Comparison of the Reversed-Phase and Ion- Exchange Contributions to Retention on Polybutadiene Coated Zirconia and Octadecyl Silane Bonded Silica Phases Xiqin Yang, Jun Dai, Peter W. Carr* Chemistry Department University of Minnesota
Outline Mixed-mode retention on silica and zirconia based stationary phases Reversed-phase vs. ion-exchange interactions Retention models in mixed-mode retention mechanism One-site vs. two-site model Three plots: log k vs. n CH2, log k vs. log [C + ] m, and k vs. 1/[C + ] Relative contribution of ion-exchange retention: k IEX / k Conclusions One-site model is WRONG! Two-site model is more accurate.
Mixed-Mode Separation on ODS Phases A + H 3 C CH 3 (CH 2 ) 17 Si CH 3 A + H 3 C O O - OH O O O O Si Si Si CH 3 (CH2)17 Si CH 3 O O Si Bonded C 18 Chains Reversed-Phase () Interactions Ionized Silanol Groups Ion-Exchange (IEX) Interactions Mixed-mode retention mechanism!
Mixed-Mode Separation on PBD-ZrO 2 Adsorbed Lewis base anion O - A + A + O - PBD coating O P OH A + A + O - OH O H 2 O OH OH H 2 O H 2 O O Zr O Zr O Zr O Zr O Zr O Zr O Zr O Zr O PBD Coating Reversed-Phase () Moieties Lewis Base Anions Ion-Exchange (IEX) Sites Mixed-mode retention mechanism!
Columns ODS columns Ace Inertsil ODS3 Eclipse XDB Zorbax Extend Zorbax SB Zorbax Rx Alltima Ace Alltima PBD-ZrO 2 Ace: 5 µm, 100 Å, 300 m 2 /g, 15.5% C Alltima: 5 µm, 100 Å, 350 m 2 /g, 16% C PBD-ZrO 2 : 4.1 µm, 500 Å, 11 m 2 /g, 2.0% C
κ-κ Plot - log k on different columns 0.90 1.5 log k'on Ace 0.70 0.50 0.30 0.10-0.1 0 R 2 = 0.838 s.d.=0.153 log k'on PBD-ZrO 2 1.0 0.5 0.0-0.5 R 2 = 0.325 s.d.=0.565-0.3 0-0.2 0 0.3 0 0.8 0 1.3 0 1.8 0 log k'on Alltim a -1.0-0.2 0 0.3 0 0.8 0 1.3 0 1.8 0 log k'on Alltim a Condition: 72 % MeOH, 25 mm phosphate, ph=6, 35 C, 1 ml/min. Solutes: 11 antidepressant drugs Poor correlations in the κ κ plot indicate changes in selectivity PBD-ZrO 2 is really different.
One-Site Model for Mixed-Mode Retention ADDITIVE Free Energy ln k ' = φ obs ln( ) + φ: the phase ratio for the single type of site. G RT o + G o IEX RT Reversed-Phase Interactions Ion-Exchange Interactions + Cationic solute + + - - - - - Silica Substrate U. D. Neue et al. J. Chromatogr. A, 925(2001), 49-67
Two-Site Model for Mixed-Mode Retention Two INDEPENDENT Interaction Sites ' obs ' k = k + k ' IEX Pure Ion-Exchange Interactions Pure Reversed-Phase Interactions + + - - - - Silica Substrate Sokolowski, A.; Wahlund, K. G. J. Chromatogr. 189 (1980), 299 A. Nahum and C. Horvath, J. Chromatogr. 203 (1981), 53-63
Ion-Exchange Retention Characteristics log + k ' = Constant log[ C ] IEX m One-site model log k ' = A' + logk' log[ C + ] obs m Two-site model log k ' = log( k' + k' obs IEX ) Slope of log k obs vs. log [C + ] m Retention Mechanism One-site Two-site only 0 0 IEX only -1-1 dominant -1 ~ 0 IEX dominant -1 ~ -1
Reversed-Phase Retention Characteristics For a homologous series of solutes: log k' = A + BnCH B: independent of the homologous used 2 (CH 2 ) n CH 3 NH 2 CH 2 (CH 2 ) n CH 3 Alkylbenzenes One-site model p-alkylbenzylamines log k ' = logk' + logk' obs IEX B: independent of the homologous used ' ' ' Two-site model log log( ) k = k + obs k IEX B: NOT independent of the homologous used
Effect of Counterion Concentration 2.0 1.6 PBD-ZrO 2, y = 2.854-0.954 x log k' 1.2 0.8 Alltima, y = 0.902-0.159 x 0.4 0.0 1.0 1.2 1.4 1.6 1.8 log [NH4 + ] Ace, y = 0.401-0.017 x Conditions: 55/45 MeOH/ammonium phosphate buffer, ph = 6.0, T= 35 C Solute: p-butylbenzylamine One-site model is WRONG (slope is NOT 1)! Ion-exchange contribution is not large on ODS phases!
Effect of Number of Methylene Units log k'obs 2.0 1.6 1.2 0.8 0.4 0.0 Alltima y = 0.29x + 0.73 0 2 n CH2 4 6 log k'obs 2.0 1.6 1.2 0.8 0.4 0.0 PBD-ZrO 2 y = 0.19x + 0.75 y = 0.30x - 0.53 y = 0.27x - 0.19 0 2 n CH2 4 6 : alkylbenzenes; : p-alkylbenzylamines Mobile phase: 55 % MeOH, 25 mm ammonium phosphate, ph 6.0. Other experimental conditions: 1 ml/min; 35 C; 254 nm. One-site model is WRONG Two-site model is more accurate Ion-exchange retention dominates on PBD-ZrO 2
k obs = k ' k'obs of p-propylbenzylam ine Two-Site Model ' 40 30 20 10 0 + Constant [C + ] m PBD-ZrO 2 : y =463x +1.4 Alltima: y =11.9x +2.0 Ace: y =1.3x +1.2 0.01 0.03 0.05 0.07 1/[N H 4 + ] Intercept 55 % MeOH, ammonium phosphate, ph =6, 1 ml/min, 35 ºC k'iex/k' of p-propylbenzylamine = k ' IEX = k ' k ' 100% 80% 60% 40% 20% 0% Ace Alltima PBD-ZrO 2 15mM 25mM 35mM 50mM [NH 4 + ] Quantitative estimation of the relative contributions of and IEX Much more IEX on PBD-ZrO 2 causes selectivity difference k' ;
log k IEX vs. log [NH 4+ ] Alltima log k obs vs. log [NH 4+ ] k ' IEX = k' obs k' log k IEX vs. log [NH 4+ ] 1.8 y = -0.16x + 1.53 1.2 y = -0.93x + 1.82 1.4 0.8 log k'obs 1.0 0.6 log k'iex 0.4 0.0 0.2-0.2 1.1 1.3 1.5 1.7 log[nh 4 + ] -0.4-0.8 1.1 1.3 1.5 1.7 log [NH 4 + ] : propylbenzylamine; : butylbenzylamine; : pentylbenzylamine; : hexylbenzylamine Estimation of k IEX by using two-site model is good!
Conclusions One-site free energy addition retention model is incomplete Two-site mixed-mode retention model is more correct The dominant of IEX on PBD-ZrO 2 accounts for the dramatic different selectivity towards basic solutes compared to ODS phases. A plot of k vs. 1/[C + ] m is a very useful tool for quantitatively estimating relative contributions of reversed-phase and ion-exchange interactions to the retention of basic solutes.
Acknowledgements National Institutes of Health University of Minnesota ZirChrom Separations