BEH (Ethylene Bridged Hybrid) Technology

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.

Selectivity Choice

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.

With a seemingly endless number of method parameters to try, developing a new chromatographic method can be an overwhelming, and time-consuming experience.  Essential to any method development strategy is finding a suitable column that delivers the desired separation while resulting in a robust and reliable separation.

Waters Method Development kits consist of several UPLC^® columns, encompassing a broad range in selectivity, to accommodate your method development approach and enable methods to be developed efficiently and effectively. 

 Reversed-Phase and HILIC Method Development Strategies

A. With a wide range of UPLC column selectivities available, screening multiple columns with a single mobile-phase condition can quickly lead to a successful result.

B. Method development for morphine-related compounds using a novel reversed-phase and HILIC approach.

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. 

The successful transfer of a method requires careful consideration and understanding of key parameters such as column dimensions, system volumes and configurations, injection volume and gradient profile.  In addition to the proper geometric scaling of all method parameters, the successful transfer of an analytical method requires the preservation of chromatographic column selectivity and resolving power, regardless of particle size. 

Method Transfer kits are designed to preserve the integrity of a separation as it is transferred between UPLC^® and HPLC platforms.  Based on the concept of maintaining column length [L] to particle size [dp] ratio [L/dp], these kits provide an ACQUITY UPLC^® column with an HPLC column of equivalent selectivity and resolving power.  Using the ACQUITY UPLC columns calculator, methods can be fully transferred from HPLC to UPLC or from UPLC to HPLC. 

Each kit contains one UPLC column, one HPLC column.  The ACQUITY UPLC columns calculator can be downloaded from the ACQUITY UPLC Online Community at www.waters.com/myuplc. 

Transfer Between LC Technology Platforms with Ease

A separation of clozapine and its related compounds is used to demonstrate the exception of scalability between different size chromatographic particles and instrument platforms to facilitate a successful method transfer.

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.

The reproducibility of the chromatographic column has a critical impact on the long-term reliability and robustness of an analytical method, and its reproducibility lies completely outside the user's control.

With exceptional batch-to-batch and column-to-column reproducibility, Waters well-established particle and column manufacturing process control provides confidence in the long-term reliability of your analytical method.  ACQUITY UPLC^® Method Validation kits include three batches of chromatographic media [derived from different base particles] to judge the quality, reliability, and consistency of your chromatographic method. 

Minimize Risk with Dependable Column Performance

Three different batches of bonded material derived from three different base particles demonstrate the solid reproducibility that can be expected from ACQUITY UPLC columns, assuring the long-term reproducibility of your analytical method.

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.

*Patent No. 5,374,755

Waters offers a selection of premier supplies, perfectly suited for the ACQUITY UPLC^® system:

Filters

We recommend that you filter buffers and samples using a 0.2 µm filter prior to use. Filtration protects the column and instrument components from a buildup of particulate matter, improving column lifetime and minimizing system down time. Use 0.2 µm GHP Filters for filtering aqueous, non-polar solvents and proteins.

* Requires solvent filtration apparatus, WAT200543 or equivalent

Vials

We recommend vials with pre-slit silicone/PTFE septa for UPLC^® systems. Pre-slit septa are designed for proper venting, resulting in reproducible sample draw volumes. Waters offers two lines of certified vials: LC Certified Vials and LCMS Certified Vials.

Sirocco Protein Precipitation Plate

Sirocco^TM Protein Precipitation Plate enables high-throughput "in-well" protein precipitation. The unique filter system -- with vented cap mat and patented valve technology -- allows you to process samples "in-well" and collect clean filtrate from smaller plasma volumes without transfer steps.

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 pK_a.

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

VanGuard™ Pre-Columns are the result of over two years of product development and are the first guard column devices designed for routine use at pressures up to 15000 psi [1000 bar] in applications run on the ACQUITY^® UltraPerformance Liquid Chromatography system. VanGuard Pre-Columns feature a 2.1 mm ID x 5 mm length, ultra-low volume design which efficiently protects UPLC^® column performance. This patent-pending design does not compromise the UPLC holistic design approach to higher efficiency, greater resolution and increased throughput since the same ACQUITY UPLC^® column stationary phases and column frits are used in VanGuard Pre-columns. Since the VanGuard Pre-Column connects directly to the inlet of the ACQUITY UPLC column, extra-column volumes are minimized and mobile-phase leaks due to additional connections are all but eliminated.

Key Features and Benefits of VanGuard Pre-Columns

Minimal Chromatographic Effects with VanGuard Pre-Columns

VanGuard Pre-Columns are uniquely designed to protect and prolong ACQUITY UPLC column performance while contributing minimal chromatographic effects.