LAB REPORT

Exercise 6

Biologically Important Molecules: Carbohydrates, Proteins, Lipids and Nucleic Acids

Student: Jim Goetz Instructor: Dr. Spiros P Katsifis

Lab Section 12

February 6, 2012

Purpose:

-Perform tests to detect the presence of carbohydrates, proteins, lipids and nucleic acids -Explain the importance of a control in biochemical tests -Use biochemical tests to identify an unknown compound

Background:

Characteristic subunits and groups impart different chemical properties to macromolecules. Because of this, these macromolecules can be identified by solubility v. non-solubility.

Procedure 1 Perform Benedicts test for reducing sugars

Introduction Benedicts test allows for the detection of the presence of reducing sugars (sugars with a free aldehyde or ketone group). All monosaccharides are reducing sugars since all of them have active carbonyl group. Some disaccharides that are exposed to a carbonyl group are also reducing sugars but less reactive than monosaccharides. By mixing the sugar solution with Benedicts solution and heating them, a redox reaction will occur. The copper (II) sulphate present in Benedicts solution reacts with electrons from aldehyde or ketose group of reducing sugars to form cuprous oxide, a red brown precipitatate.

Material Methods: The materials used in order to detect starches was: test tubes, distilled water, Benedicts solution, starch were used.

Negative Control: H20 Positive Control: starch

1. Obtain seven test tubes and number them 1-7 2. Add to each tube the materials to be tested. Add 2mL of Benedicts solution to each tube. 3. Place all of the tubes in a gently boiling water- bath for 3 min and observe colorchanges during this time. 4. After 3 min, remove the tubes from the water- bath and give the tubes ample time to cool to room temperature. Record the color of their contents.

Procedure 2 Perform the iodine test for starch

Introduction Iodine test is used to test for the presence of carbohydrates. Iodine solution reacts with starch whereas it produces a deep purple color. The iodine test is used to distinguish starch from monosaccharides, disaccharides and other polysaccharides. Due to its unique coiled geometric configuration, it reacts with iodine to produce a blue black color and tests positive. A yellowish brown color indicates that the test is negative.

Material Methods:

The materials used were iodine, potato juice, onion juice, sucrose solution, glucose solution, reducing sugar solution, reducing starch solution were used in this experiment

Negative Control: H20 Positive Control: Serum Albumin

1. 2.

Obtain seven test tubes and number them 1-7 Add to each tube the materials to be tested.

3. 4.

Add seven to ten drops of iodine to each tube. Record the color of the tubes

Table 1 Solutions and Color Rxn For Benedicts Test For Reducing Sugars Iodine Test For Starch

Iodine Color Tube Solution 1 10 drops onion juice Benedicts Color Rxn no chg blue->black w/ white 2 10 drops potato juice 3 10 drops sucrose solution 4 10 drops glucose solution 5 10 drops distilled water precip. no chg blue-> brown no chg yellow->blue no chg no chg no chg no chg yellow->blue Rxn no chg

6 10 drops reducing sugar sol blue 7 10 drops starch solution no chg

Conclusion: Reducing sugar is present in potato juice, glucose solution and reducing sugar solution while starch is present in potato juice and starch solution.

Discussion Color chnges indicates a solution, contains reducing sugar. Honey, glucose solution and reducing sugar change color because the copper(II) sulphate presence in the Benedicts solution reacts with electrons from aldehyde and ketone group to form cuprous oxide, a red brown precipitate. The samples that change from yellow to blue show starch is present.

Procedure 6.3 Perform the Biuret test for protein

Negative Conrol: H20 Positive Control: protein solution

Introduction The Biuret test is a chemical test used to detect the presence of peptide bond. Biurets reagent is made of sodium hydroxide and copper (II) sulphate. In a positive test, copper (II) ion is reduced to copper (I) ion, which forms a complex with the nitrogen and carbon of the peptide bond in an alkaline solution. The blue reagent turns violet in the presence of protein and turns to pink when combined with short chain polypeptides. In this test, it will be positive when there is a reaction between the copper (II) ions and the amino group in the peptide bond.

Material Methods:

The materials used in this experiment were: test tubes, sodium hydroxide (NaOH), Buirets regent, egg albumen, honey, amino acid solution, distilled H20, and protein solution.

1. 2. 3. 4. 5.

Obtain five test tubes and number them 1-5. Add materials listed Add 2 mL of 2.5% sodium hydroxide (NaOH) to each tube Add three drops of Biuret regent to each tube and mix Record the color of tubes contents

Table 2 Solutions and Color Rxns For the Biuret Test For Protein Tube Solution Color green/ 1 2 mL egg albumen 2 2 mL honey 3 2 mL aa sol 4 2 mL distilled H20 5 2 mL protein sol. yellow no chg purple no chg purple

Conclusion: Protein present in egg albumen, amino acid solution and protein solution.

Procedure 4 Solubility of lipids in polar and non-polar solvents

Material Methods:

The materials used in this experiment were: test tubes, acetone, and vegetable oil

1.

Obtain two test tubes. To one of the tubes, add 5 mL of water. To the other tube, add 5 mL of acetone

2.

Add a few drops of vegetable oil to each tube

Procedure 5 Perform the Sudan IV test for lipid

Material Methods:

In this experiment, test tubes, Sudan IV, salad oil, distilled water, honey and a lipid solution were used.

Introduction: The Sudan test detects hydrocarbon groups that are in the molecule. The hydrocarbon group is non polar and sticks tightly together with a polar surrounding. This hydrophobic interaction the basis for Sudan test. As lipid molecules are insoluble in water, (they consist mainly of carbon and hydrogen, with few oxygen containing functional groups) they are considered as hydrophobic. If the result is visible orange appear, it is positive for lipids.

1. 2. 3.

Obtain five test tubes and number them from 1-5 Add the materials listed Add five drops of water to tube 1 and five drops of Sudan IV to each of he remaining tubes. Mix the contents of each tube. Record the color f the tubes contents.

Table 3 Solutions and Color Rxns For The Sudan IV Test For Lipids Description of Tube Solution 1 1 mL salad oil + H20 2 1 mL salad oil + Sudan IV 3 1 mL honey + Sudan IV 4 1 mL distilled water + Sudan IV 5 1 mL known lipid solution + Sudan IV Rxn no change red no change no change red

Conclusion: Lipids are present in salad oil + Sudan IV and lipid solution + Sudan IV

There is no change in test tube 1and 6. In test tube 2, the salad oil layer turns red which indicates that lipid is present. In test tube 3, two layers form, which is light orange on the top and dark orange on the bottom. The density of honey is higher than water where the light orange layer is water and dark orange is honey. In test tube 4, the solution turns a cloudy orange. In test tube 5, it shows presence of lipids due to the red color change.

Procedure 6.6 Perform the grease spot test for lipids

Introduction Lipids involve molecules that dissolve in non-polar solvents but not in polar solvents.

Material Methods:

In this experiment, a brown paper bag, eye dropper, salad oil, condensed milk and water were used.

1. 2.

Obtain a piece of brown wrapping paper or brown paper bag Use an eye dropper to add a drop of salad oil near a corner of the piece of paper

3. 4. 5. 6.

Add a drop of water near the opposite corner of the paper Let the fluids evaporate Look at the paper as you hold it up to the light Test other food products and solutions available

Table 4 Materials and Grease Spot Reaction As A Test For Lipid Content Food Product Salad Oil Condensed Milk Description of Grease Spot Reaction Translucency Translucency

H20

No change

Conclusion Salad oil and condensed milk are oil based as they leave a translucency on the brown bag. This indicates the presence of lipids. The water leaves no grease spot and tests negative for lipids

References:

http://www.jce.divched.org/JCESoft/CCA/CCA5/MAIN/1ORGANIC/ORG18/TRAM18/ B/0591308/MOVIE.HTM

http://edis.ifas.ufl.edu/hs375

http://www.sciencedirect.com/science/article/pii/016666229280231P