Salt Effects

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 O H H
d- d-
H O O H O H O O H O
C C
H H H
H O
O

Acid in its neutral form Acid in its ionized form

is more hydrophobic is less hydrophobic 3
 Solvation with Eluent Components

CH3CN/H2O CH3OH/H2O
H CH3
H H O H HO
d- d-
O H O O H O O O
C
H d- d- H H
H H CH3 OH
H O O
O

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. 4
 Solvation with Eluent Components

Benzoic acid in MeOH/H2O Benzoic acid in MeOH/H2O

5
no buffer pH= 2.5
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 7

no buffer pH= 2.5
 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 Na2HPO47H2O + 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=2.6 VR=3.5

VR=2.8

VR=3.6

pH=4.1
pH=3.1
pH=2.6

pH=1.3

2 3 4

Time (min.) 11
 Retention Increase of a Basic Compound
Using HClO4 as Acidic Modifier

Total ClO4- pH
[mM]
47 1.4
41 1.5
20 1.8
14 2.0
7 2.2

0 10
Time (min.) 12
A Concentration Versus pH ?

Total ClO4- pH Rt.

[mM] (min.)
47 1.4 7.5
41 1.5
20 1.8
14 2.0
B 7 2.2 5.2
0 10

Total ClO4- pH Rt.

[mM] (min.)
100 2.0 9.9
89 2.0
79 2.0
70 2.0
55 2.0 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 pH=2.0
2
pH=1.8
k

1.8 pH=2.0 pH=2.0

1.6 pH=2.2

1.4

1.2

1
0 20 40 60 80 100

Conc. ClO4- (mM)

Variable pH HClO4
Variable pH KH2PO4 adj. w/ HClO4
17
pH=2.0 KH2PO4 adj. w/ HClO4 and NaClO4
 Effect of Counteranion Concentration
on Retention

pH = 1.91
4-ethylpyridine
1.2 pH = 1.91

pH = 1.91 Variable pH

0.9 Constant pH
pH = 1.73
2-ethylpyridine
pH =1.91
pH = 2.10
k

0.6
Chromatographic Conditions
Column: 15 cm x 0.46cm Zorbax XDB-C18
Eluent: 90% Aqueous / 10% MeCN
0.3
Aqueous: Water + xHClO4 and HClO4 +xNaClO4
Flow rate: 1 ml/min

0
0 0.03 0.06 0.09 0.12

ClO4- [M]
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

k s - k us
k -
k us
Solvated K [A ] 1

Solvation-desolvation equilibria is dependent on buffer (counteranion) concentration

19
 Neutral, Acidic and Basic Compounds

O
H3C CH3
CH3

O NH CH3

Metoprolol pKa 9.7 OH

(base) SO3 H
p-toluenesulfonic acid pKa<2.5
(acid)
O OH

H2N
OH

HN
HO
SO3 H
CH3
Phenol Benzene sulfonic acid pK<2.5
a
Labetalol pKa 8.7 (neutral)
(acid)
(base)

How will the retention change for neutral and acidic analytes 20
with an increase of perchlorate concentration?
 Effect of Salt Concentration on
Retention of Neutral, Acidic and Basic
Compounds
5 phenol
labetolol (base)
4 p-toluenesulfonic acid

3 benzene sulfonic acid

phenol (neutral)
2 labetolol
k

metoprolol (base) metoprolol

1
p-toluene sulfonic acid (acid)
0 Chromatographic Conditions
benzene sulfonic acid (acid) Column: 15 cm x 0.46cm Zorbax
-1 Eclipse XDB-C18
Eluent: 70% Aqueous / 30% MeCN
0 10 20 30 40 50 60 70 Aqueous: Water + xHClO4 + yNaClO4
ClO 4- [mM] 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
Compounds pKa
B
A: Theophylline >9
B: 2,4 Lutidine 6.7 C
C: Benzylamine 9.3 A+B D
D:Phenylethylamine 9.8 eCN
+ 50 mM NaClO4
0M
: 3

C
ClO 3.0 )

A+C D
4)
(ad (pH =

B
+ 10 mM NaClO4
j. H
2O
70 H

A+B D

+ 5 mM NaClO4
D

C
23
no salt added
1 2 3 4
 Structures of Beta Blockers
CH3

OH OH
H3C NH
H3C O
CH2
O CH3
O HN CH3
O
O NH CH3
CH3
OH H3C NH

Propranolol Alprenolol
pKa = 9.45 Acebutolol
pKa = 9.70
H
pKa = 9.67 H3C
OH
N CH3

HN

OH OH OH O
H3C
H3C NH O NH O H2N

O OH
H3C
CH3 CH3
Pindolol Atenolol
pKa = 8.8 Nadolol pKa = 9.55
pKa = 9.67
O
O OH H3C CH3

H2N
O NH CH3
HN
HO
OH
Labetalol CH3 Metoprolol 24
pKa = 8.7 pKa = 9.70
 Separation of -Blockers Using Different
Concentrations of Perchlorate Anion

pH 3.02
0.59 mM ClO4-

A nadolol pKa 9.67

pH 3.01 B propanolol pKa 9.45
5.6 mM ClO4- C atenolol pKa 9.55
D pindolol pKa 8.8
pH 3.02
10.6 mM ClO4- E metoprolol pKa 9.7
F alprenolol pKa 9.7
pH 3.02 G o-chloroaniline pKa 2.64
50.0 mM ClO4-

Chromatographic Conditions
Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 25
Eluent: 70% Aqueous / 30% MeCN, Aqueous: Water + HClO4 + xNaClO4, pH= 3.0
Flow rate: 1 ml/min, Wavelength: 225 nm
 Effect of Different Acidic Modifiers on
the Retention of 3,4-Dimethylpyridine
1.4 3,4 dimethylpyridine

1.2 1

1 2
0.8 Chromatographic Conditions 1. Perchlorate
Column: 15 cm x 0.46cm Zorbax XDB- C18
k

Eluent: 90%Aqueous /10%MeCN

0.6 Aqueous: 1. Water + x HClO4 pH=1-3
2. Trifluoroacetate
2. Water + y TFA, pH=1-3
0.4 3. Water + z H3PO4 pH=1.6-3 3. Dihydrogen
Flow rate: 1 ml/min
phosphate
0.2 3
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
 Effect of Different Salts on the
Retention of Acebutolol

4.5
4
3.5 BF4-
3 H2PO4-
2.5 CF3COO-
k

2 PF6-
1.5 ClO4-
1
0.5
0
0 20 40 60 80 100
-
Conc. A [mM] 27
Effect of Different Counteranions
on -Blocker Retention

30 mM PF6-

A
B C
F
D E
A C 30 mM BF4-
B F
D A - atenolol
E
B - nadolol
30 mM CF3OO- C - acebutolol
A C
B F D - metoprolol
D
E E - labetalol
A+
C F 30 mM H2PO4- F - propanolol
B D
E
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