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1 Biomolecules Lab #3. Today Quiz #2 – 15 minutes Objectives and Introduction to Biomolecules – 20 minutes Safety Today’s Lab Instructions – 10 mins Experiments.

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Presentation on theme: "1 Biomolecules Lab #3. Today Quiz #2 – 15 minutes Objectives and Introduction to Biomolecules – 20 minutes Safety Today’s Lab Instructions – 10 mins Experiments."— Presentation transcript:

1 1 Biomolecules Lab #3

2 Today Quiz #2 – 15 minutes Objectives and Introduction to Biomolecules – 20 minutes Safety Today’s Lab Instructions – 10 mins Experiments - ~ 70 minutes Clean Up Summary

3 Objectives Describe the distinct features of three of four classes of organic molecules Differentiate between monomers and polymers Identify reducing sugars, polysaccharides, lipids, amino acids and proteins using diagnostic reagents Conduct nutritional assessments from case studies

4 OBJECTIVES Describe the functions of the major classes of biomolecules Explain how dehydration synthesis causes molecular bonds Compare the different bonds that help define the major classes of biomolecules Describe the reactive groups of the major biomolecules & explain how they relate to the basic chemical structure of each class of biomolecules Use diagnostic reagents to identify biomolecules & explain the chemical basis for the tests Design & present an experiment to test for the presence of biomolecules in food materials Explain how the chemistry of biomolecules relates to FDA food labels, & how you can use this knowledge to make wise food choices

5 5 Biomolecules: The Basics Organic molecules are necessary for biological processes. Organic molecules contain carbon. 4 major types of organic molecules: –Carbohydrates –Lipids –Proteins –Nucleic Acids (We’ll look at these in a future lab.) Similarities Between Biomolecules: –All Contain Carbon –Most Contain H, O, N, S and/or P –ALL Life on Earth is made up of Carbon

6 Building Blocks of Life Each Macromolecule is comprised of building block molecules called monomers: –Monosaccharides  carbohydrates –Fatty Acids and Glycerol  Lipids –Amino Acids  Protein Macromolecule: A giant molecule formed by the joining of smaller molecules, usually by a condensation reaction. Monomer - The subunit that serves as the building block of a polymer. Polymer - A long molecule consisting of many similar or identical monomers linked together.

7 Functional groups The components of organic molecules that are most commonly involved in chemical reactions are known as functional groups Each functional group behaves consistently from one organic molecule to another Provide a way to differentiate between different biomolecules _5.gif

8 Hydroxyl Group –Found in alcohols (ex. ethanol) and sugars –Polar - increases the solubility of molecules –OH (not OH - hydroxide ion) Where are the Hydroxyl groups in the glucose and in the ethanol? ethanol

9 Carbonyl Group Found in Carbohydrates: Two types –Aldehyde terminal –Ketone internal –One or the other on every monosaccharide

10 In fatty acids AND amino acids Acidic properties Double Bond between Carbon and Oxygen Single Bonds: Carbon to Hydroxyl Written as COOH or C02H Carboxyl Group

11 Amino Group –In every amino acid –Nitrogen atom attached to two hydrogen atoms Amino Group

12 Monomer – a single molecule that can combine to form polymers Polymer – long chains of monomers

13 Dehydration Synthesis and Hydrolysis Dehydration Synthesis –Monomers are connected by an enzymatic removal of water –One monomer provides a hydroxyl group and the other provides a hydrogen Result is the release of a water molecule Hydrolysis –Covalent Bonds connecting monomers into polymers –A Hydrogen atom and hydroxyl group from a split water molecule attaches where the bond used to be 1 H(+) Atom 1 Hydroxyl Group (split from a water molecule) Attach where the bond used to be

14 Examples of Dehydration and Hydrolysis Which one of these is also called a condensation reaction? Examples of Dehydration and Hydrolysis Which one of these is also called a condensation reaction?

15 Carbohydrates (Saccharides) Four major functions: –Supply carbon for synthesis of other biomolecules –Structural components in cells and tissues –Fuel (glucose) –Store glucose (polysaccharides) Three types: –Monosaccharides –Disaccharides –Polysaccharides Defined by carbonyl group –Aldehyde –Ketone

16 Monosaccharides (Basic Unit of Carbohydrates)  Simple sugars  Classified by the number of carbons they contain  Trioses  Pentoses  Hexoses  May Exist as Rings (Stable) or Linearly (Unstable) glucose

17 Disaccharides  Consist of 2 monosaccharides  Linked via a condensation reaction (Dehydration Synthesis)  Linked by a Glycosidic Bond

18 Polysaccharides Chains of linked monomers Amylose –Linear chain of several hundred glucose molecules Amylopectin –Branched chains of several thousand glucose molecules Starch in plants Glycogen in animals Amylopectin –Branched chains of several thousand glucose molecules Starch in plants Glycogen in animals

19 19 Reducing Sugars: Contain a free aldehyde or ketone group (carbonyl) Reducing sugars reduce something else –Includes: All monosaccharides Disaccharides where one aldehyde or ketone group is free

20 Testing for Saccharides –Benedicts Test Tests for the presence or absence of reducing sugar –A reducing sugar is a sugar with free aldehyde or ketone group Free- not used in the bond between monosaccharides. All monosaccharides are reducing sugars. Positive test results red-orange- yellow color

21 Testing for Saccharides Lugol’s Test –Indicates the presence or absence of starch –Positive test results black/purple color All our tests today will be qualitative not quantitative (specific numerical measurement)

22 22 Lipids Nonpolar molecules –Composed of long chains of carbon and hydrogen Lipid molecules include : –Fats, Oils, Steroids, Phospholipids Monomers (for triglycerides) –glycerol and fatty acids Major functions include : –Energy storage –Material for membranes

23 23 Lipids Saturated vs. Unsaturated –Saturated lipids- All bonds between carbon & hydrogen are single bonds No carbon-carbon double bonds Straight Chains Solid at Room temperature –Unsaturated lipids- Some carbon-carbon bonds are double bonds –Kinked Chains where double bonds are present Kinks prevent molecules from packing tightly together Plants and Fish Fats (oils) are liquid at room temperature

24 Saturated Fats Unsaturated Fats have fatty acids with no carbon- carbon double bonds There is a hydrogen at every possible location The molecule has straight chains Saturated fats are solid at room temperature Most animal fats are saturated Contributes to cardiovascular disease have fatty acids with one or more carbon-carbon double bonds The molecule has kinked chains where there is a carbon-carbon double bonds Plants and fish fats, known as oils, are liquid at room temperature The kinks provided by the double bonds prevent the molecules from packing tightly together

25 25Lipids Triglycerides are solid lipids (fats) that are stored in human tissue OR liquid lipids (oils) that are stored in plant tissue. –Store more energy than any other molecule Triglycerides are formed by attaching 3 fatty acid molecules to a glycerol molecule through a condensation reaction. –Functional group for glycerol (an alcohol) = hydroxyl group (OH) –Functional group for fatty acid (a carboxylic acid) = carboxyl group (COOH)

26 Phospholipids Make up the cell membrane Consist of: –a polar head (hydrophilic) –a nonpolar tail (hydrophobic)

27 Testing for Lipids Sudan IV –A nonpolar dye –Bonds to other nonpolar molecules –Positive test results Cloudy emulsion of dispersed lipid droplets which are red color due to the Sudan IV

28 28 Proteins Structural support, storage, transport of other substances, and intercellular signaling –ALL Enzymes are Proteins All Proteins have a complex 3D Shape Monomers = amino acids (there are 20 amino acids used by biological organisms) Amino acids contain: –An amino (NH 3 ) group –A carboxyl (COOH) group.

29 29 Proteins and Amino Acids When amino acids are linked together via a condensation reaction, they form a protein. The chemical bond between each pair of amino acids is called a peptide bond. –Carboxyl Group of one Amino Acid joins the amino group of another amino acid Polypeptides: polymers of proteins –Proteins consist of one or more Polypeptides folded and coiled into a specific formation

30 30 Proteins and Amino Acids When long chains of amino acids form proteins, the chemical properties of the amino acids cause the protein to take on more complex shapes: Proteins are used for: –Structural support –Transport of substances –Cell signaling –Enzymes All enzymes are made of protein. Enzymes regulare metabolism by selectively accelerating chemical reactions

31 Testing for Proteins Biuret Test –React with peptide bonds –Positive test results purple Ninhydrin –Tests for single amino acids –Reacts with free amino groups –Positive test results Purple- straight chain amino acid Yellow- ring chain amino acid (proline)

32 Lab Safety – Biology 111 Lab 3 - Biomolecules Goggles and closed-toed shoes are required! Goggles and closed-toed shoes are required! Gloves need to be worn when handling all reagents. Refer to the lab manual for safety notices. Some materials such as Ninydrin and Ninhydrin-collidine are corrosive and toxic. Other chemicals are flammable. Gloves need to be worn when handling all reagents. Refer to the lab manual for safety notices. Some materials such as Ninydrin and Ninhydrin-collidine are corrosive and toxic. Other chemicals are flammable. Closely monitor all hot plates. Immediately turn off when not in use and use hand protectors when handling any beakers placed on hot plates. Keep all solvents away from heat sources. Closely monitor all hot plates. Immediately turn off when not in use and use hand protectors when handling any beakers placed on hot plates. Keep all solvents away from heat sources. Chromatography solvent is highly flammable with toxic, volatile fumes. Keep tightly closed when not in use and keep away from all heat sources. Chromatography solvent is highly flammable with toxic, volatile fumes. Keep tightly closed when not in use and keep away from all heat sources. Dispose of any solutions containing the following reagents in the appropriately labeled containers on the side counter: Dispose of any solutions containing the following reagents in the appropriately labeled containers on the side counter: –Dispose of chromatography solvent, ethanol, and Sudan IV in the solvent waste container. –Dispose of Lugol’s solution, Benedicts reagent, and Biuret’s reagent in the base waste container. –Dispose of ninhydrin filter papers in the ninhydrin waste bucket. Keep waste containers closed when not disposing of waste, and do not fill past the indicated line on waste containers. Inform your TA of any full waste containers. Keep waste containers closed when not disposing of waste, and do not fill past the indicated line on waste containers. Inform your TA of any full waste containers. Do not eat or drink in lab. Do not eat or drink in lab. Ask your TA if there is doubt about any procedure. Ask your TA if there is doubt about any procedure.

33 33 Lab Activity: Testing for the presence of organic molecules Each table will work as a single group. –Within each group, students will perform different tests. –When all tests are complete, group members will teach each other how to conduct all of the tests. Procedure How does the test work? What is a positive / negative result?

34 34 Lab Activity: Testing for the presence of organic molecules Organization for each table (8 students): –Benedict’s Test for Reducing Sugars- 2 students –Lugol’s Test for Starches- 2 students –Sudan IV Test for Lipids- 1 student –Biuret Test for Proteins- 2 students –Ninhydrin Test for Amino Acids- 1 student

35 35 Benedict’s Test Tests for the presence of reducing sugars (reacts with the free aldehyde or ketone groups) You will fill in the data for Table 3-1. –You will need 8 test tubes to test the 8 solutions in Table 3-1. Make sure you gently shake the solution bottles before you use them. 10 drops = 1 mL Use parafilm to mix your solutions with the Benedict’s reagent in the test tubes. –WARNING! BENEDICT’S REAGENT IS A CORROSIVE BASE. DO NOT COME IN CONTACT WITH THIS CHEMICAL AND DISPOSE IN BASE WASTE CONTAINER.

36 36 Benedict’s Test A positive test result is any color change. Color changes range from: –Blue > Green > Yellow > Reddish Orange Orange and Red indicate the strongest reducing sugars.

37 37 Lugol’s Test Tests for the presence of starches You will fill in the data for table 3-1. –Procedure begins on pg. 30 –You will need 8 test tubes to test the 8 solutions in Table 3-1 (these are the same solutions used in the Benedict’s test). –Make sure you gently mix the solution bottles before you use them. –10 drops = 1 mL –Use parafilm to mix your solutions with the Lugol’s reagent. –WARNING! LUGOL’S IS A TOXIN (ESPECIALLY FOR PEOPLE WITH IODINE ALLERGIES OR WILSON’S DISEASE). DO NOT COME IN CONTACT WITH THIS CHEMICAL AND DISPOSE IN BASE WASTE CONTAINER.

38 38 Lugol’s Test A positive test result is a blue-black color, which indicates the presence of starch. Monosaccharides and Disaccharides will test negative for Lugol’s.

39 Emulsification Test for lipids – Use Ethanol + Sudan IV A non-polar dye Bonds to other non-polar molecules Positive test results red color Positiv e Negativ e

40 40 Sudan IV Test A positive test is a red stain left on the filter paper after rinsing.

41 Thin Layer Chromatography Mixed lipids can be separated Yields a chromatogram Mixed molecules interact with the solvent (mobile phase) and the silica gel (stationary phase) to separate as solvent moves up Relationship between distance moved by molecule to distance moved by solvent is specific for the matrix conditions & solvent Relationship is given by R f Origi n Solvent front Triglyceride Fatty acids Phospholipi ds s

42 42 Biuret Test Tests for the presence of proteins by reacting with peptide bonds You will fill in the data for Table –Procedure begins on pg. –You will need test tubes to test the solutions –Make sure you gently mix the solution bottles before you use them. –10 drops = 1 mL –Use parafilm to mix your solutions with the Biuret reagent. WARNING! BIURET IS A STRONG BASE. AVOID CONTACT AND DISPOSE OF IN BASE WASTE CONTAINER.

43 43 Biuret Test A positive test is a violet color. –Any other color change is a negative result.

44 44 Ninhydrin Test Tests for the presence of free amino acids by reacting with their amino groups. You will fill in the data for table. Make sure you gently shake the solution bottles before you use them. The test will be done on a piece of filter paper folded into eights. –Use pencil to label each test area. –Dry each solution on the filter paper before you test it with the ninhydrin solution. –Dry the filter paper again after the ninhydrin is added. WARNING! NINHYDRIN IS POISONOUS. AVOID CONTACT AND DO NOT INHALE.

45 45 Ninhydrin Test A positive test is either a purple/violet color OR a yellow color. –Yellow indicates the presence of a specific amino acid, proline. –Purple indicates the presence of any other free amino acid.

46 Ninhydrin Ninhydrin - Collidine

47 47 Nutrition Activity When the students at your table have shared all of their data and taught each other these different tests: –Read the section on Nutritional Analysis You may do this individually or with others. –You need to know: How to convert pounds (lbs) to kilograms (kg) How to calculate BMR (know the equation) Have a basic understanding of the Prudent Diet

48 Clean up Dispose of any solutions containing the following reagents in the appropriately labeled containers on the side counter: Dispose of any solutions containing the following reagents in the appropriately labeled containers on the side counter: –Dispose of chromatography solvent, ethanol, and Sudan IV in the solvent waste container. –Dispose of Lugol’s solution, Benedicts reagent, and Biuret’s reagent in the base waste container. –Dispose of ninhydrin filter papers in the ninhydrin waste bucket. Keep waste containers closed when not disposing of waste, and do not fill past the indicated line on waste containers. Inform your TA of any full waste containers. Keep waste containers closed when not disposing of waste, and do not fill past the indicated line on waste containers. Inform your TA of any full waste containers. Rinse all used test tubes (after dumping contents in proper waste container) and mortar and pestles and return to the middle of the bench. Rinse all used test tubes (after dumping contents in proper waste container) and mortar and pestles and return to the middle of the bench. Replace all bottles and other equipment to the middle of the bench in an orderly manner. Replace all bottles and other equipment to the middle of the bench in an orderly manner. Make sure all hot plates are off Make sure all hot plates are off Clean all pipettes and return them to the “clean pipette” container Clean all pipettes and return them to the “clean pipette” container

49 49 Learning Objectives Be able to identify the three classes of organic molecules based on their functional groups. Know the difference between a monomer and a polymer. –Know the monomers for the organic molecules discussed. Know each of the diagnostic tests performed in class –Know the general procedure –Know how each test works –Know what positive and negative results look like. Nutritional Analysis- You need to know: How to convert pounds (lbs) to kilograms (kg) How to calculate BMR (know the equation) Have a basic understanding of the Prudent Diet

50 Chemical Tests: Test Test for Color before Color after Detects Benedicts Lugols Sudan IV Biuret Ninhydrin

51 Biomolecules Summary Biomolecule Building Blocks Type of Bond Binding Mechanism Products Carbohydrates Simple Sugars GlycosidicDisaccaridesPolysaccharides Lipids Gylcerol Fatty Acids DehydrationTriglyceridesPhospholipidsSteroids Proteins Amino Acids PeptidePolypeptidesProteins

52 TestTest forColor beforeColor afterPositive control, negative control BenedictsReducing sugars Pale bluePale blue – none, green – low, brown – intermediate, orange red - high Glucose solution, distilled water LugolsStarchYellowBlue - blackPotato, distilled water Sudan IVLipidsLight redRed stained oil droplets Any oil, distilled water BiuretPeptide bonds Very pale blue VioletMilk, distilled water NinhydrinFree amino acids No ColorOn drying, may be either purple or yellow Amino acid methionine, distilled water


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