BEH (Ethylene Bridged Hybrid) Technology


The 1.7 µm Ethylene Bridged Hybrid [BEH] particle is one of the key enablers behind UPLC® technology. It is available in three different pore sizes [130Å, 200Å and 300Å] and several bonded phases for reversed-phase and hydrophilic interaction chromatography and is applicable from small molecule to large biopharmaceutical analysis. Due to the intrinsic chemical stability of hybrid particle technology, a wider usable pH range [pH 1-12] can be employed, enabling a versatile and robust separation technology for method development.

BEH particle technology is available in HPLC particle sizes [2.5, 3.5, 5 and 10 µm] in the XBridge™ family of HPLC columns, enabling seamless transfer between HPLC and UPLC technology platforms.



ACQUITY UPLC BEH C18 and C8 Columns


Providing unprecedented levels of peak asymmetry, efficiency and chemical stability, the 1.7 µm ACQUITY UPLC® BEH C18 and C8 columns are versatile, high-performance separation columns suitable for a diverse range of analytes.  With the ability to operate between pH 1-12, these trifunctionally-bonded alkyl columns can employ the power of pH to impact the retention, selectivity and sensitivity of ionizable compounds while delivering exceptional low- and high-ph stability.



EPA Method 8330 HPLC separations usually require temperature control of ± 1 °C since large shifts in selectivity can occur with small changes in temperature. The increased resolution of the UPLC technology allows for a robust and less temperature-sensitivity [up to ± 5 °C] separation.




The ACQUITY UPLC® BEH HILIC column utilizes an unbonded BEH particle to improve the retention of very polar species as well as provide an orthogonal separation mode for mixtures of polar and ionizable compounds. 


The ACQUITY UPLC BEH HILIC column is optimized and tested to produce efficient, reproducible separations under UPLC® HILIC conditions and are designed to overcome a major weakness of HILIC stationary phases: chemical stability. The rugged BEH particle’s wide usable pH range overcomes this chemical instability and results in long column lifetimes.


Separation of Very Polar Bases Using ACQUITY UPLC BEH HILIC Columns


Opioid pain relievers such as morphine are used to manage chronic and acute pain.  These types of drugs are extremely potent, resulting in low doses and concentrations in body fluids.  The study of the efficacy and metabolism of morphine and its metabolites wihin the body requires selective and sensitive analytical methods such as Hydrophilic Interaction Chromatography (HILIC).  In the HILIC separation shown above, the more polar metabolite morphine-3-ß-glucuronide elutes AFTER the more non-polar parent compound, morphine.




ACQUITY UPLC® BEH Phenyl columns incorporate a trifunctionally bonded phenyl-hexyl ligand to provide industry-leading pH stability, batch-to-batch reproducibility and exceptional peak shape compared to other commercially available phenyl columns. The ACQUITY UPLC BEH Phenyl column is designed to provide complementary selectivity to straight-chain alkyl columns, particularly for analytes containing aromatic rings.


Caffeic Acid Derivatives in Echinacea Purpurea Separations



The active components in various Echinacea preparations can be divided into three major groups: caffeic acid derivatives, polysaccharides, and lipophilic components. Studies have shown that there is a variation in the quality of popular Echinacea-containing products. Thus, scientific monitoring of Echinacea’s active ingredients is desired. Shown here are sub-four-minute separations of the primary caffeic acid derivatives—polyphenolic compounds that have been shown to possess antioxidant properties.


ACQUITY UPLC BEH Shield RP18 Columns


ACQUITY UPLC® BEH Shield RP18 columns incorporate a hydrophilic carbamate group within a C18 chain to produce exceptional peak shape for basic compounds as well as alternate selectivity compared to straight-chain alkyl columns.


Fast Analgesics Separations



Over-the-counter analgesics are designed purely for pain relief. Because of their low cost, effectiveness and safety, analgesics are recommended as a first line option by most physicians. Shown here are separations of seven popular analgesics in less than 1.5 minutes. Note the change in selectivity provided by the BEH Shield RP18 embedded polar ligand.




Hydrophilic Interaction Chromatography


Since 2003, Wates has developed innovative stationary phases for Hydrophilic Interaction Chromatography [HILIC] to overcome the challenge of retaining extremely polar compounds.  Based on Waters novel ethylene bridged hybrid [BEH] particle technology, ACQUITY UPLC® BEH Amide columns utilize a chemically stable, trifunctionally-bonded amide phase, enabling a new dimension in stability and versatility for HILIC separations.


Designed to retain polar analytes and metabolites that are too polar to retain by reversed-phase [RP] chromatography, ACQUITY UPLC BEH Amide columns facilitate the use of a wide range of mobile phase pH [2-11] to facilitate the exceptional retention of polar analytes spanning a wide range in polarity, structural moiety and pKa.


In addition to enhanced retention of polar compounds, these columns provide increased mass spectrometry response, direct compatibility with sample preparation eluates [PPT, LLE and SPE] as well as orthogonal selectivity compared to reversed-phase materials, making HILIC an attractive alternative separation technique. 


Separation of Water Soluble Vitamins



Carbohydrate Analysis


One of the most abundant and diverse classes of compounds analyzed by HILIC are carbohydrates [e.g., monosaccharides, disaccharides, oligosaccharides and polysaccharides].


The ACQUITY UPLC BEH Amide column is an exceptional tool for the analysis of carbohydrates, providing the following benefits:


  • The 1.7 µm particle size enables high resolution, high speed analysis of carbohydrates in complex sample matrices while maintaining or improving chromatographic resolution.
  • Increased chemical stability enables the use of high pH and high temperature to collapse reducing sugar anomers, shorten analysis times and improve MS detection.
  • BEH particle technology, in combination with a trifunctionally-bonded amide phase, provides exceptional column lifetime, thus improving assay robustness.
  • Unlike amine-based columns used for carbohydrate analysis, the ACQUITY UPLC BEH Amide column is not susceptible to Schiff base formation, thus improving quantitation accuracy.
  • High pH mobile-phase compatibility facilitates MS detection without the need for derivatization, post column addition or complexing with a metal cation, thus dramatically simplifying sample pre-treatment before LC/MS analysis and improving assay sensitivity.


UPLC/MS Analysis of Saccharides




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