1. 1 Salt Effects Lecture 5 Yuri Kazakevich Seton Hall University

2. 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. 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 form is less hydrophobic H OH C OOH H O H H OH H O H HO H C OO H O H -- 

4. 4 Solvation with Eluent Components CH 3 CN/H 2 O CH 3 OH/H 2 O -- -- 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. H OH H O H HO H C OO H O H -- 

5. 5 Solvation with Eluent Components Benzoic acid in MeOH/H 2 O no buffer Benzoic acid in MeOH/H 2 O pH= 2.5

6. 6 Solvation with Eluent Components Salicylic acid in MeOH/H 2 O no buffer Salicylic acid in MeOH/H 2 O pH= 2.5 Note 20 times difference in signal intensity

7. 7 Solvation with Eluent Components Benzoic acid in MeCN/H 2 O no buffer Benzoic acid in MeCN/H 2 O pH= 2.5

8. 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 Additives Buffer components: Salt, Acid

9. 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 Na 2 HPO 4 7H 2 O + xH 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C

10. 10 Aniline pK a =4.6 VRVR Retention of Aniline as a Function of pH

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

12. 12 Total ClO 4 - pH [mM] 47 1.4 41 1.5 20 1.8 14 2.0 7 2.2 Retention Increase of a Basic Compound Using HClO 4 as Acidic Modifier Time (min.)

13. 13 Concentration Versus pH ? A B Total ClO 4 - pHRt. [mM] (min.) 47 1.47.5 41 1.5 20 1.8 14 2.0 7 2.25.2 Total ClO 4 - pHRt. [mM] (min.) 100 2.09.9 892.0 79 2.0 70 2.0 55 2.07.7

14. 14 Schematic of Chaotropic Process

15. 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 - PF 6, BF 4, CCl 3 CO 2 -, CF 3 CO 2 -

16. 16 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. 17 pH=2.2 pH=2.0 pH=1.8 pH=2.2 pH=2.0pH=1.8 pH=2.0 Effect of Counteranion Concentration on Retention Variable pH HClO 4 Variable pH KH 2 PO 4 adj. w/ HClO 4 pH=2.0 KH 2 PO 4 adj. w/ HClO 4 and NaClO 4

18. 18 Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax XDB-C18 Eluent: 90% Aqueous / 10% MeCN Aqueous: Water + xHClO 4 and HClO 4 +xNaClO 4 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 00.030.060.090.12 ClO 4 - [M] k pH = 2.10 pH =1.91 pH = 1.73 pH = 1.91 4-ethylpyridine 2-ethylpyridine Effect of Counteranion Concentration on Retention

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

20. 20 OH Phenol (neutral) CH 3 SO 3 H p-toluenesulfonic acid pK a <2.5 (acid) SO 3 H Benzene sulfonic acid pK a <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 pK a 9.7 (base) Labetalol pK a 8.7 (base)

21. 21 phenol p-toluenesulfonic acid benzene sulfonic acid labetolol metoprolol Chromatographic Conditions Column: 15 cm x 0.46cm Zorbax Eclipse XDB-C18 Eluent: 70% Aqueous / 30% MeCN Aqueous: Water + xHClO 4 + yNaClO 4 pH= 3.0 Flow 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. 0 1 2 3 4 5 010203040506070 ClO 4 - k phenol (neutral) metoprolol (base) p-toluene sulfonic acid (acid) labetolol (base) benzene sulfonic acid (acid) [mM]

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

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

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

25. 25 pH 3.02 0.59 mM ClO 4 - pH 3.01 5.6 mM ClO 4 - pH 3.02 10.6 mM ClO 4 - pH 3.02 50.0 mM ClO 4 - Separation of β-Blockers Using Different Concentrations of Perchlorate Anion Chromatographic Conditions Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 Eluent: 70% Aqueous / 30% MeCN, Aqueous: Water + HClO 4 + xNaClO 4, pH= 3.0 Flow rate: 1 ml/min, Wavelength: 225 nm

26. 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 020406080 Conc. Counteranion [mM] k 1 2 3 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 HClO 4 pH=1-3 2. Water + y TFA, pH=1-3 3. Water + z H 3 PO 4 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

27. 27 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 020406080100 Conc. A - [mM] k BF 4 - H 2 PO 4 - CF 3 COO - PF 6 - ClO 4 - Effect of Different Salts on the Retention of Acebutolol

28. 28 A B C DE F A + B A A A - atenolol B - nadolol C - acebutolol D - metoprolol E - labetalol F - propanolol B C D E F B C D E F C D E F 30 mM PF 6 - 30 mM BF 4 - 30 mM CF 3 OO - 30 mM H 2 PO 4 - Time (min.) Effect of Different Counteranions on β-Blocker Retention

29. 29 Proposed Retention Mechanism 50/50 MeCN/Water MeCN PF 6 -

30. 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