Review Colombini, Modugno 147 J. Sep. Sci. 2004, 27, 147 – 160 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Maria Perla Colombini Francesca Modugno Chimica e Chimica Industriale, Via Risorgimento 35, 56126 Pisa, Italy Characterisation of proteinaceous binders in artistic paintings by chromatographic techniques This review discusses the application of chromatographic techniques (GC, HPLC and Py-GC) for the characterisation of proteinaceous materials in artistic paintings. The focus is on the various analytical steps that are needed to determine these natural materials in paint samples, from sampling and sample pre-treatment, including vari- ous methods of hydrolysis and derivatisation for GC and HPLC, to approaches for data evaluation. Key Words: Proteins; Paintings; Binder; Mass spectrometry; Pyrolysis; Received: May 30, 2003; revised: September 2, 2003; accepted: November 7, 2003 DOI 10.1002/jssc.200301625 1 Introduction Safeguarding our cultural heritage is an important duty to ensure that future generations have the opportunity to appreciate artistic masterpieces. Conservation must therefore be seen as one of the most exciting new fields where progress in science can be legitimately compared with the creativity and technical knowledge of the past. Science can provide tools for the chemical, physical, and structural characterisation of the materials used to create a work of art, and on the basis of this knowledge it can highlight ongoing degradation processes. Such knowl- edge, alongside the development of new technologies and materials for the prevention of environmental damage, enables restoration and conservation work to be planned. As far as paintings are concerned, the chemical charac- terisation of their constituent materials provides art histor- ians and conservators with precious information not only on the state of conservation but also on the painting tech- nique adopted by the artist. This helps to complete our knowledge of the artefact and thus contributes to the choice of materials and methodologies to be used in restoration. A painting generally consists of several layers: the support (plaster, canvas, wood, paper, parch- ment), the ground layer or priming (for instance gypsum and animal glue), one or more paint layers where the pig- ment is dispersed into an organic binder to obtain the coloured, film-forming paint capable of adhering to the ground layer, and eventually an outer layer of varnish. Since ancient times, painters have tried out a variety of organic materials for use as paint, and the empirical attempts to develop the best recipes have led to the acknowledged use of a few natural materials for this pur- pose. In Europe, these are essentially proteinaceous bin- ders, polysaccharidic gums, and siccative oils [1]. Other natural organic materials widely used in paintings are plant resins and waxes, mostly serving as varnishes, sur- face coatings, or binders, and organic dyestuffs, used as colouring matter [1]. However, the characterisation of all these materials is beyond the scope of this review, which aims to focus on a description of current practice in the characterisation of proteinaceous paint media. The proteinaceous binders used by artists in the tech- nique known as tempera are mainly collagen glues derived from animal skins or bones, egg, and milk or casein. These binders can be used on their own, mixed together, or in a mixture with siccative oils in the tech- niques known as tempera grassa. Animal glues were obtained by boiling animal tissues such as bones, skin, and cartilaginous parts of animals and fish. The constituent protein is collagen, charac- terised in the peptidic chain by a high content of glycine and proline and by the presence of hydroxyproline, an amino acid which is absent in the other proteinaceous materials commonly used as paint media. Apart from its use as a paint medium and adhesive, animal glue was mixed with gypsum for the preparation of the priming in easel paintings. Animal glue has a relatively high solubility in water, compared to other proteinaceous media, even after ageing. Whole egg and egg yolk have been the most widespread media in tempera paintings, and have also been used as fixatives and consolidants in restoration. Since egg yolk contains a consistent fraction of lipids (dried whole hen egg contains about 45% protein, 41% lipids, and 2% cho- lesterol [2]), as it dries it forms a particularly resistant and elastic film, whose permeability and solubility decreases Correspondence: Maria Perla Colombini, Chimica e Chimica In- dustriale, Via Risorgimento 35, 56126 Pisa, Italy. Phone: +39 050 2219305. Fax: +39 050 2219260. E-mail: perla@dcci.unipi.it.