Agilent 300SB-C18 Manuals

Agilent ZORBAX RRHD 300SB-C18 1.8 µm is a new reverse phase media for UHPLC analysis of proteins and peptides. By utilizing StableBond technology, it provides a stable 300Å pore-size media under acidic conditions for robust and reproducible protein separations.

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Poroshell 300SB-C18 is a revolutionary chromatography media - superficially porous particles - that produce very fast, high resolution, RP-HPLC separation of proteins, DNA and other macromolecules.

Poroshell 300SB-C18 is a tool for fast, high-resolution protein separations. It consists of a thin layer of porous silica on a solid core, allowing for protein separation in minutes or seconds with high resolution. Bonded with the patented StableBond C18 phase, it provides reliable separations and unmatched column stability. It can also separate at high temperatures with minimal bleed, resulting in clean MS signals at ultra-high speed. Poroshell 300SB also has low backpressure, enabling separation at very high flow rates. This is a tool to revolutionize proteomics and protein analysis.

Antibodies are a group of proteins that are the key to directed immunological interaction. They can bind to an antigen (protein, glycoprotein, DNA, etc.) with extreme specificity. This property makes antibodies very valuable for use in diagnostics, general research, and for therapeutics. Treatment of intact antibodies with various chemicals and enzymes allows the specific separation of the heavy and light chains, removal of sugar moieties, and/or cleavage of the polypeptide chains. Separation of the peptide fragments (mapping) after cleavage with a proteolytic enzyme of high specificity, such as Lys-C, gives a characteristic and reproducible pattern of peaks which can be collected, sequenced, and run through a mass spectrometer (MS). This application note demonstrates the utility of using superficially porous chromatographic media (Poroshell) to achieve substantial improvements in analysis turnaround times when running high-resolution peptide maps. Figure 1 shows comparative peptide maps of a human monoclonal antibody, Lys-C digest. Note the time scales of the separations. The Poroshell maps take one-sixth of the turnaround time and show essentially the same number of peaks. See Table 1. High Speed and ultra High Speed Peptide Mapping of Human Monoclonal IgG on ZORBAX Poroshell 300SB-C18, C8, and C3 Application Biochemical Cliff Woodward, Robert Ricker, Kurt Forrer, Patrik Röethlisberger

A new reverse phase media, Agilent ZORBAX RRHD 300SB-C18 1.8µm, was used for the separation of a typical protein biopharmaceutical, insulin.

This document describes the analysis of trypsin-digested monoclonal antibody and bovine serum albumin by UHPLC using Agilent ZORBAX RRHD 300SB-C18 1.8 µm reversed-phase media. The media demonstrates robustness with different acidic eluents, temperatures, and flow rates, and exhibits good reproducibility.

This application note describes a method for the rapid separation of recombinant human erythropoietin using the Agilent ZORBAX 300SB-C18 1.8 µm Rapid Resolution High Definition (RRHD) column. This method has increased sensitivity, reduced separation time and improved run-to-run reproducibility, which is in great demand by biotechnology and pharmaceutical companies. The C18 StableBond coating technology provides increased sensitivity, exceptional pH, and thermal stability. The use of a 1.8-µm column designed for UHPLC systems significantly reduces analysis time, critical for increasing the efficiency of QC for protein primary structure analysis. The data presented here focus on the fast separation of various recombinant human erythropoietin isoforms. Like many other biomolecules, recombinant EPO protein exhibits heterogeneity due to modifications that occur during manufacturing. Methods are optimized for flow rates, gradient and reproducibility under acidic conditions that contain trifluoroacetic acid.

G4240-62001 HPLC-Chip is the standard chip for one-dimensional separations. It is comprised of a 40 nL enrichment column and a 75 μm x 43mm separation column which is packed with Zorbax 300SB-C18 5 μm material. The HPLC-Chip is made from biocompatible polyimide and the functionality of this chip is equivalent to conventional nanospray LC/MS. ZORBAX 300SB-C18 is a micro particulate column packing designed specifically for reversed-phase HPLC of peptides and proteins. The StableBond packing is made by chemically bonding a sterically protected octadecyl stationary phase to a specially prepared, ultra-high- purity, ZORBAX, porous-silica microsphere. The special ZORBAX silica support is designed to reduce or eliminate strong adsorption of basic compounds. The densely covered, sterically protected diisobutyl n-octadecyl stationary phase is chemically stable and gives longer column life. As a result, ZORBAX 300SB-C18 is a stable, reversed-phase packing at low pH that can be used for peptide maps, and separation of synthetic and natural peptides and proteins. It is particularly well suited for use with aggressive mobile phases (e.g., pH < 2, high ionic strength, ion-pair additives, TFA, etc.) since the steric protection of the bonded phase resists degradation caused by such mobile phases. This characteristic is particularly important for use in methods that need long-term stability and reproducibility. ZORBAX 300SB-C18 is well suited to applications that utilize weak acidic ion-pair additives as the mobile phase, such as those used for peptide analysis with TFA.