Main evolutions in liquid chromatography

High-Performance Liquid Chromatography (HPLC) is a key analytical technique used in a wide range of industries for the analysis of food, environmental and pharmaceutical matrices. This separative technique can be used for purely qualitative analyses, as well as for quantitative applications. In recent years, the technologies used in HPLC (stationary and mobile phases, instrumentation, etc.) have evolved very rapidly, to meet certain user requirements. Three main avenues of improvement have been pursued.

HPLC's primary development focus is on increasing analysis throughput, in order to achieve separations five to ten times faster than before, as well as the possibility of achieving separations with higher separation power (high resolution). This can be achieved by using innovative chromatographic supports such as monolithic columns, columns filled with porous particles of less than 2 μm or columns filled with superficially porous particles of less than 3 μm. It is also important to bear in mind that increasing the temperature of the mobile phase leads to a significant improvement in chromatographic performance. However, in order to reap the full benefits of fast and/or high-resolution chromatography, suitable instrumentation is required, and significant developments have been made in this direction over the last ten years.

Another area of development concerns the use of alternative chromatographic methods. Reverse phase liquid chromatography (RPLC) is by far the most widely used method today, but it may be worthwhile to use alternative separation methods to modulate the selectivity and retention of certain problematic compounds. Two such approaches are hydrophilic interaction chromatography (HILIC) and supercritical chromatography (SFC). The first of these two approaches is particularly suited to the analysis of highly hydrophilic compounds (sugars, amino acids) and/or ionizable compounds (pharmaceutical molecules), which are difficult to analyze using RPLC. The second is suitable for a wider range of analytes, including highly lipophilic compounds (lipids, fat-soluble vitamins, etc.) that are difficult to elute with conventional RPLC.

Finally, the last area of development is more specific to the pharmaceutical field, and concerns the analysis of therapeutic proteins and monoclonal antibodies with molar masses between 5 and 150 kDa. For this type of analysis, historical techniques such as steric exclusion chromatography (SEC) and ion exchange chromatography (IEX) are now facing stiff competition from the new large-pore chromatographic supports available in RPLC, offering greater compatibility with mass spectrometry (MS).

The aim of this article is to outline the major developments in liquid chromatography, so that...