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Salt Effects

Salt Effects. Lecture 5. Yuri Kazakevich Seton Hall University. 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

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Salt Effects

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  1. Salt Effects Lecture 5 Yuri Kazakevich Seton Hall University

  2. 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

  3. H O H O H H H d- d- H O O H O H H O O O C C H H H H O O Solvation Solvation is the association of the analyte with the solvent molecules primarily by the formation of hydrogen bonds. Acid in its ionized form is less hydrophobic Acid in its neutral form is more hydrophobic

  4. H H d- d- O H H O O O C H H H H O O Solvation with Eluent Components CH3CN/H2O CH3OH/H2O d- d- • 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.

  5. Solvation with Eluent Components Benzoic acid in MeOH/H2O no buffer Benzoic acid in MeOH/H2O pH= 2.5

  6. Solvation with Eluent Components Salicylic acid in MeOH/H2O no buffer Salicylic acid in MeOH/H2O pH= 2.5 Note 20 times difference in signal intensity

  7. Solvation with Eluent Components Benzoic acid in MeCN/H2O no buffer Benzoic acid in MeCN/H2O pH= 2.5

  8. 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.

  9. 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 Na2HPO4•7H2O + xH3PO4 Flow rate: 1 ml/min Temp: 25oC

  10. Aniline pKa=4.6 VR Retention of Aniline as a Function of pH

  11. VR=3.5 VR=2.6 VR=2.8 VR=3.6 Time (min.) Retention increase of 4-Ethylpyridine with TFA as Acidic Modifier

  12. 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 Time (min.)

  13. A Concentration Versus pH ? 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 B 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-

  16. 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.

  17. pH=2.0 pH=1.8 pH=2.2 pH=2.0 pH=2.0 pH=1.8 pH=2.0 pH=2.0 pH=2.2 Variable pH HClO4 Variable pH KH2PO4 adj. w/ HClO4 pH=2.0 KH2PO4 adj. w/ HClO4 and NaClO4 Effect of Counteranion Concentration on Retention

  18. 4-ethylpyridine pH = 1.91 pH = 1.91 1.2 Variable pH pH = 1.91 Constant pH 0.9 pH = 1.73 2-ethylpyridine pH =1.91 pH = 2.10 k 0.6 0.3 0 0 0.03 0.06 0.09 0.12 ClO4- [M] Effect of Counteranion Concentration on Retention Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax XDB-C18 Eluent: 90% Aqueous / 10% MeCN Aqueous: Water + xHClO4 and HClO4 +xNaClO4 Flow rate: 1 ml/min • 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

  19. Buffer (Salt) Concentration • Ionic compounds are solvated Desolvated Solvated Solvation-desolvation equilibria is dependent on buffer (counteranion) concentration

  20. C H 3 Metoprolol pKa 9.7 SO H 3 (base) p -toluenesulfonic acid pKa <2.5 (acid) O H Benzene sulfonic acid pKa <2.5 Labetalol pKa 8.7 Phenol SO H 3 (acid) (base) (neutral) Neutral, Acidic and Basic Compounds How will the retention change for neutral and acidic analytes with an increase of perchlorate concentration?

  21. 5 labetolol (base) phenol 4 p-toluenesulfonic acid 3 phenol (neutral) benzene sulfonic acid 2 k labetolol metoprolol (base) 1 metoprolol p-toluene sulfonic acid (acid) 0 benzene sulfonic acid (acid) -1 0 10 20 30 40 50 60 70 [mM] - ClO 4 Effect of Salt Concentration on Retention of Neutral, Acidic and Basic Compounds Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax Eclipse XDB-C18 Eluent: 70% Aqueous / 30% MeCN Aqueous: Water + xHClO4 + yNaClO4 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 the basic compounds.

  22. Chaotropic Approach for Basic Compounds of Different pKa • Retention of o-chloroaniline governed by ionization. • Retention of phenylethylamine governed by chaotropicity.

  23. Separation of Basic Compounds Using Chaotropic Approach CompoundspKa A: Theophylline >9 B: 2,4 Lutidine 6.7 C: Benzylamine 9.3 D:Phenylethylamine 9.8

  24. Structures of Beta Blockers Propranolol pKa = 9.45 Alprenolol pKa = 9.70 Acebutolol pKa = 9.67 Pindolol pKa = 8.8 Atenolol pKa = 9.55 Nadolol pKa = 9.67 Metoprolol pKa = 9.70 Labetalol pKa = 8.7

  25. Separation of β-Blockers Using Different Concentrations of Perchlorate Anion pH 3.02 0.59 mM ClO4- pH 3.01 5.6 mM ClO4- pH 3.02 10.6 mM ClO4- pH 3.02 50.0 mM ClO4- Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 Eluent: 70% Aqueous / 30% MeCN, Aqueous: Water + HClO4 + xNaClO4, pH= 3.0 Flow rate: 1 ml/min, Wavelength: 225 nm

  26. 3,4 dimethylpyridine 1.4 1 1.2 2 1 0.8 k 0.6 0.4 3 0.2 0 0 20 40 60 80 Conc. Counteranion [mM] Effect of Different Acidic Modifiers on the Retention of 3,4-Dimethylpyridine 1. Perchlorate 2. Trifluoroacetate 3. Dihydrogen phosphate Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax XDB- C18 Eluent: 90%Aqueous /10%MeCN Aqueous: 1. Water + x HClO4 pH=1-3 2. Water + y TFA, pH=1-3 3. Water + z H3PO4pH=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

  27. 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] Effect of Different Salts on the Retention of Acebutolol

  28. 30 mM PF6- 30 mM BF4- 30 mM CF3OO- 30 mM H2PO4- Time (min.) Effect of Different Counteranions on β-Blocker Retention A C B F D E A C F B A - atenolol B - nadolol C - acebutolol D - metoprolol E - labetalol F - propanolol D E C A B F D E A + B C F D E

  29. Proposed Retention Mechanism 50/50 MeCN/Water MeCN PF6-

  30. 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.

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