Plants & Civilization
Lab #2:
Organic Molecules
Organic Molecules
Living organisms (including plants!) contain
organic compounds such as:
1.
2.
3.
4.
Carbohydrates
Lipids
Proteins
Nucleic Acids
¾ Each macromolecule
is made up of subunits
held together by covalent
bonds.
¾Each displays different
structural properties.
1. CARBOHYDRATES
• Macromolecule made up of:
C, H, O in a 1:2:1 ratio
• Monosaccharides = “simple sugars”
• Classified by # of carbons (i.e. six carbon
monosaccharide is called a hexose).
• Polar & water soluble
CARBOHYDRATES: Types
1. Monosaccharides
2. Disaccharides
3. Polysaccharides
Monosaccharides
Example:
glucose
Monosaccharides – have a terminal
ALDEHYDE (-CHO) group
or
http://fderad.club.fr/glucose.gif
an internal KETONE (-C=0) group
Disaccharides
monosaccharide + monosaccharide=
dissaccharide
[e.g. fructose + glucose = sucrose]
http://web1.caryacademy.org/chemistry/rushin/StudentProjects/CompoundWebSites/1999/Sucrose/sucrose_structure.gif
“Reducing Sugar”
Monosaccharide
or
A disaccharide, with an
available aldehyde or ketone group.
• These “functional groups” (-CHO) or
(-C=O) can reduce weak oxidizing agents,
such as copper.
Benedict’s Test
• Test detects presence of reducing sugars
(i.e. monosaccharide or disaccharide w/ free –CHO or
–C=O group).
• Utilizes the ability of the free aldehyde or ketone
group of the simple sugar to reduce cupric
(Cu2+) ions of Benedict’s Reagent into cupric
oxide.
• Benedict’s Reagent is blue in color. If reducing
sugar is present, then it will turn green to
reddish (since cupric oxide is green to red).
http://www.umanitoba.ca/faculties/science/biological_sciences/lab2/biol
ab2_2.html
Polysaccharides
Examples:
1. Starch
2. Glycogen
3. Cellulose
• Cannot use Benedict’s Test to detect
these polysaccharides! Little free
aldehydes present in these molecules.
• So we use a different test…
Lugol’s Test
• Lugol’s test detects presence of STARCH,
a polysaccharide.
Lugol’s Iodine reacts with the coiled structure of Starch
causing it to change color from a
dark yellowish-brown
to a
violet-black color.
Lugol’s Test
Glycogen – similar structure to starch, but
not exact
Therefore…..
Glycogen displays an intermediate reaction
with Lugol’s Iodine (i.e. not quite dark
violet-black color change).
More about Glycogen
• Glycogen is a common carbohydrate
(polysaccharide) found in animals.
• It is an energy “storage molecule” found in cells.
¾ You will observe a DEMO slide of a cell containing
glycogen. Note: where is the glycogen stored in the
cell (inside nucleus or in the cytoplasm)?
¾ You will observe a DEMO slide of a cell treated with
Amylase, an enzyme that breaks down glycogen. Is
glycogen still present in this cell?
Lugol’s Test for Starch
http://www.sidwell.edu/sidwell.resources/bio/VirtualLB/Macro/macro.html
2. Lipids
• Organic molecules that are not water soluble.
• Soluble in non-polar solvents (e.g. acetone).
• Examples:
– Fats
– Steroids
– Phospholipids
– Triglycerides (glycerol + 3 fatty acids)
Lipids
Solid lipids at room temperature = fats
Liquid lipids at room temperature = oils
Lipids
Example:
Triglyceride = glycerol + 3 fatty acids
Note: triglycerides are
found mostly in adipose
tissue for high energy
storage.
http://www.biology.lsu.edu/introbio/Link2/triglyceride.gif
Test #1 for Lipids
• Sudan IV tests for presence of lipids.
• Red, non-polar dye that is selectively
absorbed by lipids.
– If lipids are present, they will appear as
floating red droplets or as a floating layer on
the surface.
Test #2 for Lipids
Grease Spot Test for Lipidsbased on ability of lipids to make translucent
spot on unglazed paper.
• Not as reliable as Sudan IV test, but may
help to verify results of Sudan test.
More on Lipids
• Lipids, or fats, are found in cells & serve
as a possible energy reserve.
¾You will observe a DEMO slide of Adipose tissue
(i.e. fatty tissue) containing lipids. Note: where are
the lipids are located within the cell (nucleus or
cytoplasm)?
¾ You will also observe a DEMO slide of Adipose
tissue treated with lipase, an enzyme that breaks
down lipids. Are lipids still present in this cell?
3. Proteins & Amino Acids
• Proteins are made up of amino acid chain held
together by peptide bonds (a polypeptide).
• Amino acids – contain an amino group (=NH2)
and a carboxyl group (-COOH).
http://fajerpc.magnet.fsu.edu/Education/2010/Lectures/10_Proteins_files/image016.jpg
Biuret Test for Proteins
Biuret Reagent – (made up of CuSO4, copper sulfate)
forms a complex with the peptide bonds of
adjoining amino acids causing the reagent
to change color.
• Biuret must bind to 4-6 peptide bonds in order
for reaction to occur (i.e. only detects proteins,
not single amino acids).
¾changes from blue to violet color.
Biuret’s Test for Proteins
Negative
Positive
http://iws.ccccd.edu/biopage/BioLab/Unit%205/Unit%205%20index.htm
Ninhydrin Test for
free Amino Acids
• Ninhydrin Test – detects presence of
free amino acids, NOT proteins, by
reacting with the free amino group (-NH2).
¾Turns violet if amino acids are present.
¾Exception: if proline (an a.a.) is present, it
will turn yellow, instead of violet.
4. Nucleic Acids
• DNA & RNA are made up of nucleotide subunits.
– DNA contains deoxyribose sugar.
– RNA contains ribose sugar.
• Tests for presence of DNA =
Dische Diphenylamine Test
We will NOT be doing this test today!!!
¾ You will observe a DEMO slide of a cell containing DNA. Note:
where is the DNA located within the cell (nucleus or cytoplasm)?
***Think about what you already know about DNA (genetic material)!
¾ You will also observe a DEMO slide of cell that has been treated
with DNAse, an enzyme that breaks down DNA. Can you still
detect the presence of DNA in this cell?
Testing for Organic Molecules
Biochemical tests were designed to
identify these molecules based on their
unique molecular characteristics.
Each test MUST have:
(1) The UNKNOWN to be tested (your samples)
(2) Controls (both positive & negative)
Testing for Organic Molecules
Before starting….
You must do INITIAL OBSERVATIONS!
Before you add any reagents to your test
samples & controls you MUST observe
your samples…
» What do they look like (color, consistency, clarity,
etc.)?
» Smell like?
» NO TASTING!!!
» RECORD these observations.
Testing for Organic Molecules
Must haves:
1. Unknown samples
2. Positive & Negative Controls
3. Reagents used for detecting specific
molecules.
4. Initial observations
Question: Why use controls?
Answer: ???
¾
Need controls to validate the procedures & to assist in
determining results of your experiment.
CONTROLS
Positive Control- a sample known to contain the molecule
that you want to detect.
Example: if you want to use the Biuret Test for proteins, your
(+) control would contain a protein, such as egg whites, so that the
sample would change from a blue to a violet color.
Negative Control – a sample that does not contain the
target molecule that you want to detect.
Example: In the case of Biuret Test, your (-) control would be a
solution, such as distilled H2O, that would not react with the Biuret
reagent to change color.
Compare the results of your (+) & (-) control tests to the
results of your unknown sample. This will help you to
determine whether you had a positive reaction.
TODAY’S PLAN:
Summary
• Working in groups of 4 (by table), you will make
observations & perform several biochemical
assays on ALL samples to help determine the
identity of your 8 ‘unknowns’ (household items).
–
–
–
–
–
Use Benedict’s Test to I.D. – simple sugars
Use Lugol’s Test to I.D. – starch
Use Sudan IV & Grease Spot Tests to I.D. – lipids
Use Biuret Test to I.D. – proteins
Use Ninhydrin Test to I.D. – amino acids
• Observe & sketch several DEMO slides
illustrating organic molecules found in cells/tissues.
TODAY’S PLAN:
Summary
Caution! Caution! Caution! Caution!
Because you will be working with chemicals & unknown
samples you MUST:
MUST
•
•
•
•
Wear gloves
Wear goggles
Follow all lab rules carefully.
Work slowly & carefully to complete the exercises & to avoid
spills & other avoidable accidents.
PAY CLOSE ATTENTION to the
LAB MANUAL INSTRUCTIONS!
Ninhydrin is a chemical used to detect amino acids.
It should be handled carefully in the FUME HOOD only!!!