321L

Carbohydrate Chemistry 1 Week Version Background

Winter 2011 Dr. Tippetts

Five to seven carbohydrate tests will be performed to identify two given carbohydrate unknowns. Positive and negative results will be observed The color of any precipitate should be recorded. In each test, there will be up to 10 known carbohydrates tested. These 10 known carbohydrates are: Six monosaccharides a: Two pentoses: ribose (rib) and arabinose (ara) b: Four hexoses: fructose (frc), glucose (glc), galactose (gal), and mannose (man) Three disaccharides: lactose (galglc), sucrose (glcfrc), and maltose (glcglc) One polysaccharide: starch Three of the carbohydrate tests will use 10% carbohydrate solutions. One of the tests will use 1% carbohydrate solutions. For stock solutions, the 10% and 1% solutions for known carbs have already been prepared in the lab. The protocol will instruct in the use of 10% or 1% carbohydrate solutions.

Step 1: Prepare two 10% unknown solutions, which will be used in two tests today and more tests next week. Treat these as stock solutions! (see page 3) Step 2: Perform the Benedicts test on 10 known and the unknown carbohydrate solutions Step 3: Perform the Barfoeds test on 10 known and the unknown carbohydrate solutions Step 4: Perform the Seliwanoffs Test. Use the 10% solutions. Do this test in the safety hood!!! Step 5: Prepare a 1% stock solution of both your unknowns, which will be used in the Bials Orcinol test. Step 6: Perform the Bials Orcinol Test. Use the 1% carbohydrate solutions in this test. Do this test in the safety hood!!! Step 7: Perform the glucose oxidase test on your unknowns, to test, or to confirm the presence of glucose in your unknowns Perform the starch iodine test on your unknowns, to test, or to confirm the presence of starch in your unknowns. 1

Protocol
Step 1: Preparation of the two 10% UNKNOWN solutions Prep each unknown solution separately. Weigh out 0.5 grams unknown and place the 0.5 grams into a clean test tube ** PLEASE DO NOT USE CUVETTES ** Dissolve the 0.5 grams of the unknown into 5.0 mL of DI H2O (0.5 g / 5.0 mL = 10% w/v) You can use a pipet or a graduated cylinder to measure the 5.0 mL Label the test tube Unknown #______ Stock 10% IMPORTANT: Treat the test tubes as a stock bottle for the 10% unknown #_____ Once both unknown solutions are prepared, reagents will not be added to this test tube Step 2: Benedicts Test This test differentiates reducing agent saccharides from non-reducing agent saccharides Test all 10 known carbohydrates and 2 unknowns (need a total of 12 test tubes) Use 2 to 3 drops of the 10% solutions (note: for starch, its ok to use 1% solution) Positive results: red precipitate (Cu2O) Negative results: solution remains blue There should be 8 reducing carbohydrates in this experiment.

1. Use test tubes. PLEASE DO NOT USE CUVETTES Youll need 12 test tubes total, labeled with the appropriate carbohydrate being tested. 2. Add 2 mL Benedicts reagent to each tube. 3. Add 0.2 mL (2-3 drops via dropper) of the 10% carbohydrate solution to the appropriate tube. 4. Mix well. Place test tubes into boiling water bath 5. Note any changes in color of the solutions; note any precipitate formation

Step 3: Barfoeds Test This test differentiates reducing agent from non-reducing saccharides (like Benedicts test) This test will also demonstrate is a difference in a reducing agent monosaccharide from reducing disaccharide This is because Barfoeds takes longer for some saccarides. If a saccharide was negative on the Benedictss test, it should also be negative on the Barfoeds test. If a saccharide was positive with Benedicts, but appears negative on this Barfoeds test, rely on the Benedicts test results to correctly describe the saccharide as a reducing agent. Test all 10 known carbohydrates and 2 unknowns (need a total of 12 test tubes) Use 2 to 3 drops of the 10% solutions (note: for starch, its ok to use 1% solution) Positive results: (+++) small to moderate red precipitate (Cu2O) relatively quickly (1-2 minutes) Slight or slow positive: (+) less amount of red precipitate, or the same amount of red precipitate as monosaccharides, but it takes longer to form (5-6 minutes). Negative results: () no red precipitate, and the solution remains blue 1. Youll need 12 test tubes total, labeled with the appropriate carbohydrate being tested. 2. Add 2 mL Barfoeds reagent to each tube. 3. Add 0.2 mL (2-3 drops via dropper) of the 10% carbohydrate solution to the appropriate tube. 4. Mix well. Place test tubes into boiling water bath (record a start time; see step 6) 5. Note any changes in color; note any precipitate formation 6. If a change (a precipitation) occurs, note the approximate time of any change occurring. If and when the solution forms a small red clump, it can be recorded as positive. Once this clump forms, the tube can be removed from the hot water bath. 7. Allow tubes to sit for around 15 minutes before recording a negative result in the data table. 8. Be sure to note the approximate time AND relative amount difference (to better enable ID of unknown) Note: for this test, the time should not be treated as an exact measurement, but rather 3

treated as an estimate to differentiate a faster reaction from a slower reaction. Step 4: Seliwanoffs Test This test differentiates both fructose and the fructose containing disaccharide from the other saccharides. This test also differentiates fructose from the disaccharide of fructose and glucose, using observations of the amount of precipitate and/or the time occurs. Test 5 known carbohydrates and 2 unknowns (a total of seven test tubes) 5 known carbs tested: glucose, fructose, sucrose, maltose, and starch (label the test tubes) Use the 10% stock solutions (note: for starch, it is ok to use the 1% stock solution) IMPORTANT: The heating will be done in the safety hood Positive results: dark orange, cloudy solution Negative results: light orange solution 1. Use 7 test tubes. PLEASE DO NOT USE CUVETTES Youll need 6 test tubes total, labeled with the appropriate carbohydrate being tested. 2. Add 2 mL Seliwanoffs reagent to each tube. 3. Add 0.2 mL (2-3 drops via dropper) of the 10% carbohydrate solution to the appropriate tube. 4. Mix well. Place test tubes into boiling water bath in the hood (record a start time; see step 6) 5. Note any changes in color; note any precipitate formation 6. Note the approximate time of any change occurring (if it occurs) 7. Allow tubes to sit for 1 minute before recording a negative result in the data table. 8. Be sure to note the time AND amount difference (to better enable ID of unknown); note any other colors present

Step 5: Preparation of the two 1% UNKNOWN solutions Have two clean test tubes, one per each unknown. Use the 10% unknown solution prepared last week as the starting material. Transfer 1.0 mL of this 10% unknown solution into a clean test tube. Add 9.0 mL DI H2O to the 1.0 mL of the 10% unknown solution Label this test tubes 1% unknown (#) Stock 1% unknown #______ IMPORTANT: Treat this test tube as a stock bottle for the 1% unknown. Once this solution is prepared, reagents will not be added to this test tube

Step 6: Bials Orcinol Test This test will differentiate a pentose from a hexose. Also, by noting the approximate time of a color change, and the specific color changes that are initially observed, this test can differentiate arabinose from ribose. Test 6 known carbohydrates and 2 unknowns *(a total of 8 test tubes) 6 known carbs: arabinose, ribose, glucose, fructose, sucrose, and starch IMPORTANT: Use the 1% stock solutions IMPORTANT: The heating will be done in the safety hood

Positive result: for pentoses, the solution will turn a dark color relatively quickly Note: some hexoses, and some disaccharides will form precipitate; their solution turn a relatively light brown color at first; if your unknown mimics the results of the known hexose in this experiment, this result should not be used to identify the unknown. Negative results: solution initially remains light orange NOTE: ALL EIGHT SAMPLES WILL EVENTUALLY CHANGE TO A DARK COLOR. If your group allows all the samples being tested to become the same color, this will not enable using this test to identify an unknown; Therefore it is not recommended that you wait until the end of this test to make an observation (unlike the other tests).

Step 4: Bials Orcinol Test (continued) SHORTER NOTE: Note the color change as it happens! Note both the color, and which pentose had a positive result faster; was there a slight color difference between the pentoses 6 known carbs tested: arabinose, ribose, glucose, fructose, sucrose, and starch 1. Use 8 test tubes. 2. Add 0.2 mL (2-3 drops via dropper) of the 1% carbohydrate solution to the appropriate tube. 3. Add 2 mL Bials reagent to each tube. DO THIS IN THE HOOD. 4. Mix well. 5. Important: Allow the water bath to reach a boil before placing the test tubes in the water bath. 6. Place test tubes into boiling water bath (record a start time; see step 7) 7. Note any changes in color and any precipitate formation and the approximate time of the change. Record the color or the change as an observation.

8. Note the approximate time of any change occurring (if it occurs) 9. Allow the tubes to sit for 3 minutes before recording a negative result in the data table.

10. Be sure to note the approximate time AND amount difference (to better enable the identification of the unknowns) Which pentose changed color first? Note the color that is present at the time of change. The color of all of the carb solutions that are being tested will eventually end up being similar to each other at the end of this experiment. 11. For ambiguous positive results, repeat this test with your unknown(s), and arabinose and ribose, to confirm and/or differentiate which pentose your unknown likely is.

Step 7:

Glucose Oxidase and Starch Iodine Test

Perform these tests to confirm the unknown as glucose or starch. You should use a positive and negative control for glucose oxidase