A Review of Waters Hybrid Particle Technology. …

1 Waters Corporation, 34 Maple Street, Milford, Massachusetts, 01757-3696 USA508-478-2000 Fax: Review of Waters Hybrid Particle technology . Part 2. Ethylene-Bridged [BEH technology ] Hybridsand Their Use in Liquid Chromatography2A New Level of PerformanceFirst introduced in 1999 in XTerra columns, Waters patentedorganic/inorganic Hybrid Particle technology [HPT] surmountedsignificant limitations of silica-based reversed-phase packing materials,particularly their hydrolytic instability at high pH. In 2005, second-generation HPT, branded as BEH technology , embodied in newACQUITY UPLC BEH Columns and in Waters XBridge HPLC columns, marks a new milestone in technology columns demonstrate outstandingcapability, especially for basic compounds, using traditionalconditions.

2 But now you have the freedom to broaden operation to adramatically wider pH range of 1 12, typical of polymerpackings, yet attain peak shape, efficiency, retention properties andhigh temperature stability equal to, or better than, that of the best silica-based reversed-phase an order-of-magnitude improvement in high pH stability anda higher level of chromatographic performance, BEH technology columnsdefine the new benchmark for LC method with SilicaBefore 1999, silica-based packings were predominant inreversed-phase HPLC columns because of their high chromatographicefficiency and excellent mechanical strength.

3 They have continued toevolve via significant process improvements made in the last two example, almost all new column brands in the last 15years have adopted the use of high purity, highly efficient, small- Particle , spherical silica, which reduces peak tailing due to metal in traditional approaches to bonding, , using trifunctional2or sterically hindered monofunctional silanes,3have greatly increased theresistance to hydrolysis of silyl groups, attached to the surface via siloxanebonds, in low-pH [1-3] mobile phases. Embedding a polar functionalgroup [ , a carbamate4] in a bonded alkyl chain was discovered toimprove peak tailing factors for basic analytes as well as to impart uniqueselectivity for certain classes of important analytes such as all these enhancements, one key chemical property of silica-basedpackings still limits their performance and operation: the hydrolytic instability of silica, especially above pH 8.

4 In high-pH mobile phases, silicaparticles begin to hydrolyze, resulting in loss of efficiency and peak distortion due to voids in the packed Particle technology [HPT]The XTerra family of reversed-phase HPLC columns featured thefirst commercially available Hybrid organic/inorganic packing materialsincorporating Waters patented Hybrid Particle technology . They providedthe first viable alternative to silica-based column high purity methyl Hybrid material is prepared in a highlyreproducible process analogous to that used today to produce high-puritysilica with one important difference.

5 It is formed from the mixedcondensation of twohigh purity monomers (Figure 1): tetraethoxysilane[TEOS, the SiO4tetrahedral subunit precursor] and methyltriethoxysilane[MTEOS, an organosilanewhich adds carbon throughout the backbone].The XTerra substrate retains certain key advantages of silica:purity, mechanical strength, highly spherical shape, ability to tailor particlesize, pore dimensions, surface area, and surface chemistry [usingtraditional bonding reagents , C18-, C8-, or phenyl-chlorosilanes].While chromatographic evaluations of bonded-phase XTerra columns demonstrate performance ( , efficiency, selectivity, and retentivity)comparable to that of silica-based reversed-phase materials, they alsohighlight key advantages: decreased tailing factors for basic analytes.

6 Significant improvement in high-pH ,6 OHSiOOOSiOOSiOOHOSiOOOSiOSiOOHSiOSiOOOSi SiSiOOSiOOSiSiSiSiSiSiSiHOSiOSiOOSiOOSiO HOOSiSiSiSiOOSiSiOHOOSiOHOSiSiOSiOOHOSiS iOHOOSiOOSiSiSiOHOOSiOOSiOHOSiSiSiSiH3CH 3 CCH3H3CH3CH3 CCH3CH3H3 CCH3CH3CH3H3CH3 CCH3CH3+ Patent: 6,686,035 B2 Methylpolyethoxysilane(MPEOS)Tetraethoxy silane(TEOS)Methyltriethoxysilane(MTEOS) Figure 1: XTerra Hybrid Particle SynthesisA Review of Waters Hybrid Particle technology . Part 2. Ethylene-Bridged [BEH technology ] Hybridsand Their Use in Liquid Chromatography3 Unlike other high-pH-stable packing materials [ , organicpolymers, graphitic carbon, alumina, titania, and zirconia]

7 , XTerra columnswere the first commercially available option that allowed the use ofconventional bonding reagents and convenient reversed-phase separationconditions withoutdrawbacks such as unpredictable selectivity, decreasedmechanical stability, bed swelling/shrinking, or inferior peak shape due tomass transfer issues [typical of polymers or organic coatings on inorganicphases] or secondary interactions [especially on highly active inorganicsurfaces such as alumina, zirconia or titania]. There are several benefits of carrying out separations at highpH.

8 Some compounds are not stable in acid and require a basic mobilephase. For many basic analytes, so predominant in the pharmacopoeia,using high-pH mobile phases provides a powerful tool for optimization the mobile-phase pH is two units above the pKaof thebase, it will be primarily in its neutral [unprotonated] form. In this state,peak tailing arising from interaction with silanols is minimized, retention isno longer strongly dependent on mobile phase pH, and analyticalmethods become more rugged and at a pH thatensures basic molecules are in their neutral form also dramaticallyincreases the amount of sample that can be loaded before peakdistortion occurs, a great benefit to preparative HPT DevelopmentWaters is committed to improving separation technologies.

9 Andbelieves Hybrid Particle technology is an important tool to achieve this impressive worldwide acceptance of the XTerra column brand validates the HPT approach to improving peak shape and extending thehigh pH stability of chromatographic sorbents. Since the XTerra launch, Waters has continued its HPTresearch, exploring a variety of organofunctional silane of these new Hybrid materials contained a terminal organofunctionalgroup ( , R = alkyl, aryl, vinyl); others had an inter-nal bridging organofunctional group ( , = alkylene, arylene).

10 From our initial evaluations, the one particleformulation that stood out above the rest in terms of chromatographicperformance was the ethylene-bridged have since invested several years of research and devel-opment to ensure that we would bring to market this BEH technology Particle in an optimized form. Based upon crucial customer feedback, threegoals for this second-generation Hybrid Particle were considered paramount: maximize efficiency; further improve high-pH stability; improve column four years of research, we are proud to introduce oursecond-generation BEH technology Hybrid technology IntroductionBEH technology synthesis creates particles that ensure extremecolumn performance and long column lifetime under harsh operatingconditions.