4 Ion Pairing

8/4/2019 4 Ion Pairing

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Ion Pairing ChromatographyIon Pairing Chromatography


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Paired Ion ChromatographyPaired Ion Chromatography

Typical reversed phase chromatography is obtained

for ionic molecules.

Adding PIC reagent causes increased retention for

ions of opposite charge and decreased retention for

ions of the same charge as the PIC reagent.

Increasing the PIC reagent lipophilicity causes

increased retention of paired ions.

Increasing PIC reagent concentration increasesretention of paired ions up to a limiting value.


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Paired IonPaired Ion EquilibriaEquilibria


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Ion Pairing for Reverse PhaseIon Pairing for Reverse Phase

SeparationsSeparations


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Mechanism of IonMechanism of Ion--PairPair

ChromatographyChromatography

Ion-pair reagent

Mobile phase

Acidic compound


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Typical IonTypical Ion--Pair ReagentsPair Reagents

Pentane sulfonic acid Hexane sulfonic acid

Heptane sulfonic acid

Octane sulfonic acid

Conc. 1 10mM

Tetra butyl ammonium Tetra hexyl ammonium

Conc. 1 10mM

For Acids For Bases


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Antihistamines and DecongestantsAntihistamines and Decongestants


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Packing: Bondapak C18Column: 4 mm ID x 30cm

Solvent: Methanol/H2O with 0.005M PENTANE

Sulfonic Acid & 1% HOAc (50/50)

Flow Rate: 2.0 ml/min

Detector: UV, 254 nm, 0.1 AUFS

1. Maleic Acid

2. Phenylephrine

3. Phenylpropanol-

amine

4. Naphazoline

5. Phenacetin

6. Pyrilamine

PIC BPIC B--55


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1. Maleic Acid

2. Phenylephrine-HCl

3. Phenylpropanolamine-HCl

4. Naphazoline-HCl

5. Phenacetin

6. Pyrilamine Maleate


Solvent: Methanol/Water with 0.005M HEXANE




PIC BPIC B--66


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Solvent: Methanol/Water with 0.005M HEPTANE




1. Maleic Acid

2. Phenylephrine

3. Phenylpropanolamine

4. Naphazoline

5. Phenacetin6. Pyrilamine

PIC BPIC B--77


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Column: Bondapak C18

4 mm ID x 30 cmSolvent: MeOH: H2O, PIC B-7

Sample: Water Soluble Vitamins

Paired Ion ChromatographyPaired Ion Chromatography


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PairedPaired--Ion ChromatographyIon ChromatographyWater Soluble VitaminsWater Soluble Vitamins

1) Niacinamide

2) Pyridoxine

3) Riboflavin

4) Thiamine

Column: Bondapak C184 mm ID x 30 cm

Solvent MeOH: H2O, PIC B-5

Sample: Water Soluble Vitamins


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ELUTION

VOLUME

(ml)

Ratio ofRatio of

PIC BPIC B--55to PIC Bto PIC B--77

vs.vs.RetentionRetention


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PairedPaired--Ion ChromatographyIon ChromatographyWater Soluble VitaminsWater Soluble Vitamins

1) Niacinamide

2) Pyridamine

3) Riboflavin

4) Thiamine

Column: Bondapak C184 mm ID x 30 cm

Solvent: MeOH:H2O, PIC B-5

& B-7, 50:50

Sample: Water SolubleVitamins


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Structure of Peptides in SolutionStructure of Peptides in Solution

COO --

+ H3N

NH3 +

COO --

COO -- NH3 +

OH


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ION SUPPRESSION (H+)ION SUPPRESSION (H+)

COO

+ H3N

NH3 +

COO

COO NH3 +

OH

H

H

H


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ION Pairing (HOOCCF3)ION Pairing (HOOCCF3)

COO

H3N

NH3

COO

COO NH3

OH

H

H

H

OOCCF3

OOC

CF3

O

O

C

F3

+

+

- +

-

-

- +

- +

+ -


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PICPIC-- Good NewsGood News

Ions can be chromatographed by reversed

phase chromatography.

Ions and neutral compounds can usually be

done in the same run. Dramatic improvements in selectivity can be

made by choice of PIC reagents, particularly

between neutral and ionic solutes.


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PICPIC-- Bad NewsBad News

Equilibration is slow for PIC reagents;

dedicated columns are often recommended.

Beware of PIC reagent precipitation in

methanol, acetonitrile, etc. Beware of trace contaminates in chemicals

used as pairing reagents.


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Water Soluble VitaminsWater Soluble VitaminsCompounds: USP Tailing

1. L-ascorbic acid 1.12

2. Nicotinic acid 1.27

3. Thiamine 1.20

4. Pyridoxal 1.13

5. Pyridoxine 1.04

6. Folic acid 1.10

7. Caffeine 1.10

8. Riboflavin 1.14

Time (Min)1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

3

4

5

87

6

V0 = 1.37 min

Conditions

Columns: 4.6 x 150 mm, 5 m

Mobile Phase A: 0.1% TFA

Mobile Phase B: ACN

Flow Rate: 1.4 mL/min

Gradient: Time Profile

(min) %A %B

0.0 100 0

4.0 97 3

6.0 85 15

15.0 80 20Injection Volume: 10.0 L

Temperature: 30 oC

Detection: UV @ 260 nm

No ion-pairing agent necessary!!


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Isocratic Separation ofIsocratic Separation of

Catecholamines and MetabolitesCatecholamines and Metabolites

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

1

2

3

4

5

7

6

V0 = 1.83 min

Conditions

Column: PolarityTM 4.6 x 150 mm, 5 m

Mobile Phase: 88% A; 2% B; 10% C

Flow Rate: 1.0 mL/min

A: H2O

B: ACNC: 100 mM CH3COONH4, pH 5.0

Injection Volume: 10.0 L

Temperature: 30 oC

Detection: UV @ 280 nm

Compounds USP Tailing

1. Norepinephrine (NE) 1.21

2. Epinephrine (E) 1.20

3. Dopamine (DA) 1.21

4. 3,4-Dihydroxyphenylacetic acid (DOPAC) 1.00

5. Serotonin (5-HT) 1.10

6. 5-Hydroxy-3-indoleacetic acid (5-HIAA) 0.97

7. 4-Hydroxy-3-methoxyphenylacetic acid (HVA) 0.97

No ion-pairing agent necessary!!


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4 Ion Pairing

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