This knowledge note describes the translation of gradient liquid chromatography (LC) methods between different column formats using the Avantor® ACE® LC Translator. When converting methods from HPLC to UHPLC or between columns of different dimensions, main taining chromatographic selectivity, peak resolution, and overall performance can be challenging. The ACE LC Translator applies fundamental chromatographic principles to scale key parameters such as flow rate, injection volume, and gradient segment time while accounting for differences in column volume and system dwell volume. Through automated calculations based on the original method and target column specifications, the tool enables accurate and efficient method translation with minimal user input. An example demonstrating the translation of an HPLC method on a 150 × 4.6 mm column to a 50 × 3.0 mm UHPLC column illustrates how the approach preserves separation performance while improving efficiency.

This knowledge note outlines practical strategies for reducing solvent consumption, energy use, and environmental impact in liquid chromatography while increasing analytical throughput. By translating conventional HPLC methods to shorter columns with smaller particles or UHPLC formats, laboratories can significantly reduce mobile phase usage and analysis time while maintaining chromatographic selectivity and separation performance.

This white paper presents the theoretical background and practical guidance required for successful method translation in liquid chromatography. By carefully selecting column dimensions and particle sizes while maintaining column efficiency through the L/dp (column length to particle size) ratio, existing HPLC methods can be effectively translated to shorter columns or UHPLC formats without compromising chromatographic performance. The paper introduces key equations used to scale injection volume, flow rate and gradient profiles, and explains how these parameters influence run time, pressure and solvent consumption. In addition, important system-related factors such as dwell volume, extra-column dispersion and detector sampling rates are discussed. Understanding and controlling these variables allows chromatographers to accurately reproduce the selectivity, resolution and efficiency of the original method while achieving significant improvements in analysis speed, solvent usage and overall laboratory productivity.

In 2018 N-nitrosodimethylamine (NDMA) was detected in a batch of valsartan at levels exceeding ICH acceptable intake limits for mutagenic impurities. Since then, the analysis of nitrosamines has become an intense focus point for the pharmaceutical industry. The identification and low-level determination of nitrosamines in potentially affected materials is challenging and requires the application of highly sensitive analytical techniques. This white paper reviews the evolution of the regulatory landscape and discusses the development of analytical methods for the determination of nitrosamine impurities referenced by regulatory authorities. The development of a separation of these compounds from the active pharmaceutical ingredient (API) is discussed, together with application of mass spectrometry (MS) to ensure that the required detection limits can be reached. Additionally, the potential for interference, notably from N,N-dimethylformamide (DMF), is considered, along with strategies for mitigating the risks of inaccurate quantification that arises.

Recent regulatory investigations have identified potentially mutagenic azido impurities in several sartan-containing pharmaceutical products, increasing the need for reliable analytical methods capable of detecting these compounds at trace levels. Impurities such as AZBT, AZLS, and AZBX have drawn particular attention because of their potential impact on drug safety and regulatory compliance. This application note describes an LC-MS/MS method for the selective determination of three azido impurities using the Avantor® ACE® Excel 3 C18-PFP column. By combining the hydrophobic retention of a C18 stationary phase with the enhanced shape selectivity, π-π interactions, and dipole-dipole interactions provided by pentafluorophenyl (PFP) functionality, the method delivers effective chromatographic separation and sensitive detection of structurally related compounds. Gradient elution coupled with multiple reaction monitoring (MRM) enables rapid, reproducible analysis. The results demonstrate that the ACE C18-PFP column provides excellent selectivity and chromatographic performance for azido impurity monitoring, support ing pharmaceutical quality control and regulatory compliance in the analysis of sartan-based drug products.

Peptide and protein analysis requires chromatographic methods capable of delivering high resolution, reproducibility, and efficient separation across a broad range of molecular weights. Reversed-phase high-performance liquid chromatography (RP-HPLC) remains one of the most widely used techniques for the characterization and quality assessment of biomolecules. This application note demonstrates the gradient separation of a mixture of peptides and proteins using the Avantor® ACE® 3 C4-300 column. The 300 Å pore size and C4 stationary phase provide suitable retention and mass transfer characteristics for biomolecu les ranging from small peptides to larger proteins. Under gradient elution conditions, compounds including oxytocin, bradykinin, angiotensin I and II, insulin variants, ribonuclease A, cytochrome C, and lysozyme were effectively resolved with excellent pea k shape and chromatographic performance. The results highlight the capability of the ACE 3 C4-300 column to support reliable peptide and protein separations, making it a valuable tool for biopharmaceutical analysis, quality control, and research applications.