**4. Preparation of proteinoids by thermal condensation polymerization**

L-glutamic acid was heated to the molten state (180°C) in an oil bath, under nitrogen atmos‐ phere. The molten mass was stirred at 180°C for 30 min. To this, different contents of additional L-amino acids were added to give a total monomer weight of 5-5.01 g, as specified in Table 1, and kept at 180°C under nitrogen. The mixture was mechanically stirred at 150 rpm for 3 h. The product is a highly viscous orange-brown paste, which hardens to give a glassy mass when cooled to room temperature. Then, water (10 mL) was added to the crude product, and the mixture was stirred for 20 min. The solution was then intensively dialyzed through a cellulose membrane (3500 Da MWCO) against distilled water. The content of the dialysis tube was then lyophilized to obtain a yellow-white proteinoid powder.


**Table 1. Amino acid content of the different proteinoids. a**In all proteinoids made by thermal condensation polymerization the total monomer content was 5-5.01 g; **<sup>b</sup>**made by microwave-assisted polymerization.

#### **4.1. Polymerization kinetics study at different temperatures**

Polymerization kinetics was studied by collecting proteinoid samples from the reaction vessel at different time periods of the polymerization at 180, 190 and 200°C. The samples

were then analyzed by both ninhydrin test for the determination of the primary amine groups content and Biuret test for the determination of the amide groups content of the various proteinoids [49]. The results shown in Figure 3 refer to the synthesis of Prot3, consisting of L-glutamic acid and L-phenylalanine. Similar results were observed for the other systems, Prot1-8, as well. It can be seen that at all temperatures, the polymerization takes place mainly over the first 100 min. After that, both ninhydrin (A) and Biuret (B) tests show no significant drop of the Ninhydrin signal indicating the amount of free amines or rise of the peptide bonds shown by the Biuret signal. This figure also shows that as the temperature of the reaction was raised, the rate of the reaction increased. However, the preferred reaction temperature is 180°C, as the yield of the reaction is higher since the amino acids decompose faster at the higher temperatures.

**Figure 3.** Thermal polycondensation kinetics of (L)glutamic acid and (L)phenylalanine to yield Prot3 at different tem‐ peratures by two tests: ninhydrin test (A) and Biuret test (B).
