Agilent 300SB-C8 Manuals

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The 300SB-C8 is a high-performance microparticulate 300Å C8 packing reversed phase column from Agilent that can be used at low pH for intact protein analysis and peptide mapping.

The use of a column packing having reduced particle size can improve efficiency of a separation or, alternatively, increase loading capacity.

This document compares the use of micro and regular flow cells with ZORBAX 300SB-C8 (3.5 µm) column for peptide and protein analysis. It highlights the reduction of analysis time without compromising efficiency. By reducing the flow cell size, the effects of extra-column volume are minimized with small particle-size packing.

This paper introduces a method for separating proteins using ZORBAX Poroshell 300SB columns under low pH conditions at high temperatures. The method uses a linear gradient elution method with a flow rate of 1.0 mL/min and a column temperature of 40 °C to 75 °C. The results show that the resolution of the peaks is significantly improved as the column temperature is increased.

ZORBAX Poroshell superficially porous particle technology allows the fastest, most rugged HPLC separation of proteins to date. A 0.25-µm porous silica crust formed around a nonporous 4.5-µm diameter silica core results in extremely hard, spherical, 5-µm ZORBAX Poroshell particles. Molecules diffusing the short distance into and out of the thin porous crust are rapidly separated. While the inherent properties of ZORBAX Poroshell columns make them usable with most modern HPLC instruments, the use of superficially porous particle columns of small diameter (2.1 mm, 1.0 mm, and 0.5 mm i.d.) for the separation of proteins and peptides places certain demands on the instrument configuration. This technical overview briefly addresses temperature flow rate and gradient adjustment and then details several of the more important parameters that can influence separation performance. These include: detector peak- width setting, extra-column volume, and flow-cell volume. Optimization of the Agilent 1100 HPLC System for Superior Results with ZORBAX Poroshell Columns Technical Overview ZORBAX Poroshell Column Features ZORBAX Poroshell 300SB columns come in a vari- ety of internal diameters and phases, all being extremely useful for the fast separation of proteins and peptides. Poroshell particles are extremely hard, 5-µm silica spheres, each consisting of a 0.25-µm porous crust formed around a nonporous 4.5-µm core. See Figure 1. With ZORBAX Poroshell packed columns, protein and

Antibodies are a class of proteins that play a key role in immunological interactions. They bind to antigens (proteins, glycoproteins, DNA, etc.) with high specificity. Antibodies have high value in diagnostics, research, and therapeutics. IgG is a class of antibodies consisting of two heavy chains (50 kDa each) and two light chains (25 kDa each) attached through disulfide bridges. While the carboxy-terminal domains are conserved, the amino-terminal domains have variable amino acid sequences, resulting in the molecule's specificity and diversity. Most antibodies are glycosylated, further increasing molecular diversity. Treatment of intact antibodies with chemicals and enzymes allows specific cleavage of the heavy and light chains, removal of carbohydrate moieties, and cleavage of the polypeptide chains, enabling the study of a particular antibody's structure. Rapid HPLC method development and analysis using ZORBAX Poroshell technology are valuable tools for investigating antibody structure. Rapid HPLC analysis of monoclonal antibody IgG1 light chains using ZORBAX Poroshell 300SB-C8.

This application note describes a method for achieving highly resolved ultrafast separations of reduced and alkylated monoclonal antibodies using an Agilent ZORBAX sub-2 µm Rapid Resolution High Definition (RRHD) 300SB-C8 reversed-phase column under optimized chromatographic conditions. The StableBond C8 phase and RRHD column technology, in combination with robust gradient elution conditions, enabled efficient, fast, and effective separation of light chain and two heavy chain monoclonal antibody variants in under 4 minutes. Additionally, alternate mobile phase compositions were employed, making separations flexible for ultra fast LC/MS analysis. At elevated operating pressures and temperature, the stable C8 phase and rugged column packing technology delivered highly reproducible separations and delivered excellent chromatographic run-to-run results.