Protein Reversed-Phase Columns

Reversed-Phase Chromatography

Reversed-phase chromatography of proteins, performed on columns packed with porous or coated, solid-core particles possessing wide pore size particles ( e.g., 300Å), and functionalized with short ligand length chemistries (e.g., C₄), is a separation technique based on the ability to separate samples based on relative hydrophobic differences of the proteins in solution. Gradients of increasing organic solvent concentration are frequently used to affect separations in the presence of ion-pairing reagents (e.g., 0.1% TFA or 0.1% formic acid) that minimize undesired ionic interactions. In general, the hydrophobicity of the protein or protein subunit determines the elution order, with the least hydrophobic proteins eluting first. Factors such as particle composition, pore size, ligand type and density, as well as separation conditions (e.g., gradient duration, separation temperature, flow rate) all play important roles in obtaining a separation that meets application requirements.

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Reversed-phase protein columns, guards, and standards

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Column flexibility, high throughput, and maximum component resolution

BioResolve RP mAb Polyphenyl Columns

These 450Å, 2.7 µm columns were developed in response to shortcomings of existing reversed-phase columns designed for LC or LC-MS analysis of intact mAb or mAb subunits.

Contain silica-based, solid core particles with defined 450Å pore coating for outstanding protein component resolution, recovery, and low injection-to-injection carryover.
Use innovative polyphenyl ligand and bonding technology (patent pending) to deliver superior intact mAb and subunits separations in LC (0.1% TFA) or LC-MS (0.02% TFA or 0.1% FA) applications.
Deliver near equivalent performance on HPLC, UHPLC, and UPLC instrumentation.
QC tested with a mAb subunit standard (i.e., Reduced IdeS-digested NIST mAb Reference Material 8671) to help ensure batch-to-batch column consistency.
“More Than Just a Column.”

BEH Particle Technology

Designed for Protein Separations

The separation of large molecular weight biological compounds can be challenging. For more than 20 years, chromatographers have benefited from performance characteristics that Waters reversed-phase columns have provided for protein separations. Waters Ethylene-Bridged Hybrid (BEH) Particle Technology can help overcome performance limitations experienced using 100% silica-based materials for challenging protein separations.

300Å pores
C₄ligand
Minimal undesirable secondary interactions
Minimal detectable carryover at elevated temperature separations

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zoom	Reproducible Protein Separations Well characterized, state-of-the-art bonding procedures Low pH and high temperature stability Quality-control tested with diverse protein mixture Consistent batch-to-batch reproducibility Available 1.7 μm particles for UPLC and nano UPLC applications

Protein Separation with nano- and microflow LC-MS

Microflow LC-MS for protein analysis is greatly simplified with the 150 µm iKey Separation Device due to plug-and-play design and fitting-less fluid connections. The iKey offers an up to 40x increase in sensitivity compared to 2.1mm I.D. UPLC.

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For nanoflow LC-MS protein analysis, ACQUITY UPLC M-Class Columns are specifically designed for low dispersion nano LC. The nano- to microscale LC-MS ACQUITY UPLC M-Class Columns and Trap Columns enable nano- and microscale separations under UPLC conditions at 15,000 psi, fully leveraging the separation power of sub-2-µm particle technology.

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BioSuite pPhenyl Reversed-Phase Chromatography (RPC) HPLC Columns

BioSuite RPC Column offerings include a phenyl (pPhenyl) chemistry bonded to a pH stable, methacrylic ester-based polymeric resin. The 1000Å pore size of the pPhenyl base matrix accommodates proteins up to 5,000,000 Daltons.

The pPhenyl RPC chemistries are available in 21.5 x 150 mm columns for “lab-scale” isolations while a 2.0 x 75 mm column is well suited for narrow-bore HPLC and LC-MS applications.

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Symmetry300 C4 HPLC and UHPLC Columns

Symmetry300 C₄ particles are 100% silica-based and are synthesized using ultrapure organic reagents resulting in high-purity material with very low silanol activity for outstanding peptide and protein separations and recoveries.

300Å pore for peptide and protein applications
Fully end-capped to minimize undesired secondary interactions
Alternative separation selectivity compared to Waters BEH C₄, 300Å hybrid material
QC tested with peptide samples to help ensure excellent batch-to-batch consistency

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Delta-Pak Columns

Delta-Pak HPLC Columns are based on highly stable, bonded, endcapped 5 μm or 15 μm spherical 100% silica-based particles. These columns are offered in two different pore sizes (i.e., 100Å or 300Å) with either C₄ or C₁₈ ligands to address different selectivity and retentivity needs. They are available in different 15 μm cartridge and column configurations, providing consistent and predictable scale-up capabilities for milligram-to-gram scale purification.

MassPrep Protein Standard

This intact protein validation mix is designed for verification of HPLC/UPLC instruments. This standard is also used to evaluate the performance of columns used for protein analysis, specifically the ACQUITY UPLC and XBridge Protein BEH C₄ Columns. The MassPrep Protein Standard contains proteins that have a wide range of isoelectric points, molecular weights, and hydrophobicities.

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