Exercise 2 - Functional Groups, Organic Molecules, Buffers, and Dilutions
Introduction
An overwhelming majority of the elements listed on the periodic table are naturally occurring. A much
smaller proportion of those are found in living systems in anything other than trace amounts. Six of
those elements are most abundant (CHNOPS):
Carbon (C)
Oxygen (O)
Hydrogen (H)
Phosphorus (P)
Nitrogen (N)
Sulfur (S)
Other elements of biological significance include sodium, potassium, calcium, magnesium, iron, and
chlorine. Atoms of these elements combine through bonding in a variety of ways to form molecules.
This exercise will examine some of the basic combinations of atoms that form molecules. Basic
principles of pH and buffers, as well as dilutions will also be covered.
Materials
Equipment
spectrophotometers
molecular model kits
cuvettes
cuvette racks
Kimwipes
Test tubes and racks
10 ml pipettes
pipette pumps
50 ml beakers
marking pencils
Reagents and Solutions
Bogen’s Universal Indicator
1M NaOH
1M HCl
pH 4 buffered solution
pH 4 unbuffered solution
colored dye stock solution, 100%
distilled water
unknown dye solutions
Part A: Functional Groups and Biologically Important Molecules
Most biological molecules are held together by covalent bonds. Covalent bonds result in relatively
stable molecules that do not dissociate in aqueous (water) environments. These stable molecules can
serve as monomers (building blocks or subunits) for the synthesis of larger dimers (2 monomers) or
polymers (chains of many monomers).
Biological molecules are classified according to their functional groups. Functional groups are clusters
of atoms bonded to carbon backbones and are most commonly involved in chemical reactions. They
impart particular characteristics to larger molecules to which they are attached. For example, any
molecule with a carboxyl group behaves as an organic acid like fatty acids or amino acids. Those with a
hydroxyl group are considered alcohols (e.g. glycerol). Carbohydrates contain a carbonyl group (either
an aldehyde if it’s at the end of the molecule or a ketone if not) along with a number of hydroxyl groups.
Table 2.1 illustrates some of the more biologically important functional groups. In this table, each line
represents one covalent bond. Single and double bonds can exist. Each functional group bonds to a
carbon backbone, often symbolized by the letter “R” (e.g. R-OH would be a molecule containing a
hydroxyl functional group). Each functional group must have at least one covalent bond available for
attachment to this carbon backbone.
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 13
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
Table 2.1 Biologically Important Functional Groups
Carbonyl
Aldehyde
__
__ C __ H
__
__
__
O
O
__ C __
Phosphate
H
H
O
__
__ S __ H
__
__
__ O __ P __ OH
__ N __ H
__ C __ OH
Sulfhydryl
__
Amine
__
O
__ OH
Carboxyl
Ketone
__
Hydroxyl
O
Procedure
1. Fill in Table 2.2 using the periodic chart in your text.
Table 2.2 Elements Represented in Molecular Model Kits
Element
Atomic
Symbol
Atomic
Number
# of Valence
Electrons
# of e-s
needed to fill
valence shell
Carbon
Hydrogen
Nitrogen
Oxygen
Phosphorus
2. Obtain a molecular model kit
3. Examine the colored balls to determine the number of holes in each. Each ball represents an
atom of a particular element. The holes represent the valence (bonding capacity) of the atom.
Using the information in Table 2.2, you should be able to determine which elemental atom is
represented by each ball
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
14
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
4. Use the molecular kit to construct models of each of the functional groups in Table 2.1. Use the
appropriate colored ball to represent each atom. The grey “sticks” are bonds. Use the longer
“sticks” to bend to create double bonds. When building functional groups, you will always have
one free end of a “stick” that represents the attachment point of the functional group to the
carbon backbone (“R”). Pay attention to the content and shape of each functional group
Circle and label the functional groups within these biologically important molecules in Fig. 2.1.
Fig. 2.1 Some Biologically Important Organic Molecules
H
C
H
__ __
OH
H
C
OH
__ __
__ __
OH
C
H
H
OH
H
OH
__ __
__ __
__ __
__ __
__ __
glucose ring
(hydroxyl)
O
H
OH
__ __
OH
H
C
________
C
__ __
H
H
__
H
____
__
H
H
OH
H
______
__
____
__
OH
O
H
H
__
OH
OH
C
__
C
H
__
H
C
__
OH
OH
____
C
C
____
H
______
H
OH
C
__
H
__
__
H
HO
H
__ __
H
__
C
O
__ __
__ __
__
HO
__
__ __
OH
__
C
OH
__
__
__
H
C
fructose chain
(hydroxyl, ketone)
__
O
__
__
C
__
H
glucose chain
(hydroxyl, aldehyde)
__ __
H
OH
H
fructose ring
(hydroxyl)
H
__
H
__ __
C
OH
H
C
OH
OH
__ __ __
__
__
glycine
(amine, carboxyl)
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
__
H
__ __
__
__ __
C
__
C
OH
C
__
N
O
__
H
__
H
__
H
H
H
glycerol
(hydroxyl)
15
OH
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
Part B: Buffers
The pH of blood and other body fluids is relatively insensitive to the addition of acids or bases. This is
due to the presence of buffers in living systems which help to maintain homeostasis by maintaining
normal pH levels. The pH of a solution can be determined in a variety of ways, including the use of pH
meters, litmus paper, and chemical reagents. In this exercise, we will use the chemical reagent Bogen’s
Universal Indicator to determine pH of specific solutions.
Bogen’s Universal Indicator changes color at specific pH end points:
Pink = pH 4
Yellow = pH 6
Green = pH 7
Blue = pH 9
Violet > pH 9
In order to determine the effect of buffers on pH, we will attempt to raise the pH of an unbuffered acid
solution by adding small amounts of a base. For comparison, we will repeat this procedure with a
buffered acid solution. Once both solutions are basic, we will attempt to return them to the original pH
by adding small amounts of acid.
Procedure
1. Obtain two 50 ml beakers and label them A and B
2. Pipette 10 ml of an unbuffered pH 4 solution into beaker A
3. Pipette 10 ml of a buffered pH 4 solution into beaker B
4. Add 3 drops of Bogen’s Universal Indicator to each beaker
5. Note the color. __________ Is this color expected? __________
6. Slowly add 1M sodium hydroxide (NaOH) one drop at a time to beaker A, swirling the beaker
between each drop. Do until you detect a permanent color change to violet
7. Record the number of drop required to change the color to violet in Table 2.3
8. Repeat the last two steps with beaker B
The test you just performed illustrated the effect of a buffer when you attempted to increase the pH
(make it more basic). Did the buffered solution require more or less (circle one) drops to change the
pH? Do you suppose buffers would resist pH changes in either direction? __________
Continue the procedure from above
9. Slowly add 1M hydrochloric acid (HCl) one drop at a time to beaker A, swirling the beaker
between each drop. Do until you detect a permanent color change to pink
10. Record the number of drops required to change the color to pink in Table 2.3
11. Repeat the last two steps with beaker B
Table 2.3 The Effect of Buffer on pH Change
Beaker
Contents
A
unbuffered, pH 4 solution
B
buffered, pH 4 solution
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
# drops to violet
# drops back to pink
16
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
Part C: Dilutions
Part C1: Basic Dilutions
During scientific experiments, it is often necessary to dilute the solution provided (the stock solution).
For example, such a dilution might be made to reduce chemical concentrations so the rate and intensity
of reactions can be controlled.
A stock (100%) dye solution and distilled water will be used in this lab.
How would you go about preparing 10 ml each of 75%, 25%, and 10% solution from an available stock
solution of 100%?
The algebraic equation C1V1 = C2V2 provides our tool to answer this question, where
C1 = concentration (%) of stock solution
V1 = volume (ml) or stock required to prepare the solution (you typically are
solving for this variable)
C2 = concentration (%) of dilution you wish to prepare
V2 = volume (ml) of dilution you wish to prepare
Procedure
1. Use the algebraic equation to determine volumes of 100% stock (ml) and distilled water (ml)
required to create 10 ml each of 0%, 10%, 25% and 75% dilution. Record your answers in Table
2.4.
Table 2.4 Volumes Needed to Prepare Dilutions
Concentrations – C2
10%
25%
75%
Volume of stock solution (ml) - V1
Volume of water (ml)
Total volume of dilution (ml) - V2
2. Obtain 3 test tubes and a test tube rack
3. Prepare the three dilutions from Table 2.4 by pipetting the correct amount of stock in the test
tube first and then diluting the stock with the correct amount of distilled water. There should
be the same amount of liquid in each test tube when you are finished
4. Obtain 5 cuvettes on a cuvette rack
5. Transfer distilled water (0% dye solution) to the first cuvette up to about ¾ full. Distilled water
is used as a blank solution to calibrate the spectrophotometer
6. One at a time and in order of increasing concentration, transfer enough of the other 4 solutions
so that each cuvette is approximately ¾ full
7. Set the spectrophotometer to a wavelength of 450nm
8. Read the % light transmittance for each dye solution you prepared and record your results in
Table 2.5
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
17
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
Table 2.5 % Light Transmittance Associated with Various Concentrations of Dye
Dye Solution
% Concentration of Dye
% Light Transmittance
1
0 (DH2O only)
100
2
10
3
25
4
75
5
100 (stock)
Unknown A, B, C, D (circle yours)
What relationship exists between concentration of dye and % light transmittance?
Part C2: The Standard Curve
Procedure
1. Plot the 0%, 10%, 25%, 75%, and 100% data from Table 2.5 on Fig. 2.2
2. Attempt to draw a “best fit” line through the scatter of data points. Do not simply connect the
dots. Make your line pass through the “average” spread of the dots. This line represents a
standard curve and illustrates the relationship between percent concentration of a dye solution
and percentage of light transmitted. Use this standard curve to complete Part C3
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
18
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
% Light Transmittance
Fig. 2.2 Standard Curve Relating Dye Concentration to % Light Transmittance
Dye Concentration (%)
Describe the kind of relationship you see:
Part C3: Determination of Unknown Dye Concentration
Procedure
1. Select a cuvette of unknown dye concentration (letters A-D) from the samples available
2. Record the letter of your unknown in Table 2.5
3. Use the calibrated spectrophotometer to read the % transmittance of your unknown dye
concentration solution. Record in Table 2.5
4. Determine the concentration of your unknown by finding the value of % transmittance on the Yaxis of Fig. 2.2 and drawing a perpendicular line down from that point to where it crosses the Xaxis. That intersection point is the percent dye concentration of your unknown. Record that in
Table 2.5
5. Return your unknown cuvette to your instructor and tell them your result
6. Rinse out the rest of the cuvettes and place them on the cuvette rack. Do not scrub them with
a test tube brush as it will scratch and render them useless
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
19
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
Practice Problems and Review Questions
1. Given a stock solution of 2.0% dextrose, how would you prepare 10 ml of each of the following
solutions?
a. 0.1% dextrose solution
b. 1.0% dextrose solution
c. 0.5% dextrose solution
2. Given a stock solution of 5.0% sodium chloride (NaCl), how would you prepare 20 ml of each of
the following solutions?
a. 2.0% sodium chloride solution
b. 0.5% sodium chloride solution
c. 3.0% sodium chloride solution
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
20
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
3. Given a stock solution of 10% dextrose, how would you prepare 5 ml of a 0.9% dextrose
solution?
4. Given a stock solution of 0.9% dextrose, how would you prepare 5 ml of a 0.5% dextrose
solution?
5. Given a stock solution of 0.5% dextrose, how would you prepare 5 ml of a 0.004% dextrose
solution?
6. How would you prepare 25 ml of a 15% dye solution beginning with a 20% stock dye solution?
7. How would you prepare 9 liters of a 50% dye solution beginning with a 60% stock dye solution?
Express your answer in ml.
8. How would you prepare 600 ml of a 20% starch solution beginning with a 50% stock starch
solution? Express your answer in liters.
9. You have 10 ml of a 60% stock dye solution. What is the maximum amount of a 12% dye
solution you could prepare?
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
21
Exercise 2 –Functional Groups, Organic Molecules, Buffers, and Dilutions
10. How would you go about preparing the 12% dye solution in question 9?
11. What are buffers and why are they biologically important?
12. List the functional groups present in each of these molecules
glucose
fructose
glycine
glycerol
13. List some possible polymers that can be formed from each of these monomers
glucose
fructose
glycine
glycerol
Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C
22