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. 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

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 Ketone
terminal
Ketone
internal
One or the other on every monosaccharide

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

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

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

13. Dehydration Synthesis and Hydrolysis
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?

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

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. Lipids Nonpolar molecules Lipid molecules include:
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. Lipids Saturated vs. Unsaturated Saturated lipids- Unsaturated 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. Lipids
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. 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 (NH3 ) group
A carboxyl (COOH) group.

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. 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!
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.
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 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.
Do not eat or drink in lab.
Ask your TA if there is doubt about any procedure.

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. 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. 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. 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. 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. 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
Positive
Negative

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 Rf
Origin
Solvent front
Triglyceride
Fatty acids
Phospholipids s

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

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. 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. 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 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.
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.
Make sure all hot plates are off
Clean all pipettes and return them to the “clean pipette” container

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 Products Biomolecule Building Blocks Type of Bond
Binding Mechanism
Products
Carbohydrates
Simple Sugars
Glycosidic
Disaccarides
Polysaccharides
Lipids
Gylcerol Fatty Acids
Dehydration
Triglycerides
Phospholipids
Steroids
Proteins
Amino Acids
Peptide
Polypeptides

52. Test
Test for
Color before
Color after
Positive control, negative control
Benedicts
Reducing sugars
Pale blue
Pale blue – none, green – low, brown – intermediate, orange red - high
Glucose solution, distilled water
Lugols
Starch
Yellow
Blue - black
Potato, distilled water
Sudan IV
Lipids
Light red
Red stained oil droplets
Any oil, distilled water
Biuret
Peptide bonds
Very pale blue
Violet
Milk, distilled water
Ninhydrin
Free amino acids
No Color
On drying, may be either purple or yellow
Amino acid methionine, distilled water