Berthelot was born in Paris, into a middle-class family, and attended secondary school at the prestigious Collège Henri IV.
He received his baccalauréat ès lettres in 1847 and his baccalauréat ès sciences in 1848.
During his time at the Collège, he became close friends with Ernest Renan, who went on to become a well-known historian and philologist.
Their friendship greatly influenced Berthelot's philosophical and historical interests, as evidenced by their lifelong correspondence.
While studying at the Collège Henri IV, Berthelot attended lectures at the Collège de France and became drawn to chemistry.
He went on to earn a degree in physical sciences from the Collège de France in 1849, before returning in 1851 to work as an assistant to Antoine-Jérôme Balard, a chemist.
Berthelot's doctoral dissertation, titled "The Combinations of Glycerin with Acids and the Synthesis of Immediate Principles of Animal Fats," was completed in 1854.
This dissertation was based on Michel-Eugène Chevreul's research on the chemical composition of fats, which led Berthelot to synthesize numerous new fats and introduce the terms monoglyceride, diglyceride, and triglyceride.
Berthelot's scientific career began with his dissertation, which sparked a longstanding dispute with French atomists, particularly Charles-Adolphe Wurtz.
Wurtz interpreted Berthelot's results in terms of type theory, which distinguished between atoms and molecules.
In contrast, Berthelot advocated for an older dualistic theory that represented organic compounds as oxides and salts. Berthelot pursued pharmaceutical studies after completing his first doctorate, earning a second doctoral degree in pharmacy in 1858.
He was appointed to a professorship of organic chemistry at the École de Pharmacie when it was created in 1859. Berthelot also taught a course at the Collège de France, where he secured the creation of a chair in organic chemistry in 1865, thanks to Balard's efforts.
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