4 ion pairing
TRANSCRIPT
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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
Packing: Bondapak C18Column: 4 mm ID x 30cm
Solvent: 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
PIC BPIC B--66
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Packing: Bondapak C18Column: 4 mm ID x 30cm
Solvent: 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
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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