Advancements in Two-Dimensional Liquid Chromatography for Biological Samples Enhancing Peak Capacity and Speed through High-Temperature Techniques

The need for improved separation power in Liquid Chromatography (LC), particularly for complex multi-constituent biological samples, is becoming increasingly critical. Recent advancements have led to a growing interest in Two-Dimensional Liquid Chromatography (2DLC), which offers significant advantages over traditional One-Dimensional Liquid Chromatography (1DLC) by enabling higher peak capacities and resolution. This review explores the potential of 2DLC, highlighting its ability to enhance separation by combining orthogonal separations and offering peak capacity that, under ideal conditions, is the product of the peak capacities of both dimensions. Despite its impressive separation power, comprehensive 2DLC is hindered by long analysis times, often extending several hours. Recent innovations in high-temperature LC techniques have addressed this challenge by drastically reducing the time required for the Second Dimension of 2DLC to approximately 20 seconds, enabling rapid separations with improved peak capacities exceeding 2000. These developments have led to significant reductions in analysis time, allowing complex biological samples with over 200 detectable peaks to be analyzed in under 30 minutes. This shift toward fast, high-temperature LC is transforming the capabilities of 2DLC, making it an invaluable tool for the analysis of biological samples. Additionally, the review touches on complementary techniques like NMR spin relaxation spectroscopy, which aids in characterizing protein dynamics, offering further insights into protein conformational changes on the picosecond-nanosecond timescale.