Multiple Glass Transitions and Freezing Events of Aqueous Citric Acid

[Image: see text] Calorimetric and optical cryo-microscope measurements of 10–64 wt % citric acid (CA) solutions subjected to moderate (3 K/min) and slow (0.5 and 0.1 K/min) cooling/warming rates and also to quenching/moderate warming between 320 and 133 K are presented. Depending on solution concentration and cooling rate, the obtained thermograms show one freezing event and from one to three liquid–glass transitions upon cooling and from one to six liquid–glass and reverse glass–liquid transitions, one or two freezing events, and one melting event upon warming of frozen/glassy CA/H(2)O. The multiple freezing events and glass transitions pertain to the mother CA/H(2)O solution itself and two freeze-concentrated solution regions, FCS(1) and FCS(2), of different concentrations. The FCS(1) and FCS(2) (or FCS(22)) are formed during the freezing of CA/H(2)O upon cooling and/or during the freezing upon warming of partly glassy or entirely glassy mother CA/H(2)O. The formation of two FCS(1) and FCS(22) regions during the freezing upon warming to our best knowledge has never been reported before. Using an optical cryo-microscope, we are able to observe the formation of a continuous ice framework (IF) and its morphology and reciprocal distribution of IF/(FCS(1) + FCS(2)). Our results provide a new look at the freezing and glass transition behavior of aqueous solutions and can be used for the optimization of lyophilization and freezing of foods and biopharmaceutical formulations, among many other applications where freezing plays a crucial role.