1

Salt Effects

Lecture 5

Yuri KazakevichSeton Hall University

2

• 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

Effect of Modifier Type and Concentration of Salt on HPLC

Separations

3

Solvation

Solvation is the association of the analyte with the solvent molecules primarily by the formation of hydrogen bonds.

Acid in its neutral form is more hydrophobic

Acid in its ionized formis less hydrophobic

H

O H

COO H

HOH

H

O H

HO

H

H O

H

COO

HO

H

-

4

Solvation with Eluent Components

CH3CN/H2O CH3OH/H2O

--

• 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.

OO

CH3HO

H

H

CH3 OHO

H

OH

H

O H

HO

H

H O

H

COO

HO

H

-

5

Solvation with Eluent Components

Benzoic acid in MeOH/H2Ono buffer

Benzoic acid in MeOH/H2OpH= 2.5

6

Solvation with Eluent Components

Salicylic acid in MeOH/H2Ono buffer

Salicylic acid in MeOH/H2OpH= 2.5

Note 20 times difference in signal intensity

7

Solvation with Eluent Components

Benzoic acid in MeCN/H2Ono buffer

Benzoic acid in MeCN/H2OpH= 2.5

8

• 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.

Retention of Basic Compounds in a Low pH Region is Affected by Salt Concentration and Type of Acid

Eluent AdditivesBuffer components: Salt, Acid

9

Effect of pH on Retention Factor of Bases

Low Retention: Fully ionized Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax XDB-C18Eluent: 90% Aqueous / 10% MeCNAqueous: 10 mM Na2HPO4•7H2O + xH3PO4

Flow rate: 1 ml/minTemp: 25oC

10

Aniline pKa=4.6

VR

Retention of Aniline as a Function of pH

11

Retention increase of 4-Ethylpyridine with TFA as Acidic Modifier

Time (min.)

3 42

pH=4.1

pH=3.1

pH=2.6

pH=1.3

VR=3.5VR=2.6

VR=2.8

VR=3.6

12100

Total ClO4- pH

[mM] 47 1.4 41 1.5 20 1.8 14 2.0 7 2.2

Retention Increase of a Basic CompoundUsing HClO4 as Acidic Modifier

Time (min.)

13

Concentration Versus pH ?A

B100

Total ClO4- pH Rt.

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

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

14

Schematic of Chaotropic Process

15

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-

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• 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.

Anionic Chaotropes in Reversed Phase HPLC

17

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

0 20 40 60 80 100

Conc. ClO4- (mM)

k

pH=2.2pH=2.0

pH=1.8

pH=2.2

pH=2.0 pH=1.8

pH=2.0

pH=2.0 pH=2.0

Effect of Counteranion Concentration on Retention

Variable pH HClO4

Variable pH KH2PO4 adj. w/ HClO4

pH=2.0 KH2PO4 adj. w/ HClO4 and NaClO4

18

Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax XDB-C18Eluent: 90% Aqueous / 10% MeCNAqueous: Water + xHClO4 and HClO4 +xNaClO4

Flow rate: 1 ml/min

Variable pH

Constant pH

• The increase in retention is independent of the pH if the analytes are fully ionized• The increase in retention is attributed to an increase of the perchlorate concentration

0

0.3

0.6

0.9

1.2

0 0.03 0.06 0.09 0.12

ClO4- [M]

k pH = 2.10pH =1.91

pH = 1.73

pH = 1.91

pH = 1.91

pH = 1.91 4-ethylpyridine

2-ethylpyridine

Effect of Counteranion Concentration on Retention

19

Buffer (Salt) Concentration

• Ionic compounds are solvated

ususs k

AK

kkk

1][

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

Solvated

Desolvated

20

OH

Phenol(neutral)

CH3

SO3H

p-toluenesulfonic acid pKa <2.5(acid)

SO3H

Benzene sulfonic acid pKa <2.5(acid)

Neutral, Acidic and Basic Compounds

How will the retention change for neutral and acidic analytes with an increase of perchlorate concentration?

Metoprolol pKa 9.7

(base)

NHO

OH

OCH3 CH3

CH3

Labetalol pKa 8.7(base)

O

NH

NH2

OH

OH

CH3

21

phenol

p-toluenesulfonic acid

benzene sulfonic acid

labetolol

metoprolol

Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax Eclipse XDB-C18Eluent: 70% Aqueous / 30% MeCNAqueous: Water + xHClO4 + yNaClO4

pH= 3.0Flow rate: 1 ml/min

Effect of Salt Concentration on Retention of Neutral, Acidic and Basic

Compounds

• 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 the basic compounds.

-1

0

1

2

3

4

5

0 10 20 30 40 50 60 70

ClO 4-

k

phenol (neutral)

metoprolol (base)

p-toluene sulfonic acid (acid)

labetolol (base)

benzene sulfonic acid (acid)

[mM]

22• Retention of o-chloroaniline governed by ionization.• Retention of phenylethylamine governed by chaotropicity.

Chaotropic Approach for Basic Compounds of Different pKa

2 4 6 8 10

pK =9.83a

pK =2.64a

AB

pH=1.58 )(25 mM ClO4

-

BA

pH=1.84 )(10 mM ClO4

-

o-Chloroaniline

Phenylethylamine

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1 2 3 4

70 H

O (

pH

= 3

.0)

2(a

dj.

HC

lO4)

: 3

0 M

eCN

+ 10 mM NaClO4

+ 5 mM NaClO4

+ 50 mM NaClO4

no salt added

A

B

C

DA+B

CD

B

A+C

DA+B

C

D

Compounds pKa

A: Theophylline >9B: 2,4 Lutidine 6.7C: Benzylamine 9.3D:Phenylethylamine 9.8

Separation of Basic Compounds Using Chaotropic Approach

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Structures of Beta Blockers

O

NHOH

CH3

CH3

Propranolol pKa = 9.45

AlprenololpKa = 9.70

O NH

CH2

OH

CH3

CH3

NH

OO

O

NH

OH

CH3

CH3

CH3

CH3

NH

ONH

OH

CH3

CH3ONH

OH

OH

OH

CH3

CH3

CH3O

NH

O

NH2

OH

CH3

CH3

O

NH

NH2

OH

OH

CH3

NHO

OH

OCH3 CH3

CH3

Labetalol pKa = 8.7

Metoprolol pKa = 9.70

Pindolol pKa = 8.8 Nadolol

pKa = 9.67

AtenololpKa = 9.55

Acebutolol pKa = 9.67

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pH 3.020.59 mM ClO4

-

pH 3.015.6 mM ClO4

-

pH 3.0210.6 mM ClO4

-

pH 3.0250.0 mM ClO4

-

Separation of β-Blockers Using Different Concentrations of Perchlorate Anion

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-C18Eluent: 70% Aqueous / 30% MeCN, Aqueous: Water + HClO4 + xNaClO4, pH= 3.0Flow rate: 1 ml/min, Wavelength: 225 nm

26

Effect of Different Acidic Modifiers on the Retention of 3,4-Dimethylpyridine

3,4 dimethylpyridine

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80

Conc. Counteranion [mM]

k

1

2

3

1. Perchlorate

2. Trifluoroacetate

3. Dihydrogen phosphate

Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax XDB- C18Eluent: 90%Aqueous /10%MeCNAqueous: 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

• Retention factor differs using different acidic modifiers • Perchlorate is a stronger chaotropic agent• Analyte more desolvated at equivalent counteranion conc. of different acids

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0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 20 40 60 80 100Conc. A- [mM]

k

BF4-

H2PO4-

CF3COO-

PF6-

ClO4-

Effect of Different Salts on the Retention of Acebutolol

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AB C

D E F

A + B

A

A

A - atenololB - nadololC - acebutololD - metoprololE - labetalolF - propanolol

BC

D E

F

BC

D EF

C

DE

F

30 mM PF6-

30 mM BF4-

30 mM CF3OO-

30 mM H2PO4-

Time (min.)

Effect of Different Counteranions on β-Blocker Retention

29

Proposed Retention Mechanism

50/50 MeCN/Water

MeCN

PF6-

30

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.

Conclusion