Chromatographic Strategies for Nanoplastic Analysis: Bridging the Gaps Beyond FT-IR and Raman

Conventional spectroscopy methods like Fourier transform infrared (FT-IR) and Raman lack the resolution to fully detect nanoplastics (<1000 nm), leaving major analytical gaps. Chromatographic approaches offer complementary solutions: pyrolysis–gas chromatography-mass spectrometry (py-GC-MS) enables polymer identification and quantification, while asymmetrical-flow field-flow fractionation (AF4), often coupled with multiangle light scattering (MALS), separates particles by size and provides structural information. When combined, AF4 and Py-GC-MS deliver two-dimensional insights—particle size distribution and chemical identity—making these techniques promising for environmental nanoplastic studies. Challenges remain in coupling workflows, overcoming low detection limits, and improving recovery in complex matrices, but innovations such as large-volume injection (LVI) and optimized carrier systems are expanding the potential of chromatography-driven nanoplastic analysis.

LCGC International spoke to Maria Hayder, corresponding author of a recently published paper (1) discussing a novel workflow for nanoplastic analysis in environmental water samples, incorporating AF4-MALS and Py-GC-MS in an offline combination.

What challenges arise when using conventional spectroscopy techniques like FTIR and Raman for detecting nanoplastics, and how can chromatographic methods help overcome them?

FT-IR and Raman are a state of the art for microplastic analysis, but they have too little size resolution for nanoplastics. FT-IR can be used down to 10-20 μm, Raman—down to a few hundred nm. Nanoplastics are particles smaller than 1000 nm, so neither of these can cover this size range fully. A search for appropriate size-measurement technique for nanoplastics is ongoing. Pyrolysis-GC-MS can be very useful for identification and mass-based quantification of polymers constituting nanoplastics but does not discern between the particle sizes. AF4 is a chromatography-related technique which separates particles based on their sizes but is not specific to particle chemistry. These two together could provide size- and polymer analysis of even the smallest nanoplastics.

Can you explain how asymmetrical-flow field-flow fractionation (AF4) separates particles and why it's suitable for nanoplastic analysis?

Instead of a chromatographic column, AF4 separation takes place in an empty channel, whose bottom part is a made of a semi-permeable membrane. While one carrier liquid flow brings particles towards the detector, a perpendicular flow, called crossflow, pushes them down, towards the membrane. Because of higher diffusion, the smaller the particle, the higher in the channel the further from the membrane it moves. Because the detector flow is a laminar flow, further from the membrane means faster flow. And so smaller particles elute faster, and larger—later.

AF4 working size rage is generally said to be 1 nm to 1000 nm, though it is not so straightforward. This is the size range which covers the definition of nanoplastics. The second advantage of AF4 in nanoplastic analysis is that the separation occurs based on size, so we can track the size distributions. When adding the MALS detector, which is often done for AF4, we can measure the radius of gyration. It is non-destructive, so we can collect the fractions and analyze them further. Moreover, AF4 is characterized by low shear forces, so it could allow us to mildly separate natural agglomerates without destroying them. Finally, the low-molecular-weight constituents of the matrix can be removed through the membrane during the separation, which cleans the sample, which we have observed in our experiments.

Why is pyrolysis-gas chromatography–mass spectrometry (Py-GC-MS) particularly effective for identifying and quantifying plastic polymers?

Plastic particles are lumps of organic compounds. It is therefore difficult to distinguish them from the organic matrix. During the pyrolysis step, the polymer is broken down into small fragments. These compounds can later be separated and identified by MS, and we can reconstruct which polymers were present in the sample. In theory, it should be quite straightforward to quantify the polymers. In practice there are, of course, problems. Especially for nanoplastics, where FT-IR and Raman are of limited (or no) use, Py-GC-MS has been so far one of the most promising ways for polymer quantification.

Describe the technical difficulties in coupling AF4 with Py-GC-MS for nanoplastic analysis and suggest how these might be addressed.

The first difficulty comes with the carrier liquid used in AF4. This is usually an aqueous solution of inorganic salt or surfactant. Py-GC-MS does not go well with involatiles, and (without certain adjustments) does not accept water. So, we tested a few volatile salts as a basis for AF4 carrier liquid and found one that works well (some other salts caused particle-membrane interactions in the AF4, which prevents separation) that can later be evaporated.

It would be, of course, handy to have the two instruments coupled on-line. I don’t think it’s technically impossible but quite challenging. Firstly, AF4 separation takes around an hour, while our Py-GC-MS method is about 30 minutes. It would be difficult to have a classical two-dimensional separation for more than two fractions then. Secondly, at the exit of the AF4 the analyte is largely diluted in the carrier liquid. Nanoplastics are generally present in low mass concentrations in environmental samples, which makes it difficult to reach the detection limits of our instrumentation. In this situation, further diluting the sample in my opinion doesn’t make sense. We would then have two either preconcentrate the sample (which takes time) or add an additional step between AF4 and Py-GC-MS to evaporate the water and so re-concentrate the analyte, which is what we did.

What role does multiangle light scattering (MALS) play in the AF4 workflow, and how does it enhance particle size characterization?

MALS is a detector placed behind AF4, whose function is to measure the radius of gyration of the particle. Although AF4 separates particles based on their sizes, the elution time may also depend on their charge, shape or interactions with the membrane. MALS measures the size independently, giving more reliable information. It’s not an ideal measurement either, though. In such a complex matrix as wastewater, the matrix may bias the measurements.

Discuss the importance of low detection limits and high sensitivity in chromatographic techniques for analyzing NPs in environmental matrices.How does large-volume injection (LVI) improve the detection of nanoplastics in AF4-based methods?

Nanoplastic concentrations in the environment are usually too low for currently available instrumentation (exactly how low is “low” we don’t know yet). There are different ways of preconcentration mentioned in the literature, but they add an additional step of work and increase losses. Without some preconcentration, though, it is doubtful we could find any nanoplastics in environmental samples with either AF4 or Py-GC-MS. LVI allows us to analyze 10 mL of the sample in one AF4 run. The particles are then preconcentrated in-line, so that we can reach the detection limits of our detectors and Py-GC-MS, which wasn’t possible with the conventional microliter injection.

What are the key differences between microplastics and nanoplastics in terms of chromatographic detection and separation?

For Py-GC-MS (or any other destructive mass concentration analysis), the difference lies rather in the sample preparation, because the mass-based approach is size-independent and so the same for micro- and nanoplastics. The differences are more pronounced when we try to measure the size (or number) of particles. Then, for nanoplastics AF4 can be used and has been suggested in the literature more often than other types of chromatography. For microplastics, which are particles ranging from micrometer to as much as 5 mm, chromatographic techniques are not popular. 

What are the main advantages and limitations of using an offline workflow combining AF4-MALS with Py-GC-MS in environmental analysis?

The main advantage is that it brings two-dimensional information: both particle size distribution and chemical nature. That’s a great strength because for nanoplastics so far only very few workflows have been developed, and they are mostly based on mass-quantification only. Secondly, AF4 provides partial in-line sample cleanup from the matrix, which facilitates Py-GC-MS. As biggest limitations I see the low throughput, as the whole protocol takes a lot of time, and so far a low recovery. I also think a lot more work must be done on Py-GC-MS of samples which include (micro- or) nanoplastics and environmental matrix residues before such a workflow can truly show its full potential.

How would you improve recovery rates for nanoplastics in chromatography-based workflows, particularly in complex samples like wastewater?

First, I would play with the AF4 method. I think we might do better with the choice of the carrier liquid, though it was already difficult to achieve the recovery we have now. Then, I think the step of sample resuspension between the two analytical steps brings in a lot of losses. Maybe different protocols of resuspension could be tried. One of the problems with recovery, though, is that we have no relevant reference materials for nanoplastics so we cannot even reliably assess what the losses are for different types of nanoplastics.

References

1. Hayder, M.; Veclin, C.; Ahern, A. et al. Integrating AF4 and Py-GC-MS for Combined Size-Resolved Polymer-Compositional Analysis of Nanoplastics with Application to Wastewater. Anal. Chem. 2025, 97 (28), 15216-15224. DOI: 10.1021/acs.analchem.5c01766