Sustainable Practices in HPLC: The Role of Miniaturization

High-performance liquid chromatography (HPLC) is an analytical chemistry technique used to separate liquid mixtures of compounds or analytes. The method employs a liquid "mobile phase" to transport samples through a permeable, solid "stationary phase" within a chromatography column. Based on their physicochemical properties, such as polarity, size, and charge, analytes in a sample will interact differently with these phases, leading to distinct retention times that enable separation and subsequent identification or purification.

Despite its invaluable role in analytical science, it is important to acknowledge that the environmental impact of HPLC is not insignificant, largely due to its energy consumption and solvent waste generation.

In recent years, sustainability has become a pressing concern, prompting analytical chemists to evaluate and modify their methods in alignment with the 12 principles of green chemistry, without detrimentally affecting data quality.¹ Moreover, increased sample throughput can often serendipitously reduce the environmental impact of a method, hence better eco-efficiency. This article explores the concept of miniaturization in HPLC, highlighting its role in reducing waste, energy use and improving the overall environmental footprint of chromatography.

The importance of miniaturization in HPLC

Miniaturization in HPLC refers to reducing the scale of various aspects of the method, including solvent usage, instrumentation, HPLC columns, method duration and waste production. Given that many common HPLC mobile phase solvents are derived from non-renewable feedstocks, hazardous in nature, energy-intensive to manufacture and generate greenhouse gas emissions at disposal, decreasing their consumption can significantly improve sustainability. Additionally, reducing analysis time not only decreases solvent use but also minimizes the energy required to run the instrument (for temperature control, degassing, detection, etc). Switching an instrument off, as soon as it is feasible to do so, is also an easy energy-saving approach — since instruments in “standby” mode often consume surprisingly large amounts of energy.

Strategies for miniaturization in HPLC

There are multiple ways miniaturization can be applied to HPLC. Below are just some examples of the application of miniaturization:

Using narrower internal diameter columns to decrease solvent consumption

A straightforward way to reduce solvent use is to use a column with a narrower internal diameter (ID), operated at a lower flow rate. This approach reduces solvent and sample volumes while maintaining peak capacity and selectivity.

For example, in our lab, scaling down a bovine serum albumin digestion method from a 4.6 mm ID column at 1.68 mL/min to a 3.0mm ID column at 0.714 mL/min, and further to a 2.1 mm ID column at 0.35 mL/min, resulting in a reduction of mobile phase volume by 57.5% and 79.2%, respectively, within the same timeframe.

It is worth noting that since sample volume contributes a significantly lower proportion of total HPLC waste (typically <1%), decreasing column ID with the same injection volume could likely be used to increase detection sensitivity, provided there is sufficient resolution of peaks in the separation.

Employing high-efficiency, short columns

By utilizing columns with smaller stationary phase particles, and a reduced column length, resolution and selectivity can be maintained, whilst reducing solvent consumption. For example, scaling an isocratic method from a 150 x 4.6 mm, 5 µm particle size column to a 100 x 3.0 mm, 3 µm column or a 50 x 3.0 mm, 1.7 µm column maintained similar separation performance while achieving substantial solvent savings of 71.6% and 85.7%, energy reductions of 56.8% and 85.1%, and run-time decreases of 60.2% and 88.5% respectively.

Notably, while this example does showcase some of the merits of ultra-high-performance liquid chromatography (UHPLC), it also demonstrates that significant sustainability improvements are achievable even with standard HPLC instrumentation.

Chromatographers must be aware of the inherent pressure increases associated with using columns with smaller particle sizes, since their instrumentation specifications may limit suitable column choices. Nevertheless, analysts can be reassured by the above example demonstrating that significant sustainability improvements are attainable for users of HPLC and higher-pressure UHPLC instruments alike.

Using superficially porous particles

Another way to enhance HPLC efficiency is by utilizing columns with superficially porous particles (SPPs). Unlike fully porous particles, SPPs offer lower permeability, allowing for faster separations with less solvent usage.

Our laboratory reduced both run time and solvent consumption by over 50% in a method for separating aspirin and related substances by switching from a fully porous to an SPP column of the same dimensions, stationary phase chemistry and particle size. The increased efficiency of SPP columns enables further reductions in column length, mobile phase, back pressure and energy consumption.

Adopting ultra-short column formats

Pairing high-efficiency stationary phases with the kinetic chromatographic benefits of ultra-short columns (e.g. 10 mm) can facilitate separations with narrower peaks and shorter retention times, thus decreasing analysis times and drastically reducing solvent consumption. Typical HPLC run times occur over several tens of minutes, although sub-minute methods are attainable with ultra-short column formats and appropriate detector scan rates. Modern detector scan rates are now sufficiently high, that even liquid chromatography-tandem mass spectrometry methods with peak coelution can offer adequate sensitivity when used in multiple reaction monitoring scanning mode.

In one example, we improved a 100 x 2.1 mm, 3 µm C18 gradient method for the separation of 25 per- and polyfluoroalkyl substances and related compounds. The original method had a 13.2-minute cycle time and consumed 5.3 mL of mobile phase per injection. By employing a 10 x 2.1 mm, 2 µm HTP-MS column with an alternative stationary phase selectivity, we reduced the cycle time by 88% (to 1.6 minutes) and mobile phase consumption by 70% (to 1.6 mL), demonstrating the potential for sustainability gains with ultra-short columns.

Limitations of widespread application of miniaturization for HPLC

There are some instances where miniaturization is not always feasible, for example in certain preparative and process scale setups requiring HPLC columns with high loadability, reductions in column dimensions would be counterproductive. However, smaller-scale analytical HPLC, where loadability is often less of a concern, is by far the more prevalent technique, so in most cases, miniaturization remains incredibly relevant.

The existence of a technology alone does not make it a viable option for every lab’s operating budget. This is showcased by the fact that plenty of analytical labs still run traditional 400-bar limited HPLC instruments as opposed to UHPLC systems capable of higher pressures (>1500 bar), owing to significant upfront costs (tens of thousands of USD). This is understandable but does lessen the extent to which end users can miniaturize their methods, compared to if systems with lower internal volumes and higher-pressure capabilities were at their disposal. It is worth noting, however, that modern UHPLC systems are becoming more energy-efficient than their outdated counterparts. Hence, when considered alongside the potential savings of high-purity solvents and higher throughput of miniaturized methods, this may encourage more organizations to purchase UHPLC systems on both financial and sustainability bases.

Despite UHPLC systems having advantages over their lower-pressure counterparts, this can lead to the misconception that miniaturization is not achievable on standard HPLC instruments. This is indeed not the case; significant reductions in solvent and energy consumption can often be achieved through method translation and instrument volume optimization on both HPLC and UHPLC systems. Analysts needn’t let their green aspirations be deterred by their pressure limits! Of course, there are some limitations to consider, such as that standard HPLC instruments aren’t suited to all miniaturized column dimensions (e.g. 2.1 mm ID columns in general), but with reasonable volume optimization to limit system dispersion, a “sweet-spot” between separation efficiency, resolution, sensitivity and solvent and energy consumption can still be found via utilization of 3.0 mm ID columns in such cases.

Embracing sustainable practices in HPLC

The push for sustainability in analytical chemistry necessitates reevaluating traditional HPLC methods and embracing miniaturization strategies. By employing smaller column dimensions, high-efficiency stationary phases, superficially porous particles, ultra-short columns and extending column longevity, laboratories can drastically cut down on the consumption of solvent, energy and raw materials. These improvements not only align with the principles of green chemistry but also enhance method throughput and efficiency.

The available technological advancements in HPLC make it clear that substantial sustainability improvements are within reach for both HPLC and UHPLC users. As awareness of environmental impact grows, chromatographers must continue to innovate and refine their methods, ensuring that analytical excellence goes hand in hand with responsible resource management.

Reference: 1. Anastas, P. T.; Warner, J. C. Green Chemistry: Theory and Practice. Oxford University Press. 1998.