Green Chromatography Advances Highlighted at HTC-18

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There was a special session focusing on green chromatography on Thursday 30 May 2024 at the International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-18) in Leuven, Belgium.

The session kicked off with a talk from Martina Catani from the University of Ferrara, Italy on “Greening Preparative Liquid Chromatography for The Purification of Biopharmaceuticals.” Catani commented that therapeutic peptides are effective medical treatments, and production has significantly developed in the past 10 years, particularly when using solid-phase synthesis. However, the purification processes have not seen similar progress, she added, and remain expensive, time-consuming, and less sustainable. The traditional method using single-column preparative liquid chromatography in reversed-phase (RPLC), involves removing impurities from peptides, which results in large amounts of acetonitrile (ACN) waste—a toxic substance harmful to humans and the environment, and there is a demand for “greener purification” methods, she said.

Catani focused on two specific strategies to enhance sustainability in biopharmaceutical purification. The first strategy replaced ACN with “greener” solvents such as ethanol, isopropanol, and dimethyl carbonate (DMC). These solvents are noted for environmental benefits but are rarely used in liquid chromatography, according to Catani. The second strategy used advanced multicolumn countercurrent preparative liquid chromatography (MCSGP) systems that recycle overlapping chromatogram sections to automate the process and significantly reduce solvent use. The session concluded that using MCSGP for purifying the therapeutic peptide, Icatibant, reduced solvent consumption by over 80% compared to traditional single-column methods.

This was followed by a presentation by David McCalley from the University of the West of England, United Kingdom with a talk covering “Per Aqueous Liquid Chromatography: A Green Alternative for Separating Polar and Ionogenic Solvents.”

McCalley stated that the use of acetonitrile can be minimized for hydrophilic interaction chromatography (HILIC), with short, small internal diameter columns (10 x 0.21 cm) featuring sub-2 micron particle diameters. Isocratic analysis with retention factors up to 5 allows 700-1000 samples for each liter of acetonitrile to be analysed. McCally commented that Aacetonitrile is considered less toxic environmentally compared to most normal phase solvents, but, there is a growing interest in further reducing or eliminating its use.

MCalley’s team investigated the lower end of the HILIC retention curve, using less than 10% organic solvent using per aqueous liquid chromatography (PALC), to separate polar and ionized solutes while maintaining acceptable peak shapes. This technique proved effective across various stationary and mobile phase combinations, according to McCalley.

McCalley highlighted that PALC does reduce some column efficiency compared to HILIC for the same solutes and stationary phases, but it still achieved up to 11,000 plates for certain solutes using a 10 cm column. The separation mechanism in PALC is complex, involving ionic retention of protonated basic solutes on silanol groups in silica-based columns and hydrophobic retention on siloxane groups on bare silica, leading to significant retention of some non-polar solutes, he said. Other mechanisms, such as polar neutral retention, may also play a role, according to McCalley. The balance of these interactions within the mixed retention process influences the peak shape, with the best peak shapes observed for polar neutral compounds or organic acids, he concluded.

Next, Leo Lebanov from Australian Centre for Research on Separation Science (ACROSS) and ARC Training Centre for Hyphenated Analytical Separation Technologies (HyTECH), University of Tasmania the gave a talk called “Application of the Biodegradable Biosolvent ‘Cyrene’ in Pharmaceutical Solvent Analysis using HS-GC-MS”

Lebanov said there is a shift towards using renewable resources and biomass in chemical production processes that aims to reduce reliance on fossil fuels and make them more sustainable. Bioderived solvents, such as Cyrene, offer a greener alternative to toxic petroleum-based solvents such as NMP and DMF to align with the Twelve Principles of Green Chemistry, according to Lebanov.

Cyrene is derived from sawdust and is a sustainable and less toxic solvent with potential applications in organic synthesis, nanotechnology, and sample preparation, he said. Although it is not commonly used in separation science, Lebanov’s team explored Cyrene as a potentially novel solvent for gas chromatography-mass spectrometry (GC–MS) in pharmaceutical analysis.

In pharmaceutical synthesis, controlling residual solvents is crucial. Traditional solvents such as DMSO and DMF, which are commonly used in GC–MS, pose toxicity concerns. Cyrene with a high boiling point of 227°C, is biodegradable and a safer alternative, according to Lebanov.

This study is believed to be the first using Cyrene in GC–MS sample preparation, Lebanov said. By using AGREE software, the method's environmental impact improved, with the greenness coefficient rising from 0.58 to 0.73. Lebanov concluded that Cyrene enabled the effective analysis of various solvents, maintaining comparable sensitivity and detection limits to DMSO, and has the potential to be a viable substitute in chromatographic applications.