OBSERVATIONS ON THE WETTING OF REVERSED-PHASE HPLC PACKINGS
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1 1997 Waters Corporation p OBSERVATIONS ON THE WETTING OF REVERSED-PHASE HPLC PACKINGS T. Walter, P. Iraneta, and M. Capparella Waters Corporation, 34 Maple Street, Milford, MA USA Presented at HPLC'97, Birmingham, UK June 1997 Poster #WT208
2 Abstract 1997 Waters Corporation p. 2 The behavior of reversed-phase HPLC packings is strongly dependent on the interaction of the mobile phase with the stationary phase. One important aspect of this interaction is wetting, or spreading of the mobile phase on the stationary phase surface. The penetration of the mobile phase into the pores of porous packings is strongly affected by the degree of wetting, as well as by the pressure difference across the interface, and the pore size distribution of the packing. Because most of the surface area of porous packings resides in the pores, the extent of penetration of mobile phase into the pores affects retention times. We have developed chromatographic protocols to observe the wetting of reversed-phase packings, and determined the extent of wetting using water/methanol mobile phases of varying water content. Results are presented for a range of C18- and C8-silica packings, showing the effects of different pore size distributions and bonded phase coverages. Results will also be presented for novel reversed-phase packings containing an internal polar group (prepared by bonding silica with 3- (chlorodimethylsilyl)propyl-n-octylcarbamate or -N-octadecylcarbamate), which are wetted even by completely aqueous mobile phases.
3 1997 Waters Corporation p. 3 Observations Chromatographers have observed complete loss of retention when working with low organic mobile phases. Vo? 40 min 40 min When we investigated this phenomenon we found that retention times were stable for over 20 hrs (77 injections) using 100% aqueous mobile phase. However, when flow was stopped, then restarted, retention was lost. This observation suggested that the mobile phase is extruded from the pores when pressure is released from the column.
4 1997 Waters Corporation p. 4 Conclusion Flow must be stopped for an extended period of time to dewet the stationary phase. Once wet, stationary phases remain wet in lower methanol content mobile phases than are required to rewet once dewetting has occurred. Three stationary phase characteristics that reduce the degree of dewetting after stopping flow in low organic mobile phases: low coverage (however, this often results in poor peak shapes for basic compounds) larger pore size embedded polar groups (which can also provide unique selectivity) Stationary phases containing polar embedded groups do not dewet even when flow is stopped for extended periods of time.
5 1997 Waters Corporation p. 5 Wetting Theory: Pressure Required to Force a Fluid into Pores Young and Laplace Equation: P = 4 γ cosθ c d Contact Angle: where: P c γ = = capillary pressure surface tension d = capillary or pore diameter θ = contact angle θ θ θ > 90 non-wetted surface θ < 90 wetted surface
6 Wetting Theory: 1997 Waters Corporation p. 6 Water vs. Methanol on C -Silica 18 Water on C 18 : d = 67Å γ = 72.8 dynes/cm θ = * (not wetting) Pc ~ 2200 psi ~110 1 atm Methanol on C 18 : d = 67Å γ = 22 dynes/cm θ = 39.9 * (wetting) Pc ~ 1500 psi ~40 1 atm *B. Janczuk, T. Bialopiotrowicz and W. Wojcik, Colloids and Surfaces 36 (1989)
7 1997 Waters Corporation p. 7 Effect of Dewetting on V 0 (D 2 O) % Dewetting V 0 decreases as stationary phase dewets R-square = #pts = 4 y = x R-square = #pts = 9 y = x This observation suggests that the mobile phase is extruded from the pores as the stationary phase dewets. 3.2 µmol/m² C µmol/m² C Column Void Volume (ml) % Dewetted % 3. 28% Vo \ l /
8 Testing Resistance to Dewetting 1997 Waters Corporation p. 8 Wet Stationary Phase Equil 30 min in MeOH then in MeOH/H2O (50:50) Equilibrate (30 min) in Low Organic Mobile Phase Low Organic Mobile Phases: A. 100% Aqueous Buffer 20 mm K 2 HPO 4, ph 6 B. 95% Aqueous Buffer MeOH:Buffer (5:95) C. 90% Aqueous Buffer MeOH:Buffer (10:90) Obtain Retention Factors before stopping flow V 0 sulfanilamide procainamide 40 min Stop Flow for 1 Hour then resume flow in low organic mobile phase Stationary Phase Dewets resume flow note loss of retention Obtain Retention Factors after stopping flow Vo sulfanilamide procainamide 40 min
9 1997 Waters Corporation p. 9 Effect of Flow Stop Time on Dewetting % Dewetting after Restarting Flow 100 The largest change in retention occurs within the first 1 min of flow stoppage However, for low coverage materials retention continues to decrease as flow stoppage times increase Flow Stop Time in 100% Aqueous (minutes) 2.0 µmol/m² C µmol/m² C µmol/m² C8
10 1997 Waters Corporation p. 10 Experimental Sample: 100 µg/ml Sulfanilamide 100 µg/ml Procainamide in 80:20 D 2 O/methanol Structures: H 2 N O S O NH 2 H 2 N O N H N CH 3 CH 3 HPLC Analysis Conditions: 1 ml/min flow rate except for high pressure runs 25 C column temperature 254 nm detection wavelength 5 µm particles in 3.9 x 150 mm columns 10 µl injection volume (n=3) D 2 O used as V 0 marker Mobile Phases 20 mm K 2 HPO 4, ph 6 (100%) Methanol/ 20 mm K 2 HPO 4, ph 6 (5:95) Methanol/20 mm K 2 HPO 4, ph 6 (10:90) Calculations: V Re tention Factor = % Dewetting = %Rewetted k before V V notes: before stopping flow (before) after stopping flow (after) k r o k before o after kafter = x100 k before x100
11 Effect of Pore Diameter on Stationary Phase Dewetting 1997 Waters Corporation p. 11 % Dewetting after Stopping Flow Less Dewetting with Larger Pore Size Pore Diameter (Å) Packing Material: C18 Bonded High Purity Silica µmol/m² Mobile Phases: 100% Aqueous 95% Aqueous 90% Aqueous CH 3 O Si C 18 H 37 CH 3
12 1997 Waters Corporation p. 12 Effect of C Coverage on 18 Stationary Phase Dewetting % Dewetting after Stopping Flow Less Dewetting with Lower Coverage C18 Coverage (µmol/m²) Packing Material: C18 Bonded High Purity Silica Å Pore Diameter Mobile Phases: 100% Aqueous 95% Aqueous 90% Aqueous CH 3 O Si C 18 H 37 CH 3
13 % Dewetting after Stopping Flow 100 Effect of Embedded Polar Group 1997 Waters Corporation p on Stationary Phase Dewetting CH 3 O Si (CH 2 ) 3 O C NH C 18 H 37 CH 3 C18 C8 O embedded polar group with Shield RP Phases Symmetry RP18 RP8 SymmetryShield Less dewetting with an embedded polar group(shield RP) even with: high coverage (3.1 µmol/m²) small pore size (65 Å) Mobile Phases: 100% Aqueous 95% Aqueous 90% Aqueous
14 To Rewet a Stationary Phase 1997 Waters Corporation p. 14 Once Dewetted: Use a mobile phase containing > 40 % methanol or other polar organic solvent this works by reducing the contact angle Use pressure to force aqueous mobile phase back into pores not practical because column outlet is at atmospheric pressure
15 1997 Waters Corporation p. 15 Rewetting Stationary Phase by Changing the Contact Angle % Rewet at 1 ml/min Flow Procainamide Sulfanilamide > 40% methanol required to rewet Symmetry C µmol/m² 67 Å Pore Size % Methanol in Water used to Rewet Stationary Phase
16 1997 Waters Corporation p. 16 Rewetting C 18 Stationary Phase using Pressure % Rewet in 100% Aqueous R-square = #pts = 4 y = x > 2.2 Kpsi required to rewet Symmetry C µmol/m² 67 Å Pore Size Pressure at Column Inlet (Kpsi)
17 1997 Waters Corporation p. 17 Pressure Gradient across Column inlet 5 e.g. 5 Kpsi at 3 ml/min Flow outlet Pressure Gradient (Kpsi) Stationary phase wet in water not wet in water Because of pressure gradient across column only about 62% of the column length is above 2.2 Kpsi inlet Column Length (mm) outlet
18 1997 Waters Corporation p. 18 Stationary Phase Remains Wet until Flow is Stopped (Dewetting Hysteresis) % Wetted in 100% Aqueous Wet using Pressure Symmetry C µmol/m² 67Å Pore Size Remains wet even at lower pressures Kpsi ml/min Column Inlet Pressure and Flow Rate Time Sequence >
19 Packing Materials used in Evaluation 1997 Waters Corporation p. 19 Packing Material Surface Area¹ (m²/g) Base Silica Pore Volume¹ (cm³/g) Avg. Pore Diameter¹ (Å) C18 Coverage (µmol/m²) Bonded Silica Surface Area¹ (m²/g) Pore Volume¹ (cm³/g) Avg. Pore Diameter¹ (Å) A -C B -C C -C D -C E -C F -C G -C H -C I - RP J - RP K - C * 0.513* 67.0* * values obtained for similar batches ¹measured by multipoint N 2 sorption Symmetry and SymmetryShield are trademarks of Waters Corporation Waters Corporation. Printed in the U.S.A. 6/97 WT208
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