LABORATORY # 4: CHEMICAL ANALYSIS
Required materials – Chemical analysis
1. Potato, apple, onion and peanut
2. Disposable transfer pipettes or eye droppers
3. Mortar and pestle
4. Hot plate & beakers with water
5. Test tubes and test tube holders
6. Knives or scalpels
7. 5 or 10mL pipettes
8. Spatulas
9. Paraffin
10. Solutions
a. Iodine
b. Benedict’s
c. Biuret reagent
d. Sudan IV stain
e. Tween-20 detergent (to help breakup or emulsify the larger fats)
11. Positive controls
a. 5% glucose solution
b. 5% Sucrose solution
c. 1% Cornstarch solution
d. Diluted egg white solution
e. Milk
f. Butter
Introduction
Living things are made up of four kinds of macromolecules: carbohydrates, lipids, proteins and
nucleic acids. In this lab, you will learn some basic procedures in identifying components of
these macromolecules
Each group is comprised of some very large molecules made up of identifiable smaller subunits.
The subunit of the carbohydrate is the sugar or monosaccharide. Linking monosaccharides
together produces disaccharides and polysaccharides. We refer to monosaccharides and
disaccharides as simple sugars and we call polysaccharides complex sugars. Glucose, fructose
and galactose are the three monosaccharides found in nature. Joining them together makes the
dissacharides lactose (glucose + galactose), sucrose (glucose + fructose) and maltose (glucose +
glucose). Complex polysaccharides include the storage polysaccharides starch and glycerol and
the structural polysaccharide cellulose.
The subunit of the protein is the amino acid. Long stretches of amino acids linked together are
called polypeptides. Chemical modifications to these polypeptide chains results in complex
folding patterns and eventually creates a functional protein. There are 22 amino acids available
in nature to make proteins. Each of these only differs in the composition of their functional
group called an “R” group. Like proteins, the nucleic acids DNA and RNA are also made up of
long stretches of a subunit known as a nucleotide to create polynucleotide chains. A nucleotide
is comprised of three components: a five carbon (pentose) sugar, a phosphate group (attached
to the 5th carbon of the sugar) and a nitrogen-containing base (attached to the 1st carbon).
While DNA and RNA are similar, there are some important differences. For example, DNA lacks
a critical oxygen at the second carbon of the pentose sugar. Plus the bases differ between RNA
and DNA.
Lipids differ a little from the model of repetitive subunits. The lipid category of macromolecules
contains the triglycerides (i.e. oils and fats), the phospholipids and steroids. The triglycerides
do have a repetitive unit – three fatty acid “tails” that chemically attach to a glycerol
“backbone”. In a phospholipid, one of these fatty acids is replaced by a phosphate group “head
group”. Steroids are completely different and are based on the chemical known as cholesterol.
Procedures:
Simple tests for confirming the presence of starch, maltose, glucose, triglycerides and proteins
are given below. Use these tests to compare the chemical composition of a potato, a potato
with saliva, an apple, an onion and a peanut.
WARNING: IF YOU ARE ALLERGIC TO PEANUTS OR ANY OF THESE OTHER FOODS – PLEASE
INFORM THE PROFESSOR SO THAT PROPER PRECAUTIONS MAY BE TAKEN TO ENSURE YOUR
SAFETY
BEFORE YOU START: Turn on the hot plate and set it to medium heat
FOR THESE TESTS, YOU WILL NEED TO EXTRACT THE JUICE FROM YOUR FOUR FOOD SAMPLES.
CUT SMALL PIECES USING A SCALPEL AND USE YOUR MORTAR AND PESTLE TO GRIND UP
EACH SAMPLE INTO A PASTE OR A JUICE – WASHING THE MORTAR AND PESTLE BETWEEN
FOODS. YOU WILL NEED TO EXTRACT AT LEAST 5.0 mls OF JUICE FROM EACH FOOD. ADD A
SMALL AMOUNT OF WATER TO YOUR MACERATED PEANUTS.
PLACE THE JUICE INTO FOUR LABELLED TEST TUBES AND USE THEM FOR THE FOLLOWING
TESTS OUTLINED BELOW.
1. Test for starch: In this experiment, an iodine solution (i.e. potassium iodide/IKI) will
react with starch and turn a blue-black color. The highly coiled starch molecules react
with the iodine solution, whereas uncoiled molecules do not.
a. Add 1.0mL of water to a series of clean test tubes labelled 1 – 8
b. Add 1.0mL of water to test tube #1
c. Set up the remaining test tubes by adding either 1.0mL of your juice or 1.0mL of
the indicated controls
d. Add five drops of IKI solution to each tube and note any precipitation or color
change in your lab notebook
You should have the following tubes:
Test tube #1: water + IKI
Test tube #2: 1% Starch + IKI
Test tube #3: 5% Sucrose + IKI
Test tube #4: 5% Glucose solution + IKI
Test tube #5: Apple solution + IKI
Test tube #6: Onion solution + IKI
Test tube #7: Potato solution + IKI
Test tube #8: Peanut solution + IKI
Analysis of Results: Design a chart and record your results for the starch test for each of your
test tubes. Include in your chart the following columns: “Test tube contents”, “Observed Color
Results”, “Positive (+) reaction and Negative (-) reaction"; and "Conclusions".
In the first column marked “Test tube contents”, label rows 1-8 with the appropriate samples
In the second column labeled “Observed Color Results”, indicate the results of the color
change. If no change occurred – write no change observed
In the third column, indicate a (+) for a positive reaction and a (-) for a negative reaction
In the fourth column, summarize your conclusions as to why a color change happened or not
2. Test for monosaccharides and disaccharides: In this experiment, Benedict’s reagent
reacts with free aldehyde groups (-C-H) and changes from blue to green when heated in
the presence of maltose (a disaccharide) and changes from blue to yellow orange or red
when heated in the presence of glucose (a monosaccharide).
a. Add 1.0mL of water to a series of clean test tubes labelled 1 – 8
b. Add 1.0mL of water to test tube #1
c. Set up the remaining test tubes by adding either 1.0mL of your juice or 1.0mL of
the indicated controls
d. Add 1.0mL of Benedict reagent to the test tube and mix well (i.e. a 2:1 ratio of
juice/water to Benedict’s)
e. Heat all test tubes for 5 to 10 minutes in a beaker of water placed on a hot plate
set to medium heat. Do NOT let the water boil and be careful not to burn
yourself
f. Observe the color change and discuss your reasons why the color changed
You should have the following tubes:
Test tube #1: water + Benedicts
Test tube #2: 5% glucose solution + Benedicts
Test tube #3: 5% sucrose solution + Benedicts
Test tube #4: 1% starch solution + Benedicts
Test tube #5: Onion solution + Benedicts
Test tube #6: Potato solution + Benedicts
Test tube #7: Apple solution + Benedicts
Test tube #8: Peanut solution + Benedicts
Analysis of Results: Design a chart and record your results for the Benedict’s test for each of
your test tubes. Include in your chart the following columns: first column “Test Tube Contents”;
second column "Unheated Reaction"; the third column "Color of Heated Reaction"; the fourth
column "positive (+) reaction and negative (-) reaction"; and the fifth column "Conclusions".
In the first column marked “Test tube contents”, label rows 1-8 with the appropriate samples
In the second column labeled “Unheated Reaction”, indicate the results of the Benedict's test
BEFORE the reaction was heated.
In the column marked "Color of Heated Reactions", indicate the results of the Benedict's test
after the reaction was heated.
In the fourth column, indicate a (+) for positive benedict's reaction and a (-) for negative
benedict's reaction.
In the fourth column, summarize your conclusions as to why a color change happened or not
3. Test for proteins: In this experiment, the copper ions of Biuret reagent reacts with the
amide group (N-H) of an amino acid and turns a violet color in the presence of proteins
and pinkish in the presence of shorter chains of amino acids. The more number of
these reactions, the more intense color results, thus allowing you to distinguish
between large proteins and smaller polypeptides.
a. Add 1.0mL of water to a series of clean test tubes labelled 1 – 8
b. Add 1.0mL of water to test tube #1
c. Set up the remaining test tubes by adding either 1.0mL of your juice or 1.0mL of
the indicated controls
d. Add 1.0mL of each juice to another series of labelled, clean test tubes.
e. To each tube, add 1.0mL of Biuret reagent and mix well
f. Observe and record the color changes for each tube if present
g. Discuss why you got the color you did.
You should have the following tubes:
Test tube #1: water + Biuret reagent
Test tube #2: egg solution + Biuret reagent
Test tube #3: 0.1% albumin solution + Biuret reagent
Test tube #4: milk + Biuret reagent
Test tube #5: Onion solution + Biuret reagent
Test Tube #6: Apple solution + Biuret reagent
Test Tube #7: Potato solution + Biuret reagent
Test Tube #8: Peanut solution + Biuret reagent
Analysis of Results: Design a chart and record your results for the Biuret’s test for each of your
test tubes. Include in your chart the following columns: first column “Test Tube Contents”;
second column "Observed Color Results"; the third column "positive (+) reaction and negative
(-) reaction"; and the fourth column "Conclusions".
In the first column marked “Test tube contents”, label rows 1-6 with the appropriate samples
In the second column labeled “Observed Color Results”, indicate the results of the color
change. If no change occurred – write no change observed
In the third column, indicate a (+) for a positive reaction and a (-) for a negative reaction
In the fourth column, summarize your conclusions as to why a color change happened or not
4. Test for lipids: In this experiment, a lipid soluble stain called Sudan Black IV will stain the
lipid droplets in your subject black, while not staining water-soluble substances. To
make a positive control using butter, take a small amount of butter and mix it into a
paste using water
a. Obtain 6 clean test tubes
b. Add 1.0mL of water to test tube #1
c. Add 1.0ml of a butter/water paste to tube #2
d. Add 1.0mL of each of your juices to tubes #3 to #6
e. Add several drops of a non-ionic detergent like Tween-20 to each tube and mix
f. Add 5 drops of Sudan IV to the tubes
g. DO NOT MIX. Look for any color change that may occur at the interface between
the juice and the Sudan
You should have the following tubes:
Test tube #1: water + detergent + Sudan IV
Test tube #2: butter + detergent + Sudan IV
Test tube #3: Onion solution + detergent + Sudan IV
Test Tube #4: Apple solution + detergent + Sudan IV
Test Tube #5: Potato solution + detergent + Sudan IV
Test Tube #6: Peanut solution + detergent + Sudan IV
Analysis of Results: Design a chart and record your results for the Sudan IV test for each
of your test tubes. Include in your chart the following columns: first column “Test Tube
Contents”; second column "Observed Color Results"; the third column "positive (+)
reaction and negative (-) reaction"; and the fourth column "Conclusions".
In the first column marked “Test tube contents”, label rows 1-6 with the appropriate
samples
In the second column labeled “Observed Color Results”, indicate the results of the color
change. If no change occurred – write no change observed
In the third column, indicate a (+) for a positive reaction and a (-) for a negative reaction
In the fourth column, summarize your conclusions as to why a color change happened
or not
Test for Lipids Using the microscope:
1.
2.
3.
4.
5.
take a small amount of butter and mix it with a few drops of Tween-20
spread this mixture on a clean microscope slide
add a few drops of Sudan IV
cover with a coverslip and observe under the microscope
look for sudan-stained lipid droplets
AT THE END OF YOUR LAB:
1.
2.
3.
4.
5.
DISPOSE OF YOUR GROUND UP FOOD IN THE TRASH
DISPOSE OF THE 5.0 ML PIPETTES IN THE TRASH
DUMP THE TEST TUBE CONTENTS INTO THE SINK
RINSE OUT YOUR TEST TUBES AND PLACE THEM IN THE USED TEST TUBE CONTAINER
TURN OFF YOUR HOT PLATES!!!
For reference:
http://faculty.uncfsu.edu/jraynor/BIOL%20200%20Online%20Lab/Biological%20Molecules%20L
ab.htm