1. 1 Comparison of the performance of amines and ionic liquids as additives in RPLC for the analysis of basic compounds María José Ruiz-Ángel María Teresa Úbeda-Torres Ester Peris-García María Celia García-Álvarez-Coque Department of Analytical Chemistry Faculty of Chemistry Valencia, Spain

2. Amines and ionic liquids as additives in RPLC 2 B + H +  BH + pH 3−7 Ionic interaction of basic drugs with silanols in RPLC silanols alkyl-bonded reversed phase BH + CH 3 – C≡N

3. 3 ●use of an acidic pH to protonate the silanols −Si−O − + H +  −Si−OH ●use of deactivated columns, where silanols are eliminated ●addition of different reagents to the hydro-organic mobile phases to block the residual silanols Strategies to reduce the silanol effect ● amines ● ionic liquids Amines and ionic liquids as additives in RPLC

4. 4 Amines R 3 -N + H +  R 3 NH + pH 3−7 Amines and ionic liquids as additives in RPLC R 3 −NH +

5. 5 Amines and ionic liquids as additives in RPLC ●are made of a cation and an anion ●low melting temperature ●high thermal stability ●green solvents ●RPLC: behave just as dissociated salts ●dual character Ionic liquids: features N N R CH 3 + BF 4 − 1-Alkyl-3-methylimidazolium cation PF 6 − Cl − Possible anions

6. 6 Ionic liquids: behavior in RPLC PF 6 − N N Butyl CH 3 + 1-butyl-3-methylimidazolium hexafluorophosphate Amines and ionic liquids as additives in RPLC

7. 7 ● isolate the silanol suppression effect of imidazolium cations using an anion without affinity for the stationary phase (chloride), and check if they are able to effectively block the silanol sites, or if it is the combination with an appropriate anion the reason of the enhanced chromatographic peaks. ● compare the results with those obtained with triethylamine and dimethyloctylamine in order to gain more insight in the behavior of ionic liquids as silanol suppressors. Aim of the research

8. 8 Experimental: Ionic liquids Ionic liquid 1-R-3-Methylimidazolium cation NN + CH 3 R Anion m.p.. ( o C)d (g/ml)Water solubility Physical state at room temperature EMIM·BF 4 1-Ethyl- Cl − 59 1.48 partially soluble solid BMIM·BF 4 1-Butyl- - 71 1.21 soluble liquid 1-Hexyl- PF 6 − 6 EMIM·PF HMIM·BF 4 BF 4 − - 81 1.15 immiscible liquid Amines and ionic liquids as additives in RPLC EMIM·Cl BMIM·Cl HMIM·Cl BF 4 − 4 − 1-Ethyl- 1-Butyl- 1-Hexyl- 1-Ethyl- Cl − − - 70 1.03 soluble liquid 73 1.10 soluble solid 15 1.28 soluble liquid 80 1.11 soluble solid

9. 9 Experimental: Basic drugs (β-blockers) acebutolol atenolol celiprolol esmolol metoprolol oxprenolol pindolol timolol Amines and ionic liquids as additives in RPLC carteolol nadolol

10. 10 Effect of amines on the separation of β-blockers Amines and ionic liquids as additives in RPLC 20 0 0.0020.0040.0060.008 0.01 0 4 8 12 16 DMOA (M) k Triethylamine Dimethyloctylamine Column: Kromasil C18

11. 11 Ionic liquids: Behaviour in RPLC PF 6 − N N Butyl CH 3 + R+R+ PF 6 − R+R+ R+R+ R+R+ R+R+ R+R+ 1-butyl-3-methylimidazolium hexafluorophosphate Amines and ionic liquids as additives in RPLC BH + Adsorbed anion

12. 12 Effect of ionic liquids on the separation of β-blockers Amines and ionic liquids as additives in RPLC Effect of the cation k EMIM·Cl (M) 0 0.010.020.030.04 0 5 10 15 20 25 k HMIM · Cl (M) 00.010.020.030.04 0 5 10 15 20

13. 13 EMIM·Cl Similarities in behaviour BMIM·Cl HMIM·Cl TEA DMOA Amines and ionic liquids as additives in RPLC

14. 14 Interactions ● Neutralisation of the anionic free silanols by the ammonium or imidazolium cations, which hinders the electrostatic attraction of the cationic basic solutes to the silanols. ● Association of the additive cations to the alkyl-bonded stationary phase, which gets positive charge that repels the cationic solutes. ● Repulsion of the cationic solutes from the mobile phase by the excess of additive cation. Amines and ionic liquids as additives in RPLC

15. 15 Effect of ionic liquids on the separation of β-blockers Amines and ionic liquids as additives in RPLC k EMIM·Cl (M) 0 0.010.020.030.04 0 5 10 15 20 25 Association Neutralisation Retention decreases Repulsion

16. 16 Effect of ionic liquids on the separation of β-blockers Amines and ionic liquids as additives in RPLC Effect of an anion with affinity for the stationary phase

17. 17 BMIM·BF 4 Similarities in behaviour HMIM·BF 4 TEA EMIM·Cl DMOA BMIM·Cl HMIM·Cl Amines and ionic liquids as additives in RPLC

18. 18 Effect of ionic liquids on the separation of β-blockers Amines and ionic liquids as additives in RPLC Effect of an anion with affinity for the stationary phase EMIM · BF 4 (M) k 0 0.010.020.03 0 10 20 30 40 50 EMIM · PF 6 (M) 00.010.02 0 40 80 120 160 k

19. 19 retention Measurement of silanol suppressing potency peak shape Amines and ionic liquids as additives in RPLC

20. 20 Horváth equation Amines and ionic liquids as additives in RPLC Suppressing potency measured from the retention The Horváth equation cannot be applied when: ● Ionic liquids are associated to anions strongly adsorbed on the stationary phase: PF 6  (k > k 0 ). ● Ionic liquids are composed by relatively small cations associated to anions with moderate or strong affinity for the stationary phase: EMIM·BF 4 (k > k 0 ), EMIM·Cl and TEA, (k  k 0 ). Restrictions Ability of the additives to block the silanols

21. 21 Amines and ionic liquids as additives in RPLC DMOA BMIM·Cl HMIM·Cl BMIM·BF 4 HMIM·BF 4 Acebutolol 2645 147 1840 304 288 K A values Atenolol 3461  Carteolol 19125232455125667 Celiprolol 2990171104575220 Esmolol 2287239114356225 Metoprolol 2722114147561276 Nadolol 12066500 Oxprenolol 274585117642226 Pindolol 47141919 Timolol 238886184128250        Mean 2875  820185  145 1610  485 95  90 330  165

22. 22 ● Small additives can access directly to the silanols Mechanisms of protection of silanols Main mechanism of protection Amines and ionic liquids as additives in RPLC R 3 −NH + ● Bulky additives cover the stationary phase K A does not measure only the direct interaction with silanols, but the repulsion with the adsorbed additive on the stationary phase Caution!!!

23. 23 Amines and ionic liquids as additives in RPLC Suppressing potency measured from peak shape Half-widths plots The main interest in blocking the residual silanols is the enhancement in peak shape of basic drugs M.J. Ruiz Ángel, S. Carda Broch, M.C. García Álvarez-Coque, “Peak half-width plots to study the effect of organic solvents on the peak performance of basic drugs in micellar liquid chromatography”. Journal of Chromatography A 1217, 1786–1798 (2010). 1.0 61218 0.0 0.5 1.0 Time (min) Half-width (min) 61218 0.0 0.5 Time (min) Half-width (min) 4.04.24.44.64.85.05.25.4 Time (min) skewed peak 10% peak height A 12.012.513.013.514.014.515.0 Time (min) B symmetrical peak BA

24. 24 Amines and ionic liquids as additives in RPLC A, B (min) 010203040 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 A: left half-width B: right half-width No additive 15% acetonitrile A, B (min) 010203040 0 0.2 0.4 0.6 0.8 1.0 024681012 0 0.2 0.4 0.6 0.8 1.0 Time (min) A, B (min) Time (min) TEA DMOA

25. 25 Amines and ionic liquids as additives in RPLC Time (min)

26. 26 Suppressing potency measured from the half-widths Amines and ionic liquids as additives in RPLC mAmA m A + m B m B / m A No additive TEA DMOA mBmB EMIM·Cl BMIM·Cl HMIM·Cl EMIM·BF 4 BMIM·BF 4 HMIM·BF 4 0.0130.0380.0512.9 0.0190.0230.0421.2 0.028 0.0561.0 0.0170.0230.0401.3 0.019 0.0381.0 0.0200.0160.0360.8 0.0180.0220.0401.2 0.0200.0220.0421.1 0.0220.0170.0390.8

27. 27 ● The suppressing potencies measured by the changes in retention and peak shape are not correlated, since: ● The retention is affected by two opposite processes: the direct interaction of the additive cation with the silanols, which decreases the retention, and the attraction to the adsorbed anion, which increases the retention. ● The access of the basic compounds to the silanols is prevented by both cation and anion, producing an improvement in peak shape. ● The protection mechanism of ionic liquids and amines is greatly influenced by the size of the cation in the additive. The larger the cation, the more intense seems the protection of the silanol effect. ● The retention behaviour through the constant K A does not offer a good indication of the suppressing potency: additives with weakly adsorbed anions and additives of relatively small size do not reveal the suppression of the silanol effect. Conclusions Amines and ionic liquids as additives in RPLC

28. 28 ● What is then measuring K A ? We can only indicate that it is a parameter related with the capability of the additive to reduce the retention of the cationic solutes. ● Therefore, the suppression of the silanol potency should be evaluated through peak shape parameters, such as the peak broadening rate and peak asymmetry. ● Additives of small size, as TEA, EMIM·Cl and EMIM·BF 4, are less effective. ● Suppressors of large size improve significantly the peak shape. Therefore, DMOA, HMIM·Cl and HMIM·BF 4 have the most interesting features for the separation of basic compounds, since they produce the smallest broadening and the largest symmetry, combined with smaller retention. Amines and ionic liquids as additives in RPLC

29. 29 Thank you for your attention! Amines and ionic liquids as additives in RPLC