Establishing Controls
As you read through each test, fill in Table 1 of the Lab Report and answer the questions underneath it.
- Sudan III Test – Read the explanation in the lab manual.
This is a test that is both qualitative (is the substance present or not) and quantitative – how much lipid is present is indicated by the intensity of the orange color.
Here are the results – negative control on the left and positive on the right.
2 Benedict’s Test – Read the explanation in the lab manual.
Note that test tubes must be boiled to cause color change.
This is a somewhat quantitative test. What does this mean?
The 3 remaining tests in this lab are qualitative only. What does that mean?
Video of this test: Benedict’s Test – YouTube
Results – negative control on the left and positive on the right.
3 Iodine Test – Read the explanation in the lab manual.
Video of this test: Iodine Test for Starch Technical Guide – YouTube
Results – negative control on the left and positive on the right.
4 Biuret Test – Read the explanation in the lab manual.
Video of this test: Biurets test proteins – Bing video
Results – negative control on the left and positive on the right.
5Silver Nitrate (AgNO3) Test – Read the explanation in the lab manual.
Video of this test (shows more than you need – just watch reaction and notice the white precipitate: Halide ions and silver nitrate – Bing video
Results – negative control on the left and positive on the right.
Performing the Experiment
- The experiment was performed for you. The results are in the attachment called “Lab 4 Results for Unknown Foods”. Open that file. The Results Table shows results for all the unknowns analyzed by the class. Keep the file open.
- On the 2nd page of the lab report, locate #2a. Type in your Team (Group) number.
- Each person on the team made predictions on the prelab for 1 of the 4 unknowns (A, B, C, D). On the chart under 2a, you will first enter the results for each person’s unknown letter. You don’t know its identity or how sure you are yet.
To find your results, locate your unknown “code” in the left column of the Lab Results table in the file you downloaded. The letter of the code indicates which of the 4 unknowns you made predictions for. The number is your team number.
Example: C-2 is unknown C for Team 2. B-5 is unknown B for Team 5.
Ignore all other codes.
- On the 2a chart, type in a + for each positive test shown for your unknown. Leave the other cells of the table blank. (For the 2 quantitative tests, you can use +, ++, +++ to indicate intensity of the result if you wish.)
- The numbers of rows you filled in on the table corresponds to the number of people in your group. If you only have 2 or 3 people participating, leave the other rows blank. Double check your results. I’ll be checking them and grading your solution based on what should be there.
Analyzing Results
- Look at the predictions you made in Table 2 of the prelab. Based on the experimental results you just entered, determine the identity of your unknown and fill it in on the table.
If there is more than 1 possibility for your unknown, take a guess (I won’t count it wrong if it’s 1 of the possibilities). - If there was only 1 possible answer for an unknown, check off that you’re sure. If there is more than one, go to the next step.
- See 2b on the lab report. Ignore the first table since the results provided were clear. (In real life, sometimes a test is hard to interpret.)
Fill in the bottom table for any unknown(s) for which there was more than 1 possibility. Type in the letter for the unknown(s) you weren’t sure about. You took a guess for the first table. On this one, list the other possibility or possibilities.
For example, if my results indicated that my unknown could be either potato starch or cornstarch, I could have identified as potato starch on the team results table, but now I’ll list the other possibility.
Lab Report 4: Identifying Unknown Foods
Lab Report 4: Identifying Unknown Foods
Manager _____________________________ Recorder ______________________________ Lab Tech _____________________________ Computer Tech _______________________
1a. Complete the following summary tables for the 5 biochemical tests performed in this experiment. • Write in the blank the category of substance being tested for. • Indicate what was used as the negative/ positive controls. • Record your results indicating presence or absence of the substance being tested.
Test 1: Sudan III Test for ___________ Final Color Interpretation of Results Negative control: Positive control:
Test 2: Benedict’s Test for ____________ Final Color Interpretation of Results Negative control: Positive control:
Test 3: Iodine Test for ______________ Final Color Interpretation of Results Negative control: Positive control:
Test 4: Biuret Test for _______________ Final Color Interpretation of Results Negative control: Positive control:
Test 5: Silver Nitrate Test for __________ Final Color Interpretation of Results Negative control: Positive control:
1b. The Benedict’s Test is quantitative. Explain. _______________________________________________ _________________________________________________________________________________________ 1c. Which test must be boiled before the results can be recorded? ___________________________ 1d. Name a test result based on solubility. ___________________________________________________
2 Lab Report 4: Identifying Unknown Foods
2a. Team’s unknown # _________ Complete the table by indicating the identity of the unknown and results (positive or negative) for each biochemical test. Your grade is not solely dependent upon the correct identification of your unknowns. But, your conclusions must be in agreement with your results. Managers, have everyone double-check that the results match the identity of each member’s unknown. Identity of Unknown SURE NOT SURE Sudan III Test Benedict’s Test Iodine Test Biuret Test Silver Nitrate Test A B C D
2b. If you have unknowns in the “not sure” column, fill in one or both of the tables below.
(1) If you had test results that were ambiguous, complete the following table.
Unknown Test(s) with ambiguous results. Describe ambiguity.
(2) If the test results for your unknown indicated more than one possible food item, complete the following table.
Unknown Possible Food Items
Lab 4: ldentifying Unknown Foods
(U Food manufacturers are required to disclose the contents of their product(s) on a nutrition label. These labels include information such as the amounts of protein, fat, and carbohydrate (sugar versus starch). Table l1 indicates the chemical composition of the various foods that will be tested in this lab.
PROBLEM OF THE DAY
Determine the identity of each unknown food by comparing the results from each biochemical test with the information found in Table 1.
Table1: Composition of Foods Grams per 100 grams edible portion (or %) AMOUNTS IN PARENTHESES NOT DETECTED IN TESTS
7%Protein % Fats halide (0.3) (0.1) 10.9
Food % Water Calories % Carbohydrate
(0.5 0 Corn Starch* 8.3 381 90.4 starch 0 0 89.3 87.6 10.0 glucose + fructose (0.7 sugars (0.6 sugars) 10.7 starch Cola 37 Egg White Egg Yolk Almond Flour (0.2) 26.5 48 (0.1) 15.9 317 671 52.3 12 25 1.5 0 85.6 (0.1) 0 Gelatin (dry) Honey Instant Coffee 13 335 (0.3) (0.1) 81.2 glucose +fructose (0.3) 56.3 sUcrose only 21.5 glucose + fructose 73.7 starch 17.1 304 99.1 2 0 (0.2) Maple Syrup Pancake Syrup Potato Starch* 32.1 261 38 234 0 0 0 6.5 357 (0.4) 0 Table Salt 0.2 0 0 0 100 319 83.9 sUcrose 0 Table Sugar L Whole Milk source: USDA Nutrient Data Laboratory. htte://www.nal.usda.gov/frnic/foodcomp/search/ 15.6 33 88.3 60 5.3 lactose 3.2 3.3 (<0.1)
In this experiment, you will perform these 5 tests in order to identify your team’s unknowns: Sudanlill Test lipids (oils/fats) Benedict’s Test reducing sugars which include fructose, glucose, lactose, but NOT SUcrose lodineTeststarch Bluret Test proteins Slver Nitrate Test halide ion (example – chloride)
Foods marked with an asterisk will appear cloudy white in solution. To distinguish between this color and a positive silver nitrate test, compare your silver nitrate test to the remaining stock solution of your unknown. If it looks the same, the test is NOT positive.
Lab 4: Identitying Unknown Foods 15
5. Use your forceps to hold the disc under running water. Rinse off all the excess Sudan l. Blot dry. 6. Record the intensity of red color as -, +,
IMPORTANT: Each team member should have made predictions in the prelab for what his/her unknown could be. Those predictions MUST be corect if you are to comectly identify your unknowns. Before starting the lab, check each other’s’ predictions for accuracy.
++, +++. Use 8 clear test tubes to run the remaining 4 tests. Label each tube according to the test performed and whether it is the positive (+) or negative (-) control. Establishing Controls The following 5 biochemical tests will be performed on each unknown in order to determine the identity of the food item. Howeve, before you get your unknowns, perform the following tests on appropriate controls. Keep the controls to refer back to as a basis of comparison when you start testing your unknowns. Turn on hot plates to a setting of “4” NOW. You will need a boiling water bath for the Benedict’s Test. This test is perfornmed to 2. BENEDICT’s TEST: determine the presence of reducing sugars which include all monosaccharides and some disaccharides, but NOT SUcrose. Benedict’s Test is both quUantitative and qualitative becaUse it wll tell you if a reducing sugar is present as well as how much is present: blue = no reducing sugar green= trace amount, yellow = small amount, orange = large amount, red = saturated. 1. SUDAN II TEST: This test is performed to determine the presence of lipids. Sudan l is a red, nonpolar (hydrophobic) substance. Because hydrophobic substances easily mix, Sudan Ill will bind to any nonpolar substance, giving that substance a definite red color. hydrophilic substance shows little, if any, red stain. Sudan Ill will not bind to Substances, therefore the For this test, indicate the following. negative control:
For this test, indicate the following. positive control: negative control: 1. Use the 3 ml pipette to transfer 2 mL of Sample in (+) and (-) test tubes labeled Benedict’s. positive control 1. Get a flter paper disc. Use a pencil to draw two circles about the 2. Add an equal amount of Benedict’s Reagent. 3. Place tube in boiling water for 3-5 minutes, leaving the test tube clamp on. 4. Note the color of the final solution. O+ size of a penny, labeling one “+” and one “”. O (See drawing.) 2. Use 2 different capillary tubes to spot the positive and negative controls inside their circles. Let the spots dry, and apply This test is performed to 3. loDINE TEST: determine the presence of starch. If starch is presence when iodine is added, the solution will turn blue-black. If starch is not present, the iodine will not react and the solution will remaina faint yellow. a second time. 3. Once both drops are dry., place the filter paper ina dish of Sudan lI. Let it soak for 15 minutes. For this test, indicate the following. IMPORTANT: While the disc is soaking, run the controls for the other 4 chemical tests. negative control: positive control: 4. After soaking for 15 minutes, use forceps to remove the filter paper. Place it on a paper towel and camy it to a sink. 1. Put 2 mL of sample in test tube. 2. Add 5-10 drops of iodine. 3. Note the color of the final solution.
16 Lab 4: Identifying Unknown Foods
with the letter of the
4. BIURET TEST: This test is performed to If determine the presence of proteins. protein is present, the Biuret reagent will turn a pale purple color. If protein is not present. the solution will appear a pale blue color.
unknown.. (See drawing Each person will Use a different capillary tube to
sample Obtain the sample from the original tube.
O O OOn their unknown.
For this test, indicate the following.
While the disc is soaking in the Sudan 1, perform the other 4 chemical tests.
negative control:
positive control: .
Collecting Data 1. Put 2 ml of sample in test tube. 2. Add 5-10 drops of Biuret reagent. 3. Note color of finail solution. 1. Everyone, write your letter in the blank above Table 2.
5. SiLVER NiITRATE TEsT: This test is performed to determine the presence of salt. If salt is present when the silver nitrate is added, a white precipitate will sink to the bottom.
2. Record your results in Table 2. Each team member must fill in a separate table for his/her unknown. Table 2: Results for your unknown food:.
For this test, indicate the following. Final Color Test Results Test negative control: of Unknown |(+, ++, t++)| positive control: . Sudan Ill
1. Put 2 mL of sample in test tube. 2. Add 2 drops of silver nitrate (AgNOs). 3. Note if a white precipitate forms.
Benedict’s
lodine
Biuret
Performing the Experiment
Silver Nitrate NOW, perform the following 5 tests on your NOTE: Only positive results from Sudan Ill and Benedict’s Test can be recorded as ++, +++. unknowns. (1) Sudan ll Test for lipids (2) Benedict’s Test for reducing sugar (3) lodine Test for starch (4) Biuret Test for protein (5) Silver Nitrate Test for salt Analyzing Results Now determine which food substance you have been working with. 1. Compare your Table 4 results with the predictions you recorded in Table 2 in order to identifyY your unknown. Some of the predictions you made in Table 3 may be identical researchers often use real life experiences and common sense when analyzing results. You may have to do the same. 1. Everyone, thump your unknown several times to thoroughly mix the contents. 2. In order to have unknown samples for each of the tests, use a 3 mL pipette to transfer approximately 2 mL of the unknown into 4 tubes of the same colo. Some of the unknown will be left in the original tube. Keep in mind that 3. Perform all 5 biochemical tests, but set up the Sudan Ill Test first since it requires a 15 minute wait. This time, the Sudan lI Test will be performed on one disc of filter paper for the entire team. To do this, draw a circle for each person’s unknown. Label each circle 2. Report out your results for your unknown so that everyone in your group can record the information in the table under part 2a of the Lab Report.
Lab 4: ldentiying Unknown Foods 17
5. Then place all the tubes into the dirty test tube bin. CLEAN UP
6. Put all the corks in the cork container located tubes: on the side counter.
1. DO NOT empty the dish of Sudan l.
7. Throw away all used pipettes and the Sudan ll discs.
2. DO NOT empty the hot water bath.
3. Take both racks of tubes to the sink area.
8. Restock test tubes.
4. Empty all test tubes into the appropriate waste container.
small test tube rack 8 clear test tubes arge test tube rack 4 of each color
18 Lab 4: ldentifying Unknown Foods
