Reversed-phase chromatography using ion-pairing reagents such as TFA and formic acid can deliver highly resolved separations of complex peptide mixtures, (e.g., tryptic protein digests or lengthy synthetic peptide sequences) whose sequences may differ by a single amino acid. In general, the hydrophobicity of the peptide determines the elution order, with the least hydrophobic peptides eluting first.
Factors such as particle composition (silica vs hybrids), pore size (130 Å vs 300 Å), ligand density, as well as separation conditions (e.g., gradient duration, separation temperature, flow rate) all play an important role in obtaining a separation that meets application requirements.
As with other bioseparations, when using smaller particle sizes, UPLC/UHPLC column offerings provide superior component resolution and in less time compared to HPLC-based columns for this demanding application.
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ACQUITY PREMIER Peptide ColumnsWaters ACQUITY PREMIER Columns represent the latest innovative technologies and provide the highest level of chromatographic performance, flexibility, and assurance to enhance the capabilities of scientific laboratories around the world.
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ACQUITY UPLC Peptide ColumnsACQUITY UPLC Peptide BEH C18, 130 Å or 300 Å Columns offer a wide usable pH range and outstanding temperature stability for applications ranging from proteomics to peptide mapping. ACQUITY UPLC CSH C18, 130 Å Peptide Columns with a low-positive charged surface can be used with standard TFA-containing eluents or with a weaker acid modifier such as formic acid, eliminating the need to compromise between a reversed-phase eluent that delivers sharp peaks or one that minimizes reduction of the MS signal. The Peptide HSS T3, 100% silica-based particle offering is the ideal choice for the separation of small, hydrophobic peptides since retentivity is greater than that obtained using either of Waters BEH or CSH, hybrid-based peptide separation columns. Columns containing XSelect Peptide HSS T3, 2.5 µm XP or 5 µm particles are also available for UHPLC or HPLC-based applications. |
XBridge Peptide BEH ColumnsXBridge Peptide BEH C18 Columns offer a wide usable pH range and outstanding temperature stability for applications ranging from proteomics to peptide mapping. These HPLC-based columns are ideally suited for reversed-phase chromatography of a wide range of peptides including large and small, acidic and basic, and hydrophilic and hydrophobic species. These columns are available in 130 Å and 300 Å pore size offerings that provide different separation selectivities for use with challenging samples. |
XSelect Peptide CSH ColumnsCharged surface hybrid (CSH) particle based peptide columns offer an outstanding peptide mass loading capability that maintains high peak capacities using either 0.1% FA- or 0.1% TFA-containing eluents. Best suited for compounds less than 10,000 Daltons and deliver different separation selectivity than seen on BEH C18, 130 Å material. Scalable 1.7 μm, 3.5 μm, 2.5 μm XP, and preparative 5 μm that address application needs. Specifically QC tested with tryptic digests of cytochrome c using 0.1% FA-containing eluent to ensure column-to-column consistency in validated methods. |
Peptide Separation with nano- and microflow LC-MSMicroflow LC-MS for peptide identification is greatly simplified with the 150 µm iKey Separation device due to plug-and-play design and fitting-less fluid connections. The iKey offers up to 40x increase in sensitivity compared to 2.1 mm I.D. UPLC.
For nanoflow LC-MS peptide bioanalysis, 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. |
BioSuite ColumnsBioSuite C18, 100 Å, 3 μm PA-A Column
BioSuite C18, 300 Å, 3.5 µm PA-B Column
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Symmetry ColumnsSymmetry, reversed-phase, silica-based particles are synthesized using ultrapure organic reagents, resulting in high purity material with very low silanol activity. When combined with the high surface coverage of the bonded phase, outstanding peptide separations and recoveries are possible.
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Delta-Pak HPLC ColumnsDelta-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 -- 100 Å or 300 Å -- 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. The same chemistry is also available in higher-resolving 5 µm particles, making it possible to easily check the purity of isolated fractions collected from purification chromatography. |
Infographic: PREMIER Columns for Peptides
Video: Choosing the Right Products for Peptide Characterization
Video: Peptide Mapping Reproducibility
White Paper: Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces
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