Agilent Manuals

Zorbax Rapid Resolution HT columns can reduce analysis time and improve resolution.

Laboratory projects that require accurate analysis of PCR products can be divided into two classes: low throughput and high throughput.

This Application Note details how the separation of enantiomers, stereoisomers or diastereomers can be improved by recycle chromatography. The configuration of the system using two identical columns and a 2-position/6-port valve is shown, and the scope and limitations of the system are explained. An application example, which demonstrates the improvement in purity of the collected fractions when using recycle chromatography, is described.

This document introduces the Agilent ZORBAX Rapid Resolution High Throughput HPLC columns using 1.8 µm particles that provide outstanding efficiency in short column lengths for high sample throughput of liquid chromatography and liquid chromatography/mass spectrometry methods. These columns can be used for either isocratic or gradient analyses with high quality, reliable results.

Introduced 1.8-µm Totally Porous particles manufactured by Agilent Technologies, which can achieve the expected resolving power and efficiency of a 150-mm, 5-µm column in a 30-mm configuration.

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

The document introduces a fast, convenient, and cost-effective preparative method development that can be achieved by scaling up from analytical HPLC separation conditions. The peak shape and separation of three antibiotics - dicloxacillan, cloxacillan, and oxacillan - are evaluated on four different ZORBAX bonded-phase selectivities, and the best separation method is scaled up in a linear manner to a 21.2 × 150 mm, 5 µm, ZORBAX PrepHT Eclipse XDB-C18 preparative column. When following the suggested easy scale-up procedure, the preparative separation results provide high yields with excellent throughput and purity.

This document discusses the advantages of high-resolution analysis of intact antibodies, heavy chains, and light chains using ZORBAX Poroshell technology for high-velocity gradient separations. Method development is significantly improved with ZORBAX Poroshell HPLC columns, achieving up to 90% reduction in analysis time. ZORBAX Poroshell 300SB columns are available in various internal diameters and bonded phases for optimal sensitivity, resolution, and speed. The use of an HPLC instrument optimized for low-volume peaks of narrow width maximizes ZORBAX Poroshell resolution. The document also addresses the challenges in monoclonal antibody analysis.

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

This document discusses the competition between speed, resolution, and sensitivity in modern protein laboratories, and introduces ZORBAX Poroshell column technology as a powerful tool for the rapid separation and analysis of proteins. The technology allows for higher mobile phase linear velocities without losing resolution, resulting in shorter run times. The presence of a thin superficially porous layer enables rapid equilibration of slowly diffusing large molecules. The column utilizes proven StableBond technology to maintain stability at low pH and elevated temperature.

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 document evaluates the performance of new GC capillary columns and compares them to columns from established manufacturers. Tests were conducted for column bleed, retention index, film thickness, and trace level acid and base performance.

This article introduces a rapid protein identification technique using atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) time-of-flight (TOF) mass spectrometry.

This document describes an LC system for online 2D LC/MS, which uses an effective continuous salt solution gradient in the first dimension. The results will be compared to previously obtained results.