Beginner's Guide to Convergence Chromatography

The basic mechanism of any chromatographic separation is to create conditions where all the analyte molecules in the sample mixture move through the system, but at different speeds so that when they elute from the analytical column they are sufficiently separated from one another to be detected and quantified. To enact this process, chromatography has two major components - a stationary and a mobile phase. The primary role of the stationary-phase is to arrest or retain the compound movement whereas that of the mobile phase is to hold and also force the compounds to move from the entrance to the exit of the system. This is the fundamental mechanism for GC, LC or CC. The main difference between CC and LC or GC, is how the mobile phase properties influence respective chromatographic behaviors.

Role Of Mobile Phase In CC Compared To LC And GC

In GC, the mobile phase is normally an inert or unreactive gas - typically He or N₂. At the GC operating temperatures and pressures, the mobile phase cannot solvate analyte molecules or modify the stationary-phase surface. GC mobile phase acts mainly as a carrier or driver of the analyte molecules through GC column. Analyte retention and separation is imparted solely through interactions between analyte molecules with the stationary-phase. This is imparted solely through interactions between analyte molecules with the stationary-phase. This schematically depicted in Figure 3 with the blank space around the analyte molecules.

In LC, on the other hand, mobile phase plays an active role, where its molecules strongly interact with both analyte molecules and the stationary-phase. Mobile phase influences analyte retention by not only directly solvating the analytes, but also influencing analyte vs. stationary-phase interactions - by competing for stationary-phase surface (Figure 3). 

Figure 3 presents LC in both RPLC and NPLC modes. Note that one of the main differences between these two LC modes is in the composition of the mobile phase - RPLC is water-based whereas NPLC is organic-based. In RPLC, the water-based mobile phase, in conjunction with C₁₈ stationary-phase, effectively modifies the interactions that the analytes have with the stationary phase - thus playing a major role resolving sample analytes in a wide variety of compound mixtures. In RPLC method development, the first modification is often made to the mobile phase, not the stationary phase. In NPLC, by comparison, the hexane or heptane-based mobile phase takes a relatively moderate role and the separation is largely imparted by varying the stationary-phase chemistry.

The role of mobile phase in CC is somewhere between RPLC and NPLC because of the unique properties of CO₂ - supercritical or not. Compressed CO₂ is non-polar, like heptane or hexane. Based on that, CC is more like NPLC. But one key difference is that CO₂ is capable of completely mixing with polar co-solvents e.g. methanol, ethanol, acetonitrile, etc and can therefore be used in gradient modes, unlike NPLC which is almost always used in isocratic mode. Additionally, the CC mobile phases are much more tolerant of the presence of small amounts of water than NPLC, which can play a strong role in analyte elution.

In the next section a systematic comparison of CC with RPLC and NPLC is presented based on the property differences between their principal solvents.

Role of CO2 Properties In Influencing Chromatographic Behavior

CO2 miscibility with other solvents

CO₂ is a non-polar solvent with a polarity index similar to heptane (=0.1). But unlike heptane, CO₂ has a non-zero quadruple moment (-13·4 ± 0·4 × 10^–40 C m²) and it is completely miscible with highly polar organic solvents, e.g. acetonitrile (polarity index = 5.8) and methanol (polarity index = 5.1). Although it is sparingly miscible with water (polarity index = 10.2), it is miscible with methanol/water, isopropanol/water or acetonitrile/water mixtures with considerable proportions of water in them. Such a wide range of miscibility permits the CC mobile phase to extend its polarity over a wider range than NPLC and RPLC mobile phases. Table 1 presents a concise picture of the situation in terms of eluotropic (eluting strength) values and polarity indices of the solvents used in RPLC, NPLC, and CC.

Note that in Table 1 the water-based mobile phase of RPLC can employ only a limited range of the eluotropic series because of the limited miscibility of water with most of the other organic solvents. Similarly for NPLC, the hexane/heptane based mobile phase does not allow a wide eluotropic range because of limited miscibility of non-polar organics with highly polar solvents. An additional problem with NPLC is that not all organic solvents are widely miscible even with one another, resulting in incompatibility of certain mixtures. For CC, on the other hand, compressed CO₂ is miscible with all other solvents across the entire eluotropic series, opening up a wide range of mobile phase choices to influence the selectivity of separations (see Table 1). Although CO₂ is non-polar, CC is comparable to RPLC because it can have a much wider eluotropic strength - especially on the higher polarity side - compared to NPLC. For example, by combining CO₂ with methanol, the mobile phase eluotropic strengths can be programmed from 0 to 0.73E^o.

Along with extended eluotropic range, the CO₂-based mobile phase of CC is compatible with the widest number of stationary-phase chemistries. Table 2 lists the stationary phases commonly employed for NPLC and RPLC. Most RPLC separations are performed with C₁₈ stationary phases with relatively few instances using other bonded phases. Some stationary-phases listed cannot be used with RPLC at all because of their higher polarity. Similarly with NPLC, column selection is constrained by the polarity range of the mobile phase. With CC, because of its wider polarity range, the selection of all these column chemistries is possible - opening up a wider range of selectivity choices (See Figure 4). As noted by West and Lesellier, as all of these chemistries can work with the same mobile phase composition, this opens up the exciting possibility of coupling columns of very different polarities .

Another reason why CO₂ miscibility is important is that CC is compatible with a wide range of sample diluents (solvent that the sample is dissolved or diluted into). This feature of CC strongly impacts the overall workflow of the laboratory. Often, the largest bottleneck in a chromatographic laboratory is sample preparation. Most common sample preparation methods result in the analytes of interest being dissolved in a solvent incompatible with the LC system at hand. For example, many analytes are easily dissolved in – and therefore best extracted with – an organic solvent. Because high amounts of organic solvents are incompatible with RPLC, additional steps are often required to convert the organic solution or extract into something compatible with RPLC (Figure 5). CC is compatible with direct injection of samples dissolved in organic solvents, steps to evaporation of organic solvents and reconstitution (very time-consuming) of the sample into water based diluents required for reversed-phase separations are no longer required. This results in considerable cost savings in an overall assay. In addition, the analysis time can be much shorter – a significant collective impact, especially for labs running multiple RPLC systems set up to analyzing numerous samples.

In short, by combining non-polar compressed CO₂ with a co-solvent at either extreme of the eluotropic spectrum, coupled with a wider diversity of stationary phases compatible to CC, one can explore an exceptionally large selectivity space, making CC applicable to a wide variety of separation challenges.

CO2 Transport Properties

Another property advantage of CC is the low viscosity and the resultant high diffusivity of analyte molecules in CC mobile phase. From a physical property point of view, the efficiency of a chromatographic column is controlled by the analyte diffusivity in the mobile phase. The higher the diffusivity of a molecule, the faster it travels in and out of the stationary particle pores, resulting in high efficiency even at high mobile phase velocity. In CC, even after adding significant volumes of liquid modifier (e.g. for CO₂/methanol (70/30, mol/mol %), mobile phase viscosity is at least half that of LC mobile phases (see Table 3). This means that CC can be operated at much higher mobile phase flow rates, without sacrificing column efficiency. This makes CC a great candidate for high-throughput analyses.

The advantage of CC in two key application areas, fast chiral screening and as a replacement for normal-phase chromatography for achiral separations is significant. For chiral screening, analysis time is reduced from 20 minutes to only 3 minutes, a seven-fold reduction in time, with an increase in resolution. This improvement was mainly driven by the employment of solvent gradient in CC, which could not be done in NPLC. Another bonus to CC is that less solvent is consumed, resulting in substantial reductions in cost.

Replacing normal-phase organic solvents with a mobile phase composed of primarily compressed CO₂ (Figure 6) reduces the cost per analysis from approximately six dollars to only five cents per sample. The overall financial impact from shorter analysis times, reduced solvent purchasing and disposal costs is exceptional.

Role of Other Advantageous Properties

Table 4 lists the advantages of CC mobile phases over LC mobile phases. After the miscibility and low viscosity advantages, there’s also low surface tension. Low surface tension allows the mobile phase to enter pores of the stationary phase particles faster, leading to faster column equilibration.

Other properties of CO₂, which makes CC highly attractive as a chromatographic system, is that it is cheaper and safer to operate – and more sustainable. CO₂ is readily available and is not dependent on any other critical processes (e.g. that of acetonitrile as a byproduct of petrochemical industry). Commercial-grade CO₂ is carbon-neutral and is considered a green solvent. CO₂ costs much less than other organic solvents and CO₂ can be directly discharged to the atmosphere (if not recycled) without incurring any disposal costs.

CO₂ is non-flammable and non-toxic, and easier to store. It’s impossible to find a mobile phase the combines the properties of CO₂ – miscibility and low viscosity – that is as economical and eco-friendly as CO₂ which makes CC superior to LC for many applications. The cumulative benefit of all these properties, listed in Table 4, makes CO₂ a unique solvent.

Supercritical or Not?

As stated earlier, from a chromatographic point of view it does not matter in CC if the mobile phase is supercritical or not. However, it is absolutely required that the CC mobile phase is homogeneous and not a heterogeneous mixture of gas and liquid, to carry out chromatographic separation. To ensure it is homogeneous, the mobile phase in CC is maintained above a certain pressure, which can be easily set through an automated back-pressure regulator (ABPR).