Generation of macro- and microplastic databases by high-throughput FTIR analysis with microplate readers

Abstract

FTIR spectral identification is today's gold standard analytical procedure for plastic pollution material characterization. High-throughput FTIR techniques have been advanced for small microplastics (10-500 µm) but less so for large microplastics (500-5 mm) and macroplastics (> 5 mm). These larger plastics are typically analyzed using ATR, which is highly manual and can sometimes destroy particles of interest. Furthermore, spectral libraries are often inadequate due to the limited variety of reference materials and spectral collection modes, resulting from expensive spectral data collection. We advance a new high-throughput technique to remedy these problems using FTIR microplate readers for measuring large particles (> 500 µm). We created a new reference database of over 6000 spectra for transmission, ATR, and reflection spectral collection modes with over 600 plastic, organic, and mineral reference materials relevant to plastic pollution research. We also streamline future analysis in microplate readers by creating a new particle holder for transmission measurements using off-the-shelf parts and fabricating a nonplastic 96-well microplate for storing particles. We determined that particles should be presented to microplate readers as thin as possible due to thick particles causing poor-quality spectra and identifications. We validated the new database using Open Specy and demonstrated that additional transmission and reflection spectra reference data were needed in spectral libraries.

Keywords: Database; FTIR; High-throughput; Microplastics; Plastic pollution; Spectroscopy.

Fig. 1

Schematic representation of the major aspects of this study, showing (left to right) the curation of the 637 materials, the high-throughput FTIR processing of individual materials with ATR plus the 96-well plates and fabricated transmission plate cover, and lastly archiving samples with a fabricated nonplastic 96 well plate

Fig. 2

Images of particles in plates for transmission and reflection measurements. Transmission (before) and transmission (after) were compared to assess whether particles moved during the measurement. Each well held a different particle. Spectral collection mode is labeled on the left axis, and the plate number is on the top axis. Transmission plates had a custom-made well overlay from heavy aluminum foil. No particles were observed missing or crossing into another well during the transmission measurement (which can be caused by vibrations in the machine if not using the transmission plate cover)

Fig. 3

Visual instructions for creating the aluminum overlay for the transmission plates. Step 1: Trace wells and outline of transmission plate on thick plastic and transfer the plastic overlay to a piece of heavy aluminum foil with tape. Step 2: Pound a gaged stamp of the well size with a rubber mallet on top of a hard plastic platform and cut the aluminum to size with scissors. Step 3: Tape the aluminum cover to the silicon plate with small slivers of tape at the edges

Fig. 4

A long-term storage setup for particles from the plate reader using a metal 96-well plate. The well positions can be labeled along the left and top axis in the blank space. A Blueprints for the creation of the 96-well plates. B An image of the 96-well plate made from stainless steel. Placing particles in this plate allows for easy transfer to the FTIR plate reader plates when needed by maintaining the reference positions, and since it is made of nonplastic materials, it has low contamination risk for plastic pollution research

Fig. 5

Validation of the database produced using Open Specy’s out-of-the-box settings to identify the material type. The X-axis is the spectral collection mode employed in collecting the database. The Y-axis is the accuracy in percent of correct identifications of Open Specy in identifying spectra from the spectral collection mode group. The total number of spectra tested for each spectral collection mode is listed above the bars. The height of the bars is the accuracy (%). Spectra counts were not identical across the techniques because not all particles were measured in all modes, and some particles were measured more times than others

Fig. 6

Box plots for Pearson correlation coefficient for ATR, transmission, and reflection analysis, showing the superior performance of ATR due to the current contents of the Open Specy library. The X-axis is the spectral collection mode, while the Y-axis is the mean of the maximum correlation values to the Open Specy library for all replicates of each particle’s spectra. The red horizontal line demarked 0.7 correlation below which identifications are considered uncertain. The plot shows boxplots for the maximum correlation for each spectral collection mode to the library in Open Specy. Points on the plot show outliers. Edges of the box are the inter quartile range. The center line in the box is the median

Fig. 7

The Pearson correlation ATR, Reflection, and Transmission as a function of nominal particle size, showing an increasing correlation with increasing nominal particle size for ATR analysis. The X-axis is the nominal particle size (square root of particle projected area). The Y-axis is the mean of the maximum correlation values to the Open Specy library for all replicates of each particle’s spectra. Smooth lines are generalized additive models with a smoothing spline and 95% confidence intervals shown in gray. ATR, Reflection, and Transmission for each particle are colored differently and shown in the legend. ATR showed better correlations for larger particles, while transmission and reflection showed the opposite

Fig. 8

Comparison of spectra from the same materials (top axis) for each spectral collection mode (right axis). The Y-axis (unitless) is min–max normalized absorbance intensity values for each spectrum. The X-axis is wavenumbers in units cm⁻¹. When multiple spectra were collected in a single mode, they are overlaid. Images on the right axis show the spectral collection modes. Images on the top axis show the particles that were assessed