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Ion Pairing Chromatography

Paired 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 increases retention of paired ions up to a limiting value.

Paired Ion Equilibria

Ion Pairing for Reverse Phase Separations

Mechanism of Ion-Pair Chromatography


Acidic compound Ion-pair reagent

Mobile phase

Typical Ion-Pair Reagents


For Bases Pentane sulfonic acid Hexane sulfonic acid Heptane sulfonic acid Octane sulfonic acid Conc. 1 10mM For Acids Tetra butyl ammonium Tetra hexyl ammonium

Conc. 1 10mM

Antihistamines and Decongestants

PIC B-5
Packing: Column: Solvent: Bondapak C18 4 mm ID x 30cm 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. Phenylpropanolamine 4. Naphazoline 5. Phenacetin 6. Pyrilamine

PIC B-6
Packing: Column: Solvent: Bondapak C18 4 mm ID x 30cm Methanol/Water with 0.005M HEXANE Sulfonic Acid & 1% HOAc (50/50) Flow Rate: 2.0 ml/min Detector: UV, 254 nm, 0.1 AUFS 1. Maleic Acid 2. Phenylephrine-HCl 3. Phenylpropanolamine-HCl 4. Naphazoline-HCl 5. Phenacetin 6. Pyrilamine Maleate

Packing: Column: Solvent:

Bondapak C18 4 mm ID x 30cm

PIC B-7

1. Maleic Acid 2. Phenylephrine 3. Phenylpropanolamine 4. Naphazoline 5. Phenacetin 6. Pyrilamine

Methanol/Water with 0.005M HEPTANE Sulfonic Acid & 1% HOAc (50/50)

Flow Rate: 2.0 ml/min Detector: UV, 254 nm, 0.1 AUFS

Paired Ion Chromatography


Column: Bondapak C18 4 mm ID x 30 cm Solvent: MeOH: H2O, PIC B-7 Sample: Water Soluble Vitamins

Paired-Ion Chromatography
Water Soluble Vitamins

Column: Bondapak C18 4 mm ID x 30 cm Solvent MeOH: H2O, PIC B-5 Sample: Water Soluble Vitamins

1) Niacinamide 2) Pyridoxine 3) Riboflavin 4) Thiamine

Ratio of PIC B-5 to PIC B-7 vs. Retention


ELUTION VOLUME (ml)

Paired-Ion Chromatography
Water Soluble Vitamins
Column: Bondapak C18 4 mm ID x 30 cm Solvent: MeOH:H2O, PIC B-5 & B-7, 50:50 Sample: Water Soluble Vitamins 1) 2) 3) 4) Niacinamide Pyridamine Riboflavin Thiamine

Structure of Peptides in Solution


NH3 + COO -NH3 +

+ H3N

COO -OH COO --

ION SUPPRESSION (H+)


NH3 + COOH NH3 +

+ H3N OH COOH

COO H

ION Pairing (HOOCCF3)


+NH3 OOCCF3 -+ COO H

NH3 OOC + - CF3

H3N + O O C F 3

-+ COO H OH

-+ COO H

PIC - Good 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.

PIC - Bad 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.

Water Soluble Vitamins


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 20 Injection Volume: 10.0 L Temperature: 30 oC 3 Detection: UV @ 260 nm
1 2 7 8

Compounds: 1. L-ascorbic acid 2. Nicotinic acid 3. Thiamine 4. Pyridoxal 5. Pyridoxine 6. Folic acid 7. Caffeine 8. Riboflavin

USP Tailing 1.12 1.27 1.20 1.13 1.04 1.10 1.10 1.14

No ion-pairing agent necessary!!

V0 = 1.37 min

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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: ACN C: 100 mM CH3COONH4, pH 5.0 Injection Volume: 10.0 L Temperature: 30 oC Detection: UV @ 280 nm

Isocratic Separation of Catecholamines and Metabolites

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

2 1 3
V0 = 1.83 min

No ion-pairing agent necessary!!


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