKa = 9.67
O
O
OH
H3C
CH3
H2N
O
HN
NH
CH3
HO
Labetalol
pKa = 8.7
CH3
Metoprolol
pKa = 9.70
OH
24
Separation of β-Blockers Using Different
Concentrations of Perchlorate Anion
pH 3.02
0.59 mM ClO4-
pH 3.01
5.6 mM ClO4pH 3.02
10.6 mM ClO4pH 3.02
50.0 mM ClO4-
A
nadolol
pKa 9.67
B propanolol pKa 9.45
C
atenolol pKa 9.55
D
pindolol
pKa 8.8
E metoprolol pKa 9.7
F alprenolol pKa 9.7
G o-chloroaniline pKa 2.64
Chromatographic Conditions
Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18
Eluent: 70% Aqueous / 30% MeCN, Aqueous: Water + HClO4 + xNaClO4, pH= 3.0
Flow rate: 1 ml/min, Wavelength: 225 nm
25
Effect of Different Acidic Modifiers on
the Retention of 3,4-Dimethylpyridine
3,4 dimethylpyridine
1.4
1
1.2
2
1
Chromatographic Conditions
Column: 15 cm x 0.46cm Zorbax XDB- C18
Eluent: 90%Aqueous /10%MeCN
Aqueous: 1. Water + x HClO4 pH=1-3
2. Water + y TFA, pH=1-3
3. Water + z H3PO4 pH=1.6-3
Flow rate: 1 ml/min
k
0.8
0.6
0.4
3
0.2
1. Perchlorate
2. Trifluoroacetate
3. Dihydrogen
phosphate
0
0
20
40
60
80
Conc. Counteranion [mM]
• Retention factor differs using different acidic modifiers
• Perchlorate is a stronger chaotropic agent
26
• Analyte more desolvated at equivalent counteranion conc. of different acids
k
Effect of Different Salts on the
Retention of Acebutolol
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
BF4H2PO4CF3COOPF6ClO4-
0
20
40
60
Conc. A [mM]
80
100
27
Effect of Different Counteranions
on β-Blocker Retention
30 mM PF6A
A
B
C
B
D
C
E
F
D
30 mM BF4-
E
30 mM CF3OO-
A C
B
D
A+
C
B
D
E
E
F
F
F
30 mM H2PO4-
A - atenolol
B - nadolol
C - acebutolol
D - metoprolol
E - labetalol
F - propanolol
Time (min.)
28
Proposed Retention Mechanism
50/50 MeCN/Water
MeCN
PF6-
29
Conclusion
The type and concentration of chaotropic counteranions in the mobile phase
can increase the retention of protonated basic analytes by disruption of the
analyte solvation shell and increase the analyte hydrophobicity. A basic
compound must be protonated in order for ion association with the
chaotropic counteranion to occur. This increase in analyte retention is not a
pH dependent process.
The chaotropic approach for use in HPLC method development has been
shown to be beneficial for the development of fast and efficient separation
methods. Combination of the ionization effect and the chaotropic influence
on the analyte retention gives the chromatographer the flexibility for
selectivity adjustment in HPLC separations.
30