Occasionally, operators in Drug Discovery and life science laboratories observe very long and uneven drying times in polypropylene microplates dried in centrifugal vacuum evaporators. Often, this is the consequence of user error, a faulty unit, or microplate contamination. This paper outlines a set of tests performed to identify how microplate contamination can impact on the boiling point of samples, thus affecting their drying times.
To examine this phenomenon, a series of experiments were performed utilizing commercially available 24-well microplates from two manufacturers, M1 and M2. In both experiments, a set of microplates were selected from a batch to guarantee an exhaustive sample representation. The wells were filled with 6 ml of pure water and, where applicable, dried utilizing heat transfer plates at 8 mbar with a Genevac EZ-2 centrifugal evaporator (Figure 1). Thermocouple probes were inserted into multiple wells across the plate for monitoring the boiling point of the water. It is anticipated that water will boil at 4 °C at 8 mbar, provided there is zero contamination.
Figure 1: EZ-2 centrifugal evaporator.
Results and Discussion
Microplates from manufacturer M2 (see Figure 2) dried as anticipated and presented no negative effects. In this experiment, the boiling temperature of the water was maintained at around 4 °C at 8 mbar until all of the water had evaporated. Conversely, in near-identical experimental conditions, microplates from manufacturer M1 did not dry as anticipated. Figure 3 below demonstrates the volume of residual water in each well at the end of the run, while Figure 4 illustrates the graph of the boiling temperature versus pressure curve against time.
The uneven drying of the microplate deriving from manufacturer M1 might be accounted for by contaminant film formation on the surface of the water, which can hinder evaporation. Figure 4 presents the fluctuation of the sample temperature at 8 mbar. The uneven results reveal that the sample undergoing evaporation was not pure water and that film formation occurring with microplate M1 impeded this process, leading to increased sample temperature. To examine this contaminant film formation phenomena further, new microplates were taken from manufacturer M1, and were subsequently washed in acetone before usage.
Water was added to these ‘cleaned’ plates and a matching evaporative experimental procedure was performed. The results (not presented) were nearly identical to those obtained when utilizing the contaminant-free M2 microplates. In additional experiments, as a means of simulating the impact of contamination, a small quantity of an inert mineral oil was added to some microplate wells from manufacturer M2, as well as 6 ml of water. Results from this evaporation experiment are presented in Figure 5 below.
Following a comparison of the results presented in Figure 5 (induced contamination) with those presented in Figure 4 (genuine contamination), it is clear that the effects are similar. The induced contamination yielded results that closely matched those of the unwashed, contaminated plates; they exhibited increased solvent temperatures during drying, and some microplate wells were left wet. The induced contamination is explicitly more extreme than that which occurred when using the contaminated microplates from manufacturer M1. This is likely because the naturally occurring contaminants were present at lower levels than the gross induced contamination.
From these experiments, it can be concluded that microplates from manufacturer M1 were contaminated with a material forming a film on the water sample surface and acting as a cap to the well, and which prevented or inhibited evaporation. The film formation phenomena triggered by contamination helps to account for why certain microplates display abnormally long and uneven evaporation times. The intention of this study was not to determine the nature of the contaminant. Following a review of information regarding microplate production processes, it is likely that the contaminant is an anti-static agent, mold release agent, or plasticizer.
To conclude, if such an effect ever manifests, it might be advisable to perform some preliminary runs utilizing known pure solvents, to prevent contamination and long sample drying times. If observed drying times are far longer than anticipated, it is recommended to try washing plates before usage. Acetone washing is not consistently effective but has been advantageous in several cases. In cases where washing cures long drying times, plate contamination is almost certainly the cause, and another plate provider must be located.
Produced from materials originally authored by Induka Abeysena from Genevac Ltd.
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