Gerry Hendrickx Regional Sales Manager Central Europe Developments in Waters Column Chemistries : BEH Technology
XTerra Columns: First Generation Hybrid Particles Initial Launch 1999 Advantages of XTerra Columns: Decreased peak tailing factors for basic analytes vs. silica columns. Improved high-ph stability vs. silica columns Comparable pore properties to conventional silica columns. milar bonded phases (C 18, C 8, Embedded Polar, Phenyl) as silica column, with comparable selectivities. milar particle sizes as lica (2.5, 3.5, 5, 10 µm). Improved chemical stability without major changes in separation conditions & without the drawbacks of many alternative packing materials. Neue, U. D.; et. al. Am. Lab., 1999, 31, 36 Cheng, Y.-F.; et. al. LCGC, 2000, 18, 1162
Introducing 2nd Generation Hybrid: Bridged Ethane-licon Hybrid Particles Waters Patented Technology No. 6,686,035 B2 Bridged Ethanes In lica Matrix Polyethoxysilane (BPES) Anal. Chem. 2003, 75, 6781-6788 Et CH 2 CH 2 Et Et Et Et Et Et Et n 4 Et Et Et Et Tetraethoxysilane (TES) + Et Et Et CH 2 CH 2 Et Et Et Bis(triethoxysilyl)ethane (BTEE)
BEH Technology Particle Attributes Surface H H H H H CH 2 CH 2 H H H Reduced lanols Wall CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 Bridging Group CH 2 CH 2 Hydrophobic Group Hybrid Particle Attribute Surface hybrid groups reduce surface silanol concentration Internal bridging groups provide high interconnectivity Internal hybrid groups provide hydrophobicity RP-HPLC Consequence Reduced USP peak tailing factors for bases Increased chemical & mechanical stability Increased high ph stability of column
Particle Dissolution at High ph lica Particles BEH Technology TM H - H - nly 4 bonds need to be hydrolyzed (H) 4 has high solubility in water Easy to dissolve silica at ph >7 Up to 6 bonds need to be hydrolyzed Hydrophobic, lower reactivity than silica -- bonds form as others break Difficult to remove Bridge group!
Particle Property Comparisons Particle ze % Carbon, Unbonded Surface Area Pore Volume Pore Diameter Metal Content (Na, Fe, Al) Phase Ratio BEH Technology TM 1.7 µm ACQUITY UPLC TM BEH 2.5, 3.5, 5 µm XBridge TM 6.6 %C 185 m 2 /g 0.7 cm 3 /g 135 Å 10 ppm 92 m 2 /cm 3 XTerra 2.5, 3.5, 5, 10 µm 7.0 %C 179 m 2 /g 0.7 cm 3 /g 120 Å 20 ppm 91 m 2 /cm 3 Phase Ratio = (1-ε ) i V p A + s 1 ρ s As = Surface Area Vp = Pore Volume Interstitial Column Porosity (ε i ) = 0.4 Skeletal Density (ρ s ) = 2.02 g/cm 3
Accelerated Low-pH Stability Test 100 Probe: Methylparaben. %Initial Retention 90 80 70 60 XBridge C18 Zorbax SB-C18 SunFire C18 Inertsil DS-3 Luna C18(2) Ace C18 50 0 50 100 Hours in 1% TFA in H 2 ph 1.0 at 80 C Gemini C18
ph 3 and ph 7 Peak Shape Comparisons USP Tailing Factors ph 3 3 2 XBridge Family Desipramine Amitriptyline ph 7 Nortriptyline Amitriptyline 1 C8 Phenyl RP18 C18 Zorbax SB C18
Accelerated High ph Stability Test
Taking ph Stability to the Extremes Hours in 0.02N NaH ph 12.3 at 50 C to 50% Efficiency Loss 0 10 20 30 40 50 XBridge C8 XBridge C18 XBridge Phenyl XBridge Shield RP18 XTerra MS C18 Symmetry C18 Challenge mobile phase: 0.02N NaH ph 12.3 at 50 C Test mobile phase: 50/50 (v/v) acetonitrile/water at 50 C Test probe: Decanophenone
Phenyl Column Ligand Stability at Low ph %Initial Retention. 100 90 80 70 60 50 Probe: Ethylparaben 0 20 40 60 80 100 XBridge Phenyl Zorbax SB-Phenyl Luna Phenyl-Hexyl Varian Pursuit Diphenyl Zorbax Eclipse XDB Phenyl Restek Ultra Phenyl Hours in 1% TFA in H 2 ph 1.0 at 80 C
XBridge Columns Bonded Phase Step 1 %C Step 1 Coverag e Endcap Final %C 16.1 %C 3.2 µmol/m 2 Proprietary 17.5 %C 11.1 %C 3.1 µmol/m 2 Proprietary 12.8 %C 13.1 %C 3.2 µmol/m 2 Proprietary 14.6 %C Polar Group 16.6 %C 3.3 µmol/m 2 TMS 16.7 %C
Cycle Time ptimization: Building on BEH Technology First Introduced at Pittcon 2004 as ACQUITY UPLC BEH Columns Chemistry fferings Expanded at Pittcon 2005 First Introduced at HPLC 2005 Ease of Migration from HPLC to UPLC mplified Purification and Isolation
Scaling from HPLC to UPLC - Constant L/dp 5 µm 150 mm Injection = 5.0 µl Flow rate = 0.2 ml/min Rs (2,3) = 2.28 HPLC 3.5 µm 100 mm Injection = 3.3 µl Flow rate = 0.3 ml/min Rs (2,3) = 2.32 2.5 µm 75 mm Injection = 2.5 µl Flow rate = 0.5 ml/min Rs (2,3) = 2.34 UPLC 1.7 µm 50 mm Injection = 1.7 µl Flow rate = 0.6 ml/min Rs (2,3) = 2.29
Scaling HPLC to UPLC : Stability-Indicating Assay Rs = 2.37 5 Terbinafine XBridge 2.1 x 150 mm, 5 µm C 18 F = 300 µl/min Detection @ 214 nm Height (µv) Deg 5 = 6,136 9 1 2 3 4 6 10 8 7 Speed 6X 0.020 0.015 Rs = 2.2 0 5 Terbinafine 9 ACQUITY UPLC TM BEH C 18 2.1 x 50 mm, 1.7 µm F = 600 µl/ min Detection @ 214 nm Height (µv) Deg. 5 = 15,656 Sensitivity 2.5X (same inj. Volume) Rs = 1X AU 0.010 1 3 0.005 2 6 8 10 4 7 0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Minutes
New 2 nd Generation Hybrid Bridged Ethane-licon Hybrid Particles Bridged Ethanes in Hybrid Matrix -Strength, - Great Peak Shape - Wider ph Range C C C C C C Anal. Chem. 2003, 75, 6781-6788
Buffer 3 : Phosphate ph 12 AU Injection #2 0 5 Minutes 15 20 Peak Width @ 4.4% 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 XBridge C18 0 50 100 150 200 250 Time (Hours) Injection #400 Injection #650 AU AU Elution rder: Propranolol Diphenhydramine Nortriptyline Acenaphthene 0 5 Minutes 15 20 0 5 Minutes 15 20