Exploring Color Separation in Salting-Out Experiments

In “salting out” demonstrations, a mixture of water and an organic liquid separates into two layers upon addition of an inorganic salt.^1-4 Typically, acetone or various alcohols are used for the organic liquid, while NaCl, Na₂CO₃, or K₂CO₃ are used as the salt. When the salt is added to the organic/aqueous mixture, strong attractions between the resulting dissolved ions and water molecules are too strong for the organic molecules to overcome. As a result, the mixture separates into two phases with the lower density organic layer on top and the higher density water layer on the bottom. As such, this experiment is useful when discussing the topics of density,⁴ classification of matter,⁴ and intermolecular forces.^1-3

The use of various dyes in these experiments allows for colorful and interesting twists on the “salting out” experiment. Food dyes,¹ dyes in glitter,^1,3 and even phenolphthalein² have been used in this regard. Because some dyes are more soluble in the organic layer while others are more soluble in the aqueous layer, a variety of interesting two-color effects can be achieved in this experiment. As noted by Webb and Rothenberger,² this experiment naturally lends itself to inquiry-based experimentation because of the wide range of variables involved (type of organic liquid, type of salt, type of dye). Interestingly, Webb and Rothenberger have shown that the temperature of the liquid mixture can affect the color separation, making temperature is an additional variation to explore.

I routinely use this experiment in a fun way to highlight the school colors of Michigan State University (green and white) and the University of Michigan (blue and gold). Because these two schools are rivals – and because I live in Michigan – this experiment often generates a lot of interest in my classes (see Video 1). Isn’t that cool? The green dye dissolves in the alcohol-water mixture but separates into its two component colors (yellow and blue) after K₂CO₃ is added.

Video 1: Michigan vs Michigan State, Tommy Technetium YouTube Channel, May 2024.

For quite some time I have wondered why each dye dissolves in the respective layers: blue in the organic phase and yellow in the aqueous phase. The structure of each dye has provided some insight (Figure 1). Indeed, a variety of characteristics have been identified that increase the probability a substance will dissolve in the aqueous layer as compared to the organic layer in salting out experiments. The most important of these appears to be molecular size,^5-7 but other factors such as ability to hydrogen bond with water and electric charge on the dye also play a role.⁷ The yellow dye outperforms the blue dye in all three of these categories. It is smaller (534 as compared to 792 g mol^-1, Figure 1), has a greater electric charge (-3 vs. -2, Figure 1), and has more hydrogen bond acceptors (13 vs. 11, Figure 1) than the blue dye.

Figure 1: Chemical structures Blue #1 (top) and Yellow #5 (bottom). Each atom capable of accepting a hydrogen bond with water is highlighted in red. One water molecule is shown donating a hydrogen bond to a single oxygen atom in each case. Note that the overall charge of Blue #1 is -2, while that on Yellow #5 is -3.

An examination of how these factors (size, charge, and hydrogen bonding) contribute to greater water solubility is a natural springboard to discussions of intermolecular forces. Therefore, I naturally discuss these when carrying out this demonstration. First, a larger negative charge on the yellow dye than the blue dye sets up stronger ion-dipole forces with water in the former over the latter. Certainly, more sites that can hydrogen bond with water also contribute to greater solubility in water. But how might the fact that smaller molecules dissolve in water more easily connect with the topic of intermolecular forces? Check out the Video 2 to find out!

Video 2: Three Colors in One Simple Chemistry Experiment!​​​​, Tommy Technetium YouTube Channel, May 2024.

In closing, it should be mentioned that several other characteristics of molecules can be used to predict solubility in the aqueous layer in salting out experiments. Some of these additional factors include the C/H ratio, number of oxygen atoms, and number of hydrogen atoms in the molecule.⁷ Perhaps these predictive factors could be used to guide investigations in how to generate various desired color effects in these experiments. Be sure to let me know if you generate some new and interesting two-color combinations in this fascinating experiment.

Happy Experimenting!

References:

1. Kuntzleman, T., Chemical Mystery #8: Go Blue! November 2016
2. Webb, J. and Rothenberger, O., The Salting-Out Effect: A Colorful Demonstration That Leads to Student - Teacher Activities, April 2023.
3. Fleming, D., Battle of the Forces, March 2023.
4. Kuntzleman, T., The Density Bottle Strikes Again, December 2017.
5. Endo, S.; Pfennigsdorff, A.; Goss, K.-U. Salting-Out Effect in Aqueous NaCl Solutions: Trends with Size and Polarity of Solute Molecules. Environ. Sci. Technol. 2012, 46, 1496-1503.
6. Hyde, A. M.; Zultanski, S. L.; Waldman, J. H.; Zhong, Y.-L.; Shelvin, M.; Peng, F. General Principles and Strategies for Salting-Out Informed by the Hofmeister Series. Org. Proc. Res. Dev. 2017, 21, 1355-1370.
7. De Stefano, C.; Lando, G.; Malegori, C. Oliveri, P. Sammartano, S. Prediction of water solubility and Setschenow coefficients by tree-based regression strategies. J. Molec. Liq. Dev. 2019, 282, 401-406.