(M1430-01-02) Approaches for Reducing the Environmental Impact and Increasing the Throughput of Bioanalytical LC Separations

Purpose: Many analytical laboratories are increasingly focussing on approaches to reduce their environmental footprint. This is particularly important in high-throughput bioanalytical environments, where the large number of samples analysed results in significant solvent usage and waste disposal, detrimental to both the environment and running costs. Significant reductions in solvent consumption and waste generated are possible for many existing analytical LC methods through quantitative translation of the methodology to modern column technology (e.g. narrower bore / smaller format). This has a demonstrable impact on the electrical consumption per analysis, whilst providing the added benefit of improved laboratory efficiency and reduced running costs. Accurate volumetric scaling of the methodology is essential to ensure that the efficiency and selectivity of the original method is correctly preserved, thus ensuring data quality is not compromised. Additionally, this approach can also be used to enhance sensitivity for sample limited low detection level assays. Often, such savings are considered in terms of migrating methods to UHPLC, however, substantial gains can also be achieved using existing HPLC equipment. This poster explores the possibilities outlined above and demonstrates the potential gains that can be achieved for isocratic and gradient LC methods. Finally, practical considerations, such as the impact of system dispersion on data quality when moving methods to narrower bore columns, are assessed.

Methods: Example isocratic and gradient LC methods developed using “legacy” HPLC methods (i.e. 150 x 4.6 mm, 5 µm columns and 400 bar HPC instruments) were quantitatively translated using first principle calculations to modern, small format columns (50 x 3.0 mm, 1.7 µm and 100 x 3.0 mm, 3 µm), installed on both existing HPLC equipment and UHPLC systems. The existing and translated methods were run to demonstrate reproduction of the original chromatographic separations, whilst solvent use and electrical consumption recorded. A tryptic digest sample was used to demonstrate the application of this approach to bioanalytical workflows and how analytical sensitivity can be increased. The impact of extra column dead volume on small format columns was assessed through theoretical modelling, whilst the impact of data acquisition and system dwell volume were practically demonstrated.

Results: For isocratic and gradient LC methods, substantial improvements were readily achieved when translating an existing HPLC separation to UHPLC. Up to 86.7% reduction in solvent use, 86.2% reduction in electrical consumption and 89.3% reduction in run time were achieved, whilst preserving the original separations. It was also demonstrated that by selecting a column format to better utilise existing equipment, substantial improvements can be realised. For a gradient method, 71.9% reduction in solvent use and 57.3% reduction in electrical consumption and 60.4% reduction in analysis time was easily achieved on a standard 400 bar HPLC system. For the tryptic digest sample, reductions in solvent consumption of 58% and 80% were obtained by translating the method from a 4.6 mm ID column to 3.0 mm and 2.1 mm ID columns respectively. The separation was well preserved, with only a minor (5.7%) decrease in peak capacity observed for the 2.1 mm ID column. The impact of injection volume scaling was then investigated, with a 4.2 fold increase in sensitivity obtained on the 2.1 mm ID column, albeit with a further decrease in peak capacity of 11.3%. Finally, practical aspects that can impact data quality, including system dispersion, dwell volume and data acquisition rates are investigated and presented.

Conclusion: Significant savings in the amount of solvent used, waste generated and electricity consumed by bioanalytical LC methods can be readily achieved through quantitative volumetric scaling of LC methods to more modern column formats, thus reducing the environmental footprint of the analysis. The savings achievable are demonstrated using both modern UHPLC equipment and HPLC systems. The approaches highlighted are highly pertinent to laboratories seeking to reduce the environmental impact of their activities.