Convergence chromatography is the next generation of supercritical fluid chromatography. It is based on the use of carbon dioxide (CO2) as the chromatographic mobile phase. It stands out for its outstanding chromatographic performance, wide applicability, and eco-friendly characteristics. This article is attempted to explain all that you are searching for about convergence chromatography. Additionally, we have provided an exclusive section in which we explain the differences between traditional supercritical fluid chromatography (SFC) and its latest variant i.e., convergence chromatography (CC).
What is convergence chromatography
Convergence chromatography is a column chromatographic technique. It uses supercritical CO2 as the main mobile phase along with co-solvents in differing ratios. Porous stationary phase materials are packed or coated along the walls of the chromatographic column. The supercritical fluid stream carries the analyte molecules into the column under high-pressure conditions (100 to 400 times higher than atmospheric pressure).
Working principle of convergence chromatography
The working principle of convergence chromatography lies in between that applied for liquid chromatographic techniques such as HPLC and gas chromatography (GC). Convergence chromatography is specifically useful for analyzing temperature-sensitive compounds, otherwise difficult to separate through GC. Analyte separation occurs inside a convergence chromatographic column based on the affinity of the analyte molecules with the stationary phase versus their interaction with the mobile phase.
The mobile phase in convergence chromatography
CO2 is converted to a supercritical fluid above its critical temperature (31.1°C) and pressure (72.9 atm). Supercritical CO2 possesses both liquid and gaseous characteristics. It has the density and viscosity of a liquid while it has a high diffusivity and compressibility like that of a gas. Thus, supercritical CO2 is used as the main mobile phase solvent in convergence chromatography. Co-solvents such as ethanol, methanol, acetonitrile, isopropanol, etc., are often added to modulate the properties of this main solvent. CO2 is completely miscible with these polar solvents.
The flexible physicochemical properties of CO2 in the supercritical state further support polarity modulation through solvent mixing. In this way, convergence chromatography can be used for the separation and analysis of a wide range of chemical compounds, polar and non-polar inclusive.
The stationary phase in convergence chromatography
The stationary phase in convergence chromatography can be designed in a normal-phase or a reverse-phase mode, depending upon the analyte mixture. Silica or alumina-based stationary phase materials are used for retaining highly polar compounds from the sample mixture. Polymeric stationary phase materials can also be employed. In contrast to that, C-8, or C-18 octadecyl silane (ODS) packed columns can be used for the retention of non-polar components. Chiral stationary phase materials can also be developed for chiral separation through convergence chromatography.
Components of convergence chromatography
A convergence chromatographic system is based on the following essential components:
1.Carbon dioxide (CO2) cylinder
Carbon dioxide exists as a gas at room temperature. It is supplied to the CC system through a gas cylinder. The gas is heated above its critical temperature and pressure for it to attain a supercritical state. The supercritical CO2 then directly enters the CC system under high-pressure conditions.
2. Pressure pumps
High-pressure pumps are used to facilitate the flow of the mobile phase stream and the analyte mixture into the column.
3. Sample injector
The sample mixture is directly introduced into the column with an on-column injector. It becomes a part of the supercritical fluid stream.
4. Analytical column
Main separation occurs in the analytical column based on the stationary phase packing and analyte-stationary phase interactions. Specific analyte molecules get retained onto the stationary phase while the remaining pass out with the supercritical fluid. The retained molecules are then eluted one by one by changing the mobile phase composition, mixing different polarity co-eluents in it. The eluted analyte molecules finally reach the detector.
The detector detects the incoming analyte molecules and sends an electronic signal to a recorder.
Some suitable detectors for a convergence chromatographic system are :
- Mass spectrometer (MS)
- Evaporative light scattering detector (ELSD)
- Ultra-violet (UV) absorption detector
- Photodiode array (PDA) detector
The recorder plots a chromatogram as a plot of detector response versus analyte retention time. Single, sharp, well-resolved peaks are expected as a result of convergence chromatography.
Convergence chromatography versus SFC
Convergence chromatography can be called a re-named version of supercritical fluid chromatography. In reality, SFC is a state-based name, as a supercritical fluid is used in both these types of chromatography. On the other hand, convergence chromatography is an application-based name.
The word convergence literally means unifying two or more things at the same point. Therefore, convergence chromatography is named considering the convergence of existing chromatographic techniques such as HPLC, GC, and SFC in a single system. Thus, it brings the benefits of all techniques together. Similarly, convergence chromatography is a convergence of supercritical fluid with co-solvents against a diverse variety of stationary phases.
When a high concentration of any co-solvent is added in the supercritical mobile phase, the mobile phase does not stay in its pure supercritical state for the entire time it spends on the column. This deviation from an ideal supercritical behavior paved way for the development of convergence chromatography. Another difference is that the convergence chromatography operates on a slightly higher temperature and pressure than normal SFC.
The older version of supercritical fluid chromatography also suffered some technological issues. The old instruments lagged behind in handling highly compressible supercritical CO2 which badly affected method robustness. Pumping a highly compressible supercritical fluid while maintaining a constant pressure inside the column is a challenging task. Additionally, it causes a high back-pressure risk inside the analytical column. Undesirable pressure ripples generate detector noise. The latest technology and equipment available for convergence chromatography however is successful in solving all these issues.
Waters Corporation has also introduced an updated version of CC i.e., ultra-performance convergence chromatography (UPCC or UPC2). Just like ultra-high performance liquid chromatography (UHPLC) is the latest version of high-performance liquid chromatography (HPLC).
Why is convergence chromatography important
- Convergence chromatography is a fast-paced chromatographic separation technique, fascinated with advanced technology. 10 minutes of analytical separation via normal-phase liquid chromatography can be accomplished through CC in just about 9 seconds.
- It can efficiently analyze a wide range of compounds, both polar and non-polar using the latest equipment and technology.
- It offers a unique selectivity for chiral separation. It finds meaningful applications in the pharmaceutical industry for the quality evaluation of medicinal materials, pharmaceutical drugs, pesticides, etc.
- Ultra-performance convergence chromatography offers orthogonality i.e., it can be coupled with MS, FTIR or NMR for complete chemical identification. Small amounts of impurities present in complex sample mixtures can be immediately detected and separated. This saves the overall chromatographic prep and analysis time.
- Convergence chromatography can be used in the food industry for natural product analysis and purification such as the analysis of lipids (Lipidomics) and fat-soluble vitamins. No derivatization is needed for lipids in CC unlike lipid analysis in GC.
- A distinguishing feature of convergence chromatography is the use of supercritical CO2 as the main mobile phase which reduces toxic solvent consumption. Thus, it is a green, eco-friendly chromatographic technique.
In conclusion, convergence chromatography is a novel, extremely valuable chromatographic analysis technique that has successfully addressed many challenges restricting chromatographic performance in the past. Thus, it is here to stay for long and chromatographers will continue researching on it further and improving it for future use.
Here is a video tutorial on convergence chromatography.
Other interesting liquid chromatographic techniques that you would like to read are:
1. Waters (2015). Beginner’s Guide to Convergence Chromatography.
2. Zhang, Xiaoyi, Liu, Jiushi, Gao, Shiman, Chen, and Rongchang (2020). “Ultra-performance convergence chromatography, a more efficient method for chemical quality evaluation of Gaoben medicinal materials compared with the ultra-performance liquid chromatography.” Journal of Chromatography 1616(17).