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What Does Biuret Test For? - Method & Equation

Instructor: Laura Foist

Laura has a Masters of Science in Food Science and Human Nutrition and has taught college Science.

In order to test for the amount of proteins in a sample the biuret test can be used. We will learn how this test is performed and how to translate that to protein concentration.

The Biuret Test

Next time you're looking at the back of a food package take note of the nutrition facts panel. You'll see the usual things: total fat, carbohydrates (and of course sugar), sodium, and so on, all with their daily percentages. At the bottom of the list, though, is protein. You might be wondering: how did they determine how much protein is in this food you're looking at? One of the methods is through the biuret test.

The biuret test measures peptide bonds in a sample. Recall that proteins are made up of amino acid connected together with peptide bonds. So the biuret test can't test for individual amino acids, because those aren't connected with a peptide bond.

The biuret complex combines copper II with the peptide bonds, changing the color of the sample
Biuret complex

In an alkaline solution copper II is able to form a complex with the peptide bonds. Once this complex has been formed, the solution turns from a blue color to a purple color. The deeper the purple color, the more peptide-copper complexes that have been formed.

Method

First, a standards needs to be made. Typically this is done using de-ionized water for a negative protein test, and albumin egg whites for a complete positive protein test. Once we know what the blue color looks like in a negative test and the purple color in a positive test, then we can start testing unknowns. Here are the various tests listed below for you to look at:

  • Add sodium hydroxide to the sample; this makes the sample alkaline
  • Add copper (II) sulfate solution to the sample
  • Watch color change
  • Measure color using spectrophotometer

The last test (using the spectrophotometer) is so that we can measure exactly how much protein is present. We can't tell exactly what shade of purple or blue is present, so we use a machine called a spectrophotometer that can tell us the exact shade of purple present.

A tabletop spectrophotometer used to measure the shades
spectrophotometer

Beer's Law and Equation for the Biuret Test

According to Beer's Law, the absorption reading from the spectrophotometer is directly proportional to the amount of protein in the sample. In order to calculate the equation for Beer's Law, we need to first run several known samples. This gives us a line to base our samples on.

Let's say that we made up 5 samples, one with 0 mg/mL protein, 0.1 mg/mL protein, 0.4 mg/mL protein, 4 mg/mL protein, and 10 mg/mL protein. The test tubes looked like this:

Test tube colors

First, we set the spectrophotometer to 550 nm. We use 550 because this is the wavelength of color observed for a complete protein. So in other words, we're measuring how much of this wavelength is in each sample. When we measured them in the spectrophotometer we got these results:

0 mg/mL = 0.04 abs

0.1 mg/mL = 0.08 abs

0.4 mg/mL = 0.12 abs

4.0 mg/mL = 0.3 abs

10 mg/mL = 0.6 abs

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