Stop Flow in action: An alternative to fraction collection
12/08/2024
At a glance
> Fera Science, a research organisation for environmental management and agriculture, is using the Beta-RAM radio-HPLC flow detector with a Stop Flow valve for environmental fate studies.
> Stop Flow holds the HPLC under pressure whilst the eluate flow is paused temporarily to increase the residence time of analytes in the flow cell, allowing users to define a fraction time and counting time.
> In this case study, Senior Chemist Phil Lamond explains how it has improved fraction collection by simultaneously speeding up the process and reducing common errors.
Research for environmental management, conservation, and agriculture
Senior Chemist Phil Lamond (inset) is using the Beta-RAM in studies for Environmental Risk Assessments at Fera's Sand Hutton site on the York Biotech Campus.
LabLogic’s Beta-RAM is being used by Fera Science for eFate studies. Located on the York Biotech Campus, it is a joint public-private research organisation for environmental management, conservation, and agriculture that adheres to guidelines defined by the Organisation for Economic Co-operation and Development (OECD) for the testing of chemicals in soil, sediment, and water. Fera performs Environmental Risk Assessments (ERA) for companies introducing new chemicals such as pesticides and biocides. Compounds are radiolabelled with 14C or 3H and degradation profiles mapped out over predetermined timepoints using chromatography, with studies lasting up to 90 and 220 days.
The Beta-RAM’s innovative Stop Flow measurement protocol increases the residence time of the analytes in the Beta-RAM’s flow cell, taking a snapshot of the sample at regular intervals. This increases the sensitivity of the radioactivity detector and improves the signal-to-noise ratio. LabLogic's HPLC system, the Logi-CHROM, fully supports Stop Flow functionality and is stackable with the Beta-RAM 6.
Maintaining peak resolution
Senior Chemist Phil Lamond has praised the Stop Flow’s value in fraction collection for eFate studies, saying, “The bottleneck in our work is the duration of the study and labour-intensive sample preparation rather than the analysis time, so we can spend more time on improving the sensitivity of the peaks of interest instead of being concerned about sample throughput. In order to the maximise the signal, the Stop Flow process stops the HPLC system and holds it under pressure. This provides a longer residence time for more radio-decay to occur in the detector, thereby increasing the peak sensitivity. It is essential to maintain peak resolution: if the pressure of the system was to reduce, the peaks would disperse and merge together.”
A close up of the Stop Flow valve inside the Beta-RAM.
“… Stop Flow is the best way to monitor low-level radioactivity.”
Being an experienced analyst, Phil has used other methods of fraction collection before. “I think Stop Flow is the best way to monitor low-level radioactivity. I have used other systems such as TopCount and Fraction Collection with off-line LSC analysis, but those techniques are always multi-step procedures and some of the techniques used can cause photo or thermal degradation to the compounds of interest. With multi-step procedures, there is also the potential to mix up samples from different studies during the different stages.
“Because the Stop Flow process all happens on the HPLC, there is no chance of sample mix-up. Whatever you put into the tray then comes out of the detector; there is no second step, no degradation, no evaporation, no volatility, or any of those common issues with fraction collection. It’s all contained within the one analysis and that’s the beauty of it.”
Reducing a 3-day methodology to 1
“Using the Beta-RAM with Stop Flow has been essential to us. In previous research positions, I’ve tried other methods of quantifying, but there just isn’t a comparison to what the Stop Flow can achieve. It really impressed me when I first came to Fera and seeing it in action made me understand the value of it. Fraction collection is a very labour-intensive process with limited automation and before Stop Flow, it could take up to three days to obtain the results. When using Stop Flow, you also eliminate the additional time it would take to train analysts on the different instruments involved in the multi-step procedures.
“Using the Beta-RAM with Stop Flow and Laura software for chromatography analysis provides us with a GLP-compliant system; we only need to validate one instrument and one piece of software and that’s it, box ticked. With Stop Flow, we’ve got one HPLC collecting samples overnight, and we have the answer the very next morning. We’ve gained two days instantly compared to not using it, and that isn’t factoring the training of staff and calibration and maintenance of instruments in using the traditional method, so it is a significant benefit.”
Find out more
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