Multiple glass transitions and freezing events of aqueous citric acid

Abstract

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/H2O. The multiple freezing events and glass transitions pertain to the mother CA/H2O solution itself and two freeze-concentrated solution regions, FCS1 and FCS2, of different concentrations. The FCS1 and FCS2 (or FCS22) are formed during the freezing of CA/H2O upon cooling and/or during the freezing upon warming of partly glassy or entirely glassy mother CA/H2O. The formation of two FCS1 and FCS22 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/(FCS1 + FCS2). 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.

Figure 1

DSC cooling (upper blue lines) and warming thermograms (lower red lines) obtained from 10–64 wt % CA drops at the scanning rate of 3 K/min. Horizontal arrows mark the direction of programmed temperature change. T_f and T_m mark exothermic ice freezing and endothermic ice melting peaks, respectively. T_g,c marks a liquid–glass transition upon cooling and T_g,w and T_g2,w reverse glass–liquid transitions upon warming (see text for details). T_cr marks freezing (ice crystallization) upon warming. Solution concentration is indicated. Scale-bars indicate heat flow through samples.

Figure 2

Optical cryo-microscope pictures of frozen CA/H₂O solution. (a) A picture demonstrates that freezing is initiated from a single ice nucleating event. Radial protuberances of different brightness are due to the different density of ice branches and channels of freeze-concentrated solution, FCS₁ (see also panel c). (b) Freezing of concentrated solution is initiated from multiple ice nucleating events. Dark spots are ice crystals formed by vapor deposition on an upper side of a cover glass. (c) Branches of a continuous ice framework (IF) formed from a single ice nucleating event shown in panel a. Arrows show ice in contact with a cover glass and channels of FCS₁ in between ice branches. A less concentrated freeze-concentrated solution, FCS₂, envelops the entire IF/FCS₁. (d) Magnification of the ice nucleation region from Panel a. Interweaved ice branches and FCS₁ channels are seen as bright and dark spots, respectively.

Figure 3

Magnified cooling thermograms of 56, 60, and 62 wt % CA obtained at 3 K/min. The cooling thermogram of 56 wt % CA is from Figure 1. T_g1,c, T_g2,c and T_g,c mark the onset of liquid–glass transitions. A vertical filled arrow marks T_g2,c transition in 60 wt % CA thermogram. An open arrow marks a transition from anhydrous CA to CA-monohydrate.^, In the 56 and 60 wt % CA thermograms, the ice freezing peak T_f is truncated to fit the figure.

Figure 4

Magnified warming thermograms from Figure 1. T_g,w and T_g,2w mark the onset of glass–liquid transitions (see also Figures 1 and 3). T_tr2 is a historical name of the second transition observed in the past during the warming of frozen hydrocarbon solutions (see text for details). Skewed lines truncate ice melting peaks, T_m, to fit the figure. The remaining symbols have the same meaning as in Figure 1.

Figure 5

Optical cryo-microscope pictures of 62 wt % CA solution taken during cooling and subsequent warming. (a,b) Pictures are taken upon cooling at 216 and 193 K, respectively. Incipient ice crystallization is seen as brown dendritic crystals. Numerous dark spots are ice crystals nucleated on a cover glass by vapor deposition. (c,d) Pictures are taken upon warming at 220 and 230 K, respectively, where nucleation and ice growth occur most strongly. Arrows show ice crystals formed during the incipient freezing upon cooling.

Figure 6

Cooling and warming thermograms of three 60 wt % CA drops. The upper thermograms are those from Figure 1. The middle cooling thermogram is that from Figure 3. The mass of drops, from which the upper, middle, and bottom thermograms were obtained, are 5.05, 5.74, and 5.64 mg, respectively. T_g,c and T_g,w mark the liquid–glass and reversible glass–liquid transitions of a fraction of 60 wt % CA, which does not freeze upon cooling. The remaining symbols have the same meaning as in Figure 4.

Figure 7

Comparison of cooling and warming thermograms obtained from two different drops of 50 wt % CA cooled/warmed at the scanning rate of 3 K/min (upper thermograms) and 0.5 K/min. The mass of drops are 6.22 mg (upper thermograms) and 6.35 mg. The upper thermograms are from Figure 1. All symbols have the same meaning as those in the Figures above.

Figure 8

Comparison of 40 wt % CA thermograms obtained at the scanning rate of 3, 0.5, and 0.1 K/min. The upper thermograms are those from Figure 1. The middle thermograms are obtained at the cooling and warming rate of 0.5 K/min. The bottom cooling thermogram is obtained at 0.1 K/min and warming thermogram at 0.5 K/min. A sharp exothermic peak at ∼305 K is due to the transition from anhydrous CA to CA-monohydrate.^, All symbols have the same meaning as those in the Figures above.

Figure 9

Warming thermograms of 10–55 wt % CA solutions quenched in liquid N₂. T_g,22w and T_g,11w mark glass–FCS₂₂ and glass–FCS₁₁ transitions (see text for details). The circle shows a magnification of the T_g,22w transition in 50 wt % CA. Other symbols have the same meaning as those in previous Figures.

Figure 10

Comparison of the glass–liquid transition onsets in the warming thermograms of quenched (upper line, from Figure 9) and slowly cooled (3 K/min, bottom red line, from Figure 4) 50 wt % CA. All symbols have the same meaning as those in previous Figures.