Much of my research has focused on answering questions about why birds eggs possess colorful pigments. In recent years many others have begun to ask similar questions. Unfortunately, few of us have tried to understand the pigment behind the color. Luckily there are many types of simple experiments that can be run at home to help understand pigments. I've posted two videos documenting my attempts at examining pH dependent color change in red cabbage and biliverdin from an emu's eggshell.
The first titration experiment shows strongly pH dependent color. First, the pigmented liquid is made more acidic with household white vinegar. Then, the solution is made basic with household cleaning ammonia. This is a particularly good example. The experiment with biliverdin was slightly more difficult. First, I needed to extract biliverdin from the eggshell. In the lab, I have found that the protocol outlined by Gorchein et al. 2009 Biomedical Chromatography 23:602-606 works well. Today, I limited these extractions to only materials that can be found commonly at home. The eggs, are harder to come by, but emu farms are becoming more popular.
I first crushed the eggshell into a fine powder with a mortar and pestle. Then I used substituted contact cleaning solution for the EDTA buffer used to clean the shell fragments. Household ammonia can be used to adjust the pH to 7. Litmus paper can be difficult to find, but sporting good stores sell pH indicator litmus strips for testing pools and hot tubs. I was given an old centrifuge that our department was disposing of, and it came in handy here (it also helped Dave Wilson in the field). After mixing well, the samples in buffer were centrifuged. This was repeated 3 times. Then I made a solution of 8:9 isopropanol-white vinegar (in lieu of 4:1 acetonitrile-acetic acid). The ratio here will depend on the amount of acetic acid in your vinegar. Since I was familiar with the 4:1 acetonitrile-acetic acid mixture, I used smell to help optimize this mixture. This is then added to the cleaned powder then vigorously mixed. That is centrifuged for two minutes, and the blue-green supernatant is removed. Chromatography paper (held in this solution for ~ 3 hours) detected one nice blue-green band.
When I repeated the titration experiment with the biliverdin, I saw very little color change, despite changing the extract from highly acidic to highly basic. The next step will be to think about what this says about biliverdin in solution, so that we may apply it to how biliverdin behaves within the shell.
