Beginner's Guide to Convergence Chromatography

Understanding the Founding Principles of Convergence Chromatography

Expanding Selectivity for the Chromatographic Laboratory
Of the chromatographic tools available today, the two most commonly utilized are gas chromatography (GC) and liquid chromatography (LC). Due to significant advancements in the performance of systems designed to manage supercritical fluids, convergence chromatography (CC) is a viable chromatographic approach for addressing both complex and routine separation challenges.

chromatography techniques
Figure 1: Three complementary chromatographic techniques used in the analytical laboratory.

 

Each of these three chromatographic techniques has gone through numerous iterations and improvements over time, enhancing the capabilities of the chromatographer. In gas chromatography, the separation is achieved by varying the temperature of the column over the course of the analysis. This technique provides high efficiency (separation power), but with limited selectivity. Selectivity is determined solely by the interaction of the analytes with the column chemistry (stationary phase); the carrier gas (mobile phase) is inert. The advent of capillary GC, 30 years ago, affected significant performance improvements, but only small incremental advancements have occurred since.

Due to a narrow polarity range and volatility requirements, gas chromatography is limited relative to the types of analytes that can be separated. To address a more diverse range of compounds than is possible to analyze by GC, liquid chromatography, in reversed-phase mode, has proven an invaluable analytical technique.

In LC (unlike in GC), the analyte has affinity for both the stationary and mobile phases. Separation is achieved by creating a competition between these phases. This allows for more options when changing selectivity. Advancements in LC systems were small and incremental until the introduction of UltraPerformance LC® (UPLC®) in 2004.

Convergence chromatography is a new category of separation science that provides an exceptional increase in selectivity to the chromatographic laboratory. CC is orthogonal to reversed-phase LC and provides significant potential in streamlining the entire analytical workflow. In CC, the separation is achieved by manipulating the density and composition of a supercritical fluid-based mobile phase. Because of the very high diffusivity of the mobile phase, high separation efficiency can be achieved. Additionally, the diversity of stationary phase and mobile phase (co-solvent) options give the chromatographer access to the largest selectivity space available to any separation technique.

acquity upc2 system
Figure 2: The ACQUITY UPC2™ System.

 

The term “convergence chromatography” originates from a concept Professor Calvin Giddings discussed in 1964. In one of his publications, Giddings states, “One of the most interesting features of ultra high pressure gas chromatography would be convergence with classical liquid chromatography.”1 Convergence chromatography is the realization of this idea, taking the benefits of GC and merging them with the benefits of reversed-phase and normal-phase LC.

In order to harness the theories originally proposed by Giddings and realize the benefits of converging GC and LC, Waters has developed the ACQUITY® UltraPerformance Convergence Chromatography™ (UPC2®) System (Figure 2) - a holistically designed chromatographic system that utilizes compressed CO2 as the primary mobile phase. The system leverages the chromatographic principles and selectivity of normal-phase chromatography while providing the ease of use of reversed-phase LC. Built upon proven, low-dispersion, high-efficiency UPLC technology, the ACQUITY UPC2 System offers a level of reliability, robustness, sensitivity, and throughput never before possible when utilizing supercritical fluids and convergence chromatography.

 

Streamlined Workflow
Equally as important as its separation capability is how CC impacts the overall workflow of the laboratory. Often, the largest bottleneck in a chromatographic laboratory is the preparation of samples for further analysis. Most common sample preparation methods result in the analytes of interest being dissolved in a solvent incompatible with reversed-phase LC. Many analytes are easily dissolved in – and therefore best extracted with – an organic solvent. Because of this, additional steps are often required to convert the organic solution or extract into something compatible with reversed-phase chromatography (Figure 3).

sample preparation techniques
Figure 3: Examples of various sample preparation techniques, which often conclude with the sample being dissolved in an organic solvent.

 

CC is compatible with direct injection of samples dissolved in organic solvents. Evaporation and reconstitution (often the most time-consuming steps) required for reversed-phase separations are no longer required, resulting in considerable cost savings in the overall assay. In addition to the reduced time required to prepare the sample, the analysis can be significantly shorter – a significant cumulative impact, especially with multiple systems.

 

Diverse Applicability and Selectivity
CC utilizes the same eluotropic spectrum as normal-phase LC. The flexibility of this technique, however, also allows the use of some conventional reversed-phase columns such as C18, yielding similar retention characteristics to reversed-phase LC when analyzing highly lipophilic compounds. The ability to span this wide range of selectivity gives CC clear advantages.

An overlap of approximately 80-85% of compounds can be analyzed by both convergence and reversed-phase chromatography. In addition, CC can also analyze those compounds typically analyzed by normal-phase LC. The ability to analyze chiral compounds, stereoisomers, and diasteroisomers dramatically increases the utility of CC. Essentially, any compound soluble in an organic solvent is a candidate for analysis via CC. Unlike normal-phase LC, CC is compatible with gradients, enabling utilization of detection techniques such as ultraviolet (UV), photodiode array (PDA), and evaporative light scattering (ELS).

The primary mobile phase in CC is dense CO2 in either a supercritical or subcritical state. This mitigates the use of harmful solvents necessary in normal-phase LC mobile phases. Additionally, the variability expected from normal-phase LC, due to the adsorption of water to the stationary phase, is completely eliminated. The nonpolar nature of the mobile phase also makes injection in organic solvents preferable. These advantages are currently being leveraged in two application areas: fast chiral screening, and normal-phase replacement methods.

In a chiral screening example (Figure 4), analysis time is reduced from 20 minutes to only 3 minutes, a seven-fold reduction in time, with an increase in resolution. Along with taking less time, significantly less solvent is consumed, resulting in substantial reduction in cost.

UPC2 fast chiral screening
Figure 4: The utility of CC for fast chiral screening using UPC2.

 

Similar reductions in analysis time and cost per analysis occur with achiral normal-phase replacement (Figure 5). Replacing typical normal-phase organic solvents with a mobile phase composed of primarily compressed CO2 reduces the cost per analysis from just under 6 dollars to only 5 cents per sample. The overall financial impact from shorter analysis time, lower solvent purchase costs, and reduced solvent disposal costs achieved by using CC as a normal-phase LC replacement is exceptional.

achiral normal-phase replacement
Figure 5: The utility of CC for normal-phase replacement.

 

The Underlying Principle: Supercritical Fluid Chromatography 
The underlying principle of convergence chromatography is supercritical fluid chromatography (SFC). A supercritical fluid is any substance at a temperature and pressure above its critical point, where distinct liquid and gas phases no longer exist (Figure 6). Essentially, the substance takes on properties of both a gas and a liquid, including high diffusivity and low viscosity, enabling fast and efficient chromatography.

substance phase diagram
Figure 6: Phase diagram depicting the physical change of a substance from one state to another.

 

Carbon dioxide is the most common substance utilized as a supercritical fluid mobile phase. The physical state of CO2 is easily manipulated at temperatures and pressures in an acceptable range. Extreme conditions needed to put other substances into their supercritical states, as well as undesireable properties when in that state, make CO2 the best candidate (Table 1).

Substance

Critical temp °C

Critical pressure (bar)

Comments

Carbon dioxide

31

74

Physical state easily changed

Water

374

221

Extreme conditions needed

Methanol

240

80

Extreme temperature needed

Ammonia

132

111

Highly corrosive

Freon

96

49

Environmentally unfriendly

Nitrous oxide

37

73

Oxidizing agent

Table 1: Conditions required to transition a particular substance into a supercritical fluid.

 

While in reversed-phase LC, water is typically used as the weak mobile phase, in CC, supercritical CO2 is used instead. Pure CO2 has limited solvating power, so it is often mixed with different cosolvents and modifiers.

Pairing supercritical CO2 with different co-solvents opens up a significant range of selectivity options (Figure 7). Reversed-phase LC can access only a small piece of the eluotropic series, whereas normal-phase and convergence chromatography can explore a much larger range (Table 2). One caveat of normal-phase chromatography is that not all organic solvents are miscible with one another, resulting in incompatibility of certain mixtures. Supercritical CO2, however, is miscible with all other solvents across the entire eluotropic series, opening up a wide range of solvent choices to influence the selectivity of separations. Importantly, different eluotropic strengths can be obtained by using solvents outside of the normal-phase tool kit.

CC column selectivity
Figure 7: Column selectivity can be an exceptionally powerful tool when developing methods in CC. In this example, an active pharmaceutical ingredient and its related compounds were analyzed on multiple stationary phases (typical to reversed-phase and normal-phase) under a fixed set of conditions.

 

With CC, although the full range of normal-phase solvents can be accessed to increase selectivity, in many cases it is not necessary. By simply combing supercritical CO2 with an organic co-solvent at the other extreme of the eluotropic spectrum, in addition to the diverse range of available stationary phases, an exceptionally large selectivity space can be explored, making this technique applicable to a wide variety of separation challenges. By combining CO2 with methanol, for example, the mobile phase can be dialed to eluotropic strengths ranging from 0 to 0.73Eo. The realization of the analytical power of CC is made possible because of the advancements in instrumentation found in the ACQUITY UPC2™ System.

CC solvent selectivity options
Table 2: Solvent selectivity options for reversed-phase, normal-phase, and convergence chromatography. The supercritical CO2 used in CC is miscible with the entire eluotropic series, opening up a wide range of solvent selectivity choices to develop a separation.

 

Table 3 shows several of the available stationary phases commonly utilized for different chromatographic techniques. Reversed-phase and normal-phase LC utilize limited column options as compared to CC (Table 3). Most reversed-phase separations are performed on C18 stationary phases. Some columns cannot be used in reversed-phase separations at all because of the phase polarity. Normal-phase chromatography is also limited because of incompatible polarity between phases. CC allows for use of all of the column chemistries, opening up a wider range of selectivity choices.

Convergence chromatography phase options
Table 3: Stationary phase options for reversed-phase, normal-phase, and convergence chromatography. Convergence chromatography can utilize both traditional normal-phase and reversed-phase column chemistries, opening up a wide range of selectivity choices to develop a separation.

 

 

 

 


Realizing the Potential of Convergence Chromatography Realizing the Potential of Convergence Chromatography.
Using Convergence Chromatography Utilizing Convergence Chromatography in the Chromatographic Laboratory.
Example of Problems Solved by Convergence Chromatography View Convergence Chromatography Examples of Problems Solved.

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