This article provides comprehensive guidance on the maintenance, cleaning, regeneration, and storage of silica-based HPLC (High-Performance Liquid Chromatography) columns. The general considerations emphasize the importance of using in-line filters and guard cartridges to protect columns from blockage and irreversible sample adsorption. While these measures help, contamination by strongly adsorbed sample components can still occur over time, leading to an increase in back pressure, loss of efficiency, and other issues. To maximize column lifetime, especially with UHPLC (Ultra-High Performance Liquid Chromatography) columns, it is advisable to use ultra-pure solvents, freshly prepared aqueous mobile phases, and to filter all samples, standards, and mobile phases. Additionally, an in-line filter system and sample clean-up on dirty samples are recommended. However, in cases of irreversible compound adsorption or column voiding, regeneration may not be possible. The document also provides specific recommendations for column cleaning procedures, including the flushing procedures for various types of columns such as reversed phase, unbonded silica, bonded normal phase, anion exchange, cation exchange, and size exclusion columns for proteins. The flushing procedures involve using specific solvents in a series to clean and regenerate the columns. It is emphasized that the flow rate during flushing should not exceed the specified limit for the particular column, and the last solvent used should be compatible with the mobile phase. Furthermore, the article outlines the storage conditions for silica based HPLC columns, highlighting the impact of storage conditions on the column's lifetime. It is recommended to flush all buffers, salts, and ion-pairing reagents from the column before storage. The storage solvent should ideally match the one used in the initial column test chromatogram provided by the manufacturer, and column end plugs should be fitted to prevent solvent evaporation and drying out of the packing bed.

This article provides detailed instructions for the correct installation, maintenance, and troubleshooting of capillary gas chromatography (GC) columns. It emphasizes the importance of proper installation to ensure optimal performance and longevity of the column. The document covers various aspects such as column trimming, installation, conditioning, testing, storage, and ferrule selection. The installation process involves ensuring that the heated zones of the GC are cool before placing the column cage in the column oven. It is essential to avoid sharp bends or stress on the capillary column during installation and to connect the front end of the column into the GC inlet at the recommended insertion distance. The document also provides guidance on trimming the column, including the use of a ceramic wafer or capillary column cutter to achieve a clean, burr-free cut. For previously used columns, it recommends removing any capillary caps, positioning the nut and ferrule, and trimming 1-2 cm from the column. After installation, the column should be purged with carrier gas to remove any oxygen and avoid oxidizing the column. Conditioning the column involves ramping to the upper isothermal temperature limit and maintaining this temperature for a specified duration. It is crucial to maintain carrier gas flow during conditioning and not exceed the upper temperature limit of the column to avoid phase damage. The document also discusses testing column performance using a suitable method and performing a test injection to assess performance. It provides recommendations for column storage, including flame-sealing the capillary ends or using retention gaps for long-term storage. Additionally, it emphasizes the importance of routine maintenance and replacement of GC consumables to extend the column's lifetime. Ferrule selection is another important aspect covered in the article, with a variety of ferrule materials available for different applications. The characteristics of common ferrule options are presented in a table, including temperature limits, reusability, and suitability for specific detector types.

The article discusses the critical role of chromatography in the analysis and purification of proteins in biopharmaceuticals, emphasizing the importance of comprehensive characterization for ensuring their safety and efficacy. It highlights the use of Avantor® ACE® HPLC columns for the separation and purification of proteins, focusing on the analysis of intact proteins using reversed-phase liquid chromatography (RPLC) with fully porous particles. This article also details the application of different mobile phase additives, such as TFA and formic acid, and emphasizes the advantages of using type B ultra-pure silica-based columns for efficiency and peak shape in biomolecule analysis. Additionally, it addresses the challenges of analyzing intact proteins due to slow molecular diffusion and introduces the concept of solid-core (or superficially porous) particles, emphasizing their benefits over traditional porous particles for the analysis of therapeutic proteins. Furthermore, it discusses the development of Avantor® ACE® UltraCore BIO columns, specifically designed for the high-efficiency separation of large biomolecules, such as proteins, and demonstrates their effectiveness in achieving high-resolution separations, even for higher molecular weight proteins like monoclonal antibodies (mAbs). In addition, it underscores the complexity of analyzing and characterizing intact protein biopharmaceuticals, requiring a range of analytical techniques and the use of wide-pore stationary phases, operated at elevated temperatures and with relatively shallow gradients. It highlights the comprehensive range of options offered by Avantor® ACE® wide pore columns, including both fully porous and solid-core particles, bonded with a variety of complementary stationary phase chemistries to optimize selectivity during method development. The use of ultrapure and highly inert base silica is emphasized for enabling the use of lower concentrations of mobile phase modifiers without compromising analyte peak shape, particularly beneficial for LC-MS applications. Then the article concludes by emphasizing the significance of reversed-phase liquid chromatography and its compatibility with mass spectrometry as a valuable tool for the separation and analysis of intact proteins and their closely related variants in biopharmaceuticals.

The Avantor® ACE® UltraCore series encompasses High Performance Liquid Chromatography (HPLC) and Ultra High Performance Liquid Chromatography (UHPLC) columns designed to deliver high throughput and high-efficiency ultra-fast separations. Utilizing ultra-inert solid-core silica particles with monodisperse particle distribution, these columns combine the high efficiency of UHPLC with the operability of HPLC instrumentation, yielding lower backpressure and high-resolution separations suitable for a broad spectrum of analytes. The Avantor® ACE® UltraCore range includes three primary product types: • UltraCore BIO: Designed for large biomolecules (≥5 kDa), these columns offer exceptional performance in separating biologically derived compounds. • UltraCore: Ideal for standard small organic molecules, providing rapid separations for both synthetic and natural mixtures. • UltraCore Super: Equipped with encapsulated bonding technology for small organic molecules in extreme pH conditions, optimal for high pH buffer requirements. The Avantor® ACE® UltraCore columns present a versatile and high-efficiency solution for chromatographic separation needs, accommodating a wide range of molecular sizes and providing enhanced resolution and reduced analysis time. Their adaptability to both HPLC and UHPLC systems, combined with the advantages of solid-core technology, makes them an invaluable tool in analytical and preparative chromatography.

The document discusses the regeneration of Partisil/Partisphere ion-exchange columns in chromatography. It mentions that column efficiency can diminish with use due to the accumulation of sample and/or mobile phase impurities at the head of the column. This can lead to a change in back pressure, lower column efficiency, and sometimes a change in selectivity. The document outlines a procedure that may restore column performance. The document also provides everyday practices to enhance the lifetime of a column. These include using only high-purity HPLC solvents and buffers, using freshly prepared mobile phases and buffers, filtering mobile phases to remove particulates, using appropriate sample clean-up procedures, using a guard column or pre-column filter, and working within the pressure and flow rate limitations of the column. For the regeneration of Partisil/Partisphere SAX, SCX, WAX, and WCX columns, the document suggests passing 20 column volumes of various mobile phases through the column. These include a buffer wash, distilled water, an acid wash, a chelating wash, a methanol wash, and a buffer for separation. The document emphasizes that not all of these wash steps are required for every column clean-up and that some chromatographers require only a combination of certain steps.

The document is a white paper on Hydrophilic Interaction Liquid Chromatography (HILIC) analysis method development. HILIC is a type of chromatography that uses an organic/aqueous mobile phase and a polar stationary phase. In HILIC, water is a strong solvent, and unlike in Reversed Phase Liquid Chromatography (RPLC), increasing the proportion of water in the mobile phase reduces the retention time of the analyte. The paper discusses when to consider HILIC analysis methods, the advantages of HILIC, and the challenges often encountered due to the lack of understanding of HILIC mechanisms compared to RPLC. It also provides a systematic flowchart for intelligent solutions for HILIC analysis method development, which includes a three-step approach for chromatography analysis method development. The first step involves gathering as much information as possible about the analyte (e.g., pKa, log P, log D). The second step involves analyzing the sample under different pH conditions using three HILIC columns in either isocratic or gradient mode to identify the suitable column/pH combination for the analyte. The third step involves optimizing the separation by investigating other parameters such as temperature and ionic strength, and assessing the robustness of the method. The paper emphasizes that the selection of the appropriate stationary/mobile phase combination, based on the differences between the HILIC stationary phases and the mobile phase pH, can provide high selectivity in the analysis. This step-by-step approach can help users develop an efficient analysis method.

The document "Understanding the Relationship Between Particle Size, Performance, and Pressure" explores the impact of particle size on chromatographic performance and system pressure. The study highlights how smaller particles can improve separation efficiency by providing higher resolution and faster analysis times. However, this comes at the cost of increased backpressure, which can challenge the system's hardware and require higher operating pressures. The document discusses the balance needed between particle size, column dimensions, and system pressure to optimize performance without exceeding the pressure limits of chromatographic systems. It outlines the advantages of using superficially porous particles (SPPs) over fully porous particles (FPPs) in achieving high efficiency with lower backpressure. The study also emphasizes the importance of selecting appropriate column dimensions and flow rates to manage system pressure while maintaining optimal performance. In conclusion, understanding the interplay between particle size, performance, and pressure is crucial for optimizing chromatographic separations, ensuring system longevity, and achieving high-quality analytical results.

Ultra-high performance liquid chromatography (UHPLC) systems are integral to modern analytical laboratories, necessitating careful maintenance and operation protocols to ensure optimal performance. This document provides comprehensive guidelines for the proper shutdown and reactivation of UHPLC systems to prevent damage and maintain operational efficiency. • Shutdown: Remove the column and replace it with a union to avoid blockages. Flush the system with a compatible solvent mix, clean mobile phase reservoirs to prevent microbial growth, flush the pump with storage solvent, and clean the autosampler, including the needle and injection port. • Reactivation: Inspect the system for wear or damage, gradually reintroduce mobile phases starting with a weak solvent, reinstall the column securely, and perform system checks on baseline stability, pressure consistency, and detector performance. By adhering to these guidelines, laboratories can ensure the longevity and reliability of their UHPLC systems, maintaining high analytical performance and minimizing downtime. These procedures help prevent common issues such as blockages, contamination, and component wear, thereby supporting efficient and accurate analytical operations.

Method development for complex low molecular mass (LMM) samples using reversed-phase (RP) separation conditions presents significant challenges due to the presence of many unknown analytes over wide concentration ranges. This guide aims to optimize method parameters-column length (L), temperature (T), flow rate (F), and final mobile phase conditions (Øfinal)-to maximize separation peak capacity. Validated by prior research, this protocol benefits laboratories dealing with metabolomics, natural products, and contaminant screening. This practical guide provides a structured approach to maximizing peak capacity for complex LMM separations. It complements computational optimization strategies and offers a step-by-step method development process. The Snyder-Dolan test is highlighted as essential for determining the need for gradient or isocratic elution and guiding column length decisions. The decision tree framework helps analysts prioritize variable optimization to develop effective separation methods for complex samples.