Freezing Point Depression Near Body Temperature with Eicosane and Azobenzene

Co-Authored by Dean J. Campbell*, Kayla Lippincott*, Audrey Stoewer*, Trevor Tran*

*Bradley University, Peoria, Illinois

It is well known that homogeneous mixtures of chemical substances can have a lower freezing point or melting point than the pure components of the mixture. Here in the U.S. Midwest in the winter, sodium chloride or other water-soluble species are spread on roads and sidewalks to help keep them ice-free, with varying levels of success.¹ There have been studies of whether various aqueous salt solutions could have sufficiently low melting points as to be liquid under certain conditions on Mars.² The addition of sodium oxide to silicon dioxide lowers its melting point, enabling it to be melted more easily for making glass.³

Our General Chemistry courses have long done some variation of a freezing point depression lab, using solvents such as water, t-butanol, and most recently, stearic acid. The stearic acid lab experiment that we use is adapted from one published online by Beyond Benign,⁴ which in turn is adapted from a paper published in the Journal of Chemical Education.⁵ Stearic acid is an attractive solvent for this lab because it is has relatively low-toxicity and a melting point of about 69°C.^4,5 Since this melting point is not very far above room temperature, it requires only a moderate amount of heat and no cooling below room temperature, so the lab is also not very energy intensive. In the Journal of Chemical Education paper and its supplemental material, some of the melting points for fatty acid mixtures were near the temperature of the human body.⁵ This inspired us to look around our labs and stockrooms for reagents that in their pure forms had melting points that were greater than body temperature, but when combined, could be melted by body heat. The result would be a freezing-point demonstration that would require neither hot plates nor ice baths, just a warm body. An additional criterion we sought was for the component reagents to be of different colors so that mixtures would appear to be different colors than the pure material. Our search among what we had available yielded eicosane, a colorless linear alkane with the formula C₂₀H₄₂. A sample from Chem Service, Media, PA, had a melting point of 31-38°C listed on the bottle, and a sample from Sigma-Aldrich, St. Louis, MO, had a melting point of 35-37°C listed in its Product Specifications. The compound selected to depress the melting point of the eicosane was azobenzene, an orange compound with the formula C₁₂H₁₀N₂; our sample from Sigma-Aldrich, St. Louis, MO, had a melting point of 66°C listed on the bottle. It is important to note that although the azobenzene is orange, it does not appear to be as intensely colored as other compounds (most of its light absorbance is in the ultraviolet range).⁶ The advantage of this is that solid mixtures of eicosane and azobenzene appear to be orange, but intermediate in color between the white solid pure eicosane and the darker orange pure azobenzene. Addition of a small amount of azobenzene to the eicosane produced solutions that could be melted by body temperature.

In one series of experiments, azobenzene and eicosane were weighed out and combined in test tubes, which were then placed in hot water to melt and combine the compounds. Then the mixtures were solidified, scraped out of the test tubes, and transferred to NMR spectroscopy tubes. These tubes are narrow with thin glass walls to optimize heat transfer in and out of the samples. The NMR spectroscopy tubes containing the samples were placed in water baths at various temperatures to measure their melting points. It was important to not raise or lower the temperature of the baths too quickly, as it does take time for samples to convert between cloudy solids and transparent liquids. Table 1 below lists mass combinations of eicosane and azobenzene, the mass % azobenzene in eicosane, the molal concentration of the azobenzene dissolved in eicosane, and the estimated melting points of these mixtures. A graph of eicosane and azobenzene melting points as a function of molality of azobenzene is shown in Figure 1.

Table 1. Eicosane and azobenzene mixtures and their melting points.

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Figure 1. Graph of melting point of mixtures of eicosane and azobenzene as a function of the molality of azobenzene in the mixture.

In another series of experiments, azobenzene and eicosane were weighed out and combined directly into the NMR spectroscopy tubes or microcentrifuge tubes. These tubes were heated with hot water to melt the components and placed on a vortex mixer to combine them. The tubes were placed in a variable temperature heating bath (Fisher Scientific Isotemp 1016D) by pushing them through sheets of plastic foam floating on the surface of the bath. The temperature of the bath was raised slowly, with usually at least 30 minutes passing before entering a new temperature into the system. The samples in their tubes were lifted briefly out of the bath and visually assessed as being solid, liquid, or a mix of both. Phase diagrams for the eicosane and azobenzene system, describing phase as a function of temperature and % mass azobenzene, are shown in Figures 2 and 3 below.

These samples and their melting points have been used in three ways in educational settings. In one approach, the data described in Table 1 was used as a question in the freezing point depression lab in our General Chemistry 2 course. Students were given the mass compositions and melting points, instructed to calculate the mixture molality, and then instructed to plot melting point as a function of molality, see Figure 1 above. The curve decreases and then increases as the concentration of azobenzene increases. Students were asked to indicate which section of the curve showed that the freezing point best decreased linearly with molality.

In a second approach, a video was made to demonstrate freezing point depression of the eicosane and azobenzene mixture. NMR spectroscopy tubes containing pure eicosane, pure azobenzene, and a mixture of the two were all carefully placed into a thick glove near the palm of a human hand for several minutes. The heat produced by the hand was sufficient to melt the mixture before samples of pure eicosane or pure azobenzene. Video 1, which shows this demonstration, has been linked to course websites for viewing by students.

Video 1. Mixture of eicosane and azobenzene melted by body temperature, whereas the pure compounds are not melted by body temperature. ChemDemos YouTube Channel (accessed 10/20/2021)

In a third approach, students in a Materials Chemistry course were asked to each weigh and add eicosane and azobenzene to two NMR spectroscopy tubes.  The tubes were heated in the variable temperature bath and the physical state of the mixtures they contained were recorded, and the data were given to the students to produce phase diagrams for the eicosane and azobenzene system. To show phase as a function of temperature and % mass azobenzene, many students used a spreadsheet scatter plot. The experimental data for the samples were arranged in three columns: % mass azobenzene, temperature, and physical phase of the sample. The notation “s” for solid, “m” for mix of liquid and solid, and “l” for liquid were used. The data in the columns were then sorted alphabetically by the phase column, grouping samples of the same phase together. All of the samples of a single phase were used as a data set with the same data point colors and shapes in an x-y scatter plot of % mass azobenzene on the x-axis and temperature on the y-axis.  Each of the data sets for the other phases (with their own data point colors and shapes) were also placed on the same scatter plot. The data produced from this activity are plotted in Figure 2 below.

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Figure 2. Phase diagram for eicosane and azobenzene, based on sample components weighed and then placed into NMR tubes.

The data in the phase diagram were a bit erratic. Students noted that it was challenging to get the solid eicosane and azobenzene into the narrow NMR spectroscopy tubes. This could quite possibly be a source of the error in the data. With this in mind, a couple of research students were asked to weigh and add eicosane and azobenzene to plastic microcentrifuge tubes. Though the plastic walls of the microcentrifuge tubes were not quite as thin and transparent as the glass in the NMR spectroscopy tubes, the samples and their physical states were visible. The microcentrifuge tubes were more robust and, more importantly, had a wider opening and were thus easier to fill than the NMR spectroscopy tubes. The phase diagrams produced from this new trial are shown in Figures 3 and 4. The points are made rather wide in these scatter plots to aid in visualization of the various phases. The melting point data from Figure 1 are also added to Figure 3. The eutectic point for the eicosane and azobenzene system appears to be at about 10% azobenzene by mass.

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Figure 3. Phase diagram for eicosane and azobenzene, based on sample components weighed and then placed into microcentrifuge tubes. Melting point data from Figure 1 has also been added.

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Figure 4. Phase diagram detail for eicosane and azobenzene, based on some of the microcentrifuge samples from Figure 3 measured at smaller temperature intervals.

Even in these trials there was some error. A sample identified as a mix in one trial might appear as a liquid in a subsequent trial.  Possible sources of error could be variations in the water bath temperature, superheating or supercooling, and variations in the composition of the mixture during melting and freezing.

To develop these activities further, it is desirable to switch to “greener” chemical compounds. When considering the Twelve Principles of Green Chemistry, this chemical system is aligned with the principle of Design for Energy Efficiency in that it requires relatively little heating or cooling to demonstrate phase transitions.⁷ However, eicosane and especially azobenzene have some toxicity.^8,9 Even though their mixtures are confined to closed containers once they are prepared, less-toxic alternative compounds should be sought. By utilizing less-toxic compounds, the Green Chemistry principles of Less Hazardous Chemical Synthesis and Designing Safer Chemicals might be achieved.⁷ Both compounds must be nontoxic, nonpolar, and melt at a temperature just above body temperature. Perhaps a fatty acid similar to stearic acid might suffice to be a safe, colorless component compound.^5,10 The other compound should be colorful yet not too intensely dark in order to distinguish the color of the pure compound from the mixture. β-Carotene might have sufficiently low toxicity to warrant further study.¹¹ In any case, the eicosane and azobenzene system described here can be used to illustrate concepts of freezing point depression and phase diagrams.

Safety All reagent containers should be clearly labeled. The NMR spectroscopy tubes are narrow and have thin glass walls, making them somewhat fragile. Azobenzene is listed as harmful by inhalation or if swallowed, is a possible carcinogen, and is dangerous to the environment. Eicosane is listed as possibly being fatal by inhalation or if swallowed. Consult the Safety Data Sheets for more information on these compounds.^8,9  In one trial, the eicosane and azobenzene mixtures were combined into plastic cuvettes. When warmed, the liquid mixtures seemed to dissolve through the bottom of the plastic (possibly polystyrene) cuvettes in a dramatic example of “like dissolves like.”

Acknowledgements This work was supported by Bradley University and the Mund-Lagowski Department of Chemistry and Biochemistry with additional support from the Illinois Heartland Section of the American Chemical Society and the Illinois Space Grant Consortium. We would also like to thank the Bradley University Materials Chemistry course for working on the phase diagram assignment.

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