Build Instructions

Collapse

• 1

  Making Benedict's Reagent

  The easiest way to test the functionality of amylase is to see if it can actively breakdown a di- or polysacchiride into a simple sugar, and that is by utilizing the Benedict's test. This test includes the use of Benedict's reagent, which will change color from a transparent, blue solution to a range of colors between green and brick-red.

  In order to make 100 mL of Benedict's Reagent (BR), you will need:

  - 17.3g of Na3C6H5O7 (sodium citrate)

  - 10g of Na2CO3 (sodium carbonate anhydrous)

  - 1.73g of CuSO4 * 5H2O (copper sulfate pentahydrate)

       *these chemicals should be weighed out either on watch glasses or on weigh boats*

  - distilled or RODI water

  - one 100mL volumetric flask OR measuring cylinder

  - two 80mL beakers

  - two glass stirring rods

  - a small funnel

  - a bottle or container with a close-fitting seal to store the BR in after making it

  - a hot plate or Bunsen burner

  - two scoopulas
  

  Once you have these items, the procedure for making the BR is as follows:

  1. add 17.3g of sodium citrate and 10g of sodium carbonate to one 80mL beaker

  2. add 50mL of distilled or RODI water to the beaker and stir the contents together (can be accelerated by using a burner)

  3. in the other 80mL beaker, add 1.73g of copper sulfate pentahydrate

  4. add 10mL of distilled or RODI water to the beaker and stir the contents together

  5. slowly add the copper sulfate pentahydrate solution to the sodium citrate-sodium carbonate solution, making sure to stir the solutions together after each small increment is added

       *for the following steps, take care to not exceed 100mL of mixture, as this will tamper with ruin the batch

  6. rinse the stir rod and beaker used for the copper sulfate pentahydrate solution using distilled or RODI water and pour the rinse into the combined solution to ensure that all of the copper sulfate pentahydrate is added

  7. add the combined solution to a 100mL volumetric flask or measuring cylinder using a funnel

  8. rinse the beaker and the funnel using distilled or RODI water and pour the rinse into the volumetric flask or measuring cylinder

  9. fill the remaining solution with water until it reaches the 100mL mark exactly

  10. cap the solution and mix the contents together thoroughly by inverting multiple times

  11. transfer the solution into a separate container labeled "Benedict's Reagent" and seal tightly before storing
  

  Once you have done this, you now have Benedict's Reagent and are able to carry out a Benedict's Test to test the presence of reducing sugars and, subsequently, the activity of amylase. It is best to use Benedict's Reagent promptly after making it, but if you need to store it for later, I recommend a sealed container and refrigeration to ensure its stability and preservation.

• 2

  Testing Surfactants with Benedict's

  Since Cu2O nanoparticles (the product of the Benedict's reaction that provides the brick-red color of a positive result) are unstable, we will be attempting to stabilize these with several surfactants. We will be using several surfactants in the presence and absence of the starch 1) to test the ability of each surfactant to remain stable within Benedict's reagent (BR) and 2) to ensure that the starch mixture won't react with BR on their own without amylase. 

  Below is the list of surfactants used and the quantities used to make the corresponding concentration in 50mL of water:

  A) Polysorbate 20     -     0.028mL (0.0005M)

  B) Polysorbate 80     -     0.031mL (0.0005M)

  C) Glycerol monostearate     -     0.0090g (0.0005M)

  D) Triton X-100     -     0.084mL (0.0005M)

  E) Lauric acid     -     0.0101g (0.001M)

  F) Dodecanol     -     0.0093g (0.001M)

  G) Diaminododecane     -     0.0100g (0.001M)

  H) Brij L-4     -     0.095mL (0.0005M)

  J) Polyvinylpyrrolidone (PVP)     -     0.0556g (0.0005M)

  In order to test the ability of each surfactant to remain stable in BR and to stabilize Cu2O nanoparticles, we will perform two tests parallel to one another. For each surfactant, we will make a solution of just BR and surfactant and we will make a solution of BR, surfactant, and starch. The results of the former test ideally should show that the BR remains unchanged, meaning that the surfactant did not react with the BR. The results of the latter test ideally should show minimal color change, maintaining its predominant light blue color. 

  To test the stability of the surfactants in the BR, the procedure is as follows:

  1. add 4mL of BR to a 20mL glass vial

  2. using a hot plate, bring the solution to a boil

  3. remove the vial and add the premeasured Polysorbate 20 (A)

  4. mix the solution and observe any change in the solution

  5. cap and label the vial and store it in a cool, dark place

  6. repeat steps 1-5 for the remaining surfactants (B-J)

  To test the starch in BR, the procedure is as follows:

  1. add 4mL of BR to a 20mL labeled glass vial

  2. using a hot plate or Bunsen burner, bring the solution to a boil

  3. add the premeasured Polysorbate 20 (A, see above)

  4. add 0.4mL of test sample (made by combining 100mL of DI water, 4.5850g flour, and 1.287mL of fruit pectin)

  5. mix the solution together and observe any color change

  6. return to the hotplate or Bunsen burner and bring to a boil

  7. remove the vial and mix the solution, observe any color change

  8. cap the vial and store it in a cool, dark place

  9. repeat steps 1-8 for the remaining surfactants (B-J)

  Once both of these procedures are complete, leave the solutions sitting for 24 hours to observe if the color has changed in that period of time. Be sure not to subject the solutions to too much movement or environment change while they are resting, as this may disturb the solutions and could disrupt the stability of the BR and the nanoparticles. Take pictures before and after to see any change.

  After these mixtures were left overnight, we found that a few of the surfactants would not be viable for the test and excluded them from further testing. We moved forward with Polysorbate 20, Polysorbate 80, glycerol monostearate, Triton X-100, and PVP.

• 3

  Benedict's Reagent, Successful surfactants, and Reducing Sugar

  This part of the experiment happened in two stages: testing to see if Benedict's Reagent (BR) would still function when diluted; and seeing how the surfactants are at stabilizing the copper oxide nanoparticles to prevent them from oxidizing. 

  The first part was done under the thought that if there is less BR present in the reaction, the rate of reaction will be slower and the nanoparticles produced from the reaction would be smaller. We wanted to see how far we could dilute the BR and still have a positive result when introducing it to a reducing sugar, so we tested a 1:2 part dilution, a 1:5 part dilution, and a 1:10 part dilution.

  To test the BR dilutions, the procedure is as follows:

  1. using a pipette, add 5mL of distilled or RODI water to a 20mL glass vial labeled 1:2 dilution

  2. using a pipette, add 5mL of BR and mix together to make the 1:2 part BR dilution

  3. using a pipette, add 8mL of distilled or RODI water to a 20mL glass vial labeled 1:5 dilution

  4. using a pipette, add 2mL of BR and mix together to make the 1:5 part BR dilution

  5. using a pipette, add 9mL of distilled or RODI water to a 20mL glass vial labeled 1:10 dilution

  6. using a pipette, add 1mL of BR and mix together to make a 1:10 part BR dilution

  7. to make the test sample (1M honey solution), add 70mL of water to a beaker

  8. add 14g of pure honey to the beaker

  9. stir until the honey has completely dissolved in the water

  10. using a Bunsen burner or hot plate, bring the 1:2 part BR dilution to a boil
  

  11. remove the vial and add 1mL of the 1M honey solution

       *I chose to photograph and videotape the reactions to see what they looked like and how long it took for each one, but that is not necessary for the procedure, though it is nice to look back at for logistics and filing*

  12. repeat steps 10 and 11 with the 1:5 and the 1:10 part BR dilution

  For all of these tests, the BR reacted with the honey solution to produce a dark orange color. The particulate matter was the most fine in the 1:10 part dilution, so we are moving forward with that, as it has the smallest nanoparticles produced with the most diluted sample of BR.

  For the next stage of this experiment, we are testing the surfactants that we tested with starch earlier (see above instructions) and their ability to stabilize the copper oxide nanoparticles in solution after the BR reaction takes place. This was done using Polysorbate 20, Polysorbate 80, Glycerol monostearate, Triton X-100, and PVP, each mixed into BR, followed by the addition of a 1M corn syrup solution.

  To test the surfactants in a BR and corn syrup solution, the procedure is as follows:

  1. add 45mL of distilled or RODI water to a 100mL beaker
  

  2. add 5mL of BR to the beaker (this will make a 1:10 BR solution)

  3. vacuum the gas out of the BR solution (*this step is recommended, as it helps to stabilize the copper oxide from oxidizing further and precipitating out of solution*) 

  4. immediately after vacuuming, add 10mL of the sample into five 20mL glass vials with a lid and screw the lid on tightly

  5. weigh out all of the surfactants (0.5mM of each)
  

       a. Polysorbate 20 - 0.0061g

       b. Polysorbate 80 - 0.0066g

       c. Glycerol monostearate - 0.0179g

       d. Triton X-100 - 0.0324g

       e. PVP - 0.0111g

  6. add 50mL of water to a 100mL beaker

  7. add 9g of corn syrup and stir until the corn syrup is completely dissolved in the water

  8. label each of the five 20mL glass vials with the surfactant to be used

  9. bring the 'Polysorbate 20' BR solution to a boil using a Bunsen burner or a hot plate

  10. remove from heat to add the premeasured Polysorbate 20 to the vial and mix

  11. add 1mL of the 1M corn syrup solution to the vial and mix

       *I chose to videotape the reactions to see what they looked like and how long it took for each one, but that is not necessary for the procedure, though it is nice to look back at for logistics and filing*

  12. repeat steps 9-11 for the remaining surfactants

  13. take pictures of the completed reaction to compare the solution to a later time to prove stability
  

  14. store in a dark place overnight or for 24h

  15. take pictures of the mixtures again to see if each of the solutions' nanoparticles were stabilized by the present surfactants

View all 5 instructions