BEH SEC Particle Technology

These SEC columns are based on Waters’ Ethylene Bridged Hybrid (BEH)-based particle technology and diol-bonded surface that provides a stable chemistry with minimal non-desired secondary interactions for proteins and peptides size based separations. There are formats for UPLC, UHPLC, and HPLC.

XBridge BEH SEC Technology

Waters series of XBridge Protein BEH SEC, 125 Å, 200 Å, and 450 Å Columns containing 2.5 and 3.5 μm were developed to complement our existing line of UPLC-based SEC offerings for use where UHPLC or more traditional HPLC-based instrumentation and methods. These SEC offerings are based on the same Waters Ethylene Bridged Hybrid (BEH)-based particle technology and diol-bonded surface coating as used in our successful line of UPLC-based SEC columns. This process offers chromatographers the option and ability to easily transfer methods based on laboratory instrumentation and component resolution or sample throughput needs.

The image to the right shws how size-exclusion chromatography (SEC) separates compounds primarily based

on their relative size in solution. Calibration curves on Waters HPLC-based, SEC Columns of different pore size, using defined protein and peptides of known molecular weight, help chromatographers select the most appropriate SEC column for their specific application.

XBridge Protein BEH SEC Columns

Outstanding Protein SEC Separations

XBridge Protein BEH SEC, 125 Å, 200 Å, and 450 Å Columns containing 2.5 or 3.5 μm particles were developed to complement the existing line of UPLC-based SEC offerings for use on HPLC or UHPLC instrumentation for protein size-exclusion chromatography (SEC). These 2.5 or 3.5 μm SEC chemistries are based on the same Waters Ethylene Bridged Hybrid (BEH)-based particle technology and diol-bonded surface coating as used in our successful line of UPLC-based SEC columns.

Each manufactured batch of XBridge Protein BEH SEC, 125 Å, 200 Å, and 450 Å material is extensively QC tested to help ensure unmatched batch-to-batch and column-to-column consistency as desired when used in validated methods.

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ACQUITY UPLC BEH SEC Technology

Waters ACQUITY UPLC Technology allows scientists the ability to exceed what is obtainable using UHPLC or traditional HPLC separations by improving data quality while increasing sample throughput and productivity. Chromatographers can now choose the most appropriate UPLC-based, Protein BEH SEC Column (i.e., 125 Å, 200 Å, or 450 Å pore size) to satisfy their need to analyze biotherapeutic peptides and proteins for internal or regulatory purposes.

The image to the left shows how size exclusion chromatography (SEC) separates compounds primarily based on their relative size in solution and not absolute molecular weight Calibration curves on UPLC-based SEC columns of different pore size, using defined proteins and peptides help chromatographers select the most appropriate SEC column for their specific application.

ACQUITY UPLC BEH SEC Columns

UPLC SEC Column Separations

The ACQUITY UPLC BEH SEC Columns are specifically designed and QC tested with BEH protein standards, ensuring unmatched batch-to-batch consistency for increased confidence in validated methods. The SEC-based offerings are ideally suited for the accurate determination of low levels of protein aggregates contained in therapeutic monoclonal antibodies up to 10X faster than traditional HPLC-based SEC. By comparison (data not shown), the BEH C4, 300Å UPLC Columns separate proteins of various sizes, hydrophobicities, and isoelectric points while maximizing recovery and minimizing protein carryover.

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BioSuite Ultra-High Resolution (UHR) Columns

Size-exclusion columns (silica-based)

BioSuite ultra-high resolution (UHR), high resolution (HR), and standard size-exclusion column packings use a rigid yet “wettable” silica-based media that is stable from pH 2.5–7.5. As indicated in the calibration curve tables, the exclusion limits of BioSuite SEC packings are determined by the particle and pore size of the silica-based material. Particle size of the SEC packing media as well as column length are important parameters that determine separation efficiency. BioSuite 4 μm particle size, UHR Columns provide maximum separation efficiency, followed by BioSuite HR Columns and BioSuite Standard SEC Columns. To maximize column life of analytical (i.e., 4.6 mm or 7.8 mm I.D.) or preparative (i.e., 21.5 mm I.D.) SEC Columns, use of BioSuite Guard Columns is recommended.

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Protein-Pak SEC Columns

Protein-Pak SEC Columns contain silica-based, diol-bonded, 10µm particles with either 60A, 125A, or 300A pores and are well suited for analytical- or lab-scale purification of biomolecules.

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BEH SEC Protein Performance Standards

Benchmarking, Method Development, and Troubleshooting

The BEH SEC Protein Standards are specifically designed to help aid in the benchmarking of each set of columns. Each standard contains carefully chosen proteins unique to that chemistry, which has been worked out meticulously over time. These standards are used by Waters to QC the respective material used to produce the UPLC, UHPLC, and HPLC columns. They are also well suited for benchmarking a new column and for monitoring the SEC column performance during repeated use.

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