9 Lab Protocol: Biologically Important Molecules
Test for proteins, sugars, and starch in milk and milk products:
You have begun your journey into learning about the fundamentals of chemistry, chemical bonds, and the functional groups that make up biologically important molecules, or biomolecules. This week’s experiments will allow you to use various chemical reagents to test milk and milk products for the presence of protein, reducing sugars, and starch. Your job is to use the results gathered from each chemical test to try and draw conclusions about the identity of each product.
At the front of the lab, you will find 6 beakers labeled 1 – 6 filled with either a type of milk or milk product. With your group, you will test for the presence of biomolecules in each of the products and attempt to identify the unknown substances based on the results from each test.
The possible substances you are testing are:
- Milk plus protein
- 2% Lactose-free Milk
- Almond milk
- Soy Milk
- Oat Milk
- Regular Milk
Planning your experimental procedures:
As budding scientists, you are learning the process of science while doing it. One skill you will need to practice is planning out the steps of your scientific protocol.
- Skim through the entire protocol – a quick scan of steps and materials is recommended.
- Survey all the materials present at your lab table to assess what lab equipment you will be using. Make sure you are using appropriate lab safety procedures.
- Scan your Student Data Sheet to see what is required as a final product of this lab experiment. Ensure that all group members will be participating. Write down a hypothesis to test for the presence of various biomolecules in milk and milk products.
- Check in with your instructor before you begin to ensure you have not missed any important information.
- As a group, label 6 test tubes with numbers 1-6 and place 5 mL of each milk product from either the front or back of the lab, depending on where your instructor has placed them, into each of the 6 tubes. Take the samples back to your table and begin your experiments. You will use these as your source of sample for each of the followings tests. You will also use distilled water for as a negative control for each test.
Test 1: Using Biuret reagent to test for presence of protein.
The biuret reagent tests for the presence of proteins in a sample. It contains cupric ions that react with peptide bonds in proteins. When added to a sample that contains protein, it will turn purple. If no protein is present, it remains blue.
- Label 8 wells in the ceramic spot plate and then add 50 uL of each sample in the wells. Make sure to include a well for your negative control (water) and your positive control (egg albumin), then label them 1-6.
- Add 250 uL of Biuret reagent to each well. As you add reagent, you may use the pipette tip to mix before discarding it. Make sure to change your pipette tip between each well.
- Record your results in your student data sheet. Take note of the original color of the sample and the color after. Describe the color change specifically in your table.
Test 2: Benedict’s Reagent to test for presence of a reducing sugar.
Benedict’s Reagent is used to detect reducing sugars, such as glucose and lactose, in a sample. Reducing sugars are sugars that can donate electrons to another molecule. Sucrose is a non-reducing sugar because it does not have any free aldehyde or ketone groups. When heated in the presence of reducing sugars, Benedict’s changes color from blue to yellow to orange to red; depending on how much sugar is in the sample.
For this test, you will conduct the experiment in a set of test tubes.
- At your table, you have a hot plate with a beaker of water. Always assume the hot plate is hot! Ensure the hot plate is on by turning the dial to the highest setting and bring the beaker of water to a boil.
- Label another series of test tubes 1-6. Label one test tube with H2O for your negative control and one with glucose as your positive control.
- Add 500 uL of each sample to the test tubes. Then add 1 mL of Benedict’s Reagent to each tube.
- Once your water is boiling, add the test tubes with sample and reagent to the boiling water bath for 2 minutes. Be careful not to drop the test tubes in the water so they do not crack or break. Use a test tube clamp.
- After 2 minutes, remove the test tubes using test tube clamp and place in an ice bath to stop the reaction.
Note that if polysaccharides and disaccharides are heated too long, the glycosidic bond may be broken, resulting in the production of monosaccharides. This may result in a delayed positive for Benedict’s test. All Monosaccharides and some Disaccharides are considered reducing sugars. Lactose & Maltose are both considered a reducing sugar. Sucrose is not considered a reducing sugar based on the structure of the Glycosidic bond. All Polysaccharides are not considered reducing sugars, as they are complex sugars.
Blue color = no reducing sugars
Green color = low level of reducing sugars
Orange color = moderate level of reducing sugars
Red color = high level of reducing sugars
Negative control = distilled water
Postive control = 1% glucose solution
Test 2: Iodine Test to test for the presence of starch.
In the presence of starch, iodine reacts to turn a sample dark blue or black. If there is no starch present, the iodine remains the original brownish yellow color. You will use the ceramic spot plate for this experiment.
- Add 500 uL of each to a well: distilled water, starch solution, and each of the 6 unknown milk products. Make sure your spot plate is labeled properly.
- Use the dropper bottle with potassium iodide (KI) solution to add 2 drops of reagent to each well.
- Record observed results in your Student Data Sheet.
Starch + Iodine = Blue-Black color
Negative Control = Distilled Water
Positive Control = 1 % (w/v) Starch
