Making HPLC More Sustainable

There is a lot we can do to optimize our methods.

Often, we are only aware of a fraction of the possibilities, as technical options are usually available only to those who have worked for several years with a specific instrument.

However, there are several actions we can take that are simple in nature and will greatly benefit not only the environmental impact but also the time, money, and sample required.

I want to give you an (almost) exhaustive list of opportunities I am aware of for optimizing your HPLC workflows.

I created this list because there are plenty of opportunities, but you should be able to review it quickly to identify where you might find unused potential in your lab:

I compiled the following:

  • Technical Components
  • Column and Gradient Selection
  • Alternative Eluents
  • Approach Optimization
  • Solvent Recycling

I’ll start with Technical Components & Set-up, because I think these are low-hanging fruit: the practices are straightforward, and add-ons can be quickly purchased and installed—even by non-experts.

Before we start, big shout out to Michael Meyer who significantly helped with collecting these ideas!

Technical Components & Setup for a More Sustainable HPLC

Let us start with some technical tips and gadgets that will greatly benefit your work, health and the environment:

SCAT filter units for eluent and waste bottles are an effective way to improve both safety and sustainability in the lab.

  • Special GL45 screw caps with capillary feedthroughs and secure fittings prevent solvent evaporation and reduce exposure risks.
  • Patented filters include a time indicator for replacement intervals, helping you change filters only when needed — avoiding unnecessary waste while maintaining safety.
  • The filter’s valve design ensures no impact on system pressure.
  • Filters are available in sizes tailored to all common bottle volumes.

Why it matters: Reduced solvent evaporation lowers VOC emissions and solvent waste, and using the right size filter minimizes unnecessary material use.

Protecting the Column and Reducing Waste

Inline filter frits for the eluent system protect the column from particles and contamination, extending its lifetime and reducing the need for costly replacements.

  • Single-use frits are available, but reusable frits made of steel mesh or titanium can be cleaned using ultrasonic baths — a more sustainable choice that reduces solid waste.

Why it matters: Column replacements are expensive and resource-intensive to manufacture. Protecting them saves money and reduces environmental impact.

Minimizing Dead Volume with Smart Capillaries

Dead-volume-free capillary connections (e.g., Thermo Viper / nanoViper) significantly improve efficiency and sustainability.

  • Enable fast, reproducible gradients, reducing run times and solvent use.
  • Fingertight sealing holds at pressures above 1000 bar, removing the need for constant screwing, re-tightening, and troubleshooting leaks.
  • Universal fitting design works with any column head or detector port, sealing via a PTFE ring at the capillary tip.
  • Unlike PEEK capillaries, these can be used without restriction for aggressive solvents such as THF.
  • Only defined lengths are available — ensuring consistent method performance and avoiding revalidation when replacing capillaries.

Why it matters: Lower dead volume and reproducible fittings improve separation quality, save solvent, and reduce the number of re-runs caused by leaks or poor connections.

Preparing the System Before Runs

Proper preparation reduces errors, prevents re-runs, and extends the lifetime of expensive components — saving time, money, energy, and solvents.

  • Degas and filter mobile phases where necessary.
    Air bubbles in the solvent lines can lead to automatic system shutdowns or measurement errors. Filtration removes particles that might damage the pump or column.
  • Purge all solvent channels daily, even if using the same mobile phase, to remove air via the bypass.
    Neglecting this can cause pressure fluctuations, baseline noise, or system failure during runs.
    However, purging all channels daily may be excessive if certain channels are not in use — could waste solvent unnecessarily.
  • Replace flushing solvents daily to prevent microbial growth.
    Biofilm buildup can cause carryover and blockages, leading to time-consuming cleaning or costly capillary replacements. Of note, this is especially important for aqueous solvents, but purely organic phases like acetonitrile will be somewhat less vulnerable.
  • Use guard columns (pre-columns) to trap contaminants from samples.
    A guard column costs ~€50, while an analytical column can range from €500–€3000 — a clear case of prevention being cheaper than cure.
  • Ensure proper column equilibration with the mobile phase before starting sequences.
    Insufficient equilibration reduces reproducibility, potentially requiring re-runs (and wasted solvent, time, and energy).
  • Follow calibration cycles for target compounds and confirm validity with standards (e.g., peak area calibration). This avoids both re-runs and the much worse risk of reporting incorrect results — which can waste entire project weeks.

System Care After Sequences

Post-run maintenance protects both the column and the instrument, reducing the frequency of repairs and replacements.

  • After using acidic, basic, or high-salt mobile phases, flush both the column and the system with appropriate solvents before shutdown.
  • Automate detector shutoff when no immediate follow-up runs are planned.
    This saves lamp hours (extending their lifespan) and reduces overall energy consumption.

Adapting Methods to Your Column Dimensions

When using a column with different dimensions from the one specified in a method (e.g., from a paper), adjustments are essential to maintain performance.

  • Tools like the Restek Pro EZLC Method Translator allow you to enter both the original and your target column specifications (dimensions, particle sizes, injection volumes, gradients).
  • The tool then calculates a reliable starting point for method development — reducing trial-and-error work and saving substantial time and resources.

Modern Column Selection and Method Adaptation

Why Column Choice Matters for Sustainability

Liquid chromatography consumes significant amounts of solvents, particularly methanol and acetonitrile — globally estimated at over 150,000 tons per year. Producing and disposing of these solvents has a considerable carbon footprint. Even small efficiency improvements can significantly reduce waste when multiplied across thousands of runs.

One of the most direct ways to reduce HPLC’s environmental impact is by reducing column internal diameter (i.d.) and optimizing method parameters accordingly. This cuts mobile phase use, lowers waste, and can even improve sensitivity.

Narrow-Bore Columns: Small Changes, Big Savings

  • 4.6 mm i.d. → 3.0 mm i.d.
    ~60% solvent reduction per injection.
  • 4.6 mm i.d. → 2.1 mm i.d.
    Up to 80% solvent reduction, ideal for LC–MS where low flow rates improve ionization efficiency.

Advantages:

  • Lower flow rates = less solvent consumed and disposed of.
  • Reduced stationary phase and packing material in manufacturing.
  • Potential increase in sensitivity for sample-limited assays.

Considerations:

  • 2.1 mm columns are more sensitive to extra-column dispersion; best used with low-dispersion systems.
  • 3.0 mm columns are more forgiving and widely compatible with standard LC setups.

Translating Methods to Smaller Columns

When switching to a smaller i.d., you must scale flow rates and injection volumes to maintain performance:

  • Flow rate: Scale proportionally to the square of the column radius to keep the same linear velocity.
  • Injection volume: Scale to match the reduced column volume — unless a boost in sensitivity is desired.

Practical tip: Use free online method translation tools (e.g., Agilent Method Translator, Restek Pro EZLC) to calculate correct flow rates, gradient times, and injection volumes for your new column size.

Reducing column length (L) while maintaining particle size lowers analyte retention time and shortens the run. Halving the column length can cut mobile phase consumption by ~50%. If the particle size (dP) is also halved (keeping L/dP constant), the same separation efficiency can be maintained — meaning you save time and solvent without losing performance.

Benefits include:

  • Shorter equilibration and re-equilibration times.
  • Reduced instrument energy use (less run time).
  • Faster turnaround for results and higher sample throughput.

Translating Legacy Methods to Modern Formats

Many older methods still use 250–150 mm, 4.6 mm i.d. columns packed with >5 μm particles. These can often be translated to shorter columns with smaller particles while keeping the same stationary phase chemistry to preserve selectivity and peak spacing.

Example solvent savings:

  • Translating an isocratic method from a 250 × 4.6 mm, 5 μm column to a 50 × 3.0 mm, 1.7 μm UHPLC column → 85.7% less solvent per injection.
  • Translating the same method to a 100 × 3.0 mm, 3 μm HPLC column (400 bar system) → 71.6% less solvent without upgrading to UHPLC.

Energy savings:

  • Optimized HPLC method: 56.8% less energy.
  • UHPLC method: 85.1% less energy (higher efficiency at elevated flow rates).

Very Short Format Columns

For some applications, excess separation efficiency can be traded for speed and solvent savings.

  • 20–30 mm UHPLC columns can dramatically cut run times and solvent use.
  • Changing stationary phase chemistry (e.g., from C18 to C18-PFP) can improve resolution enough to allow shorter columns without losing separation quality.

Example: Translating a 150 × 4.6 mm, 5 μm C18 method to a 30 × 2.1 mm, 1.7 μm C18-PFP column gave better resolution than the original method while greatly reducing solvent use.

Ultra-Short Cartridges for Targeted Analysis

With LC–MS, full separation isn’t always needed — as long as analytes are retained long enough to separate from interferences.

  • 10 mm cartridge columns can reduce analysis time by 88% and solvent use by 70%.
  • Efficiency drops (e.g., 6.7× lower than a 100 × 2.1 mm column), so structural isomers may not resolve — but for screening or preselection, they can be a powerful sustainability tool.

Practical Tips for Implementing Shorter Columns

  • Keep the same stationary phase chemistry when translating methods to avoid selectivity shifts.
  • Use method translation calculators to adjust flow rates, gradients, and injection volumes correctly.
  • Consider hybrid strategies: use short columns for prescreening, reserve full methods for confirmatory analysis.

Choosing the Right Column for the Job

The right column choice can dramatically improve both efficiency and sustainability. Still:

  • Standard lab setups often use 75–100 mm length columns with 2.1 mm internal diameter for general work.
  • For specific applications, such as broad-range fermentation analysis, longer polymer-based columns (e.g., 300 mm × 7.8 mm) may be better — allowing full analyte separation in a single injection.
    This reduces the need for multiple runs, saving solvents and instrument time.

Tip: Always know the minimum column specifications required for your analytes. Consult:

  • Experienced colleagues or application specialists from reputable suppliers (e.g., Phenomenex, Waters, Macherey-Nagel).
  • Company representatives often visit research facilities monthly — arranging short on-site consultations can be very productive.

Some suppliers provide online column search tools based on analytes; in the past, mobile app solutions were discussed, though availability should be verified before relying on them.

Solid Core Particles

Solid core (superficially porous) particles improve kinetic performance compared to fully porous silica.

Key benefits:

  • Higher efficiency: A solid core column with the same dimensions and particle size as a fully porous column can nearly double separation efficiency.
  • Faster runs: Reduced surface area and permeability lead to lower retention times — in one example, run time dropped by 50%, cutting solvent and energy use in half.
  • Improved sensitivity for impurities due to sharper peaks.
  • Similar back pressure to fully porous columns — compatible with conventional HPLC systems.

Practical tip: The extra efficiency can be traded for sustainability by using shorter solid core columns (50–75 mm), further reducing run time, solvent use, and energy consumption without sacrificing resolution.

UHPLC and Particle Size Advantages

Modern UHPLC systems (>1,500 bar) support smaller sub-2 μm fully porous particles or superficially porous particles in narrow-bore columns. These allow:

  • Higher efficiency separations in less time.
  • Shorter gradients without loss of resolution.
  • Lower mobile phase consumption.

Note: While UHPLC hardware is optimized for low dispersion, the principles of narrow-bore column scaling also apply to conventional HPLC.

Shorter and Smarter Gradients

Most HPLC users stick with the gradient they first developed — or copied from a paper — without questioning if it’s the most efficient. But gradients are one of the biggest levers for sustainability: shorten them, optimize them, and you save solvent, energy, and time every single run.

Why gradients matter

A gradient isn’t just a ramp of mobile phase composition. It determines:

  • How long your run takes (and therefore how much solvent is consumed).
  • How well compounds are separated (resolution vs. speed trade-off).
  • How much equilibration is required before the next injection.

Unoptimized gradients often mean unnecessary “flat” baseline time, overlong separation windows, or excessive re-equilibration — all of which waste solvents and hours.

What shorter gradients can achieve

Scientific studies show that halving run times by adjusting gradient slopes can save 40–60% mobile phase per injection without losing separation quality. For example:

  • Switching from a 30-minute gradient to a 15-minute gradient with optimized slope often maintains resolution but halves solvent consumption.
  • Reducing post-gradient re-equilibration from 10 minutes to 3 minutes can save even more over hundreds of runs.

In practice, this means one lab running 50 injections a week could save multiple liters of acetonitrile per month — simply by trimming dead time.

2 Tips to optimize gradients

  1. Remove unnecessary plateaus
    Look at your chromatogram: are there long stretches where nothing elutes? Those are prime candidates for trimming.
  2. Steepen the slope strategically
    Instead of a slow, linear gradient, use segmented ramps: shallow slopes where resolution is critical, steeper slopes where analytes elute cleanly apart.

A note on regulations vs. flexibility

If you’re working under pharmacopeial methods (e.g., in pharma QC), gradient changes aren’t always allowed — regulatory frameworks often require methods to be followed exactly, even if they’re almost obsolete and wasteful. In academic or research settings, however, you usually have much more freedom. Don’t be afraid to question inherited methods: trimming gradients or re-optimizing equilibration can save solvents and time without breaking any rules.

UHPLC can make a difference Modern UHPLC systems (ultra-high pressure LC) allow the use of smaller particles and shorter columns while maintaining separation efficiency. This means gradients that once took 30–40 minutes on a 5 µm, 250 mm column can often be completed in 10–15 minutes on a 50–100 mm, sub-2 µm column — with equivalent or even better resolution, and a fraction of the solvent use.

Flow Rate Optimization

UV Detection and Flow Rate: Practical Implications

  • UV detectors are concentration-sensitive: peak height depends on analyte concentration, while peak area reflects the total amount passing through the detector.
  • Lower flow rates increase the residence time of analytes in the detector cell, which can increase peak area without changing the amount injected.
  • Peak height changes with flow rate in more complex ways, but for small adjustments, the impact on resolution is minimal.

Sustainability takeaway:
Reducing flow rates without increasing total rune time decreases solvent use and can increase sensitivity for certain analyses, potentially allowing you to inject smaller sample volumes and shorten gradients.

Greener Alternatives to Conventional HPLC Eluents

Switching solvents is one of the most direct ways to make HPLC more sustainable – but it’s also one of the most complex. The choice of organic modifier in a mobile phase affects everything: separation efficiency, system back pressure, detector compatibility, method robustness, and even instrument wear.

For decades, acetonitrile (ACN) has been the default choice but greener alternatives do exist — from bio-based alcohols like ethanol to emerging solvents like ethyl lactate and Cyrene. While every substitution has to be well considered, if established they are much greener and in several cases even improved process and data quality. Moreover, incidents like the “Acetonitrile Crisis” showed how a bottleneck in ACN supply can limit research processes and drive prices significantly higher.

Therefore, the sections below outline realistic solvent alternatives, their pros and cons, and practical tips for implementation – so you can make informed changes without sacrificing chromatographic performance.

Why Focus on Acetonitrile (ACN) Reduction or Replacement?

Acetonitrile (ACN) is one of the most widely used organic solvents in reverse-phase HPLC due to its:

  • Low viscosity → lower back pressure.
  • Low UV cut-off (~190 nm) → ideal for UV detection.
  • Strong elution strength in reversed-phase separations.

However, ACN has drawbacks:

  • Derived mainly from petrochemical sources, with fluctuating supply and high production footprint.
  • Toxic to aquatic life and requires controlled waste disposal.
  • Supply shortages (as in 2008–2009) can disrupt laboratory workflows.

Reducing or replacing ACN can therefore lower environmental impact, improve laboratory resilience, and sometimes reduce costs. Below are realistic, technically tested alternatives.

Ethanol (EtOH)

Pros:

  • Lower toxicity than ACN and MeOH.
  • Easier disposal, biodegradable.
  • Compatible with both UV and MS detection for many applications.

Cons & considerations:

  • Higher viscosity → higher back pressure, especially in aqueous mixtures.
  • Higher UV cut-off (~210 nm) → potential noise increase and reduced sensitivity at low wavelengths.
  • May require increased column temperature to reduce viscosity and improve performance.

Practical tips:

  • Use columns packed with superficially porous particles to offset high back pressure.
  • Pre-heat column or mobile phase to 40–60 °C to lower viscosity.
  • Works well for analytes soluble in ethanol — in some cases, analytes can be extracted directly into ethanol (e.g., UV filters from cosmetics).

Propylene Carbonate (PC)

Pros:

  • Polar aprotic solvent, potential ACN replacement in RP-LC.
  • Commercially available in high purity at reasonable cost.
  • Produced via relatively green synthesis routes.

Cons & considerations:

  • Not fully miscible with water → requires ternary mobile phases (often PC + EtOH + water).
  • Higher viscosity and density → increased back pressure.
  • Higher boiling point and lower vapor pressure → may reduce MS sensitivity.

Ethyl Lactate

Pros:

  • Biodegradable, non-toxic, food additive status.
  • Fully miscible with water and many organic solvents.
  • Low cost and renewable origin.

Cons & considerations:

  • Not stable under strong acidic or alkaline conditions.
  • Higher UV cut-off than ACN → not ideal for UV detection at short wavelengths.
  • Not widely available in HPLC-grade purity.

Example:
A mobile phase of 87% water, 10% ethyl lactate, 3% acetic acid achieved baseline separation of three pharmaceuticals on a C18 column at 60 °C in under 3 minutes.

100% Aqueous Mobile Phases

Pros:

  • Non-toxic, safe, no disposal concerns.
  • No UV absorbance above ~190 nm → excellent for UV detection.

Cons & considerations:

  • Low elution strength at room temperature for non-polar compounds.
  • Risk of phase collapse/dewetting with standard C8/C18 columns — use polar end-capped or polar-embedded stationary phases.
  • Often requires elevated temperature to improve elution strength.

Cyrene

Pros:

  • Produced from renewable biomass (sawdust).
  • Biodegradable in <28 days.
  • Low toxicity, high flash point, safe handling.

Cons & considerations:

  • Immiscible with water — requires ethanol or another co-solvent.
  • High viscosity and boiling point (227 °C) → high back pressure, possible MS limitations.
  • UV detection below 350 nm problematic due to background noise.
  • Not currently available in HPLC-grade.

Example:
In RP-HPLC, ternary mixtures of Cyrene + ethanol + buffer allowed separation of metronidazole and moxifloxacin at 50 °C on a monolithic column with acceptable back pressure (~130 bar).

Practical Strategies for Using Alternative Eluents

  1. Start with partial replacement (e.g., replacing 20–30% ACN with EtOH) to evaluate performance.
  2. Adjust temperature to manage viscosity-related back pressure.
  3. Use compatible columns — superficially porous or monolithic formats often handle viscous solvents better.
  4. Validate sensitivity impact — higher UV cut-off solvents may require detection at higher wavelengths or MS-based detection.
  5. Test method robustness — alternatives may affect retention time stability under small variations in pH, temperature, or composition.

Smarter Method Development

  • Dry-run gradient simulation: If your software allows, simulate gradients before running them to spot obvious inefficiencies.
  • Start isocratic where possible:
  • Tip: Start Isocratic Where Possible
    • If your separation doesn’t need a gradient, isocratic runs are almost always more sustainable.
    • Lower solvent use: Composition stays constant, so you avoid the long high-organic washes and re-equilibration phases that gradient methods require.
    • Shorter equilibration: Switching from one run to the next is faster because the column is already at the correct mobile phase composition.
    • Easier solvent recycling: Isocratic methods make post-detector recycling straightforward.
    • Practical check: If your analytes elute within a narrow retention window in a gradient, try an isocratic method with that composition — you may get equivalent resolution in less time, using less solvent.
  • Short equilibration: Validate the shortest possible re-equilibration time — many default methods leave unnecessary idle times between runs.

Optimizing Sample Preparation

When people think about making HPLC greener, they usually start with the instrument — smaller columns, alternative solvents, clever fittings. But sample preparation is often the hidden giant in both time consumption and environmental footprint. It’s easy to overlook because it feels like “the routine bit before the actual analysis.” Yet, if you optimise your prep, you can save hours of work each week and cut waste dramatically.

Why optimising it matters:

  • Sustainability: Less solvent, fewer consumables, and smaller hazardous waste volumes.
  • Time savings: Trimming just 2 minutes per sample in a 100-sample sequence means over 3 hours gained — enough to run an extra sequence or wrap up earlier.
  • Data quality: Cleaner, more consistent samples mean fewer failed runs and less rework.

Practical changes that deliver:

  • Miniaturise volumes: Use micro-extraction techniques (e.g., SPME), smaller vials, and scaled-down filters.
  • Direct injection: If your matrix is clean enough, skip concentration/dilution steps.
  • Batch preparation: Prep all daily samples at once to avoid repeated purging, solvent refills, and cleaning cycles.
  • Greener solvents: Swap chlorinated or high-toxicity solvents for ethanol or ethyl acetate when possible.
  • Smarter filtration: Pre-centrifuge to avoid filter clogging; filter only when necessary.

Using Combination Method

If you plan to use an HPLC–MS (LC–MS) approach, there are often opportunities for savings that go beyond just getting great data. LC–MS setups are already among the most sensitive analytical systems available — often 10–1000× more sensitive than UV detection — which means you don’t always need the long columns, high flow rates, or large sample volumes that standard HPLC methods rely on.

Why This Matters for Sustainability

  • Smaller sample amounts: Lower detection limits mean you can inject far less sample — good for precious, hard-to-make, or hazardous materials.
  • Shorter columns and runs: Since MS can identify compounds by their mass, you don’t always need full baseline separation, so you can shorten gradients or use smaller formats.
  • Less solvent waste: Lower flow rates directly reduce mobile phase use and disposal volumes.

Capillary HPLC for LC–MS

Capillary HPLC reduces column internal diameter to 100–500 μm, running at 0.4–100 μL/min instead of the 1–2 mL/min of a typical analytical column.

In practice, this means:

  • Over 90% less solvent per run.
  • Sharper peaks for the same mass injected — giving better sensitivity without extra sample prep.
  • The ability to run dozens of injections before you’ve even used as much mobile phase as a single standard HPLC run.

Solvent Recycling: Turning Waste Back into Usable Mobile Phase

In isocratic HPLC, the mobile phase composition stays constant throughout the run. This means that the solvent exiting the detector is identical in composition to the one entering the column — apart from minor changes due to analyte and impurity carryover.

This makes it straightforward to collect the “clean” post-detector solvent and feed it back into the mobile phase reservoir after appropriate filtration and (if necessary) degassing.

In gradient elution, however, the mobile phase composition changes over time, so the effluent is constantly varying in proportion of organic and aqueous components. Recycling would require real-time composition matching — impractical in most lab setups and potentially risky for method reproducibility.

Solvent Recycling Systems

Basic principle of solvent recycling can summarized as: divert the post-detector solvent to a collection system.

There are two main approaches:

A. Manual Recycling (No Computer Support)

  • Effluent is collected in a clean container during known “blank” parts of the chromatogram — typically before the analyte of interest elutes.
  • This requires knowledge of retention times and precise timing of collection to avoid contamination.
  • Solvent is then filtered (0.2 μm or finer) and reintroduced to the mobile phase reservoir.

Pros:

  • Low cost, no specialized equipment.
  • Easy to implement for single-analyte methods with stable retention times.

Cons:

  • Requires careful manual operation.
  • Not suitable for complex mixtures with unpredictable peaks.

Automated Recycling (Computer-Controlled)

  • Integrated with the HPLC’s data system or an external controller.
  • The detector signal triggers a divert valve that sends the flow either to waste (when peaks are eluting) or to the recycling reservoir (during baseline).
  • Many systems allow the user to set a threshold absorbance value — when the signal drops below this, solvent is automatically routed for recycling.

Pros:

  • Reduces human error and contamination risk.
  • Ideal for high-throughput labs running the same method repeatedly.
  • Can recover a significant percentage (often >80%) of mobile phase for reuse.

Cons:

  • Requires investment in hardware and software integration.
  • Still best suited for simple isocratic methods with stable baselines.

Practical Considerations for Safe Recycling

  • Filter carefully: Use high-quality solvent-resistant filters (PTFE or PVDF) to avoid introducing particulates back into the system.
  • Monitor solvent quality: Over multiple cycles, trace impurities can accumulate — periodically replace all solvent to maintain method performance.
  • Use compatible containers: Ensure collection and storage vessels are chemically resistant to your mobile phase.
  • Degas before reuse: Prevent bubble formation in pumps and detectors.