Due to the challenges in visually identifying microplastics, secondary analyses should be used to confirm the identity of suspected polymeric material. The method employed is often dictated by the equipment available and while any chemical characterization of the polymers recovered is useful, some techniques are more robust than others. The European Commission suggests that a subsample (5%—10%) of particles with a size between 100 mm and 5 mm and all particles between 20 and 100 mm should be subjected to further verification techniques. Postvisual analyses have shown misidentification of microplastics in wild-caught animals of up to 70% [82,147,150]. It should be noted here that errors in identification often include unmatched spectra that could not be assigned with confidence to a known polymer type; confidence thresholds for spectra matches are usually set at 70%-75% [77,83,89]. Confirming the identity of suspected plastics may be carried out in a number of ways depending on the funds and equipment available to the researcher. Perhaps the simplest technique is the use of a hot needle to observe melting points [38,52,60,86]. While both cheap and fast, this method does not allow for the accurate identification of the polymer; however, the temperature range at which melting occurs does provide a specific range of potential plastics. A converse method is to exclude nonplastics rather than identifying the plastics present; oven and freeze drying removes water from organic material causing it to wither. This increases the likelihood of nonplastic material being identified and removed from mixed samples [151,152]. Combining these two techniques provides a cheap, if laborious, method of plastic identification.
Another low-cost technique involves the examination of microplastics under a polarized light microscope to observe the birefringent properties of the suspected polymer. The birefringence of a polymer is the result of its chemical structure and manufacturing methods which results in unique anisotropic properties; by passing polarized light through a sample, unique spectra are created, from which it is possible to confirm the identity of plastic materials . As with the hot needle technique, this method require plastics to be viewed individually; while initial costs are low, the time taken makes it prohibitive for large samples. More complex—and costly—methods can also be used to infer resin constituents, plastic additives and dyes.
Often, these techniques require the purification of the potential microplastic prior to analysis. The removal of biofilms and organic and inorganic matter adhered to the surface will avoid impeding polymer identification and the removal of nonplastic particles . Following purification, suspected plastics are submitted to analytical techniques including Fourier-transformed infrared spectrometry (FTIR) in transmittance or reflectance; attenuated total reflectance (ATR); Raman spectrometry for color pigment spectra and pyrolysis-gas chromatography combined with mass spectroscopy (Pyr-GC-MS), which analyzes particles using their thermal degradation properties and can be used to analyze polymer type and organic plastic additives simultaneously . Alternate analytical methods include high- temperature gel-permeation chromatography (HT-GPC) with IR detection; SEM-EDS and thermo-extraction and desorption coupled with GC-MS [150,155,156].
If coupled with microscopy, FTIR and Raman can be used to identify microplastics with a size >20 mm [123,149]. Raman spectroscopy combined with microscopy has a higher resolution (approx. 1-2 mm) [100,149] and can be used to locate particles within biological tissues . FTIR and Raman have been recommended for determining resin constituents [123,149]. There is minimal sample preparation, other than clean up, required for FTIR. However, FTIR and Pyr-GS-MS are both destructive. Raman is nondestructive as it does not require the sample to be flattened or manipulated. The disadvantages of Pyr-GS-MS is the manual placement of the particle in the instrument, which can incur size limitations and only one particle can be run per sample. However, qualitative and quantitative analyses are being developed [141,157]. A drawback of chemical analysis is that the isolation of small, highly degraded samples increases the chances of misidentification and producing noisy spectra in which the vital fingerprint areas are obscured, although this can be improved by the use of microscope-aided instrumentation (micro-FTIR and micro-Raman), which is designed to target and read responses from samples of a smaller size.