Ultralow flow liquid chromatography and related approaches: A focus on recent bioanalytical applications

Ultralow flow LC employs ultra-narrow bore columns and mid-range pL/min to low nL/min flow rates (i.e., ≤20 nL/min). The separation columns that are used under these conditions are typically 2-30 μm in inner diameter. Ultralow flow LC systems allow for exceptionally high sensitivity and frequently high resolution. There has been an increasing interest in the analysis of scarce biological samples, for example, circulating tumor cells, extracellular vesicles, organelles, and single cells, and ultralow flow LC was efficiently applied to such samples. Hence, advances towards dedicated ultralow flow LC instrumentation, technical approaches, and higher throughput (e.g., tens-to-hundreds of single cells analyzed per day) were recently made. Here, we review the types of ultralow flow LC technology, followed by a discussion of selected representative ultralow flow LC applications, focusing on the progress made in bioanalysis of amount-limited samples during the last 10 years. We also discuss several recently reported high-sensitivity applications utilizing flow rates up to 100 nL/min, which are below commonly used nanoLC flow rates. Finally, we discuss the path forward for future developments of ultralow flow LC.