Laboratory Manual for Physiological Chemistry
Spring 2009
Table of Contents
Handling Emergencies ............................................................................................ 2
Safety Regulations ................................................................................................. 3
General Laboratory Procedures............................................................................... 4
Introduction to Organic Compounds ....................................................................... 7
Isolation of Chlorophyll and Carotenoid Pigments from Spinach ............................. 26
Chemical Properties of Aliphatic and Aromatic Alcohols .......................................... 34
Oxidation and Structure of Carbonyl Compounds................................................... 42
Optical Isomers.................................................................................................... 50
Carbohydrates ..................................................................................................... 58
Acid-Base Reactions with Carboxylic Acids and Esters ............................................ 63
Synthesis of Aspirin .............................................................................................. 67
Synthesis & properties of Soap ............................................................................. 75
Isolation and Characterization of Casein from Milk................................................. 79
Amylase: The Activity of an Enzyme ..................................................................... 85
Interaction of UV Light with Matter ....................................................................... 91
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1
Laboratory Rules
and Guidelines
Handling Emergencies
While we will do everything possible to ensure a safe environment, accidents can
occur. In case of the following emergencies, always inform the instructor and do the
following:
Burns - Flush with cool tap water.
Chemicals in the eye - CALL FOR HELP. Force the eye open, flush with water for
at least 20 minutes.
Chemicals on the skin - Flush with water. Rinse acid spill with sodium hydrogen
carbonate solution; bases with boric acid.
Chemicals on your clothes -REMOVE YOUR CLOTHES. You do not want the
chemicals to reach your skin.
Clothing on fire - STOP-DROP-ROLL. Use a fire blanket or shower only if you are
standing within arm's reach.
Cut in skin - Rinse immediately with water. Inspect for glass. Get medical
attention.
2
Safety Regulations
1) Acceptable eye protection must be worn at all times. Wearing contact lenses in the lab is
strongly discouraged and may be forbidden by your instructor.
2) Bare feet and sandals are not allowed in the lab. Spilled chemicals and broken glass on
the floor can result in serious injuries.
3) Shorts and short skirts are not allowed. Neither midriff nor shoulders may be exposed
whether standing straight, reaching, or bending over.
4) Each student must know the location of the safety equipment: fire extinguishers, eye
washes, safety shower and the exit.
5) Horseplay and/or carelessness are prohibited.
6) No unauthorized experiments are to be performed.
7) Work is permitted only at the assigned time unless otherwise authorized by the instructor. In
any case, NEVER work alone.
8) Chemicals and equipment are not to be removed from the laboratory.
9) Do not sit on bench tops.
10) Eating, drinking, smoking or chewing anything is not permitted in the laboratory.
11) Never pipet by mouth; always use a pipet bulb.
12) Be cautious when testing for odors. Always wave fumes towards your nose with your
hand. NEVER smell a chemical directly.
13) Always add acid to the water or base, never do the opposite.
14) Never aim the opening of a test tube or flask at yourself or anyone else.
15) Never leave reactions unattended if they involve heating or rapid reactions.
16) Only the lab manual and lab notebook should be at lab counter. Book bags, outerwear, etc.
are to be placed out-of-the-way in the location indication by your instructor.
17) Report any injury, however minor, to the instructor at once.
18) Always use tongs to handle hot objects.
19) Loose clothing must not be worn in the laboratory. Open sweaters/hoodies, loose sleeves,
excessively “flowy” blouses.
20) Hair below chin must be tied back, while working in the laboratory.
21) Broken glass will be disposed of in the glass disposal boxes, not in the regular trash.
22) Dispose of waste properly, as directed by your instructor.
23) Clean up all spills immediately.
24) Always check glassware for chips and cracks.
I realize that these RULES WILL BE ENFORCED for my safety and for the safety of my lab partners
and that failure to observe these rules, in addition to resulting in unacceptable safety hazards and the
loss of working time, will result in expulsion from the laboratory. I acknowledge that I have received
a written copy of these regulations and have been given the opportunity to discuss them with the
instructor.
NAME __________________________________COURSE_______SECTION_____
SIGNATURE_____________________________________DATE_______________
3
General Laboratory Procedures
The following procedures are intended to prevent contamination of chemicals, and to promote safety,
smooth laboratory operation and laboratory efficiency.
Read labels
carefully
Some chemicals have very similar names. In the case of acids and bases, do not
assume that the reagent bottles are in the correct places.
Eyedroppers/
Pipets
Never use an eye dropper or pipette in a reagent bottle unless there is a dropper with
the bottle for use only with that bottle. Your seemingly clean eyedropper may actually be
dirty, and end up contaminating an entire bottle of reagent. If you need to dispense a
chemical with an eyedropper, pour a small amount into a beaker and use the
eyedropper from there.
Unused or
excess
reagents
Never pour them back into a reagent bottle. Contamination of the contents may result.
Dispose of the extra properly. Along with this, do not take large amounts of reagents,
since excess amounts must be wasted. You can always get more if you need it.
Obtaining
solids
Obtain solids using a spatula specified for the particular reagent, or by pouring the solid
out by rotating the bottle back and forth until the solid works its way out of the bottle or
vial.
Cleaning
Equipment
Be sure your equipment is clean before use, and be sure to return borrowed equipment
either as clean or cleaner than you found it. Wash glassware with soap and tap water.
Rinse with tap water. Occasionally, some chemicals and glassware markers must be
removed with acetone. Do a final rinse with a small amount of distilled water.
Using litmus
and other
test papers
Always do litmus paper or pH paper tests by placing the test paper on a clean watch
glass and transferring a drop of the test solution to the litmus using a stirring rod.
Vials and Jars
Always recap vials, jars and bottles immediately after use. DO NOT LEAVE THEM
UNCAPPED. Contamination of the contents may result, spillage could occur if the
container is knocked over, and many solid chemicals absorb water from the air which
makes them cake or become sticky making them unweighable.
Using
Balances
The balances are expensive pieces of equipment and must be treated with respect.
Please observe the following:
a. Do not move the balance from its spot unless specifically asked to do so.
b. Place objects on the pan gently.
c. Never place chemicals directly on the pan. Always use a container.
Clean spills on balances immediately. Ask your instructor for assistance.
Waste
Disposal
Use designated waste container to dispose waste after the experiment.
4
Laboratory Notebooks
A laboratory notebook is the most basic piece of equipment used in the laboratory. It is very
necessary to develop a proper method of using a notebook, no matter what science one pursues. It is
one of the goals of this laboratory experience to develop good notebook technique. Everyone has his or
her own style of recording data, and different instructors may demand different things. The guidelines
described here are to give you an idea of the minimum that is typically acceptable.
Your laboratory notebook is for recording data and observations while performing experiments.
Notes from class discussion may appear in the notebook, but they must be clearly distinguished from
experimental data. (see below)
The purpose of a laboratory notebook is to provide YOU OR ANYONE ELSE with an accurate
record of what YOU DID in the laboratory, not necessarily what you were supposed to do. It should
contain:
1) Title of the project/experiment performed
2)
Brief descriptions of what you did.
2)
Qualitative observations.
3)
Numerical data which is well-labeled as to what it is, and which has the proper units of
measurement.
4) Information of any reference material used for the work
ALL data and observations are to be written in your notebook, NOT in the margins of reference
materials, handouts, or laboratory manuals, and NOT on any other sheets of paper in the laboratory.
You will not copy over anything into another notebook at any time. Your notebooks will be handed in
from time to time for grading. Grading will be based in part on your adherence to these guidelines.
Concerning (1) above, you should write down what you did without copying what the instructions
said. Fragmentary sentences are fine, as long as they are clear. If you ask a question of your lab
instructor, he or she will probably ask to see your notebook to see what you did. DO NOT present the
instructions. That says what you should have done, not what you did.
Remember...
You or another person should be able to use your notebook and any sources
you cite, and exactly reproduce what YOU DID in the laboratory.
Guidelines
1. While in the lab write ONLY in your notebook. DO NOT write on scrap paper or anything else.
DO NOT transcribe your notebook in any form. The original is the only legitimate copy. You may
write in your notebook outside of lab as long as you properly date the entries and DO NOT CHANGE
ANYTHING THAT WAS PREVIOUSLY ENTERED.
2. Write what you did. This may be different from what you were supposed to do. If you have a
record of what you did, you may have a way of figuring out what went wrong if something does go
wrong.
3. The first two pages of a notebook should be reserved for a Table of Contents, which is
continually updated. Sometimes notebooks already have a table of contents section in them.
5
4. The pages must be numbered starting at 1 for the first page and continuing on each page (each
side of the sheet) until the end of the notebook. Notebook pages are numbered just like book pages
are. Some notebooks come with the pages prenumbered.
5. The date (including the month, day and year) must appear on each page of the
notebook. The date must also appear whenever data is being taken on a new date.
6. The name of the experiment must be at the beginning of the notes for that
experiment. This is usually, but not required to be, the name of the exercise. It could be your own
title for what is being done.
7. The source of the experiment, that is, the full bibliographic citation of where your procedures
are taken from, must appear at the beginning of an experiment. At any time if another source
is used, it must also be properly cited. If any data are taken from reference or other books, these
must be properly cited. Your instructor will give you an example.
8. Notebooks must be written in blue or black pen, NOT in pencil. Use only standard blue or
black ink please.
9. NO erasures should ever be made. Also, no White-out is to be used. If you write something
incorrectly, simply draw a SINGLE line through it and continue to write. Also, you should NEVER
overwrite anything. Again, simply putting a single line through the error and rewriting is the best
policy.
10. Pages must never be torn out of a notebook. The original pages must remain intact.
11. Everything must be labeled. That is, each procedure to be performed must be clearly identified.
All data and observations must be clearly labeled as to what it is, what the units of measurement
are, etc. For example, labeling the mass of an object, say a test tube, as "test tube 2.0 g" is
insufficient. "Mass of test tube 2.0 g" would have a better label. Always label your data so that
several days after recording it you will be able to know what it is. Be liberal with headings and
subheadings. Headings such as "Part 1" and "Part 2" are not sufficient. What are you doing in Part
1? You must also explicitly distinguish between experimental data and observations, notes from
group discussions, and your own conclusions or hypotheses.
12. If you use an instrument such as a balance or spectrophotometer, always include the brand
name and model number (if available). Also, many of our instruments are numbered, for
example "Spectronic 20 #5". This number should be included. This is especially important if you
discover at a later time that the data you recorded do not make sense. Perhaps there was an
instrument malfunction. If you know specifically which instrument you used, that possibility can be
checked out.
13. Your notebook does not have to be so neat and orderly that it is ready to be published in
"Notebooks Beautiful", but it should be sufficiently organized and legible so that you or someone
else could use it to reproduce your experiment or write a report.
14. Do not use your lab notebook for any other course.
15. There should be only one notebook, no other copy.
6
Introduction to
Organic Compounds
Goals for the Student:







Learn to identify organic functional groups
Learn to classify organic compounds based upon their functional groups
Learn to name organic compounds based upon their functional groups
Construct models of alkanes to view three-dimensional structure of alkanes.
Investigate the relationship between a structural formula and a three dimensional molecule
using molecular models
Construct models of isomers of alkanes with the same molecular formula.
Identify isomers, structural formula, condensed structural formula and skeletal formulas.
Introduction
In this exercise we will be introduced to organic compounds. Organic chemistry is the study of
compounds that are primarily composed of carbon and hydrogen atoms. Other prominent elements in
organic chemistry are oxygen, O, nitrogen, N, sulfur, S, and the halogens (fluorine, F, chlorine, Cl,
bromine, Br, and iodine, I). Since all organic compounds contain some amount of carbon and hydrogen
atoms, organic compounds are identified and classified by the functional groups they possess. A
functional group is a group of atoms that react in a predictable way. Compounds with the same
functional group are classified into a particular class of organic compounds and the name is derived from
belonging to that class.
Functional Groups
Table 1: Organic Functional Groups
Functional Group
Class
Characteristic
Example
C
C
Alkane
Only carbon-carbon
single bonds
H3C
CH3
C
C
Alkene
Carbon-Carbon double
bond
H2C
CH2
C
C
Alkyne
Carbon-carbon triple
bond
HC
CH
7
Aromatic
H
Six atom carbon ring with
alternating double and
single bonds
H
H
H
H
H
X
X = F, Cl, Br, or I
OH
O
SH
O
C
Haloalkane
Alcohol
Hydroxyl group (-OH)
Ether
Oxygen atom bonded to
two carbon atoms
Thiol
A –SH group bonded to a
carbon atom
Aldehyde
Carbonyl group (carbonoxygen double bond)
with –H
H
O
One or more halogen
atoms
Ketone
H3C
OH
H3C
O
Carboxylic Acid
O
O
H
Ester
Carboxyl group (carbonoxygen double bond and
–OH)
SH
O
H3C
C
Nitrogen atom with one
or more carbon groups
N
8
C
CH3
O
H3C
C
O
H
O
H3C
Amine
H
O
Carboxyl group with –H
replaced by a carbon
O
CH3
H3C
H3C
O
C
Cl
Carbonyl group between
two carbon atoms
C
C
H3C
C
H3C
O
NH2
CH3
O
O
C
N
Amide
Carbonyl group bonded
to a nitrogen atom
H3C
C
NH2
Nomenclature
Nomenclature of organic compound is governed by the International Union of Pure and Applied
Chemistry (IUPAC) system. The system is founded on two major principles; (1) determine the longest
continuous carbon chain and (2) number, name, and alphabetize all substituents. These principles have
been elaborated into the following set of nomenclature rules.
1. Straight chain compounds with only carbon and hydrogen atoms (Alkanes)
a. count the number of carbon atoms
b. add the ending –ane to the prefix corresponding to the correct number of carbon
atoms.
Table 2: IUPAC Names for the First Ten Continuous-Chain Alkanes
Number of Carbon Atoms
1
2
3
4
5
6
7
8
9
10
Prefix
meth
eth
prop
but
pent
hex
hept
oct
non
dec
Name
methane
ethane
propane
butane
pentane
hexane
heptanes
octane
nonane
decane
Molecular Formula
CH4
C2H6
C3H8
C4H10
C5H12
C6H14
C7H16
C8H18
C9H20
C10H22
2. Alkanes with substituents
a. write the name of the longest continuous chain of carbon atoms
b. number the carbon atoms starting from the end nearest the first substituent to
generate the lowest set of numbers
c. give the location and name of each substituent as a prefix to the alkane name
i. place a hyphen between the number and the substituent name
ii. alphabetize the substituents
iii. use a prefix (di-, tri-, tetra-, etc) if a substituent appears more than once and
use commas to separate two or more numbers.
Table 3: Common Substituent Names and Structures
Substituent Structure
methyl
H3C
H3C
H3C
Name
ethyl
CH2
CH2
propyl
CH2
9
H3C
H
C
isopropyl
H3C
H3C
CH2
CH2
butyl
CH2
H3C
H
C
CH2
isobutyl
H3C
CH3
H3C
C
tert-butyl (tbutyl)
CH3
X
Cl
H3C
1
CH
2
CH3
CH2
3
CH
4
CH2
5
CH3
Br
CH2
CH
CH
CH3
4
3
2
1
Cl
H3C
C
fluoro, chloro, bromo, iodo
(X = F, Cl, Br, or I)
2-chloro-4-methylhexane
CH3
6
2-bromo-5,5-dichloro-3-methylhexane
Cl
6
5
3. Cycloalkanes
a. count the carbon atoms in the ring and add the prefix cyclo to straight chain name
b. substituent rules from above apply except the first substituent is always placed on
carbon 1
c. alphabetize to determine substituent on carbon 1
H3C
CH2CH3
1-ethyl-3-methylcyclopentane
4. Alkenes and Alkynes
a. name the longest continuous carbon chain that contains the double or triple bond
i. replace the –ane ending of the alkane with –ene for an alkene and –yne for an
alkyne
b. number the longest continuous carbon chain from the end nearest the double or triple
bond
c. give the location and name of each substituent as a prefix to the alkene or alkyne name
10
i. place a hyphen between the number and the substituent name
ii. alphabetize the substituents
iii. use a prefix (di-, tri-, tetra-, etc) if a substituent appears more than once and
use commas to separate two or more numbers.
CH3
H2C
1
CH
2
CH
3
CH
4
Cl
Br
4-methyl-2-pentene
CH3
5
HC
C
CH
CH
CH3
1
2
3
4
5
3-bromo-3-chloro-1-pentyne
5. Aromatics
a. monosubstituted benzene rings are named as benzene derivatives using the substituent
name
Table 4: Common Monosubstited Aromatic Compuonds
Structure
IUPAC Name
Common Name
methylbenzene
toluene
NH2
benzeneamine
aniline
OH
hydroxybenzene
phenol
CH3
b. disubstituted benzene rings are numbered to give the lowest number to the
substituents
i. common prefixes are often used (1,2 substitution is ortho, 1,3 substitution is
meta, and 1,4 substitution is para)
c. if a benzene ring is a substituent (longest chain is more than six carbons or contains a
double or triple bond), then it is named a phenyl group
Isopropylbenzene
Cl
1,2-dichlorobenzene or
orthochlorobenzene
Cl
11
H3C
1
CH2
2
CH
3
CH2
4
CH2
5
CH2
6
CH3
7
3-phenylheptane
6. Alcohols & Thiols
a. name the longest continuous carbon chain containing the hydroxyl group (-OH)
i. replace the –ane ending of the alkane with –ol ending
b. number the longest continuous carbon chain starting at the end closest to the hydroxyl
group
c. name and number other substituents relative to the hydroxyl group
d. name a cyclic alcohol as a cycloalkanol with all cycloalkane rules applying for
substituents
e. apply aromatic naming rules for benzene rings containing a hydroxyl group. The base
name is then phenol
f. thiols are named by adding thiol to the alkane name of the longest continuous carbon
chain bonded to the –SH group
i. the location of the –SH group is indicated by numbering the main chain from
the closest end
H3C
1
OH
CH3
CH
2
CH
3
SH
CH
2
4-iodo-3-cyclohexanol
OH
I
H3C
1
3-methyl-2-butanol
CH3
4
CH3
CH2
3
CH
4
CH2
5
4-methyl-2-hexanethiol
CH3
6
7. Ethers
a. write the alkane name of the larger alkyl group as the main chain
b. name the oxygen and smaller alkyl group as a substituent called an alkoxy group
H3C
H2C
O
CH2
O
CH2
Ethoxypropane
CH3
methoxybenzene
CH3
8. Aldehydes & Ketones
a. for an aldedyde, name the longest continuous carbon chain containing the carbonyl
group by replacing the e in the alkane name with al
i. name and number any substituents on the carbon chain by counting the
carbonyl carbon as carbon 1
12
b. for a ketone, name the longest continuous carbon chain containing the carbonyl group
by replacing the e in the alkane name with one
i. number the main chain starting from the end nearest the carbonyl group
ii. name and number any substituents on the carbon chain
O
H3C
4
CH2
3
H3C
H2C
5
4
CH2
2
CH3
C
1
butanal
H
O
CH
CH2
C
3
2
1
3-methylpentanal
H
O
H3C
H2C
C
CH2
CH3
5
4
3
2
1
H3C
H2C
6
5
3-pentanone
O
Cl
CH2
C
CH
CH3
4
3
2
1
2-chloro-3-hexanone
9. Carboxylic Acids
a. name the longest continuous carbon chain containing the carbonyl group and replace e
of the alkane name with oic acid
b. number the carbon chain beginning with the carboxyl group as carbon 1
c. give the location and names of substituents on the main chain
d. for the aromatic benzoic acid, number the ring from the carboxyl group as carbon 1
O
H3C
4
H3C
H2C
3
H2C
CH2
2
CH3
C
CH2
C
1
butanoic acid
OH
O
C
OH
3,3-dimethylpentanoic acid
CH3
5
4
3
2
1
10. Esters
a. write the name of the carbon chain from the alcohol as an alkyl group
b. write the name of the carboxylic group as carboxylate with an –oate ending
13
O
H3C
CH2
O
ethyl propanoate
C
CH2
CH2
CH3
O
H3C
O
methyl benzoate
C
11. Amines & Amides
a. for amines, name the longest continuous carbon chain bonded to the nitrogen atom
and replace the e in the alkane name with amine
i. number the carbon chain to show the position of the amine group and any
other substituents
ii. in secondary and tertiary amines, use the prefix N- to name smaller alkyl group
attached to the N atom.
b. amides are named by dropping the oic acid from the carboxylic acid name and adding
amide
H3C
CH2
CH2
Propanamine
NH2
CH3
H3C
CH2
CH2
N-methyl propanamine
NH
O
H3C
H3C
H2C
H2C
CH2
CH2
C
butanamide
O
NH2
CH3
C
N
N,N-dimethyl butanamide
CH3
Also, in this exercise we will study the three dimensional structure of some alkanes, using a
molecular model kit to reinforce the nomenclature for alkanes and some of their’s derivatives. In each
type of alkane each carbon has four valence electrons and must always have four single bonds to other
carbon, hydrogen or halogen atoms. The bond arrangement of four single bonds used by carbon in
alkane is shown as below.
Bonding Pattern of Carbon
Arrangement of Bonds around Carbon
Spatial Structure and Bond Angles
C
Tetrahedral
To understand the three dimensional structure of organic compounds, models can be build using a
ball and stick model kit. In this kit, there are colored spheres which represent the atoms drilled to
receive connecting bonds. Different color spheres, black for carbon and red for oxygen, are used to
14
represents different kinds of atoms and a color code for atoms will be included in the model kit. Each of
the spheres (atoms) has the correct number of holes for bonds (wooden or plastic stick) that attach to
other spheres.
a)
(c)
(b)
The three dimensional structure of the alkane models represents very closely resembles the
approximate geometry (shape and angle) of the molecules they represent. Two structures are
identical if they are superimposable-that is, if one structure can be place “on top” of another so that
all colored spheres coincide. Methane is the first member of alkanes and three different structure of
methane is shown above, which represents the structural formula (a), three dimensional structures (b)
and ball and stick model (c).
Compounds having the same molecular formula can be represented by more than one structure and
each structure includes the same group of atoms but a different spatial arrangement of the atoms.
These compounds are called isomers. Isomers have the same molecular formula but different three
dimentional structures. One structure cannot be converted to the other without breaking and forming
new bonds.
The isomers have different physical and chemical properties. One of the reasons for the vast array
of organic compounds is the phenomenon of isomerism. Many biological reactions are very specific and
involve only one isomer.
Isomers of C2H6O
Isomers of C4H10
CH3
CH3 CH2 CH2 CH3
n-butane
CH3 CH
CH3
H
2-methyl propane
H
H
C
C
H
H
ethyl alcohol
H
O
H
H
C
H
H
O
C
H
H
dimethyl ether
Experimental
This is a two week exercise. In the first week you should complete and turn into your instructor the
nomenclature report sheet. During the second week you will perform the structure portion of the
experiment, completing and turning in the second report sheet.
The model kit includes different colored spheres representing different atoms and grey connectors
for representing bonds. Carbon atoms are black spheres and have four holes that represent the four
15
covalent bonds that carbon atoms always form. Hydrogen atoms are white spheres and only form one
bond. The green sphere represents chlorine atom and oxygen atoms are red sphere. Halogen atoms
form one bond and oxygen atoms form two bonds; spheres for these atoms will have the appropriate
number of holes.
Single covalent bonds are represented by grey connectors, which insert into holes of the atoms. To
conserve time and depending upon the number of pieces in your model kit, you may use only the stick
to represent the C- H bonding arrangement.
You will be working in groups(two/three) to construct the models of different compounds(alkane,
haloalkane, haloalcohol) using the model kits. Each model must be investigated for geometry (shape &
angle)
16
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17
REPORT SHEET-Introduction to Organic Compounds (Week 1)
Name____________________________ Partner’name _________________________
Section______________ Date_________
Name the following compounds
I. Alkanes
Br
___________________________________
___________________________________
II. Alkenes / Alkynes
_________________________________
III.
___________________________________
Aromatics
_________________________________
___________________________________
18
IV. Alcohols & Thiols
HS
OH
_________________________________
___________________________________
V. Ethers
O
O
_________________________________
___________________________________
VI. Aldehydes & Ketones
O
O
H
Br
_________________________________
VII.
___________________________________
Carboxylic Acids
O
I
O
HO
I
HO
_________________________________
I
___________________________________
19
VIII. Esters
O
O
O
O
_________________________________
___________________________________
IX. Amines & Amides
O
NH2
N
_________________________________
___________________________________
20
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21
REPORT SHEET – Introduction to Organic Compounds (Week 2)
I. Construct a model of methane, CH4
a) What is the geometry associated with this structure ?
b) What is the value of the H─ C ─ H bond angles?
____________________________
_____________________________
II. Construct a model of chloromethane, CH3Cl
a) Draw a wedge and hash mark to represent the three dimensional shape of the molecule.
b) Is the geometry the same as methane? ___________________
c) Are the hydrogen atoms equivalent (i.e., do they have identical environments with respect to the other atoms
adjacent to themselves)?__________________
III. Construct a model of chloromethanol, CH2Cl(OH)
a) Is the geometry the same as the previous two structures? ____________________
b) Are the hydrogen atoms attached to the carbon atom equivalent? ________________
c) What part of the name indicates the alcohol group? ___________________
IV. Construct a model of ethane, C2H6
a) Draw a condensed structural formula of C2H6 ______________________________
b) Draw a structural formula of C2H6
c) Are the two carbon atoms of C2H6 equivalent?
___________________________
d) Are the six hydrogen atoms of C2H6 equivalent? ____________________________
22
V. Construct a model for chloroethane, CH3CH2Cl
a) Are the carbon atoms in
CH3CH2Cl equivalent? ____________________
b) Are the hydrogen atoms in CH3CH2Cl
equivalent? _____________________
VI. Construct all possible models for dichloroethanes, C2H4Cl2
a) How many structural isomer exist for
C2H4Cl2 ? _______________________
b) Draw condensed structural formulas for each structural isomers of
C2H4Cl2.
VII. Construct all possible models for propane, C3H8
a) Draw a structural formula for C3H8 and using squares, triangles, and /or circles, indicate the carbon
atoms that are equivalent to each other
b) Are the eight hydrogen atoms of
C3H8 are equivalent? ________________
c) Is there a relationship between equivalent carbons and equivalent hydrogens? If so, state the
relationship.
VIII. Construct all possible models for chloropropane, C3H7Cl
a) How many structural isomers exist for C3H7Cl
? ________________________
23
b) Draw condensed structural formula for each structural isomer of C3H7Cl
IX. Construct all possible models for C4H10 (Hint: straight versus branched chain)
a) How many structural isomers exist for C4H10
? ________________________
b) Draw condensed structural formulas for each structural isomers of C4H10. Also, using squares,
triangles, and /or circles, indicate the carbon atoms that are equivalent to each other
24
X. Using your C4H10 models from above, remove one hydrogen atom and
replace it with a chlorine atom to make different structural isomers of
C4H9Cl.
a) How many structural isomers exist for C4H9Cl.?
_______________________
b) Draw condensed structural formulas for each structural isomer of C4H9Cl.
XI. Draw line-bond formulas for all possible structural isomers of C4H8Cl2,
which are formed by replacing hydrogen atoms in the various isomers of
C4H9Cl (exercise X. b) with a second chlorine atom.
25
Isolation of
Chlorophyll and
Carotenoid Pigments
from Spinach
Adapted from: Pavia, D. L.; Lampman, G. M.; Kriz G. S.; Engel, R. G. Introduction to Organic Laboratory
Techniques: A Microscale Approach 3rd Edition Saunders College Publishing: New York, NY, 1999 and
also Quach, H. T.; Steeper, R. L..; Griffin, G. W., J. Chem. Educ., 2004, 81, 385-387. Toni Bell (2004)
Goals for the Student:


Learn the techniques of extraction and purification of chemical compounds from natural
products.
Learn the technique of identification of different components in chemical compounds isolated
from
natural products
Introduction
Spinach, a green leafy vegetable usually can be grown as a spring and fall crop in the cooler
North American climate. Spinach is a source of Vitamin A and it is rich in iron, and calcium. The leaves
contain a number of colored pigments, generally falling into two categories: chlorophylls and
carotenoids.
Isoprene is the basic five-carbon
building block of the terpene class of
biological compounds.
head end
tail end
Carotenoids are part of a larger collection of plant derived compounds called terpenes. These
naturally occurring compounds contain 10, 15, 20, 25, 30, and 40 carbon atoms. Isoprene is the basic
five-carbon building block of the terpene class of biological compounds. Also known as 2-methyl-1,3butadiene, these units are linked in a “head to tail” fashion to build the structure of terpenes. Two
isoprenes are linked together to make one terpene unit. The branched end is the “head” and the
unbranched end is the “tail”. Carotenoids are tetraterpenes (eight isoprene units).
Spinach leaves contain chlorophyll a and b and β-carotene as well smaller amounts of other
pigments such as xanthophylls which are oxidized versions of carotenes and pheophytins which look
like chlorophyll except that the magnesium ion Mg+2 has been replaced by two hydrogen ions H+.
Chlorophylls a and b are the pigments that make plants look green. The double bonds are conjugated,
meaning they occur between every other pair of carbons, and allow capture the (nongreen) light energy
used in photosynthesis.
26
β-Carotene is a carotenoid and it causes carrots and apricots to be orange. When ingested, βcarotene is cleaved to form two molecules of Vitamin A. Vitamin A, also called retinol, plays an
important role in vision and serves as an anti-oxidant.
Chlorophyll a (left) and
chlorophyll b (right) are very
similar molecules. Can you
spot the differences? These
small changes are enough to
change their color.
-Carotene (top), carotene (middle), and
xanthophylls (example on
bottom) have very similar
structures. Can you spot
the differences? These
small changes are
enough to change their
color. Your body can
only use -carotene to
make Vitamin A. Why?
27
In this experiment we will isolate and use differences in polarity of the pigments to effect a
separation. Chlorophylls and carotenoids are slightly different in polarity. Due to their lovely color, we
will easily follow the separation visually. β-Carotene is a hydrocarbon and it is very nonpolar. Both
chlorophylls contain C ─ O and C ─ N bonds which are polar and also contain magnesium bonded to
nitrogen which is such polar bond that it is almost ionic. Both chlorophylls are much more polar than βCarotene. There is another structural difference in between the chlorophyll a and b, Chlorophyll a has a
methyl group (─ CH3) in a position where chlorophyll b has an aldehyde group (─ CHO). This makes
chlorophyll b slightly more polar than chlorophyll a.
Since spinach also contains cellulose, iron, and water soluble vitamins in addition to chlorophylls
and carotenes, we have to have a method of separating all these compounds. The most common
approach to isolating these bioactive natural products is extraction. Chlorophylls and carotenes are
relatively non-polar organic substances compared to other components; hence, they are more soluble
in organic solvents like dichloromethane or acetone. Since ‘like dissolves like’, these solvents will be
suitable to selectively extract these compounds into organic solvent and leave the other compounds
behind. To do the extraction, you will first grind up the spinach in a little bit of acetone. The green
acetone with spinach compounds is called extract. Unfortunately acetone will dissolve almost anything,
including the stuff that you do not want.
Chlorophylls and carotenes do not dissolve very well in water; they dissolve like crazy in hexane.
The other compounds do not dissolve well in hexane, but dissolve well in water. Like water and oil,
water and hexane are immiscible; they ‘do not mix’. The hexane will form a layer on top of the water
(like oil does) because hexane is less dense than water. After vigorous shaking to mix the layers
temporarily, you will allow them to separate. The lovely green chlorophylls and yellow carotenes will
leave the water at the bottom to dissolve in the hexane layer at the top. After pipetting-off hexane
layer, the different components in the pigment mixture will be analyzed by using thin layer
chromatography.
The number of compounds in the hexane extract can be quickly determined by a technique
called thin layer chromatography, which is abbreviated “TLC.” You will put a little spot of your
extract on a plastic plate coated with silica gel. Silica gel is a very polar substance. The plates are
placed in a container with a mixture of solvents. In this case, the solvents will be quite non-polar. The
solvents will begin to travel up the plate, like a wick. Some of the compounds in the hexane extract will
be more polar and will stick to the spot on the silica. Other compounds in the hexane extract will be
less-polar to differing degrees and will travel up with the solvent Since there are many levels between
totally polar and totally non-polar, the compounds can be separated by polarity. The more affinity a
compound has for the solvent, the farther up the plate it will travel.
Different compounds should rise to different heights on your TLC plate; however the exact
height a particular compound rises depends on how high the solvent is allowed to rise up the plate. If
the solvent travels higher, then the spots all travel higher too. To correct for this difference and
generate a number which can be compared to reported values or to other individual’s work, the
retention fraction or Rf. value is calculated. The retention fraction is defined to be the fractional rise
of the spot compared to the rise of the solvent. The Rf value for a compound will change if a different
developing solvent or a different type of plate is used. After you have developed your TLC plate with
your hexane extract you have to calculate Rf values for each spot on your plate. Spots with the same Rf
values within experimental error and the same appearance should be the same compound. An example,
if you had just two components in your original extract, this is what your results might look like:
28
Experimental
Extraction of pigment from leaves:
This is a VERY colorful experiment and you need to make note of the colors of things at each step.
You should draw the centrifuge tube, for example, and label the layers and the colors. Keep in mind
that the term “clear” refers to transparency while “colorless” refers to absence of color. You can have
a clear green solution or an opaque green solution. Conversely, you can have a clear colorless solution
or an opaque colorless solution.
1. Weigh about 1.5 g fresh spinach leaves (don’t use stems) and record the mass and observations
about the color. Tear the leaves into confetti-sized pieces and place these pieces along with
0.5 g of anhydrous magnesium sulfate and 1.0 g of sand into a mortar (the bowl part of the
mortar and pestle).
2. Grind with a pestle until a light green powder is obtained (about 5-10 minutes).
3. Transfer the powder mixture into a 15 mL plastic centrifuge tube with a cap.
4. Using the squeeze bottle, add roughly 1.0 mL of acetone to the mortar to rinse.
5. Transfer the rinse acetone to the centrifuge tube containing the powder with a Pasteur
pipette. Your instructor will show you how to use a pipette properly as part of your pre-lab
discussion.
6. Repeat steps 4 and 5.
7. If the volume of acetone has evaporated to less than roughly 2.0 mL, as measured using the
markings on the centrifuge tube, then add enough to make-up the volume.
8. Cap and shake the mixture. Be sure to vent the tube occasionally by pointing away from
you and others and loosening the cap. Allow the tube to stand for a few minutes so the
solid material may separate.
9. Transfer the liquid from centrifuge tube to a clean centrifuge tube with a Pasteur pipette. Write
“extract” on the second tube, along with your initials.
10. Add about 2.0 mL of hexane and 2.0 mL distilled water to the extract. Cap and shake the
mixture. Be sure to vent the tube occasionally by pointing away from you and others
and loosening the cap. Allow the tube to stand for a few minutes so the layers may separate.
Identify the hexane layer and the water layer (How can you do this if you don’t know?). Make
a labeled sketch of the tube, contents, and colors in your notebook.
11. Remove the water layer with a Pasteur pipette and transfer it to a small beaker labeled
“waste.”
12. Add another 2.0 mL of distilled water to the hexane layer in the centrifuge tube as a wash.
Cap and shake the mixture. Be sure to vent the tube occasionally by pointing away from
you and others and loosening the cap. Allow the tube to stand for a few minutes so the
29
layers may separate. Remove the water layer with a Pasteur pipette and transfer it to a small
beaker labeled “waste.”
13. Although water and hexane ‘do not mix’…in reality a little bit of water will stay in the hexane.
You can tell there is water in the hexane layer if it is a little cloudy. You must dry (remove
water from) the hexane layer by adding a drying agent called anhydrous sodium sulfate
(Na2SO4). Your instructor will show you how to use a drying agent. A couple of microspatula
scoops is usually sufficient.
14. Allow the drying agent to settle and then transfer the hexane to a small vial with a cap. Label
the vial and then proceed with thin layer chromatography.
Thin Layer Chromatography:
1. Prepare the TLC chamber by placing one half of a filter paper into the jar. Then pour the
solvent mixture, provided by instructor, over the filter paper into the beaker until it is about 0.5
cm deep. Place the lid on the chamber. This does two things:
A. it keeps all the solvent from evaporating
B. it allows the air inside the chamber to become saturated with solvent.
** Note: even slight changes in the composition or contamination of the developing
solvent will lead to differences in Rf values. Don’t let the chamber sit for long
periods of time between plates. If a chamber gets contaminated, prepare a fresh
one.**
2. Obtain a TLC plate and VERY lightly draw a pencil line (NO INK) about 1 cm from the bottom.
If you press too hard, the silica gel will come off… in which case you will to get a new TLC plate.
Using a capillary tube, make a spot of your extract on the pencil line. You may have to let the
spot to dry and then spot it again if it isn’t dark enough carefully place the spotted plate into the
chamber and replace the lid.
** Note: if your spot goes under the solvent, it will not travel up the plate. Prepare a
new plate if this happens.**
3. You will immediately see the solvent start to travel up the plate. The line of solvent moving up
is called the ‘solvent front’. Once the solvent front is roughly 1 cm of the top of the plate,
remove the plate and quickly mark the solvent front with a pencil.
4. Although most of the spots are easily visualized by the naked eye, use the UV lamp to insure
that you are noting all possible spots. Lightly circle each spot in pencil. Lightly label each spot
(A, B, C, etc.).
5. Determine Rf values for all of your spots. This will give you quantitative values for comparison.
Measure the distance from the starting line to the solvent front. Then, measure to the center of
each spot. Divide the center spot distance by the solvent front distance; this is the Rf value. The
higher the Rf value, the less polar the compound
6. Try to match them to the compounds shown below (listed in order of decreasing Rf values):
Carotenes
Pheophytin A
Pheophytin B
Chlorophyll A
Chlorophyll B
Xanthophylls
(1-2 yellow-orange spots)
(gray intense)
( gray, may only be visible under UV)
(blue-green, intense)
(green)
(as many 3 yellow spots)
30
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31
REPORT SHEET-Isolation of Pigments from Spinach
Name____________________________ Partner’s name __________________________
Section______________ Date_________
1.
Mass of spinach __________________
2.
To the right sketch the layers in centrifuge tube
and clearly label the water and hexane layers.
3.
Why must you occasionally vent the tube during shaking?
4
Draw a sketch of your TLC plate, labeling the spots. Then, fill in the table
Spot
Rf
TLC plate
32
Probable Identity
5. Were there any spots your that does not match with the probable compounds?
6. Which compound is the most polar? Which one is least polar? Explain the reason for your answer.
7. What do you think would happen if you used ink to mark the spotting line?
33
Chemical Properties
of Aliphatic and
Aromatic Alcohols
Goals for the Student:




Learn to identify the visible observations in a chemical reaction
Learn the differences in reactivity of 1o, 2o, 3o alcohols with strong oxidizing reagent
Learn the differences in reactivity of aliphatic and aromatic alcohol
Learn the chemical reactions involve converting one functional group into another
Introduction
Subclass
General formula
Examples
H
Primary
R
C
OH
2-methylpropan-1-ol
H
H
Secondary
R
C
OH
R'
butan-2-ol
R''
Tertiary
R
C
OH
R'
2-methylpropan-2-ol
H
Phenol
C
H
C
H
C
C
H
C
OH
C
H
phenol
Alcohols, aldehydes, and ketones, are three very important classes of oxygen containing
organic compounds. Alcohols are classified into primary (1o), secondary (2o) and tertiary (3o)
according to the presence of substituents in the carbon containing the hydroxyl group. Phenol is a
34
class of aromatic compounds containing a hydroxyl group attached to a benzene ring. Three
subclasses of alcohols and phenol are shown on the preceding page.
In first part of this experiment you will learn the difference between 1o, 2o, and 3o alcohols and
aromatic alcohols with respect to their reactivity with the strong oxidizing reagent sodium dichromate.
This can be easily demonstrated by noting a color change when the Cr in the +6 oxidation state of the
orange colored dichromate ion, Cr2O72-, is reduced to the green colored chromium (III), Cr3+, ion.
Simultaneously, an appropriate alcohol is oxidized to either an aldehyde (and subsequently to a
carboxylic acid) or a ketone. Of course, if there is a no redox reaction there will be no observed
color change. In general the following unbalanced reaction describes the redox reaction：
Alcohol
(colorless)
+
Cr2O72-
Carboxylic acid (or ketone)
(orange)
+
(colorless)
Cr3+
(green)
For the remaining parts of the experiment, the chemical properties of aliphatic alcohols will be
examined. Here we will compare the solubility, acid/base properties and the reactivity with iron (III)
chloride (ferric chloride) of a similar sized aliphatic alcohol with that of the aromatic alcohol (phenol).
Experimental
Oxidation with Acidic Dichromate
1. Obtain ~ 5 mL of acidic dichromate solution (prepared previously by mixing 3 mL of a 5%
sodium dichromate and 1 mL of concentrated sulfuric acid) and place ~ 1 mL (~20 drops) into
four separate, clean, dry small test tubes and note the color. Caution: the dichromate
solution can potentially burn your skin or make holes in your clothes. If you spill any
of this reagents report the spill immediately to your instructor so that it may be cleaned up in an
appropriate manner.
2. To the separate test tubes add ~ 0.5 mL (~10 drops) of ethyl alcohol, isopropyl alcohol, t-butyl
alcohol, or aqueous phenol. Mix by finger flicking the test tubes.
3. Observe and record the colors of the resultant solutions. Hint: A table of results may help you
find things quickly during a lab quiz.
4. Dispose of the solutions in the special waste container for dichromate waste.
Solubility
1. Using a spatula or forceps, place a pea sized amount of solid phenol crystals into two separate,
clean, dry small test tubes.
2. To one add 1 mL (~20 drops) of distilled water and to the other add 1 mL (~20 drops) of 3M
NaOH (sodium hydroxide). Swirl the test tubes equally and note the relative speed with which
the crystals dissolve. Record your observations.
3. Repeat this procedure using 1 mL (20 drops) of cyclopentanol instead of the phenol. In this
case you are adding a liquid to a liquid, thus solubility is noted by a single homogenous solution
and insolubility by two layers.
4. Record your observation and dispose of these solutions as indicated by your instructor.
Acid/Base Properties
1. Place 1 mL (~20 drops) of distilled water, ethyl alcohol and aqueous phenol into three separate,
clean, dry test tubes.
35
2. To each sample add one drop of Universal Indicator solution and observe the color. Compare
the solutions to the reference Universal Indicator color card and estimate the solution’s pH.
3. Record your observations and dispose of these solutions as indicated by your instructor.
Reactivity with Iron (III) chloride (FeCl3)
1. Obtain and observe the color of the iron (III) chloride solution. Place 1 mL (~20 drops) of
distilled water, cyclopentanol and aqueous phenol into three separate, clean, dry test tubes.
2. To each sample add one drop of the iron (III) chloride solution. Mix by finger flicking the test
tubes.
3. Observe and record the colors of the resultant solutions and dispose of these solutions as
indicated by your instructor.
36
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37
REPORT SHEET-Chemical Properties of Alcohols
Name____________________________ Partner’s name __________________________
Section______________ Date_________
Oxidation with Acidic Dichromate
1. a. Draw condensed structural formulas of ethyl alcohol, isopropyl alcohol, t-butyl alcohol, and
phenol.
b. Classify each of the preceding alcohols as 1°, 2°, 3°, or aromatic.
2. What is the function of the acid solution of sodium dichromate?
3. a. What did you observe when the sodium dichromate solution was added to ethyl alcohol?
b. What did you observe when sodium dichromate solution was added to isopropyl alcohol?
c. What did you observe when the sodium dichromate solution was added to t-butyl alcohol?
d. What did you observe when the sodium dichromate solution was added to the phenol
solution?
38
4. Draw condensed structural formulas for the organic products of the above reactions that occur.
If no reaction occurs, write NR.
Solubility
1. a. Did the phenol crystals dissolve better in water or in the NaOH solution?
b. Did the cyclopentanol dissolve better in water or in the NaOH solution?
c. Based on the different behaviors of phenol and cyclopentanol, what generalizations can you
make about the solubility of similar sized aliphatic and aromatic alcohols?
d. Write a full chemical equation for the chemical reaction that occurred when NaOH was added
to phenol.
e. Write a net ionic equation for chemical reaction that occurred when NaOH was added to
phenol.
39
Acid/Base Properties
1. Based on the Universal Indicator Color Chart, what is the approximate pH of:
a. The distilled water?
b. The ethyl alcohol?
c.
The aqueous phenol?
2. Write a full chemical equation for the chemical reaction that occurs when phenol is placed in
water that explains the observed pH. Is phenol an acid or a base?
Reactivity with Iron (III) chloride (FeCl3)
1. Can ferric chloride be used to distinguish aromatic alcohols from aliphatic alcohols? Explain.
40
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41
Oxidation and
Structure of Carbonyl
Compounds
Goals for the Student:



Learn about the families of representative carbonyl compounds
Study the behavior of representative carbonyl compounds toward oxidizing agents
Learn about the different functional groups in carbonyl compounds and in oxygen containing
organic compounds
Introduction
In organic chemistry, a carbonyl group is a functional group composed of a carbon atom
double bonded to an oxygen atom: C ═ O. There are several types of carbonyl compounds, depending
upon what is attached to the carbon atom in C ═ O. The aldehyde group is often written as ─ CHO,
CO
the ketone group is written as
and the carboxylic acid group is written as ─ COOH, ester
group written as ─ COOR and the amide group written as ─ CONH2. A carbonyl, group characterizes
the following types of common compounds, where CO denotes a C ═ O carbonyl group.
Compound
Aldehyde Ketone
Carboxylic acid Ester
Amide
Structure
General formula RCHO
RCOR'
RCOOH
RCOOR' RCONHR'
The aldehyde group occurs in molecules of most sugars, like glucose. The ketone group is
occurs also in one common sugar, fructose. The amide group occurs in all amino acids, the building
block of protein.
Carbonyl compounds are very reactive due to the difference in electronegativity between the
carbon and the oxygen atom. Oxygen is more electronegative than carbon, and thus pulls electron
density away from carbon to increase the bond’s polarity. The oxygen is said to carry a partial
42
negative charge or “delta minus” and will be attracted to positive species in solution; for example, a
proton in an acidified solution or the carbon of another carbonyl. The oxygen is a nucleophile. The
carbon is said to carry a partial positive charge or “delta plus” and will be attracted to negative
species in solution; for example, the oxygen of an alcohol or water. Therefore, the carbonyl carbon is an
electrophile.
In every carbonyl, the more
electronegative oxygen atom pulls
electron density away from the carbon
atom. The oxygen is said to be “delta
minus” and will be attracted to
positive species in solution. The
carbon is said to be “delta plus” and
will be attracted to negative species in
solution.
In this experiment we are going to look at both the structure and the oxidation of some
carbonyl compounds. In the first part the action of mild oxidizing agent will be examined. In the
Tollen’s Test, we will use an oxidizing agent called the “Tollen’s Reagent.” Tollen’s Reagent contains
silver diamine ion [Ag(NH3)2]+ which can oxidize some categories of carbonyl-containg compounds
to carboxylic acids. The Ag+ is reduced to metallic silver producing a silver mirror on the glassware.
The Tollen’s test reaction is shown in the following generic example:
RCHO(aq) + 2Ag(NH3)2]+(aq) + 3OH-(aq)  RCOO-(aq) + 4NH3(aq) + 2Ag(s) + 2 H2O
In the second part of the experiment ball and stick models of oxygen containing compounds
including carbonyl compounds distributed in the laboratory will be examined. From the models you
will determine what functional group family (alcohols, ethers, aldehydes, ketones, hemiacetals, or
acetals) it belongs to and draw its structure.
Experimental
Tollen’s Test
1. Obtain four small test tubes and clean thoroughly with detergent and a brush. Rinse well with tap
water and finally with distilled water; shake out excess water. To each add 1 mL (20 drops) of 5%
silver nitrate solution and 1 drop of 3M NaOH, mix well.
2. To each tube add 2% ammonium hydroxide drop by drop until the grey silver oxide (Ag2O) just
dissolves forming the soluble Ag(NH3)2+ ion solution. Be careful not to add excess ammonium
hydroxide as this decreases the sensitivity of the test.
3. To the first tube add 2 drops of 10% glucose, to the second tube add 2 drops of formaldehyde, to
the third tube add 2 drops of acetone, and to the fourth tube add 2 drops of isopropyl alcohol; be
sure to label each test tube so that you know which is which. Record your observations for each
tube. Has a reaction already occurred?
4. Mix the contents of each tube and place them into a hot water bath. After several minutes remove
the tubes and record your observations.
43
5. Pour the contents of the test tubes into a waste container designated by your instructor. Clean the
tubes with detergent and a brush. To remove any silver adhering to the test tubes, add small of
concentrated nitric acid (caution conc. HNO3 can burn skin and clothes). Add contents to the waste
container and clean the tubes.
Structures of Carbonyl Compounds
Some ball and stick models of carbonyl compounds belonging to the families of aldehydes, ketones,
carboxylic acids, ester and other oxygen containing compounds, such as alcohol, acetal, hemiacetal and
ether will be distributed in this experiment. For each model draw a line-bond formula, and give its
functional group name (i.e., alcohol, ketone, hemiacetal, etc.). Using your results from the first part of
the experiment, predict if the compound would react in the Tollen’s Test.
The important pieces are:





The white sphere represents hydrogen atom
The black sphere with four holes represents carbon atom
The red sphere represents oxygen atom
The gray sticks are for connecting carbon atoms to one another and to connect the carbon
atoms to hydrogen and oxygen atoms
A stick (bond) attached to a carbon atom or an oxygen atom and not connected to anything
else will represent the C─ H or O─ H bonding arrangement.
44
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45
REPORT SHEET-Oxidation and Structure of Carbonyl Compounds
Name____________________________ Partner’s name __________________________
Section______________ Date_________
Tollen’s Test
1. Record your observations for the Tollen’s test:
Glucose:
Formaldehyde:
Acetone:
Isopropyl alcohol:
2. Draw the structures and give the names of the compounds that gave a positive Tollen’s Test.
3. Circle the functional group in the above structures that is responsible for the positive Tollen’s
test.
46
4. What is the name of this functional group?
Structure
#
1
Line-bond Formula
Functional Group(s)
2
3
4
5
6
47
Should React in
Tollen’s Test?
7
8
9
10
48
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49
Optical Isomers
Goals for the Student:


To investigate the use of three dimensional structures to identify different types of isomers
To learn about stereoisomers, and optical isomers
Introduction
Stereoisomers are isomers that have same molecular and structural formulas but different
spatial arrangement. Optical isomerism is one form of stereoisomerism. Optical isomers are named
like this because of their effect on plane polarized light.
All optical isomers contain a carbon atom joined to four different groups. The carbon atom of
this isomers are called “asymmetric carbon atom or chiral center” and the molecule is called chiral.
Only chiral molecules have optical isomers. Some examples of optical isomers are shown below. The
chiral center is marked with a star.
Butane-2-ol
2-Hydroxy propionic acid
Or Lactic acid
2-Aminopropionic acid
or Alanine
A carbon atom with the four different groups attached which causes this lack of symmetry is
described as a chiral center or as an asymmetric carbon atom. If you cannot find a plane of
symmetry the molecule is chiral. Practice on the molecules on the next page. D-alanine, in a wedgeand-dash formula below-right, does not have a plane of symmetry and is chiral. Glycine on the
left below has a plane of symmetry and is thus achiral.
50
Glycine-plane of symmetry
Alanine-no plane of symmetry
Chemist developed methods to facilitate the visualization of 3-dimensional spatial
arrangements of atoms or groups of atoms in a 2-dimensional environment, i.e., the plane of this
paper. The most common method for presenting 3-dimensional structures in a 2-dimensional plane is
the Fischer projection. Fischer projections are formed when the observer orients a tetrahedral
structure such that the atoms or group of atoms in the vertical plane are away from the observer
(dashed wedges) while atoms or groups of atoms in the horizontal plane are towards the observer
(solid wedges).
Whenever a Fischer projection is seen
it is meant to represent the orientation of atoms or
groups of atoms attached to the central tetrahedral
carbon atom, as shown in the figure at the left. It is
important to mention that the cross in a Fischer
projection represents chiral carbon.
Using Fischer projection formula, 3-dimensional information of any molecule can be oriented
in 2-dimensional surface. Stereoisomers can be classified into several different types of isomers.
Enantiomers are stereoisomers that are two nonsuperimposable complete mirror images of each other
much as one’s left and right hands are the same but opposite. Enantiomers have similar physical and
chemical properties except for their ability to rotate the plane of polarized light in the same amount
but in opposite direction. This property is called optical activity
Other types of stereoisomers are diastereomers, which are two nonsuperimposable non mirror
images, and mesoisomers, which contain more than one chiral carbon atom but are optically inactive
because of the symmetry of the molecule; the mirror images of these compounds are superimposable
Typically a meso compound can be identified by noting that it’s Fischer projection has a mirror plane,
51
i.e., the top and the bottom halves of Fischer projection are mirror images of each other.
Diastereomers of glyceraldehydes of 2,3- dichlorobutane are shown below.
CH 3
CH 3
CH 3
CH 3
H
Cl
Cl
H
H
Cl
Cl
H
Cl
H
H
Cl
H
Cl
Cl
H
CH 3
CH 3
CH 3
Enantiomer (chiral)
CH 3
Meso (Identical, achiral)
Experimental
Construct the 3-dimensional model for the following molecules using the ball and
stick model kit containing black sphere as carbon atom, white sphere as hydrogen
atom red sphere as oxygen atom and green sphere as nitrogen atom. Make the
notes in your laboratory notebook.
I. Build the structure of 2,3-dihydroxybutanal (D- and Lglyceraldehyde).
a) Draw the Fischer projection of molecule with aldehyde groups are on the
top of the structures.
b) Label the two isomers with the type of stereoisomers, these structures represent.
II. Build the structure of the four stereoisomers for 2,3,4,trihydroxybutanal (D- and L- threose and D- and L-erythrose).
a) Draw the Fisher projection of the structures with aldehyde groups are at
the top of the structure and hydrogens and hydroxyl groups attached
to chiral carbon s point towards you.
b) Using Fischer projections for D- and L- glyceraldehyde reference
compound, label each of these Fischer projections using ,e.g., D1,
L1,etc.Identify the relationships between various pairs of models.
52
III. Build the structure of all stereoisomers for Asparagine.
a) Draw Fischer projections of all stereoisomers with carboxylic acid
at the top of the structure.
b) Label the isomers as D- or L- asparagine with reference to the Dor L- glyceraldehyde structure. Use carboxylic acid and the
amino group of asparagin as analogous to the aldehyde and
hydroxyl groups of the reference compound.
IV. Build the structures of all of stereoisomers for Tartaric
acid.
a) Determine how many structure you can build and draw the
corresponding Fischer projections for all the structures with a
star mark for each chiral center.
b) Circle the structure that would not be optically active
V. Build the structure for 2,3,4,5,6- pentahydroxyhexanal (all are isomers of glucose)
a) Construct the model so that the aldehyde group is at the top of the
molecules and the hydroxyl group on the last chiral carbon (furthest from
the aldehyde group) is pointing to the right, thus generating a D- structure
for an aldohexose sugar. Also build your model such that the hydrogens
and hydroxyl groups on each chiral carbon are oriented towards you.
b) Draw the Fischer projection corresponding to your model. Designate
each chiral carbon with an asterisk *. Determine how many chiral center
in the whole molecule. Notice the molecule, whether it formed chain or
coiled structure and how the last hydroxyl and the carboxylic group are
positioned in the model. Predict the group formed and the structure of
the molecule if the hydroxyl and the aldehyde group react together.
Identify eight different stereoisomers can be formed with D-form, one of which is the very
important monosaccharide, D-Glucose. Show your model and Fischer projection to instructor.
Compare your Fischer projection with eight possible D-hexose to determine which sugar structure
you have.
53
REPORT SHEET-Optical Isomers
Name____________________________ Partner’s name __________________________
Section______________ Date_________
I. Draw and label the Fischer projections for 2,3-dihydroxybutanal.
II. Draw and label the Fischer projections for 2,3,4-trihydroxybutanal.
a) How many chiral centers are there? _____________________________
b) How many total stereoisomers are there? ____________________________
c) How many pairs of enantiomers are there? ___________________________
d) How many pairs of diastereoisomers are there? __________________________
III. Draw and label the Fischer projections for asparagines.
54
a) How many chiral centers are there? ____________________________
b) How many total stereoisomers are there? _________________________
c) What would be general formula for determining the maximum number of stereoisomers
when n is the number of chiral center
IV. Draw and label the Fischer projections for tartaric acid. Mark all the chiral with an
*. Circle the Fischer projection and draw its mirror plane for the meso compound.
a) How many chiral centers are there? ____________________________
b) How many total stereoisomers are there _________________________
c) How many pairs of enantiomers are there? ____________________________
d) How many pairs diastereomers are there? _______________________________
V. Draw the Fischer projection for your 2, 3, 4, 5, 6- pentahydroxyhexanal and label
all chiral centers with an *.
55
a) How many chiral centers are there? ___________________________
b) How many total stereoisomers are possible based on general formula for isomers?
_____________
c) Show your Fischer projection to your instructor, which D-aldohexose did you construct?
d) is the structure is straight or coiled? ___________________________
e) What class of compounds would be made if the hydroxyl group on carbon 5 reacts with the
aldehyde group?
________________________________
f) How many atoms are in the ring of the resulting cyclic structure?________________________
56
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57
Carbohydrates
Goals for the Student:



Learn the qualitative tests to identify organic functional groups in carbohydrates
Learn the use of specific qualitative tests to distinguish between aldehydes and ketones
Learn the qualitative test for complex carbohydrate
Introduction
Carbohydrates which comprise one of the three basic classes of foodstuffs, contain carbon,
hydrogen and oxygen atoms. They are an important class of biological macromolecules, which are
found in nature both in isolation as mono-, di-, and polysaccharides and in association with other
biological macromolecules, e.g., as glycolipids and glycoproteins. They are involved in a diverse
range of physiological roles, such as molecular recognition, energy storage enzyme regulation.
Carbohydrates in our diet are our major source of energy.
*
*
D-glucose
D-fructose
Sucrose (table sugar)
Carbohydrates are classified as polyhydroxy aldehydes or polyhydroxy ketones.
Therefore, they will exhibit chemical properties associated with both alcohols and carbonyl compounds.
Some examples of carbohydrates are shown above. Carbohydrates easily cyclize to form hemiacetals
and hemiketals. Indeed, they spend most of their time in a cyclic form. When cyclized, the carbon that
was the carbonyl carbon becomes part of the ring and is called the anomeric carbon. You can easily
find the anomeric carbon on any cyclic saccharide by locating the ONLY carbon with two oxygen atoms
directly attached. Sucrose has two anomeric carbons, as indicated with asterisks in the diagram above.
If at least one of the anomeric carbons has a hydroxyl group directly attached, it can reverse
the cyclization process and form the linear aldehyde or ketone again. In the linear aldehyde or ketone
58
form, the molecule can participate in any aldehyde or ketone reaction. Does sucrose have a hydroxyl
group directly attached to either anomeric carbon? We are going to do series of analyses to examine
the reactivity of some monosaccharides, disaccharides and polysaccharides. Tests similar to these may
be used clinically to test for glucose in urine and blood.

In the Benedict’s test a reducing sugar (a sugar with a hydroxyl directly attached to an
anomeric carbon) reacts with the blue-colored Cu2+ ion in the presence of base. The copper (II)
ion is reduced to form copper (I) in a red-orange Cu2O precipitate whereas the aldehyde group
is oxidized to the carboxylic acid functional group. In addition to all aldose monosaccharides
giving a positive Benedict’s test, ketose monosaccharides, though lacking an aldehyde group,
react due to the presence of a hydroxyl group next to the ketone group. Thus hydroxyl ketones
give positive tests.

The Barfoed’s Test is a variation of the redox reaction mentioned previously. Copper(II)
acetate in acetic acid is not as reactive as the Cu2+ Benedict s reagent. Thus, one can
distinguish monosaccharides from disaccharides based on how fast the red-orange precipitate
forms. Typically, monosaccharides react within 2-3 minutes, whereas disaccharides take longer.

The Seliwanoff’s Test is used to distinguish from aldoses using the aromatic alcohol in the
presence of concentrated hydrochloric acid. This is useful for both monosaccharide ketose as
well as disaccharides ketose. A positive test is noted by a red colored solution; a yellow straw
or apricot color is not indicative of positive test.

To distinguish pentoses from hexoses one can use the Bial’s Test. Pentoses react with orcinol
in the presence of FeCl3 and conc. HCl to give a characteristic blue-green color. Non-reacting
sugars may produce a brown precipitate but the solution usually remains the yellow color of the
FeCl3.

Starch is a complex carbohydrate that interacts in the Iodine Test. It is composed of two
fractions; the linear, helical fraction n and the branched amylopectine fraction. When I 2 inserted
into the interior of the amylase fraction, a dark blue color is observed.
Experimental
You will find the following carbohydrate test solutions on your bench: glucose, galactose, fructose,
arabinose, maltose, lactose, sucrose and starch. These tests are very colorful and therefore your
notebook should be full of observations!
Benedict’s Test:
1. Prepare a boiling water bath and label eight clean small test tubes.
2. In separate test tubes add 1 mL of the Benedict’s reagent. To each test tube add 5 drops of the
test carbohydrate solution. Mix the samples.
3. Place all the test tubes at the same time into the boiling water bath.
4. Note and record the how long it takes for the red Cu2O precipitate to form; also note if the blue
Benedict’s reagent color disappears.
5. After 10 minutes remove all the tubes and keep the boiling water bath going for the remaining three
experiments. Did any sugars not produce the red precipitate? Which are reducing sugar? Which are
not?
59
Barfoed’s Test:
1. Use the boiling water bath from before and label a new set of 8 clean small test tubes.
2. In separate test tubes add 1 mL of the Barfoed’s reagent. To each test tube add 10 drops of the
test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record the long it takes for the red Cu2O precipitate to form.
5. After 10 minutes remove all the tubes. Determine which are monosaccharides, which are
disaccharides
Seliwanoff’s Test:
1. Use the boiling water bath from before and label a new set of 8 clean small test tubes.
2. In separate test tubes add Seliwanoff’’s reagent. To each test tube add 3 drops of the test
carbohydrate solution. Mix the samples
3. Place all of the test tubes at the same time into the boiling water bath. Note and record how long it
takes for the first clear red colored solution to form.
4. Remove all of the test tubes as soon as the first positive test is seen as prolong heating (in excess of
5 minutes) may cause spurious results. Which sugar solution(s) contain a ketose?
Bial’s Test:
1. Use the boiling water bath from before and label anew set of 8 clean test tubes.
2. In separate test tubes add 1 mL of the Bial’s reagent. To each test tubes add 10 drops of the
carbohydrate solution.
3. Place all the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the first clear blue-green solution to form
5. Remove all the test tubes as soon the first positive test is seen as prolonged heating may cause
spurious results. Which sugar solution(s) contain pentose?
Iodine test:
1. Place 3 drops of each test carbohydrate solution in separate wells of a clean spot plate.
2. Add 1 drop of the iodine solution to each test carbohydrate solution. Note and record the color of
each sample.
3. Did any other solutions besides the starch solution give a positive test?
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60
REPORT SHEET-Carbohydrates
Name____________________________ Partner’s name __________________________
Section______________ Date_________
61
Starch
Sucrose
Lactose
Maltose
Arabinose
Fructose
Galactose
Glucose
Carbohydrate
Benedict’s Test
Barfoed’s Test
Seliwanoff’s Test
Bial’s Test
Iodine Test
1. Complete the table of results. Be sure to include color!:
2. Suppose you saw no sign of a color change in Benedict’s test, no sign of a red precipitate after
10 minutes with Barfoed’s test, and a dark red colored solution with the Seliwanoff’s test. What
sugar(s) could you have?
3. Suppose you saw a red precipitate with the Benedict’s test, a red precipitate after 2 minutes
with the Barfoed’s test, and straw-colored solution after more than 5 minutes with the
Seliwanoff’s test. What sugar(s) could you have?
4. Suppose you saw a red precipitate with Benedict’s test, no sign of red precipitate after 10
minutes with Barfoed’s test, and a straw-colored solution after more than 5 minutes with
Seliwanoff’s test. What sugar(s) could you have?
62
Acid-Base Reactions
with Carboxylic
Acids and Esters
Goals for the Student:



Learn about two very important functional groups, carboxylic acids and esters
Learn about the hydrolysis reaction of ester, and it’s product
Learn about the industrial application of saponification
Introduction
Salicylic acid
Methyl benzoate
In the first part of this experiment you are going to evaluate the solubility of salicylic acid in cool
water and hot water. Then, the reactivity of salicylic acid with aqueous NaOH will be investigated. As
you would expect carboxylic acids should react with water to form a water-soluble carboxylate anion. In
general the following acid-base reaction occurs:
R-COOH + H2O  R-COO- (aq) + H3O+
Take care: solubility is also dependent of the size of the alkyl or aryl group attached to the carboxylic
acid functional group. Additionally, carboxylic acids react with bases to form water soluble salts as
shown below:
RCOOH + NaOH (aq)  RCOO-(aq) + Na+(aq) + H2O
Note: salicylic acid contains, in addition to the carboxylic acid functional group, a phenol group hydrogen
that can also react with the base.
The resultant sodium carboxylate/phenolate salt can further react with strong acids to reform the free
acid and phenol as shown below:
R-COO- + HCl
(aq)
Ph-COO- + HCl
 R-COOH
(aq)
 Ph-COOH
63
The use of bases and acids serves as “solubility switches” to convert an insoluble form of a
compound to a soluble form and vice versa. Observing the solubility or insolubility of reactants and /or
products serves as a means to monitor acid base reactions.
Saponification is the lysis of an ester with a strong base to form an alcohol and the salt of a
carboxylic acid. Saponification is commonly used to refer to the reaction of a metallic alkali (base) with
a fat or oil to form soap. The concept of a solubility switch will also serve as the basis for following the
base (NaOH) catalyzed hydrolysis (specification) reaction of an ester (methyl benzoate) to the
subsequent water soluble carboxylate salt (sodium benzoate) and alcohol (methanol). The resultant
mixture containing the sodium benzoate is then reacted with acid to form benzoic acid.
Experimental
Solubility and Acid-Base Reactions of Salicylic Acid:
1. Set up a boiling water bath with a 250mL beaker on a hot plate and an ice water bath in a
beaker.
2. Place a small amount of salicylic acid (pea size amount) into a clean small test tube and add a
5mL of distilled water. Stir well and record your observations in your notebook.
3. Carefully place the test tube into the boiling water bath. Record your observations.
4. Remove the test tube and cool the solution in the ice water bath. Note that you may have to
scratch the inside o surface of the test tube with a glass rod. Your instructor will demonstrate
this technique. Record your observations.
5. Add 3M NaOH to the mixture drop by drop and agitate by finger flicking three test tubes after
each drop. Be sure to keep track of the number of drops that you add. Record your
observations and write the overall equation for this reaction.
6. Finally, add to the solution as many drops of 3M HCl as you used for the 3M NaOH. Then add
several more drops of the 3M HCl. Record your observations and write the overall equation for
this reaction.
Acid-Base Reactions of Methyl Benzoate:
1. Place 3 drops of methyl benzoate into a clean test tube and add 2 mL of distilled water. Record
your observations in your notebook.
2. Add 12 drops of 2.5 M or 3M NaOH and mix the contents of the test tube. Place the test tube
into the boiling water bath for 30 minutes (or longer) and every 5 minutes stir the contents
vigorously with a clean glass rod, replacing the test tube back into the boiling water bath. Stop
the reaction when you judge the solution to be homogeneous. Record your observations and
write the overall equation for this reaction.
3. Cool the mixture to room temperature by running cold tap water along the outside of the test
tube.
4. Add 15 drops of 3M HCl mixing by finger flicking the test tube after each addition. Record your
observations and write the overall equation for this reaction.
64
REPORT SHEET-Acid-Base Rxns with Carboxylic Acids and Esters
Name____________________________ Partner’s name __________________________
Section______________ Date_________
Solubility and Acid-Base Reactions of Salicylic Acid:
1. Does salicylic acid dissolve in the cold water?
2. Does salicylic acid dissolve in hot water?
3. Does salicylic acid dissolve in aqueous sodium hydroxide?
4. Write an overall equation for the reaction of salicylic acid with aqueous sodium hydroxide.
5. What did you observe when hydrochloric acid was added to the test tube during step 6?
6. Write an overall equation for the chemical reaction that occurs in step 6.
Acid-Base Reactions of Methyl Benzoate:
1. Write an overall chemical equation for the saponification of methyl benzoate.
65
2. Are the initial products of the saponication of methyl benzoate soluble or insoluble in water?
3. What did you observe when hydrochloric acid in step 4?
4. Write an overall chemical equation for the reaction for step 4.
66
Synthesis of Aspirin
Goals for the Student:




Learn to do the organic synthesis of an ester from an alcohol and an anhydride
Learn how to purify the product in the organic synthesis
Learn how to calculate the % yield of a product
Learn different techniques to compare the purity of a synthesized product with commercial
product.
Introduction
Acetylsalicylic acid (ASA), commonly called aspirin, is widely used as medicine to reduce fever
(an antipyretic), to reduce pain (an analgesic), to reduce swelling (anti-inflammatory), and to prevent
platelet aggregation that initializes thrombosis or hemostasis (anti-clotting). Aspirin inhibits the enzymes
necessary for the formation of prostaglandins and thromboxanes (hormones) that are associated with
pain, fever, inflammation, and blood-clotting in the human body. It has been also suggested that aspirin
small amount as, 80-100mg for daily ingestion can lower the risk of stroke and heart attack in at-risk
adults.
Aspirin is an ester of acetic acid and salicylic acid. Salicylic acid, is acting as the alcohol,
because this also has a hydroxyl group attached to the benzene ring besides a carboxylic acid. Though
esters can be produced from the direct esterification of an alcohol and a carboxylic acid in the presence
of an acid catalyst, typically sulfuric acid, the present method to prepare aspirin uses acetic anhydride a
derivative of acetic acid to form more quickly an acetate ester with salicylic acid. Acetic anhydride, as a
substitute acetylating agent reacts with salicylic acid as follows:
In the first week of a two-week experiment you will synthesis aspirin (acetyl salicylic acid). The
following week you will determine the percent yield of your synthesized aspirin. Then you will analyze
the purity of your product by thin layer chromatography and by a melting point determination.
67
CHARACTERIZATION OF SYNTHESIZED ACETYLSALICYLIC ACID (ASA, aspirin):
The second week you will determine the percent yield of the synthesized product, aspirin and
perform different techniques to determine the purity of the product. Of course, this aspirin is not
suitable for oral admission or any physiological test because reagents used for this synthesis are not of
sufficient purity for ingestion.
Determination of Percent Yield:
Percent yield is defined as follows:
% yield =
mass of acetylsalicylic acid(actual yield of ASA in experiment) x 100
Theoretical yield of ASA (calculated from stoichiometric relationship)
The actual yield is the number of grams of acetyl salicylic acid that you actually made in the
laboratory. The theoretical yield is the number of grams of acetylsalicylic acid you should get based on
the stoichiometry of the chemical equation. The theoretical yield is based on the number of grams of
salicylic acid you used. First convert the number grams of salicylic acid to moles by dividing the grams of
salicylic acid by the molecular weight of salicylic acid (138 g/mol). One mole of acetylsalicylic acid is
formed for each mole of salicylic acid used. Therefore, the number of moles of acetylsalicylic acid is the
same as the number of moles of salicylic acid. To convert to grams of acetyl salicylic acid, multiply the
number of moles you calculated by the molecular weight of acetylsalicylic acid (180 g/mol)
Thin Layer Chromatography:
One way of determining the purity of your product is to do thin layer chromatography using
your product, one of the reactant (salicylic acid) and an authentic aspirin sample. This is the same
procedure you completed when you analyzed chlorophyll and carotenoids from spinach. Review that
experiment if you have forgotten.
Determination of the melting point:
You should use the Meltemp apparatus to determine the melting point of your acetylsalicylic
acid. Carefully place a thermometer in the slot on the Meltemp device. These are mercury
thermometers and are quite fragile, so handle the thermometer with care. If you break the
thermometer, notify the instructor immediately so the mercury spill can be cleaned up. Before
measuring the melting point of your product, look up the true melting point of acetylsalicylic acid in The
Handbook of Chemistry and physics. This will give you an idea of about where your product should
melt.
Ferric chloride test:
Dissolve a small amount of your product in water and test it with ferric chloride. A purple color
indicates the presence of a phenol (unreacted salicylic acid) in your product. Compare your results with
those obtained from a sample of authentic aspirin.
Acid hydrolysis of acetylsalicylic acid:
Esters can be hydrolyzed by heating them in the presence of an acid. The original alcohol and
carboxylic are generated from the acid hydrolysis.
68
Experimental
Synthesis:
1. Place ~ 50 mL of distilled water into a 250 mL beaker, add a couple of boiling chips and heat to
boiling. Also put ~20 mL of distilled water in a 50 mL Erlenmeyer flask into an ice water bath
using another 250 mL beaker (Fig. A, Note: use of hot plate will be more safe than using
burner).
2. Write your group number in pencil on a piece of filter paper. Tare the balance and weigh the
filter paper to the nearest 0.001 g; record this value in your notebook. Obtain ~ 1 g of salicylic
acid and place it into a tarred plastic weigh boat. Weigh the salicylic acid to the nearest 0.001 g
and record this value in your notebook.
3. Place the salicylic acid into a clean dry medium test tube. Add 2 mL of acetic anhydride
and 2 drops of concentrated sulfuric acid (caution: acetic anhydride produces irritation and
necrosis of tissues in liquid or in vapor state). Avoid contact with skin and eyes. Do this
addition in the hood.
4. Place the test tube containing the reaction mixture into the boiling water bath. Stir the mixture
vigorously with a clean glass rod while in the boiling water bath. Be careful not to break the
test tube.
5. After the entire solid has dissolved, remove the test tube into the ice water bath. If the crystals
do not form, induce crystallization by scratching the inside of the test tube with a glass rod.
When crystallization is complete add 10 mL of the ice cold water.
6. Fold your weighed, initialed filter paper so that it is fluted (your instructor will demonstrate this)
and place it into a short stem funnel.
7. Collect the solid on the filter paper in the funnel. Rinse the solid with 2 or 3 small (5 mL)
portions of ice cold water. Be sure to let the water drain through the filter between additions of
the rinses.
8. Carefully remove the filter paper from the funnel and spread it out on a piece of paper towel.
Set the paper towel with the filter paper in a drawer and allow it to dry until the next laboratory
period.
9. Determine the gram formula weight for salicylic acid. Using the mass of your salicylic acid,
determine the number of moles in the reaction. Determine the gram formula weight for the
acetyl salicylic acid product. Finally, calculate the mass of product expected if all of the salicylic
acid is converted to acetyl salicylic acid.
10. Your instructor will demonstrate how to prepare a sample for a melting point apparatus.
Practice taking melting points using the salicylic acid.
11. The following week carefully remove the filter paper containing the aspirin from the drawer and
weigh it to the nearest 0.001 g. Record this value in your notebook.
12. Determine the percent of the of yield of the product aspirin. Save aspirin for further analysis.
Thin layer chromatography:
69
1.
Obtain ~ 1 mL of methanol to three separate clean and dry test tubes. Into these separate
test tubes, add a small amount of your synthesized aspirin, commercial aspirin and salicylic
acid, which you have used for the synthesis. Be sure label each test tube so that you know
which is contained in each test tube. To aid in dissolving the crush the solid gently with a
clean dry glass rod. Be sure to clean the glass rod prior to using for the next sample.
2. Obtain 7 cm × 14 cm fluorescent silica gel TLC plate for your group., Follow the procedure
in pages 24, 25, and 26 to spot (4 µL) your product, authentic aspirin and salicylic acid. Be
sure to mark care fully the spot line at the bottom and label for the spots at the top. Be
sure to keep at least (10 mm) space between the spots. Your product’s spot should be on
the left most position, then the spot of authentic aspirin on the middle position and salicylic
acid spot on the right most position. Try to keep the diameter of the spot less than 2 mm.
Several quick small applications will be better than one large application.
3. Prepare a TLC developing tank similar to what you used previously. Pleace one half of a
filter paper into the jar, then add 10 mL of the TLC solvent provided by your instructor.
Make sure the filter paper is completely saturated with the developing solvent. When the
three samples have been applied, carefully place the TLC plate into the developing
chamber. Make sure that the solvent level is below your original spots.
4. After the solvent front reaches 1 cm from the top of the TLC plate, remove the plate from
the beaker. Allow it to dry under the hood. Use a UV lamp to view the spots. Carefully,
mark the spots with a pencil. Compare the spots with one another. Draw a reproduction of
your developed chromatogram in your lab notebook.
Melting point determination:
1. Place a small amount of your product into a melting point capillary tube as demonstrated by
your instructor. Place the capillary tube into the capillary slot on the Meltemp apparatus.
2. To obtain an accurate melting point it is necessary to heat the sample slowly. For the
melting point of your product, the meltemp should be set at about 40V. Turn on the power
and watch the crystals through the magnifying glass of the Meltemp device.
3. As soon as the crystals start to melt, record the temperature as precisely as possible; Keep
watching the crystals. Record a second temperature as soon as all of the crystals have
melted.
4. If your product is pure, the melting range (the difference between the two recorded
temperatures) will be small-perhaps a degree or two. If your product is not pure, you will
have a wider melting point range. If your product is pure, the melting point should be
similar to the true value for acetylsalicylic acid. If it is impure, your will have melting point
lower than the true value.
Ferric chloride test:
Dissolve a small amount of your product in ~ 1 mL of water. Add two drops of ferric chloride
solution and note the color. Repeat with an authentic aspirin sample of aspirin and with the
salicylic acid that you have used. Record your observations in your lab notebook.
Acid hydrolysis of acetylsalicylic acid:
70
Dissolve a small amount of your product in ~ 1 mL of water. Add five drops of conc. Sulfuric
acid and heat the test tube in a boiling water bath for 10-15 minutes. Remove the test tube,
cool to room temperature and test for the presence of a phenol by adding two drops of ferric
chloride.
71
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72
REPORT SHEET-Synthesis of Aspirin
Name____________________________ Partner’s name __________________________
Section______________ Date_________
Synthesis:
1. Mass of filter paper____________________________
2. Mass of filter paper plus product________________________________
3. Mass of acetylsalicylic acid (aspirin)________________________________
4. Mass of salicylic acid used__________________________________
5. Moles of salicylic acid used___________________________________
6. Moles of acetylsalicylic acid expected (show theoretical yield)(refer to the equation)
7. % yield of acetylsalicylic acid (show calculation)
8.
Write full chemical equation (line-bond formulas for both reactants and products) for the
synthesis of aspirin.
9.
After filtering your reaction mixture, what was the purpose of rinsing the product with water?
10. Why it is important for the water to be ice cold?
73
Characterization:
1. Write the literature value for the melting point of acetylsalicylic acid._______________oC
2. Write the melting point range of your product? ____________oC to ___________oC
3. Draw a reproduction of your developed chromatogram. Show the position of all spots, the
solvent front and spotting line. What conclusions can be drawn about the purity of your product
from this chromatogram?
4. What conclusion can be drawn about the purity of your product from this chromatogram?
5. What did you observe when FeCl3 was added to your product?
6. What did you observe when FeCl3 was added to authentic aspirin?
7. What did you observe when FeCl3 was added to salicylic acid?
8. Write the full chemical equation (line-bond formulas for both reactants and products) for the
hydrolysis of your acetylsalicylic acid.
9. What did you observe when FeCl3 was added to the aspirin hydrolysis solution?
10. Using all experimental data, compare the purity of your aspirin with that of the authentic aspirin
sample.
74
Synthesis &
properties of Soap
Goals for the Student:


Learn about the process of soap making
Learn the procedure for purifying and testing the of properties of a soap
Introduction
A natural soap is the sodium or potassium salt of long chain fatty acids produced by the base
catalyzed hydrolysis of triacylglycerol (the fat storage molecule in plants and animals, known clinically
as “triglycerides”). In the first part of this experiment you will prepare soap by a saponification
reaction of a small sample of oil or fat. A generalized saponification reaction is shown below:
In the second part of this experiment some of the properties (pH and solubility) in solutions of
your soap will be examined.
Experimental
Synthesis:
1. Caution must be observed as the concentrated sodium hydroxide (lye) is corrosive and can
cause burns to skin, destruction of clothing and irreversible cornea damage to the eye. At no
time are your safety glasses/goggles to be removed during this experiment.
2. Prepare boiling water bath in a 600 mL beaker; be sure to add a couple of boiling chips. Place
12 mL of oil or 10 g of fat into a 250 Erlenmeyer flask. Also prepare an ice water bath using
another 600 mL beaker.
3. Add 10 mL of ethanol (ethylalcohol) and 12 ml of 6M sodium hydroxide to the vegetable oil.
**You may add a small piece of wax crayon now, if you want your soap to have a color. Many
dyes used in crayons will be altered due to the change in pH. The proper color should return
when you rinse your soap later. Someone in your group needs to leave theirs white for the
75
tests
4. Stir the mixture with a glass stirring rod. Using a ring stand and a clamp secure the flask and
heat it in the boiling water bath. Continuously stir the mixture during the heating process to
preventing the mixture from foaming up the sides of the flask.
5. Heat the mixture in the water bath, with stirring until the odor of ethyl alcohol is no longer
detected. This may take 15 to 30 minutes. Remove the flask from bath.
6. Place the flask into the ice bath. Cool the soap solution for 10 minutes.
7. To the contents of the flask add 20 mL of a concentrated sodium chloride solution. Using a
spatula, break up the lumps of soap as completely as possible to permit contact between the
solid and the sodium chloride wash solution. Carefully decant the solution to remove the wash
solution while retaining the solid in the flask.
8. Repeat the washing and decanting step two more times. After the washing remove the last
traces of liquid by dumping the solid into the paper towels and carefully blotting the soap with
additional paper towels. Avoid touching the soap with your skin.
Analysis:
1.
Dissolve a small pea sized of your soap in a small test tube containing 5 mL of distilled water. Add
3 to 4 drops of Universal indicator. Note the color appeared. Using the Universal indicator reference
card, determine the approximate pH of your soap solution. Repeat this experiment using a purified
commercial soap (e.g., Ivory). Record color and pH value in your notebook.
2.
In three separate clean test tubes place 5 mL each of distilled water, tap water and 10% CaCl2
(calcium chloride ) . Add a small pea sized pieces of your soap to each separate test solution. Make
sure that you use equal sized amounts. Stopper each tube and shake them vigorously. Describe the
relative amounts of lather and foam that appear in each tube. Record your observations in your
notebook.
3.
Dissolve a small amount of your soap in a minimum amount of distilled water; estimate the volume
of water you used. To the dissolved soap solution add an equal volume of concentrated sodium
chloride solution. Describe and record in your note book what happens.
4.
Visit other groups and compare the texture of soaps made from different fat sources.
76
REPORT SHEET-Synthesis and Properties of Soap
Name____________________________ Partner’s name __________________________
Section______________ Date_________
1.
Write a chemical equation for the saponification of a triglycerol that contains palmitic acid, oleic
acid and linoleic acid as the three fatty acid moieties.
2.
What was the purpose of adding the concentrated salt solution to your soap preparation?
3.
a). What was the pH of your soap solution?
b). Was your soap solution acidic, basic or neutral?
c) What was the pH of the commercial soap solution?
d) Was the commercial soap solution acidic, basic or neutral?
e) Based on your result to parts 3a-3d above is it possible to have a neutral solution of a pure
soap in distilled water?
4. Described the observed behavior when your soap was added to each of the following and
shaken up:
a)
Distilled water:
77
b)
Tap water:
c)
Calcium chloride solution:
5. Write a net equation for the reaction of calcium ions with the anion of palmitic acid.
6.
What did you observed when concentrated sodium chloride solution was added to dissolved
soap?
7. What does the observation in 6, suggest about the effectiveness of ordinary soap in seawater?
8. Were there any differences in texture in soap from different fat sources? Does this agree with
your knowledge about saturated versus unsaturated fats?
78
Isolation and
Characterization of
Casein from Milk
Adopted from: “Isolation of Protein, Carbohydrate and Fat from Milk”, Mohr. S. C., Griffin, S. F., and
Gensler, W.J., in Laboratory Manual for Fundamentals of Organic and Biological Chemistry John McMurry
and Mary E. Castellion, Englewood Cliffs, Prentice-Hall, 1994 Wayne P. Anderson (4/2002)
Goals for the Student:



Learn the about the protein present in milk, and cheese
Learn the procedure to isolate the protein from the milk
Learn the techniques used to characterize the protein
Introduction
You may recall the Mother Goose nursery rhyme, “Little Miss Muffet sat on a tuffet, eating her
curds and whey…..” When milk is acidified, it is transformed into a solid component, called curd, and
a liquid component called whey. This method is used to make cottage cheese. The curds contain
butterfat and a protein called casein, which contain all of the common amino acids and is particularly
rich in the essential ones. Casein exist in the milk as a soluble calcium salt, that precipitates at pH
values below 4.6. So milk can be curdled by acids such as lactic acid that forms during natural
souring of milk. The carbohydrate, lactose, is present in the whey. In this experiment you will also
isolate casein from milk and carry out some qualitative tests for protein.
The Biuret test is generally used for protein. When the pale blue Cu2+ ion forms a complex with
adjacent amide nitrogen of the peptide backbone, a very deep violet blue color results.
The Xantoperoteic acid test is on the other is a general test for the presence of the aromatic
amino acids, tryptophan, phenylalanine and tyrosine, in proteins. Aromatic groups that have an
79
amino group (tryptophan) or a hydroxyl group (tyrosine) are easily nitrated by concentrated nitric
acid to form yellow (xantho, Greek for yellow) colored aromatic nitro compounds.
Experimental
1. Determine the mass of a 125 mL Erlenmeyer flask. Add 50 mL of milk to the flask and re-weigh
the flask to determine the mass of the milk. Check the label on the milk container and record
the amount of protein per serving in your notebook.
2. Prepare a water bath by placing 200mL of water in a 600 mL beaker. Heat the water bath to
40oC; as the temperature is critical for this experiment monitor the temperature with a
thermometer. Place the flask containing the milk into the water bath.
3. Slowly add 10 drops of glacial acetic acid to the milk while stirring with a glass rod. Continue to
add drops of glacial acetic acid drop wise until no more precipitate is formed when a drop of
acid is added. Allow the mixture to cool.
4. Filter the mixture into a 250 mL beaker by pouring it through cheese cloth that has been
fastened to the beaker with a rubber band. Squeeze out as much liquid as possible from the
solid. Then scrape the solid into a 100 mL beaker.
5. To remove any fat from the curd (do you expect any for skim milk?), add 25 mL of ethanol to
the solid in the 100 mL beaker. Stir the mixture for about 5 minutes; then let the solid settle.
The fat will dissolve in the alcohol. Decant the liquid into another beaker.
6. Under a hood, add 25 ml of a 1:1 (v/v) mixture of diethyl ether and ethanol to residue. Be sure
that that there is no flames or sparks present as the diethyl ether is extremely flammable. Stir
the mixture for about 5 minutes. Let the protein solid dry in your drawer for a week, then weigh
your solid and determine the % yield during the next class period.
BIURET TEST:
1. Add a pea sized amount of your casein to a small test tube and dissolve it in 4 mL of distilled
water. Divide this protein solution into two 2 mL portions. Save one portion for the next test.
2. Thoroughly mix 2mL of the protein solution with 2 mL of 3M sodium hydroxide solution. Add 1
drop of 1% of copper sulfate solution. Note the color and record your observations in your
notebook.
3. Continue to add the copper sulfate solution one drop at a time, note and record your
observations. Stop after adding 10 drops of the copper sulfate solution.
4. Repeat this test with a 1% casein solution.
XANTHOPROTEIC TEST:
1. To the second 2 mL protein solution carefully add 1 mL of concentrated nitric acid. Mix and note
the appearance of any heavy white precipitate.
2. Warm the mixture carefully in a hot water bath noting any change to a yellow colored solution.
80
3. Cool the mixture in a stream of cold tap water and carefully add a few drops of 3M sodium
hydroxide. A positive test is indicated by the yellow color changing into orange color. The entire
tube does not have to turn to orange. Look for the color as the sodium hydroxide is added to
the solution or on a piece of precipitate on the wall of the test tube.
4. Repeat this test with 1% casein solution.
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82
REPORT SHEET-Isolation and Characterization of Casein from Milk
Name____________________________ Partner’s name __________________________
Section______________ Date_________
Isolation:
1.
Mass of 125 mL Erlenmeyer flask __________________________________
2.
Mass of 125 mL Erlenmeyer flask +milk _________________________________
3.
Mass of milk ______________________________
4.
Mass of crude casein (may not be totally dry) _______________________________
5.
Calculate % yield (show your calculation):
6.
Determine the amount of protein in a serving of milk (use the mass of crude casein)
(show your calculation)
Biuret test:
7.
Color of 0.1% of copper sulfate solution:
8.
Color of your casein + sodium hydroxide solution:
9.
Color of your casein solution after addition of one drop of copper sulfate:
10.
Color changes observed after adding additional drops of copper sulfate:
11.
Color of 1% casein + sodium hydroxide solution:
83
12.
Color of your 1% provided casein solution after addition of one drop of copper sulfate:
13.
Color changes observed after adding additional drops of copper sulfate:
Xanthoproteic acid test:
14.
Observation for your casein mixed with concentrated nitric acid:
15.
Observation for your casein mixed with concentrated nitric acid after heating:
16.
Observation for your casein after adding 3M sodium hydroxide:
17.
Observation for 1% casein mixed with concentrated nitric acid:
18.
Observation for 1% casein with concentrated nitric acid after heating:
19.
Observation for 1% casein after adding 3M sodium hydroxide:
Milk comparison:
Did one of the varieties of milk have a higher percentage by weight of protein?
84
Amylase: The
Activity of an
Enzyme
Adapted from: “Factor’s Affecting Enzymatic Activity” in John R. Holum and Sandra L. Olmstead,
Laboratorty manual for Fundamentals of General, Organic and Biological Chemistry, 5th Ed., New York:
Wiley, 1994. Michael E. Pugh and Wayne P. Anderson (Rev. 3/2004)
Goals for the Student:


Learn about enzymes that catalyze biological reactions
Learn about the factors that influence the activity of enzymes
Introduction
Amylase, an enzyme that is found in saliva, catalyzes the hydrolysis of starch (amylase). Since
enzymes are proteins, their secondary and tertiary structures are effected by temperature, pH, and
the presence of heavy metal ions. Enzyme activity is closely associated with the structure of an
enzyme. So any change in the secondary or tertiary structure leads to a change in enzymatic
activity.
In this experiment you will examine the effect of temperature and pH on the activity of amylase.
Molecular iodine forms a complex with starch that has a characteristic deep blue color. As starch
undergoes hydrolysis to form oligosaccharides and glucose, the characteristics color of the starch –
iodine complex disappears. Therefore, loss of the deep blue color can be used to measure of the
extent of hydrolysis of starch. A second test for hydrolysis is the occurrence of a positive Benedict’s
test for the solution. Starch is not a reducing sugar, but glucose formed upon hydrolysis is a
reducing sugar.
Experimental
PART I. Effect of Temperature on Enzyme Activity
In order to make sure that the concentration s of enzyme and starch remain reasonably
constant in different parts of the experiment, use an eye dropper to measure quantities of solutions.
Assume that 20 drops represent 1.0 mL.
1. Label three medium test tubes as “0”, “rt” and “100”. Into each test tube place buffered solution
and 2.5 mL of distilled water. Place the test tube marked “0” into the ice bath, the one marked “rt”
into room temperature and “100” into a beaker of boiling water.
85
2. Place 1 mL of freshly prepared amylase solution (100 mg/100 mL) into each of three small test
tubes. Put one of these into the ice bath, another into the thermostated water bath, and the third
into the beaker of boiling water.
3. Allow the solutions to remain in the temperature baths for about 5-10 minutes to equilibrate.
Carry out the following steps in sequence for the solutions in the 0o, rt, and 100o temperature
baths.
4. Remove the test tube containing the starch and the test tube containing the enzyme from the bath
and pour the enzyme solution into the starch solution. Record the time to the nearest second, and
call this starting time 0.
5. Quickly place a piece of parafilm over the end of the test tube, mix thoroughly, remove the parafilm
and remove a small amount of the solution from the test tube using a disposable pipette. Place the
remaining solution back into the temperature bath. Place 4 drops of the solution onto a spot plate
that contain 1 drop of the iodine solution, record the color. Put any excess starch/enzyme solution
in the pipette back into the test tube in the temperature bath.
6. Take a sample from the solution using the disposable pipette exactly 1 minute following time 0.
Place 4 drops onto a second spot of the spot plate containing 1 drop of iodine solution, record the
color. Return any excess starch/enzyme solution in the pipette to test tube in the temperature bath.
7. Repeat the procedure in at the following time intervals until the color of the solution following
addition of the iodine is yellow: 2 min, 4 min, 6 min, 8 min, and 10 min. If the color of the solution
in the spot plate remains yellow for successive trials. It is not necessary to continue the run.
Part II. Effect of pH on Enzyme Activity
1. Label three medium test tubes as “5”, “7” and “9”.
2. Into the test tube marked “5”, place 2.5 mL of unbuffered starch solution and 2.5 mL of pH 5
buffer solution. Into the test tube marked “7” place 2.5 mL of unbuffered starch solution and
2.5 mL of pH 7 buffer solution. Into the test tube marked “9” place 2.5 mL of unbuffered starch
solution and 2.5 mL of pH 9 buffer solution. Place these into either the room temperature bath
or the ice bath, depending on which temperature gave the better results in part I.
3. Place 1 mL of the amylase solution into each of three small test tubes. Put these into the
temperature bath containing the starch solutions from step 2.
4. Allow the solutions to remain in the temperature bath for about 5-10 minutes to equilibrate.
5. Carry out the steps 4-7 in PART I in sequence for the solutions that buffered at pH 5, 7, 9.
86
PART III. Effect of Metal Ions on Enzyme Activity
In this part you will test the effect of a metal ion on enzyme activity, Cu2+, Fe3+, Zn2+ or other transition
metal ions or heavy metal ions may be tested.
1. Label a test tube with the identity of the metal ion in the salt solution you will use for this part.
2. Into each test tube, place 2.5 mL of buffered starch solution and 2.5 mL of the metal ion solution.
Place these into room temperature bath or the ice bath, depending on which one gave better results
in part I.
3. Place 1mL of the amylase solution into each of two small test tubes. Put them into the temperature
bath containing the metal ion solution.
4. Allow the solutions to remain in the temperature bath for about 5-10 minutes to equilibrate.
5. Carry out the steps 4-7 in PART I in sequence for the solutions containing the metal ion.
87
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88
REPORT SHEET-Amylase: The Activity of an Enzyme
Name____________________________ Partner’s name __________________________
Section______________ Date_________
For each section, record the colors of the starch/enzyme solution in the presence of iodine.
PART I. Effect of Temperature on Enzyme Activity
TIME
(minutes)
0
0oC
Room Temperature
100oC
pH =7
pH=9
1
2
4
6
8
10
Part II. Effect of pH on Enzyme Activity
Temperature __________________
Time
(minutes)
pH =5
0
1
2
4
6
8
10
89
PART III. Effect of Metal Ions on Enzyme Activity
Temperature ______________________
Metal Ion = _____________
Time (minutes)
0
1
2
4
6
8
10
CONCLUSIONS
1. Give a clear explanation of the effect of temperature on the activity of amylase. Consult
yoir class notes ideas.
2. Give a clear explanation of the effect of pH on the activity of amylase. Consult your class
notes for ideas.
3. Give a clear explanation of the effect of the metal ion on the activity of amylase.
90
Interaction of UV
Light with Matter
Goals for the Student:



To understand the factors that affect how certain types of matter interact with UV light
To gain a rough understanding of how a spectrophotometer works.
To be able to draw conclusions about protecting one’s body from UV exposure.
Introduction
Recalling what we have learned about light:
Figure 1- We can think energy as a wave. The
distance between two equivalent points on the
wave is the wavelength (.
Our sun emits the energy that sustains life on earth as we know it. If we think about the
energy as a wave, as illustrated in Figure 1, we can relate the wave and its associated energy, as
shown in Equation 1:
(1)
E = hc / 
E stands for energy. The energy is proportional to Planck’s constant (h) and the speed of light (c, also a
constant), while it is inversely proportional to the wavelength (, lambda is the symbol for wavelength)
of the energy. When the wavelength increases, the energy decreases, and vice versa.
Figure 2 shows a common diagram of an electromagnetic spectrum. You can see that radio
and TV waves are made-up of relatively long wavelengths, and so their energy is relatively low. They
pass harmlessly around us all the time. Microwaves have shorter wavelengths and have higher
energy. Microwaves make water molecules move, heating up the food you place in the microwave
oven. Infrared waves can be felt as heat, with still greater energy. We commonly think of visible
91
waves as “light.” Visible waves cause molecules in your retina to change energy states. The change
gets transmitted through your optic nerve by chemical signals to your brain, where it is interpreted as
vision. Visible waves have wavelength of roughly 700-400 nm, or about the size of a living cell; hence,
you are able to see cells in a light microscope due to visible light passing through or bouncing off them.
X-rays are smaller still; one the order of the size of atoms. A wave with a wavelength this small is very
high energy! X-rays pass right through most organic molecules. This is why we can use them medically
to see bones and other structures that contain metals. At the same time, great care must be taken to
avoid unnecessary exposure or the DNA in cells can be damaged and cause tumors. Gamma-rays (rays) have the shortest wavelength, and highest energy, of all. Short-term -ray exposure is used to
treat pre-packaged meats. The energy passes through into the meat and kills microbes, thereby
extending the shelf life of the product. What can kill microbes can kill any cell, thus -rays are very
dangerous. Fortunately, we have a protective magnetic field around our planet. Without it, life as we
know it could not exist.
Figure 2- Comparing
wavelengths (of energy in the
electromagnetic spectrum.
92
Our experiments will focus on ultraviolet light. Ultraviolet light (UV light) has wavelengths
from about 400 nm, down to about a nanometer. UV light is subcategorized by wavelength, as shown
in Table 1. This size of these wavelenghts is on the order of that of molecules, and so it can interact
with certain functional groups of organic molecules. If the organic molecule is in a living organism,
changes in the molecule can take place so that it no longer functions properly. When UV light hits the
DNA in our skin cells, changes can take place in the chemical structure such that it no longer base pairs
properly. During replication, improper base-pairing can cause a mutation. If the mutation is in a region
needed for cell survival, the cell can die prematurely. If that mutation is in one of the many genes that
control cell growth, a cancerous tumor can form.
Cells have built-in mechanisms that
protect us from UV-induced tumor formation.
There are repair enzymes that constantly scan our
Name
Wavelength Range (nm)
DNA for changes in molecular structure. When
UVA (black light)
400-320
found, the damaged piece of DNA is cut out and
UVB
300-280
replaced with a good piece. At the same time,
UVC (germicidal)
Below 280
when cell damage due to UV light exposure
occurs, our bodies produce a natural sun block called melanin. It is the brown pigment in our skin that
shows-up as a ‘tan.’ So, a suntan is the body’s response to cell damage in order to try to prevent
cancer. Premature cell death also causes the skin to thicken and wrinkle. Obviously, these mechanisms
do not always work. Liberal use of sun block is essential for people of all skin types to prevent cell death
and cancer.
Table 1- Subcategories of UV light.
Light in the laboratory: (this section is adapted from experiments written by Dr. Emeric Schultz for the
Chemistry for the Sciences 2 lab manual)
Matter that appears colored is preferentially absorbing different wavelengths of visible light.
Your eyes detect the compilation of all of the reflected and/or transmitted colors – the observed
color. As laboratory scientists, we are interested in the color absorbed, which is the complement of the
color observed. Table 1 shows the correlation between absorbed and observed visible light. For
example, tree leaves that appear green to our eyes do so because red and purple light is absorbed; the
green bounces off the leaves, enters our eyes, and triggers the chemicals in the retina to signify “green”
to the brain.
Table 1: Colors of Visible Light
Wavelength of Light Absorbed (nm)
red
purple
blue-green
green
780
orange
680
blue
630
yellow
590
violet-blue
560
greenyellow
530
green
500
bluegreen
470
blue
440
violetblue
420
violet
380
violet
purple
red
orange
yellow
greenyellow
 Absorbed Color 
 Observed Color 
A spectrophotometer is an instrument that can measure the amount of light “absorbed” by
matter that is dissolved in solution. A bare-boned schematic is shown in Figure 3. Our eyes only detects
93
a very limited range of wavelengths. A spectrophotometer can detect any wavelengths it is designed to
detect. There are UV-Visible spectrophotometers that detect from 190-800 nm. There are IR
spectrometers, and so forth. In a spectrometer, the light source emits waves that are selected by a
monochometer. The chosen range of light passes through the sample. Some of the light is absorbed
and does not reach the detector. The instrument knows how much light was sent into the sample and
compares that to the amount that is transmitted. The difference is the absorbance. A substance will
have a different absorbance, depending upon the wavelength.

Light
source
Sam


sample
wavelength selector
(monochromator)
Light detector
Figure 3- The set-up for a spectrophotometer.
A scanning spectrometer, as the name suggests, can scan through lots of wavelengths and
produces what is called a spectrum (Figure 2). [spectra is plural for spectrum]. There are two
important numbers that you will get from the scans of your samples. The first number is called the
absorbance maximum and is given the symbol max. The symbol (lambda) is universal in science as a
designation for wavelength; max is therefore the wavelength at which a dissolved species interacts best
with light (as shown by a peak in absorbance); max in Figure 4 is about 525 nm. A word about units for
wavelength. Wavelengths of different kinds of electromagnetic radiation (everything form radio waves to
X-rays) can go from being very short to very long. The most convenient units to use for electromagnetic
radiation in the visible and the surrounding ultraviolet and infrared regions are in nanometers (10-9m).
It is important for you to distinguish
between what type of light interacts with matter
(wavelength) and how much light interacts
with matter (absorbance). Take a look the
Figure 3 below and its labels (Note: although
this is a spectrum of visible wavelengths
whereas we will be looking at UV wavelengths,
the spectra are interpreted the same way).
Figure 4- An absorbance spectrum for a sample
with color.
The second number is called the
absorbance. This is the quantity that we have
been studying. It is a measure of the intensity of
the color. You know the factors that affect this
value. Absorbance by definition has no units.
This means that the units selected for the
factors that determine absorbance have to
cancel.
Matter has a wavelength or wavelengths of light that it interacts with (absorbs) best. These
wavelengths are called absorbance maxima and are labeled as max.(“lambda max”). One
wavelength can be the very best but the wavelengths around this maximum are also absorbed quite well
by the molecule. We call this set of wavelengths an absorbance band.
94
Pre-Lab Assignment
Scientists have discovered many molecules that absorb UV light and are essentially harmless
when applied to human skin. Before you come to lab, use the internet (Wikipedia is fairly
trustworthy in this case or you can use an image search in Google) to look-up and record the
structures of each of these compounds in your lab notebook:
p-Aminobenzoic acid (PABA)
Avobenzone
Cinoxate
Dioxybenzone
Homosalate
Methyl anthranilate
Octocrylene
Octyl methoxycinnamate (Octinoxate)
Octyl salicylate (Octisalate)
Oxybenzone
Padimate O
Phenylbenzimidazole sulfonic acid (Ensulizole)
Sulisobenzone
Titanium dioxide
Trolamine salicylate
Zinc oxide
Experimental
Your instructor will have set-up the Genesys spectrophotometer so that it will scan from 200-400 nm.
Baseline scan of air: the purpose of this part of the experiment is to make a control scan so
that atmospheric conditions do not affect your results.
1. Ensure the instrument sample compartment is empty.
2. Shut the door and press the “new baseline” key. The instrument will make all kinds of rude
noises, move up to the first position, then begin to scan.
3. Do not open the sample compartment until the baseline is completed.
4. Once the baseline is completed, the instrument will return the sample holder to the #2 position.
UV scan of quartz:
1. Record your visual observations about the quartz cuvet. (A cuvet is a container to hold liquid
samples for spectrometry. We can only use it is it does not interfere by absorbing the light we
are studying.) Clean it with a Kimwipe. We do not want fingerprints scattering the light!
2. Place the cuvet in the #2 position of the sample holder.
3. Shut the door, then make sure the sample position indicator in on “2.”
4. Press the “new scan” or “scan” key.
5. Do not open the sample compartment until the scan is completed.
6. Print a copy of the scan for each lab partner to tape in his or her book. Take care to not tape
over the print or the adhesive in the tape will dissolve it.
7. Describe the shape of the spectrum in your notebook. For example we can describe Figure 4:
At 700 nm, the absorbance is zero and then it increases to a maximum at around 525 nm.
Finally, the absorbance decreases to around 0.1 at 400 nm.
95

Does the quartz absorb any wavelengths of UV light? If so, what is the max? Is this
UVA, UVB, UVC, or all three? If the quartz does not absorb UV light, it is said to be
“UV transparent.” If the quartz is UV transparent, we could use it as a holder for
liquid samples and it would not interfere with our spectra.
UV scan of glass:
1. Record your visual observations about the piece of window glass. Clean it with a Kimwipe. We
do not want fingerprints scattering the light!
2. Secure the piece of glass into the #2 position of the sample holder.
3. Shut the door, then make sure the sample position indicator in on “2.”
4. Press the “new scan” or “scan” key.
5. Do not open the sample compartment until the scan is completed.
6. Print a copy of the scan for each lab partner to tape in his or her book. Take care to not tape
over the print or the adhesive in the tape will dissolve it
7. Describe the shape of the spectrum in your notebook.

Does the glass absorb any wavelengths of UV light? If so, what is the max? Is this UVA,
UVB, UVC, or all three?
UV scan of water:
1. Fill the quartz cuvet with distilled water. Take care to prevent air bubbles in your sample.
2. Record your visual observations. Clean it with a Kimwipe. We do not want fingerprints
scattering the light!
3. Place the cuvet in the #2 position of the sample holder.
4. Shut the door, then make sure the sample position indicator in on “2.”
5. Press the “new scan” or “scan” key.
6. Do not open the sample compartment until the scan is completed.
7. Print a copy of the scan for each lab partner to tape in his or her book. Take care to not tape
over the print or the adhesive in the tape will dissolve it
8. Describe the shape of the spectrum in your notebook.

Does the water absorb any wavelengths of UV light? If so, what is the max? Is this
UVA, UVB, UVC, or all three?
UV scan of DNA:
1. Fill one quartz cuvet with distilled water and one quartz cuvet with DNA solution. Take care to
prevent air bubbles in your samples.
2. Record your visual observations about the DNA solution in the cuvet. Clean each with a
Kimwipe. We do not want fingerprints scattering the light!
3. Put the cuvet with water in the first position and the cuvet with DNA solution in the second
position.
4. Shut the door and press the “new baseline” key. The instrument will make all kinds of rude
noises, move up to the first position, then begin to scan.
5. Do not open the sample compartment until the baseline is completed.
6. Once the baseline is completed, the instrument will return the sample holder to the original
position.
7. Make sure the sample position indicator in on “2.”
8. Press the “new scan” or “scan” key.
9. Do not open the sample compartment until the scan is completed.
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10. Print a copy of the scan for each lab partner to tape in his or her book. Take care to not tape
over the print or the adhesive in the tape will dissolve it
11. Describe the shape of the spectrum in your notebook.

Does the DNA absorb any wavelengths of UV light? If so, what is the max? Is this UVA,
UVB, UVC, or all three?
UV scan of sunscreen:
1. Dry the quartz cuvets. Near the top of one of them, write a small letter ‘S’ so that you can
distinguish the cuvets.
2. Record your visual observations. Clean it with a Kimwipe. We do not want fingerprints
scattering the light!
3. Place the cuvet in the #2 position of the sample holder.
4. Shut the door, then make sure the sample position indicator in on “2.”
5. Press the “new scan” or “scan” key.
6. Do not open the sample compartment until the scan is completed.
7. Print a copy of the scan for each lab partner to tape in his or her book. Take care to not tape
over the print or the adhesive in the tape will dissolve it
8. Describe the shape of the spectrum in your notebook.

Does the sunscreen absorb any wavelengths of UV light? If so, what is the max? Is this
UVA, UVB, UVC, or all three?
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REPORT SHEET-Interaction of UV Light with Matter
Name____________________________ Partner’s name __________________________
Section______________ Date_________
I. Complete the table of results. Indicate if the substance tested absorbs UVA, UVB, or UVC. For the
sunscreen, list the name of the active ingredient. Gather a few results from your labmates.
Substance
Absorbs
UVA?
Absorbs UVB?
Absorbs UVC?
Quartz
Glass
Water
DNA
Answer the following. Include your scientific evidence to support your answer where
appropriate.
II. If the windows of your home or car were made of quartz, would they protect your from UV light?
III. Do standard glass windows protect your from UV light?
IV. Does being under water (as when swimming) protect you from UV light?
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V. What common functional group is found in the active ingredients of sunscreens?
VI. Based upon your findings for question V, what functional group of DNA is likely to interact with UV
light?
VII. What purpose does this functional group in DNA serve?
VIII. How could UV damage to this functional group cause cancer?
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