Fractionating microplastics by density gradient centrifugation: a novel approach using LuerLock syringes in a low-cost density gradient maker

3.1 Luer-Lock low-cost density gradient maker

The setup of the low-cost density gradient maker is simple and based on the Luer-Lock-system. This ISO-standardized medical system offers a simple option for leak-free connections through their special and accurate fittings. Normally used for medical equipment, it is increasingly used in laboratories due to its user-friendly design and compatibility with various products such as syringes, needles and tubing. To reduce costs, it can be helpful to ask hospitals or doctors for expired Luer-Lock items, which are often provided for free.

The only modification needed is piercing a stopper with a needle. All other parts used in the gradient maker are standard issue lab materials (see Figure 1).

Figure 1:

Setup of the Luer-Lock low-cost density gradient maker.

The Luer-Lock low-cost density gradient maker is assembled vertically as shown in Figure 1. The lower syringe acts as a mixing chamber and is filled with a high-density medium. The upper syringe, filled with a low-density medium, serves as the reservoir. Due to the airtight system, the maker can produce the gradients based on the principle of pressure equalization.

For every drop leaving the lower syringe through the tube, a drop from the upper syringe enters the lower syringe via the needle, diluting the solution. Thus, the gradient maker can ensure a continuous dilution of the lower solution that leads to a continuous density distribution.

To make a gradient, the apparatus has to be prepared with all three-way-stopcocks closed. The lower syringe is filled with 5 mL of the higher density medium (e.g. a saturated potassium carbonate or sucrose solution in water). Next, the upper (bigger) syringe reservoir is filled with the lower density medium, e.g. water. A fitting stirring bar or a neodymium magnet is placed in the lower syringe, so that the liquid in the lower syringe is in motion during use and can ensure permanent mixing and consequentially continuous dilution. Then it is crucial to connect the upper and lower syringe with the pierced stopper and the three-way-stopcock so that the connection is airtight.

To make a density gradient both three-way stopcocks need to be opened, allowing the media to flow through the apparatus. It is essential that this flow begins only after both three-way stopcocks have been opened. If there is any flow before this step, it indicates a lack of airtightness, making the apparatus unsuitable for use. To prevent the mixing of the gradient in the Falcon tube, it is important to raise the end of the tubing along the level of the liquid. We recommend filling the Falcon Tube with 12–13 mL of density medium so that there is still room in the tube for the sample to be analyzed.

3.2 Testing sucrose and potassium-carbonate solutions as harmless and functional density media

After it was shown that there was no leakage, the stability and reproducibility of the gradients were tested with different density media. At first a solution of 66 % sucrose in water as dense medium and water as the dilutant was tested. Thereafter, the density of each milliliter of the water-sugar gradient was determined using refractive indices and UV–VIS spectroscopy. This used methylene blue added to the sucrose solution, which was then used indirectly to determine the sucrose concentration (see Figure 3).

For further experiments, sucrose was ruled out due to insufficient maximum density (1.32 g/cm³) for separation of high-density plastics like PET and PVC. Hence a saturated potassium carbonate solution was used as a novel high-density medium (1.53 g/cm³) as suggested recently (Gohla et al., 2021). A big advantage of this setup is that the potassium carbonate solution can act as both the extraction medium for sample preparation as well as the separation medium in gradient centrifugation.

Continuity and reproducibility of the potassium-carbonate-and-water-gradient was measured 6-fold using a “DMA46” flexural vibrating meter from Anton Paar K.G. (0–3 g/cm³, accuracy ± 1 × 10⁻⁴ g/cm³). The gradients were collected in a 15 mL Falcon tube and centrifuged in a SBS-LZ-400B benchtop centrifuge from Steinberg Systems, with an angular rotor at 5000 rpm for 30 min. For validation the gradients were fractionated before and after centrifugation using a pipette to take 1 mL fractions from the top of the gradient to the bottom. Then, the density of each fraction was determined.

3.3 Fractionation of microplastic particles by density gradient centrifugation

Finally, we tested the separation efficiency of gradient centrifugation to fractionate microplastic particles. We only tested plastic types with a density higher than seawater (>1.02 g/cm³), since they will sink and therefore accumulate in sediments (see Table 1) (Van Cauwenberghe et al., 2015). Plastic types with a lower density, such as low-density polyethylen (LD-PE), polypropylen (PP), high-density polyethylen (HD-PE) and expanded polystyrol (EPS) would float on seawater, hence they also float atop of the gradient and fractionation is therefore not possible with the density media used here.

The tested microplastic particles were produced by cutting, chopping or grating of everyday items.

The density gradients were then prepared as described previously. The fractionation was repeated 10-fold where each time 3–5 particles of each plastic type were placed on the density gradient. The density gradients were then centrifuged at 5000 rpm for 30 min. Afterwards, the microplastic particles were identified visually based on their shape or color.

In Figure 2, a summary of the process is shown that can also be used in schools. First, the gradient maker is used to produce a centrifugation-stable gradient. Setting up the gradient maker requires 15 min, making the gradient takes around 10 min. Second, microplastic particles are placed in the Falcon tube. After 30 min of centrifugation, the microplastic particles are fractionated and can assigned to a density range.

Figure 2:

Overview of the two-step fractionation process of microplastic particles.

4.1 Luer-Lock low-cost density gradient maker is suitable to produce continuous density gradients

With the Luer-Lock low-cost density gradient maker, continuous and reproducible density gradients can be produced both with a sucrose solution and water (see Figure 3) and with a saturated potassium carbonate solution and water (see Figure 4). A characteristic of a continuous gradient is its exponential decay, as can be seen in Figure 3.

Figure 3:

The sucrose – water gradient achieved by the low-cost gradient maker, separated milliliter by milliliter. (A) Methylene blue was added to the sucrose solution for visualisation (B) the refractive index for each fraction is shown as a mean of 10 measurements. The black line shows a regression for exponential decay [f = a*exp (−b*x)].

Figure 4:

Densities of each fraction of the potassium-carbonate-and-water gradient before (blue) and after (red) centrifugation.

As shown in Figure 4 there are no significant changes in the gradient before and after centrifugation. Generally, the gradients are characterized by their high stability. Therefore, the handling of the gradients is also suitable for students handling the gradients with little caution.

The initial phase of exponential density decay can be approximated by a linear decay, especially in fractions 2–9, allowing for reliable separation of microplastic particles in this density range.

4.2 Fractionation of microplastic particles by density gradient centrifugation

Preliminary results with the sucrose gradient were promising separating plastic types polypropylene (PP), polystyrol (PS), polyamide (PA), polycarbonate (PC) and polyethylene terephthalate (PET) (see Figure 5) as confirmed by ATR-IR spectroscopy (see Supplementary Information).

Figure 5:

A picture of the sucrose gradient with microplastic particles of the plastic types PP, PS, PA, PC and PET after centrifugation showing clear bands for each plastic type as confirmed by ATR-IR spectroscopy.

Furthermore, using the potassium carbonate gradient for fractionation makes the separation of PA, PC, PU, PET and PVC easily accessible. After centrifugation, the tested microplastic particles were separated into five well-defined bands (see Figure 6). The bands were in the same place in all 10 samples tested.

Figure 6:

Picture of fractioned microplastic particles after centrifugation in a saturated-potassium-carbonate-and-water density gradient showing clear bands for each plastic type, attributed to the density of each fraction.

Particles were then identified visually, and the density of the fraction was compared to the specific density of the plastic types. All five polymers formed a band in their specific density range, showing this method is viable for separation of multiple microplastic particles by plastic type. Separation efficiency is similar to other protocols using caesium chloride solution (Jakobs et al., 2023; Jing et al., 2022), making a saturated potassium carbonate solution suitable and favourable as a non-hazardous and cost-effective alternative for school context. It should be noted that the separation does not allow for a clear identification due to the partially overlapping density ranges of some plastic types. Density can also vary due to the use of additives (such as plasticizers, flame retardants, slip additives and thermostabilizers) or non-polymeric fillers (Andrady, 2015; Hale et al., 2020). Nevertheless, density separation by gradient centrifugation allows an approximate estimation of the type of the microplastic particles.

4.3 Workshop for teachers

In order to transfer the knowledge created by the students from school and extended by further development at university back to school, a hands-on experience working with this device has already been integrated in workshops for teachers. In addition to theoretical information on the topic of microplastics, the teachers also received an introduction to the analysis of microplastics in the workshop. Afterwards, the teachers had enough time to practice the methods actively and independently with the help of instruction sheets that were developed to be used with students (see Supplementary Information). A subsequent plenum discussion showed potentials for integration of the device into upper secondary chemistry classes. The workshop received a lot of positive feedback. Following the workshops, teachers reported that they had already successfully used the gradient maker and method in their laboratory lessons with minor, class-specific modifications and plan to implement it on a more regular basis.