• lipid - protein co-oxidation
• lipid oxidation
• microstructures
• protein oxidation

The 15th century in Italy marked a crucial moment in art history as Italian painters began exploring the integration of oil painting techniques, which had originated in Flanders, with traditional egg tempera methods. This blending of different media led to the creation of artworks where both oils and proteins coexisted, sometimes in separate layers, but also within the same layer of paint. However, the exact details of this transition remain unclear to this day, and little is known about the specific techniques and formulations that artists used or why they chose one over the other.
This PhD thesis centers on the study of two distinct mixed media painting techniques: tempera grassa (tg) and protein-coated pigment (pcp). Tempera grassa is essentially an emulsion where oil and pigment particles are suspended in water, with proteins present as a colloidal dispersion in the aqueous phase. In contrast, protein-coated pigment involves a layer of protein surrounding pigment particles, which are then dispersed in oil. These two techniques result in different microstructures, as they vary in how the pigment particles, proteins, and oils interact within the paint layer.
My project could not therefore be based on a systematic and statistically significant study of ancient samples. Instead, I decided to focus on model paints. Since the beginning of the project, a challenging question arose: how to prepare model paints and paint layers? For this reason, the thesis is structured around two main research pathways that have run in parallel throughout the three years of the PhD, with the first being functional to the second: 1) understanding which parameters must be controlled when
preparing model paints and 2) studying mixed media paints.
The thesis comprises five chapters. Chapter 1 presents a literature-based summary on the oxidation of lipids, the oxidation of proteins, and their cooxidation when simultaneously present. Chapter 2 of the thesis addresses the preparation of model paints, stressing the importance of controlling various parameters to achieve reproducible results. The research found that factors such as paint thickness, pigment storage conditions, type of pigment, oil type, and oil pre-treatment all significantly influenced the curing of ultramarine blue paints. One key discovery was that natural lapis lazuli, behaved differently from synthetic ultramarine blue. This was especially evident in its rheological and chemical properties, suggesting that the handling and treatment of pigments were essential to the formulation of effective oil paints. The study raises the question of whether Old Masters employed specific methods, such as coating pigments with proteins, to overcome the challenges posed by natural pigments like lapis lazuli.
Chapter 3 presents findings from advanced imaging techniques, such as confocal microscopy and synchrotron-based deep UV photoluminescence microimaging, to analyze the microstructures of tg and pcp paints. These methods revealed that in pcp, proteins seem to protect lipids from direct interaction with pigments, while in tg, the microstructure is more heterogeneous with several interfaces at play. Deep UV photoluminescence microimaging in particular appears potentially very useful for examining cross-sections of aged paint layers, potentially allowing scientists to study works of Old Masters. Chapter 4 expands on the chemical properties of tg and pcp paints using a multi-analytical approach. The results showed that lipid oxidation was the initial stage of the process, but proteins quickly became involved, leading to strong chemical interactions between the two. In pcp paints, the protein coating helped form a cross-linked polymer network, which reduced the degradation of lipids. Conversely, tg paints displayed a higher degree of oxidative damage due to their more complex and heterogeneous microstructure. In Chapter 5, proteomics methods, including electrophoresis and mass spectrometry, were applied to study the protein content in mixed media paints. The findings revealed that proteins in paints undergo cross-linking and oxidation, starting as soon as the paint is applied, with oxidation levels reaching those of aged tempera paint within just a month of formulation.
While tempera grassa remains a somewhat mysterious technique, it is often referenced in the context of mixed media painting but, there is no clear evidence that Old Masters employed this technique. Research has suggested that tempera grassa allows for slightly better color transitions compared to traditional tempera and maintains a softer consistency due to the oil's delayed drying. However, the implications of this slower drying process and the extent of oxidation in tempera grassa are not yet fully understood. Moreover, using egg yolk versus egg white in the formulation can influence the paint’s consistency, with egg yolk yielding a thicker paint with better hiding power.
On the other hand, protein-coated pigment (pcp) appears to offer several advantages. Coating pigments with egg white or egg yolk prevents issues with humidity and reduces the stiffening of the paint, which can sometimes occur with pure oil paints, due to unwanted absorption of humidity from the environment on the pigment particles. Additionally, pcp paints exhibit high yield stress, allowing for thick brushstrokes that maintain texture and have less yellowing over time compared to traditional oil paints. The slower drying time associated with egg white-based pcp could be problematic for certain techniques like layering, but it could be beneficial for wet-in-wet or alla prima methods, which require the paint to remain workable for longer. All in all, pcp appears as a promising technique that was likely used by the Old Masters, offering a practical means to modify paint properties, improve paint layer stability, and reduce yellowing over time.