Beginner's Guide to Convergence Chromatography
Introduction
Development of Convergence Chromatography
1.1 Convergence chromatography (CC) is a separations technology that uses compressed CO2 as the principal solvent in the chromatographic mobile phase, at pressures between 100 to 400 times atmospheric pressure. CO2, most often, is mixed with liquid co-solvents (e.g. methanol, ethanol, isopropanol, acetonitrile) to modulate mobile phase properties; similar to the way these co-solvents are mixed with water to modulate the mobile phase properties of reversed-phase liquid chromatography (RPLC). Those familiar with the modern version of Supercritical Fluid Chromatography (SFC) will immediately see the resemblance of CC to SFC. In reality CC is a renamed version of modern SFC. To understand what we mean, we need to take a historical look back at the development of SFC, which we do in the following paragraphs.
SFC was invented as a chromatographic technique employing solvents under supercritical conditions to expand the capability of gas chromatography (GC). To solve the problem of analyzing compounds that elute only at high temperatures but thermally decompose at those temperatures, Klesper and co-scientists [1] employed higher pressures in GC to compensate for the requirement of high temperatures. Supercritical conditions were selected for (a) the solvation power of a high-pressure gas and for (b) avoiding conditions where the mobile phases no longer flow as a single solvent (undergo gas-liquid phase separation) by varying either the pressure or the temperature or both. Later, they learned that by varying solvent density, one can modulate solvent strength, the main advantage of working at supercritical conditions. Because fluids are highly compressible under supercritical conditions, even minor changes in pressure greatly changes solvent density and hence analyte retention. In effect, one can create a solvent gradient by varying the operating pressure leading to density modulation, negating the need to mix in organic co-solvents to create a gradient. This possibility of a single non-toxic solvent separation mode drew considerable interest and excitement among analytical scientists.
The excitement, however, was short-lived as it became gradually understood that modulating density, as useful a technique as it is, may not modify solvent polarity enough to be widely applicable to the range of compounds that can be separated by other powerful chromatographic techniques like Reversed Phase Liquid Chromatography (RPLC). The fact of the matter is that CO2, which evolved as the best solvent of choice for SFC, remains a non-polar solvent even with significant density modulation. For separating most polar analytes then, it is necessary to add polar co-solvents e.g. methanol. This realization changed the course of SFC development. Although there are many important applications that still rely on density modulation alone, SFC practitioners began relying more on mixing liquid organic co-solvents and additives, just like in RPLC, to achieve separation of a wider variety of analytes.
CO2 is completely miscible with very polar solvents, (e.g. methanol, ethanol, acetonitrile) and solvent gradients reaching very high modifier compositions (e.g. 60%) are routinely employed. Such practices repeatedly brought forth questions about the supercriticality of the mobile phase. At higher modifier concentrations, and at temperatures and pressures typically used in SFC, the mobile phase is not supercritical during most of the method time. And more importantly, such deviations from supercritical conditions do not make any difference in chromatography. So why should a technology, which does not depend on the solvent reaching a supercritical condition, still be known as Supercritical Fluid Chromatography? There have been suggestions to name the modern version of SFC something else, for example Sub/Super-critical Fluid Chromatography, Simplified Fluid Chromatography, Separations Facilitated by Carbon-dioxide or simply by its acronym - SFC, but none does justice to the expanded scope of its current use in analytical laboratories.
Along with the confusion regarding its identity, SFC also faced acute technological difficulties that kept it from being employed as a serious analytical instrument. Method robustness and reproducibility were poor because older instruments could not handle a compressible solvent like CO2 reliably and repeatedly, at least on par with the modern HPLC or UPLC instruments.
That all changed when, in 2012, Waters Corporation introduced Ultra Performance Convergence Chromatography (UPC2) (see Figure 1) to address both the instrumental issues and the naming dilemma. With significantly higher instrument robustness, which will be discussed in detail in chapter 3, and a new name to set it apart from older technologies, SFC finally became a serious option for analytical studies.
Figure 1: The ACQUITY UPC2™ System.
What Does The "Convergence" In Convergence Chromatography Signify?
The word "convergence" in Convergence Chromatography was borrowed from Giddings's observation of having a technology that converges existing chromatographic technologies - GC and liquid chromatography (LC), in one system. Giddings portrayed SFC as a technique to bridge LC and GC and extend the use of mobile phases beyond the boundaries of supercriticality. Convergence chromatography is now most often used with organic co-solvents encompassing both super- and subcritical regions, expanding past the limited density modulation of CO2-only SFC. Rather than just bridge the gap between GC and LC, SFC’s potential is much greater than originally envisioned.
