When Banting and Best set out to perform their experiments, glucose levels in blood were measured with the Lewis and Benedict method. This method relied on judging how red a solution appears compared to a standard solution that provided a standard red color.
Blood is a complex mixture of cells, proteins, and small molecules. At the time, measurements of glucose, a small molecule, required that the cells and proteins were first removed from the blood. Complicating this analysis, blood quickly forms a clot when exposed to air, so the blood must be first treated with an anticoagulant.
The Lewis and Benedict method was based on the observation that when glucose is added to a solution of picric acid and sodium carbonate, then heated, a characteristic red color forms. With just a small amount of glucose added, only a slight hint of red is visible in the solution. When a large amount of glucose is added, a deep red forms.
It’s easy to see how this assay could be made quantitative. First, you would prepare a set of say, ten, standard solutions. Each standard would have a different concentration of glucose. When tested with the Lewis and Benedict method, these ten standard solutions would produce a range of red colors, from a pink (just a hint of red) to a deep red. Second, you test a blood sample, containing an unknown quantity of glucose, with the Lewis and Benedict method. Finally, you find the closest standard red to your unknown red. This procedure provides an estimate of the glucose content in your blood solution.
Lewis and Benedict’s innovation was that they combined two steps of the reaction into one. Blood cells are easily removed with a filter, or by sedimentation. Removing the protein, but not the small molecules is much more of a challenge. Lewis and Benedict found that the blood protein would precipitate, or fall out of solution, when picric acid is added to blood. Coincidentally, picric acid reacts with glucose under the correct conditions to form picramic acid salts, which have a characteristic red color.
Lewis and Benedict’s procedure required a series of steps. First, 2 ml of blood was drawn from a needle into a container with some potassium oxalate, an anti-coagulant added to prevent clotting. This blood was added to 5 ml of water and shaken vigorously. The addition of this much water to blood causes the salt content to drop, which in turn causes the blood cells to burst. This is the first step of removing the cells from the blood. Now, 15 ml of picric acid are added to the blood/water mixture. This picric acid causes the protein to precipitate, or fall out, of solution. Thus completing the second step, removal of protein from the blood. Finally, a combination of picric acid and sodium carbonate were added to the container. This container was heated to boiling, to evaporate some of the solution, then cooled and filtered through cotton into a chamber. This process leads to the formation of minute red crystals of picramic acid. Finally, the color of the solution in the chamber, which is a measure of the number of crystals of picramic acid, is compared to a standard to determine the amount of glucose in the initial blood sample.