30 Environmental Laboratory
PURE PERFORMANCE: SAVING TIME WITH ULTRA-FAST PREPARATIVE LC
How Syngenta is using UFPLC to extract and purify gram-scale by-products for regulatory tests
C
hemicals such as herbicides, fungicides, insecticides and seed treatments are widely used to maximise crop yields worldwide. Ensuring the effi cacy and safety of products requires studying the formulations in detail, including even the most minor of components. In this article, we talk to Syngenta about how they’re using Shimadzu’s Ultra-Fast Preparative LC (UFPLC) systems to speed up the extraction and purifi cation of gram- quantities of by-products that are present in technical-grade active-ingredient material at levels below 1%, so providing the material needed for regulatory tests and toxicity studies.
Isolating by-products – A major challenge
In the world of agriculture, there are increasing pressures to maintain or increase crop yields in the face of growing threats – whether from pests, diseases, competitive weed species, or the changing climate. To minimise the effect of these threats, farmers need to deploy a range of methods to promote strong and healthy crop growth. Crop protection products such as herbicides, fungicides, insecticides and seed treatments are an essential part of this effort, and Syngenta works with farmers, researchers and agriculture experts to understand the challenges faced, and to develop new or improved treatments.
As a heavily-regulated industry, crop protection products must meet numerous criteria before they can be registered for use, which includes rigorous tests to assess their safety for workers, the environment, crops and consumers. Some of these tests must be carried out on all components of technical-grade material of an active ingredient at, or close to, the 0.1% weight level. However, obtaining these ‘by-products’ in suffi cient quantity and purity is a big challenge.
Developing analytical solutions at Syngenta
Isolating these by-products in usable quantities is where separation scientists such as Louise Bacon come in. Louise works at Syngenta as the Team Lead for the Preparative and Isolation Chemistry Team, which sits within the company’s Analytical Solutions Group at Jealott’s Hill International Research
IET SEPTEMBER / OCTOBER 2023
Centre in Bracknell, UK. Her team helps their colleagues around the world with the toughest of separation challenges, with the help of preparative-scale liquid chromatography.
Louise has had several roles at Syngenta over the last 30 years, and has been working full-time on the preparative side since 2016. She describes the sorts of projects that her team gets given: “Most of our projects come from our ‘global analytical champions’, who are responsible for an active ingredient and all the associated method validation and regulatory work. As part of that, they may need our help to extract and purify milligram levels of an unknown by-product for identifi cation. But more often they will need larger quantities of a by-product for use as analytical standards, extending up to the gram-levels required for toxicity testing. For example, it’s not uncommon to have a by-product present at 0.5% by weight in a product, and that requires a toxicity test on the pure chemical in order for the active ingredient as a whole to be registered”.
It’s this need for larger quantities, she explains, that presents the biggest challenge. “We’ll liaise closely with our chemistry colleagues to work out the best route to these by-products. The preferred route is synthesis, because it’s quicker – but not everything can be synthesised, and even if it can, it usually results in an impure mixture. So we’re often asked to extract the pure by-product from these mixtures, or from extracts or washes obtained from the original synthesis of the active ingredient”.
The value of ultra-fast preparative LC
Louise and her team usually like to tackle these purifi cations through a two-stage approach: “We’ve found it works best if we do a ‘rough and ready’ large-scale purifi cation of the supplied material, for example to enrich it from 0.1% up to 5%, perhaps up to 20% if we’re lucky. We then follow this with a ‘polishing’ step to bring it up to the required purity, which is usually 90% or higher”.
This fi rst step is the most challenging, Louise explains, because it involves dealing with such large volumes of material. “To take one example, we were working with an insecticide by-product that was a highly volatile acid, supplied at 0.3–0.4% in acetic acid solution that also contained aliphatic components and salts. The request was for 800 mg, which we knew at the outset was going to be tough”, she says.
Their initial approach was to run the washings through an HPLC and collect/combine all the fractions. This was far from ideal, requiring 1000 injections with a 22 minute run time, giving litres of extracts to deal with. Not only that, but subsequent processing was also diffi cult. Lyophilisation didn’t work because the target compound was too volatile, while the alternative – extraction of
Louise Bacon (centre), with her colleagues Cyprien Bone (left) and Lorraine Ezra (right) in the Preparative and Isolation Chemistry Team at Syngenta.
the acetonitrile/water fractions from HPLC with dichloromethane (DCM) – gave mixtures that were diffi cult to evaporate down without losing material. Louise explains the upshot: “All this consumed 5 months of the team’s time, and to add insult to injury, the fi nal material was only 55% pure!”.
So when the request for a further 4 grams of the target compound came in, Louise knew they needed another, more automated approach: “The HPLC route was scarcely feasible at the smaller scale, and so obtaining more material the same way was clearly going to be impossible. But fortunately at about this time, in the middle of 2021, an opportunity came up to acquire a Shimadzu UFPLC instrument. We immediately saw how it could help us, and jumped at the opportunity!”.
Shimadzu’s Ultra-Fast Preparative LC (UFPLC) works by automatically heart-cutting the fraction containing the target compound, and trapping it on a short column, rather than leaving it to elute. Louise’s team realised that this would enable them to backfl ush off their target compound in acetonitrile, dispensing with the need for DCM extraction, and leaving them with a solution from which the acetonitrile could be gently removed without problems.
Following some work to optimise the column to avoid co-elution of the analyte with a ‘hump’ of unwanted material, the team fi nally found success, says Louise: “From 5 litres of washings, we obtained well over a gram of material, and the purity of the product was better than we’d achieved before. But more than that, we’d avoided all the manual handling, eliminated the problems with drying-down, and reduced the processing time to a matter of weeks”.
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