Chemistry Question

Structural Studies with Molecular ModelsPart I. Constitutional Isomers and Rotational Conformers
1. Draw the skeletal formulas and make models for all the constitutional isomers of
C5H12. Name each of your isomers.
2. Build a model and sight down the C2-C3 bond of 2,2-dimethylbutane. Draw the
Newman projection formula for the most and least stable conformations.
3. Make the model of 1,2-dichloroethane. In order to produce a potential energy diagram
a. Draw the Newman projection of the anti conformer to the far left.
b. Rotate around the carbon-carbon bond 360o in 60o increments, drawing
each conformer. You should produce seven conformers drawn in a line
across the page. The first and last with both be the anti conformer.
c. Estimate the relative energy of each conformer and complete the diagram.
E
Bond rotation angle
1
Structural Studies with Molecular Models
4. Write the bond-line formulas and build models of cyclobutane and cyclopentane. Use
the iSpartan app to build each as well. View down various bonds of each to
investigate, then explain why cyclopentane is less strained than cyclobutane. Use
Newman projections to illustrate your answer. Use the app to record specific dihedral
angles to support your answer.
5. Write bond-line formulas and build models of the chair and boat conformations of
cyclohexane. View down various bonds of each to investigate, then explain why the
chair conformation is more stable than the boat conformation. Use Newman
projections to illustrate your answer. Label the hydrogens as axial and equatorial in
the Newman projections.
6. Write skeletal formulas and build models for cis-1,4-dimethylcyclohexane and trans1,4-dimethylcyclohexane. Carefully twist your models to perform ring flips, draw
both chair conformations, and identify the most stable.
7. Make models of the cis and trans isomers of 1,2- and 1,3-dimethylcyclohexane.
Carefully twist your models to simulate ring flips to draw both chair conformations
and identify the most stable for each.
2
Structural Studies with Molecular Models
Part II. Enantiomers and Diastereomers
8. Write bond-line formulas and build models of 2-bromopropane. Is this compound
chiral? Why or why not?
9. Build a model of 2-bromobutane.
a. Build the mirror image of your first structure. Are these two structures
superimposable?
b. Draw each structure with care to indicate the 3D tetrahedral nature at the
chiral carbon using solid and dashed wedges.
c. Indicate the configuration of each structure using the Cahn-Ingold-Prelog
R/S notation system.
d. Use the iSpartan app on your iPad to build your models and confirm the
configuration of each.
10. Using the model of 2-bromobutane representing the R enantiomer, switch any two
groups on the chiral carbon.
a. Compare this modified model with the model of the S enantiomer. Are the
models identical or are they still enantiomers?
b. Switch two groups around the chiral carbon in either model. What is the
relationship between these structures?
3
Structural Studies with Molecular Models
11. Write bond-line formulas and build models of ALL of the stereoisomers of 2-bromo3-chlorobutane. Assign the R/S configurations of each chiral carbon in all isomers.
12. Return to the previous problem and identify each unique pair as being enantiomeric
or diastereomeric pairs.
13. Construct a model of this meso form of 2-3-dibromobutane and identify the mirror
plane in the molecule. Use this model to draw the bond-line, Newman Projection as
viewed down the C2-C3 bond, and the Fisher projection of this meso form. It is
important to assure that the mirror plane is clearly identifiable in each drawing. This
will require your rotating around bonds to use each structure type to its greatest
advantage making the mirror plane obvious to the reader.
14. Can cis-1,2-dibromocyclopentane exist as a pair of enantiomers? Make appropriate
models and check it out. Try the same thing for trans-1,2-dibromocyclopentane. Draw
these structures to illustrate the relationships observed. Assign the R/S configurations
of each chiral carbon in all isomers. Identify each unique pair as being enantiomeric
or diastereomeric pairs.
4
CHEM 2010 Laboratory
Name: _________________________________
Acid-Catalyzed Isomerization of an Alkene
Background
Cis and trans isomers of an alkene have different stabilities, in general, because
they have different amounts of steric strain. The cis isomer has its attached groups closer
together. They repel each other, causing the molecule to be less stable. The trans isomer
however has its substituents comfortably far apart. As a result, the trans isomer is more
stable.
The double bond holds the molecule rigid because of the side to side overlap of
the p orbitals that form the pi bond. This keeps the less stable cis isomer from converting
it to the more stable trans form.
The pi bond can be broken by adding an acid. The acid adds a proton to one
carbon of the double bond and leaves a carbocation on the other side. In the absence of
the double bond, there is nothing left to hold the cis geometry in place and it rotates to a
more stable conformation. If the added proton is removed by a water molecule, the
double bond is reformed. This creates a new isomeric alkene having the trans geometry.
In this experiment, we will start with a cis alkene, maleic acid, and convert it to
its trans isomer, fumaric acid, by heating it in water solution in the presence of
hydrochloric acid. Both of these substances are white crystalline powders. We can tell
them apart because they have very different melting points. The reaction requires higher
temperatures so we perform it in boiling water under conditions of reflux.
Reaction
maleic acid
fumaric acid
CHEM 2010 Laboratory
Name: _________________________________
Pre-lab-(Note there are 3 parts!)
Part 1: Study each of the linked references below-Take notes from each. Be sure to use
these and the logic within them to better understand each step of the experimental
procedure you are performing this week.
1. Heating a reaction at reflux
2. Reflux set-up: Note chemist uses a heating mantle as the heat source. We will use
a metal bowl filled with sand which is heated by the hot plate/stirrer beneath.
3. Vacuum Filtration – basics
4. Crystal Collection via Vacuum filtration
Part 2: Answer all of the Pre-lab questions below.
1. Provide the missing information for the reactant and product:
Compound
Molecular weight (g/mol)
Melting point (oC)
Maleic acid
Fumaric acid
Literature reference
2. Provide arrow to complete the mechanism for the isomerization of maleic acid to
fumaric acid.
CHEM 2010 Laboratory
Name: _________________________________
3. Calculate the theoretical yield of fumaric acid. Show Calculations!
4. What is meant by the term reflux?
5. In general, what is the temperature of a refluxing reaction?
6. A round-bottomed flask used in a heated reaction is always clamped at what part
on the flask? Why is this clamp so important?
7. While we are using a magnetic stirrer to keep our reaction solution moving to
prevent ‘bumping’. A popular method for this is to use boiling stones. What is a
boiling stone and how does it work?
Part 3: Study the experimental procedure for the Isomerization of an Alkene, using the
provided images and technique notes to visualize and think your way through it as you
go.
CHEM 2010 Laboratory
Name: _________________________________
Safety Concerns
You will be using concentrated hydrochloric acid, which causes severe burns
when it comes in contact with the skin. Be very careful of the high temperature required
to reach the melting point of fumaric acid.
Chemical Disposal
Excess maleic acid and the final product fumaric acid should be disposed of in the
disposal containers labeled Waste Container in the main hood. The liquid used as a
reaction solvent(water) can be disposed of down the drain.
CHEM 2010 Laboratory
Name: _________________________________
Experimental Procedure
1. Set up for reflux by assembling a jack, hot/stir plate, and sand bath with a stable
ring stand. Position the set-up in front of your student hood.
2. In a 100 ml round bottomed flask, place approximately 1.0 g of maleic acid,
record exact mass. Add 10 ml of D.I. water to this flask. Swirl the flask by hand
until all of the maleic acid is dissolved. Carefully add a magnetic stir bar to the
flask and clamp the flask securely above the sand bath.
3. Proceed to the main hood with a 10 mL graduated cylinder. Carefully measure an
8 ml portion of concentrated HCl into a graduated cylinder. Slowly and carefully
add this to the reaction flask.
4. Prepare a reflux condenser by attaching water tubing with tubing clamps and
assuring proper water flow into the bottom port and exiting the top port. Use
stopcock grease to assure a good seal of the condenser into the mouth of the
reaction flask. Assure cold water is flowing through the jacket of the condenser
and draining via tubing into the sink at a slow constant rate. This keeps the
condenser at the temperature of the water, providing a good condensation surface.
5. Bring to reflux by turning the hot plate to medium/high heat. Ajust the ehat as
required to reach reflu and kep the condensation occurign in the bottom portion of
the condenser. When refluxing, you should be able to see a boiling liquid in the
flask and condensing vapors dripping back down into the mix. This is “refluxing”
a term that means “to flow back.”
6. Continue refluxing for 15 minutes. As the reaction progresses, a crystalline solid
will begin to separate out of solution. This is your product, fumaric acid, which is
less soluble in aqueous acid than its isomer maleic acid.
7. While the reaction is refluxing, prepare for product collection by preparing an ice
bath and vacuum filtration apparatus. Use a large test tube to cool a few
milliliters of D.I. water to wash your product during filtration.
8. At the end of the15 minute heating period, turn off the hot plat and use the jack to
lower the sand bath away from the flask. CAREFULLY, remove the hot plate and
sand bath from the jack. Let the reaction flask cool for 10 minutes.
9. Fill your 250 ml 2/3 full with ice, and add a 50 to 75 ml of water to this to make
an ice water bath. Use the jack to support the ice bath to cool the reaction mixture
and promote crystallization. Allow to cool in ice bath for 10 minutes.
10.Set up your suction filtration apparatus and Buchner funnel with filter paper and
filter your reaction mixture. Collect the solid product. Rinse the round bottomed
flask and the product with cold D.I. water.
CHEM 2010 Laboratory
Name: _________________________________
11.Remove your product from the filter flask and dry it by pressing between pieces
of large filter paper (see technique notes).
12.Collect, weigh and determine the melting point of your product. Take images of
your product on the balanced showing the final mass as well as a close-up image
showing the quality of the product. Insert in Analysis section where appropriate.
Show your product to your instructor and obtain initials/comments on purity.
Data and Analysis
Mass of maleic acid _____________grams
Moles of maleic acid _____________moles
Mass of dried fumaric acid
Moles of fumaric acid
__________grams
__________moles
Experimental melting point range of fumaric acid product _________ °C
Percent yield of fumaric acid __________ %
Show your calculation:
Image of product on balance with mass showing:
Close up image of product:
CHEM 2010 Organic I
Laboratory
Extraction of Spinach Pigments and Analysis by
UV/Vis Electronic Absorption Spectroscopy
THEORY
Ultraviolet-visible spectroscopy is a useful tool for studying the electronic structure of
unsaturated molecules and their conjugation. The electronic absorption spectra can generally
reveal the degree of delocalization of the conjugated π system. The electronic transition between
bonding and anti-bonding orbitals in organic molecules are large and normally require higher
energy. As the number of π molecular orbitals increase in conjugated systems, the energy gaps
between the filled and unfilled orbitals decrease. Lower energy is needed to promote electrons
into an excited stated, resulting in molecules that can absorb in the visible region.
The pigments found in spinach are good examples of highly conjugated molecules that can
absorb in the visible region. Spinach contains green pigments known as chlorophylls and yellow
pigments known as carotenoids, both which are involved in the photosynthesis process. There
are several types of chlorophyll, chlorophyll a and b being the most common. The difference
between the two chlorophylls is that a methyl side-chain in chlorophyll a is substituted with a
-CHO group in chlorophyll b. Carotenoids are a class of hydrocarbons (carotenes) and their
oxygenated derivatives (xanthophylls). The yellow color due to the carotenoids is obscured by
the chlorophyll pigments. Structures of chlorophyll and β-carotene are shown in Figure 1.
H2 C
H 3C
R
N
H3 C
N
CH3
Mg N
N
H 3C
H 3C
O
O
H3 C
H3 C
CH3
O
H3 C
O
R = CH3 or CHO
H3 C
Chlorophyll
H3C CH3
CH3
CH3
H3 C
CH3
CH3
CH3
H3C CH3
β-carotene
Figure 1 Structures of various visible light absorbing pigments found in spinach
Adapted from Organic Chemistry with Vernier
1
In order to investigate the electronic absorption spectra of the pigments extracted from spinach,
the chlorophylls and carotenoids need to be separated.
Column chromatography is a purification technique used to isolate compounds from a mixture.
In column chromatography, the stationary phase is a solid adsorbent placed in a column and the
mobile phase is a solvent that is added to the top and flows down through the column. Separation
is achieved based on the polar and non-polar interactions among the compounds, the solvent (the
mobile phase), and the solid adsorbent (stationary phase).
In this experiment, you will extract the pigments from spinach leaves and isolate the chlorophylls
and carotenoids using column chromatography. Using electronic absorption spectroscopy, the
wavelengths of absorbance peaks for the chlorophyll and carotenoid pigments will be identified.
PRE-LAB READING AND VIDEOS FOR REVIEW
Ø Review each of the references above and take notes on technique and theory to be
included in this report.
General concept of Column
Microscale(Pipette) column chromatography
Spectrophotometry

Klein Textbook: Refer Chapter 16 covering conjugated pi systems with particular focus on the
characteristics of light absorption and the basis of color.
OBJECTIVES
In this experiment, you will
• Extract the pigments in spinach leaves.
• Isolate the green and yellow pigments using column chromatography.
• Identify the wavelengths of absorbance peaks for the extracted spinach pigments.
MATERIALS
Part I Extraction
fresh spinach leaves
mortar and pestle
three 12 ´ 75 mm test tubes
test tube rack
disposable Pasteur pipets and bulb
one No. 0 stopper
hot plate
250 mL beaker
Temperature Probe or thermometer
sodium sulfate, Na2SO4, anhydrous
acetone
hexane
saturated aqueous sodium chloride
cotton plug
10 mL graduated cylinder
weighing paper
balance
boiling stone
Part II Column Chromatography
disposable Pasteur pipets and bulb
2
ring stand with utility clamp
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
six medium test tubes
test tube rack
cotton plug
sand
silica
spatula
acetone/ hexane solvent mixture
micro funnel
three 100 mL beakers
2.5 cm piece of cut rubber tubing
pinch clamp
weighing paper
Part III Measure the Absorbance
LabQuest or computer
LabQuest App or Logger Pro
SpectroVis Plus spectrophotometer
glass cuvettes with lids
disposable Pasteur pipets and bulb
acetone/ hexane solvent mixture
hexane
acetone
Kem-wipes
samples from Part II
TLC RESULTS FOR SPINACH EXTRACT
Previous TLC study of the Spinach extract with a very low polarity solvent system produces the
results below. Consider the relative Rf values of the target components and use this information
to rationalize why the procedure is performed as it is.
Adapted from Organic Chemistry with Vernier
3
PROCEDURE FOR COLUMN CHROMATOGRAPHY
Part I Extraction of pigments from Spinach leaves
1. Obtain and wear goggles and gloves. Conduct this experiment in front of student fume hood.
**to save time, see step 9 and prep hot water. Set-up outside of hood area to keep that
space clear for extraction.**
2. Weigh out 1.0 g of fresh spinach leaves. Record the mass to the nearest 0.01 g.
3. Tear the leaves into small pieces and place them in a mortar. Add 5 mL of acetone/hexane
mixture (as indicated by your instructor) and grind the mixture for 3–5 minutes.
4. Create a filter pipet with a cotton plug and a disposable Pasteur pipet. Filter the green extract
into a clean test tube. Try to avoid transferring the ground leaves.
5. Wash the extract in the test tube:
a. Add 2 mL of saturated aqueous sodium chloride solution to the test tube.
b. Place a piece of parafilm over the top (do not wrap it around), hold in place with thumb,
and shake the test tube.
c. Immediately vent the test tube by lifting the parafilm edge.
d. Repeat this process of shake and vent for 1-2 minutes.
6. Place the test tube in the test tube rack and allow the mixture to separate while prepping
drying agent column (Step 7).
7. Create a drying agent filled filter pipet: Obtain a disposable Pasteur pipet and add a cotton
plug. Fill the pipet half-way with solid sodium sulfate (anhydrous). Note: Use a micro funnel
or rolled weighing paper to fill the pipet.
8. The final traces of water are removed by treating the organic solution with the drying agent.
Draw up the yellow/green organic layer from your test tube with a disposable pipette and
pass it through the sodium sulfate plug into a clean test tube. Avoid removing the aqueous
layer when your draw up your organic layer.
9. Prepare a 70°C hot water bath in a 250 mL beaker. Monitor the temperature of the water bath
using a Temperature Probe or thermometer.
10. Carefully move the hotplate and bath in front of/under your fume hood. Add a glass bead to
the test tube to prevent the solution from bumping. Use a test tube holder and carefully warm
the tube with gentle agitation. Take care to not allow it to boil aggressively and flash/froth
out of the tube. Keep tube directed toward the back of your hood, not at a neighbor or self.
Evaporate the solvent to approximately 0.2–0.3 mL (about the depth of the glass bead.)
4
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
Part II Separation of pigments via Column Chromatography
19. Assemble the pipet column. CAUTION: Silica powder is harmful if inhaled. Use only in the
fume hood.
a. Place a small piece of cotton into the bottom of the pipet to support the separation
materials. Use a piece of aluminum wire to seat he plug gently into the tapered part of the
pipette. Do not pack too tightly; enough to prevent particles from passing it but not so
densely that liquid struggles to pass through.
b. Add approx. 0.5 cm of sand to support and provide a flat base for the stationary phase
c. Add enough silica to fill the pipet about 3/4 full. Note: Micro funnel or weighing paper
may be used to help fill the column with silica. See instructor for micro-funnel.
d. Gently tap the sides of the pipet for 1 minute to pack the silica.
e. Add another 0.5 cm of sand to the top of the pipet to maintain a flat surface for loading
the mixture onto the silica.
20. Use a micro clamp to secure the pipet column to a ring stand. Place the fingers of the clamp
in the middle of the silica column for easy viewing of the top and bottom. Adjust the height
of the column on the ring stand to allow the bottom to insert into the top of a small test tube
which can be easily swept from beneath it and replaced with another.
21. Obtain six test tubes to collect the column fractions and a 50/100 mL beaker for waste.
Important: Once you start the elution process, it cannot be stopped.
You must go to completion. Read Step 22-23 carefully before continuing.
22. Prepare the column for chromatography (work in front of/under student fume hood)
a. Place a waste beaker under the pipet column. This will catch the eluent which does not
contain the target molecules.
b. Obtain approx. 10 mL of the hexane in a 10 ml graduated cylinder. Set to back of your
student fume hood, not on open benchtop, for later use as the first eluting solvent (step h
below).
c. Obtain 8-10 mL of the acetone in a 10 ml graduated cylinder. This will be used first to
pack the column, and later as the second eluting solvent.
d. Carefully load a portion of acetone into the pipet to fill the space above the column. Use a
small bulb to apply pressure and push the solvent through the silica gel to force air from
the column. Only apply pressure; tilt bulb to side while still holding slight pressure to
prevent pulling air back up and ‘cracking’ the column. This process will provide a
consistent stationary phase for separation.
e. Repeat loading acetone and pushing with bulb until all air is removed from the column.
The solvent should be clean and can be recycled through the column during this process.
Note: If air bubbles or cracks appear in the column, tap to pack and continue the solvent
flush process.
f. Important: Do not let air move into the top of the column during this procedure.
Replenish with solvent as needed. When the column is prepared for loading with the
green extract fill with solvent to give you time to pull up the extract and get ready to load
it.
Adapted from Organic Chemistry with Vernier
5
23. Load and run the colum chromatography:
a. Let the solvent line drop until the meniscus is just above top sand layer, then use a
clean pipette to load the concentrated green spinach extract onto the top of the
column.
b. Allow the green solution to adsorb onto the silica. As the extract flows through
the sand to reach the silica get, drip acetone above it only as needed to not let the
sand get dry.
c. Do not let the column get dry during this procedure
d. Once the extract is properly loaded, switch to using pure hexane to elute the
yellow layer faster. Refer to the TLC results above and consider what target
compounds are in this yellow extract which is flowing the fastest with the nonpolar hexane solvent.
***Take a good picture of the loaded column to later insert into your report
e. A yellow band will appear and begin to separate from the green band. Allow the
colorless solvent to flow into a small beaker until the yellow band nears the
bottom. Do not let the column get dry during this procedure, continuing
flushing with pure hexane.
***Take a good picture of the eluting column to later insert into your report
f. To collect the yellow extract, replace the beaker with the first test tube. Collect
the entire yellow fraction into this test tube. WATCH THE SOLVENT LEVEL Do not let air get to the silica layer during this procedure.
g. Once you have collected the yellow fraction, place another small waste beaker
under the column and change the eluting solvent to pure acetone to begin
pushing the green layer through the column.
h. Continue adding the solvent, collect the colorless portion in the waste beaker. The
green band should be moving down the column.
***Take a good picture of the eluting column to later insert into your report
24. To collect the green extract moving with the acetone, replace the beaker with a clean test
tube. Collect all of the green portion. Do not let the column get dry during this procedure
Note: All of the pigments will NOT be removed from the column.
23. Place a beaker back under the column and allow it to drain/dry during next portion of
experiment.
6
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
Part IV Measure the Absorbance
24. Locate an available laptop/Spectrovis station. Connect the SpectroVis Plus
spectrophotometer to the USB port of LabQuest or a computer. Start the data-collection
program, then choose New from the File menu.
25. Calibrate the Spectrophotometer for the yellow fraction.
a. Prepare a blank for testing the yellow fraction by filling a glass cuvette with hexane and
cover with a cap or parafilm. Note: Always wipe the cuvette thoroughly to remove any
trace amounts of solvent before placing in the Spectrophotometer. The exterior of the
Spectrophotometer is not resistant to organic solvents!
b. Place the blank cuvette in the Spectrophotometer assuring that the proper sides are in the
light path.
c. Choose Calibrate from the Sensors menu of LabQuest or the Experiment menu of
Logger Pro.
d. When the warm-up period is complete, select Finish Calibration. Select OK.
26. Determine the wavelengths of maximum absorbance for the peaks in the yellow solution.
Note: If the sample is too concentrated, dilute with the appropriate solvent.
a. Empty the blank cuvette and fill the cuvette about 3/4 full with the yellow solution and
place it in the Spectrophotometer.
b. Start data collection. A full spectrum graph of the solution will be displayed. Stop data
collection.
***Take a good picture of the spectra and insert into your report.***
c. In Logger Pro, choose Analyze and then choose Examine. Move the cursor to identify the
wavelengths of maximum absorbance for each peak and prominent shoulders. Record
these values on your image neatly.
d. Store the run by choosing Store Latest Run from the Experiment menu in Logger Pro.
e. Remove the cuvette from the Spectrophotometer and dispose of the solution as directed.
27. Analyze the green fraction.
a. Recalibrate the Spectrophotometer with acetone by repeating Step 25.
b. Repeat Step 26 using the green solution to determine the wavelengths of maximum
absorbance. Note: If the sample is too concentrated, dilute with the appropriate solvent.
***Take a good picture of the spectra and insert into your report.***
28. Dispose of waste as directed by your instructor.
Adapted from Organic Chemistry with Vernier
7
DATA ANALYSIS
1. Column Chromatography Images:
a. Insert and label images of the loaded column, column while eluting the yellow
compounds, column while eluting the green compounds as indicated above.
b. Keep them in chronological order and label.
2. Absorbance Spectroscopy:
a. Consider the reference spectra below. Use this information to analyze your
results.
8
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
3. Insert large image of each pigment spectra collected in this experiment (or one spectra if
both sample lines are overlayed).
a. Clearly indicate the color of sample and wavelengths of absorbance maxima for
each peak or obvious shoulder.
b. Using the provided refence spectra in Q2 above, identify your samples as either
chlorophylls or carotenoids. Comment on the quality of your extracted samples.
4. Combine what you learned in your pre-lab about why something has a certain color based
on light absorption/transmission/reflection with what you observed in your absorption
spectra to fully explain why your samples are yellow or green. Refer to your notes taken
in pre-lab assignment and use that logic.
Adapted from Organic Chemistry with Vernier
9
CHEM 2010 Laboratory
Name: _________________________________
Acid-Catalyzed Isomerization of an Alkene
Background
Cis and trans isomers of an alkene have different stabilities, in general, because
they have different amounts of steric strain. The cis isomer has its attached groups closer
together. They repel each other, causing the molecule to be less stable. The trans isomer
however has its substituents comfortably far apart. As a result, the trans isomer is more
stable.
The double bond holds the molecule rigid because of the side to side overlap of
the p orbitals that form the pi bond. This keeps the less stable cis isomer from converting
it to the more stable trans form.
The pi bond can be broken by adding an acid. The acid adds a proton to one
carbon of the double bond and leaves a carbocation on the other side. In the absence of
the double bond, there is nothing left to hold the cis geometry in place and it rotates to a
more stable conformation. If the added proton is removed by a water molecule, the
double bond is reformed. This creates a new isomeric alkene having the trans geometry.
In this experiment, we will start with a cis alkene, maleic acid, and convert it to
its trans isomer, fumaric acid, by heating it in water solution in the presence of
hydrochloric acid. Both of these substances are white crystalline powders. We can tell
them apart because they have very different melting points. The reaction requires higher
temperatures so we perform it in boiling water under conditions of reflux.
Reaction
maleic acid
fumaric acid
CHEM 2010 Laboratory
Name: _________________________________
Pre-lab-(Note there are 3 parts!)
Part 1: Study each of the linked references below-Take notes from each. Be sure to use
these and the logic within them to better understand each step of the experimental
procedure you are performing this week.
1. Heating a reaction at reflux
2. Reflux set-up: Note chemist uses a heating mantle as the heat source. We will use
a metal bowl filled with sand which is heated by the hot plate/stirrer beneath.
3. Vacuum Filtration – basics
4. Crystal Collection via Vacuum filtration
Part 2: Answer all of the Pre-lab questions below.
1. Provide the missing information for the reactant and product:
Compound
Molecular weight (g/mol)
Melting point (oC)
Maleic acid
Fumaric acid
Literature reference
2. Provide arrow to complete the mechanism for the isomerization of maleic acid to
fumaric acid.
CHEM 2010 Laboratory
Name: _________________________________
3. Calculate the theoretical yield of fumaric acid. Show Calculations!
4. What is meant by the term reflux?
5. In general, what is the temperature of a refluxing reaction?
6. A round-bottomed flask used in a heated reaction is always clamped at what part
on the flask? Why is this clamp so important?
7. While we are using a magnetic stirrer to keep our reaction solution moving to
prevent ‘bumping’. A popular method for this is to use boiling stones. What is a
boiling stone and how does it work?
Part 3: Study the experimental procedure for the Isomerization of an Alkene, using the
provided images and technique notes to visualize and think your way through it as you
go.
CHEM 2010 Laboratory
Name: _________________________________
Safety Concerns
You will be using concentrated hydrochloric acid, which causes severe burns
when it comes in contact with the skin. Be very careful of the high temperature required
to reach the melting point of fumaric acid.
Chemical Disposal
Excess maleic acid and the final product fumaric acid should be disposed of in the
disposal containers labeled Waste Container in the main hood. The liquid used as a
reaction solvent(water) can be disposed of down the drain.
CHEM 2010 Laboratory
Name: _________________________________
Experimental Procedure
1. Set up for reflux by assembling a jack, hot/stir plate, and sand bath with a stable
ring stand. Position the set-up in front of your student hood.
2. In a 100 ml round bottomed flask, place approximately 1.0 g of maleic acid,
record exact mass. Add 10 ml of D.I. water to this flask. Swirl the flask by hand
until all of the maleic acid is dissolved. Carefully add a magnetic stir bar to the
flask and clamp the flask securely above the sand bath.
3. Proceed to the main hood with a 10 mL graduated cylinder. Carefully measure an
8 ml portion of concentrated HCl into a graduated cylinder. Slowly and carefully
add this to the reaction flask.
4. Prepare a reflux condenser by attaching water tubing with tubing clamps and
assuring proper water flow into the bottom port and exiting the top port. Use
stopcock grease to assure a good seal of the condenser into the mouth of the
reaction flask. Assure cold water is flowing through the jacket of the condenser
and draining via tubing into the sink at a slow constant rate. This keeps the
condenser at the temperature of the water, providing a good condensation surface.
5. Bring to reflux by turning the hot plate to medium/high heat. Ajust the ehat as
required to reach reflu and kep the condensation occurign in the bottom portion of
the condenser. When refluxing, you should be able to see a boiling liquid in the
flask and condensing vapors dripping back down into the mix. This is “refluxing”
a term that means “to flow back.”
6. Continue refluxing for 15 minutes. As the reaction progresses, a crystalline solid
will begin to separate out of solution. This is your product, fumaric acid, which is
less soluble in aqueous acid than its isomer maleic acid.
7. While the reaction is refluxing, prepare for product collection by preparing an ice
bath and vacuum filtration apparatus. Use a large test tube to cool a few
milliliters of D.I. water to wash your product during filtration.
8. At the end of the15 minute heating period, turn off the hot plat and use the jack to
lower the sand bath away from the flask. CAREFULLY, remove the hot plate and
sand bath from the jack. Let the reaction flask cool for 10 minutes.
9. Fill your 250 ml 2/3 full with ice, and add a 50 to 75 ml of water to this to make
an ice water bath. Use the jack to support the ice bath to cool the reaction mixture
and promote crystallization. Allow to cool in ice bath for 10 minutes.
10.Set up your suction filtration apparatus and Buchner funnel with filter paper and
filter your reaction mixture. Collect the solid product. Rinse the round bottomed
flask and the product with cold D.I. water.
CHEM 2010 Laboratory
Name: _________________________________
11.Remove your product from the filter flask and dry it by pressing between pieces
of large filter paper (see technique notes).
12.Collect, weigh and determine the melting point of your product. Take images of
your product on the balanced showing the final mass as well as a close-up image
showing the quality of the product. Insert in Analysis section where appropriate.
Show your product to your instructor and obtain initials/comments on purity.
Data and Analysis
Mass of maleic acid _____________grams
Moles of maleic acid _____________moles
Mass of dried fumaric acid
Moles of fumaric acid
__________grams
__________moles
Experimental melting point range of fumaric acid product _________ °C
Percent yield of fumaric acid __________ %
Show your calculation:
Image of product on balance with mass showing:
Close up image of product:
CHEM 2010 Organic I
Laboratory
Extraction of Spinach Pigments and Analysis by
UV/Vis Electronic Absorption Spectroscopy
THEORY
Ultraviolet-visible spectroscopy is a useful tool for studying the electronic structure of
unsaturated molecules and their conjugation. The electronic absorption spectra can generally
reveal the degree of delocalization of the conjugated π system. The electronic transition between
bonding and anti-bonding orbitals in organic molecules are large and normally require higher
energy. As the number of π molecular orbitals increase in conjugated systems, the energy gaps
between the filled and unfilled orbitals decrease. Lower energy is needed to promote electrons
into an excited stated, resulting in molecules that can absorb in the visible region.
The pigments found in spinach are good examples of highly conjugated molecules that can
absorb in the visible region. Spinach contains green pigments known as chlorophylls and yellow
pigments known as carotenoids, both which are involved in the photosynthesis process. There
are several types of chlorophyll, chlorophyll a and b being the most common. The difference
between the two chlorophylls is that a methyl side-chain in chlorophyll a is substituted with a
-CHO group in chlorophyll b. Carotenoids are a class of hydrocarbons (carotenes) and their
oxygenated derivatives (xanthophylls). The yellow color due to the carotenoids is obscured by
the chlorophyll pigments. Structures of chlorophyll and β-carotene are shown in Figure 1.
H2 C
H 3C
R
N
H3 C
N
CH3
Mg N
N
H 3C
H 3C
O
O
H3 C
H3 C
CH3
O
H3 C
O
R = CH3 or CHO
H3 C
Chlorophyll
H3C CH3
CH3
CH3
H3 C
CH3
CH3
CH3
H3C CH3
β-carotene
Figure 1 Structures of various visible light absorbing pigments found in spinach
Adapted from Organic Chemistry with Vernier
1
In order to investigate the electronic absorption spectra of the pigments extracted from spinach,
the chlorophylls and carotenoids need to be separated.
Column chromatography is a purification technique used to isolate compounds from a mixture.
In column chromatography, the stationary phase is a solid adsorbent placed in a column and the
mobile phase is a solvent that is added to the top and flows down through the column. Separation
is achieved based on the polar and non-polar interactions among the compounds, the solvent (the
mobile phase), and the solid adsorbent (stationary phase).
In this experiment, you will extract the pigments from spinach leaves and isolate the chlorophylls
and carotenoids using column chromatography. Using electronic absorption spectroscopy, the
wavelengths of absorbance peaks for the chlorophyll and carotenoid pigments will be identified.
PRE-LAB READING AND VIDEOS FOR REVIEW
Ø Review each of the references above and take notes on technique and theory to be
included in this report.
General concept of Column
Microscale(Pipette) column chromatography
Spectrophotometry

Klein Textbook: Refer Chapter 16 covering conjugated pi systems with particular focus on the
characteristics of light absorption and the basis of color.
OBJECTIVES
In this experiment, you will
• Extract the pigments in spinach leaves.
• Isolate the green and yellow pigments using column chromatography.
• Identify the wavelengths of absorbance peaks for the extracted spinach pigments.
MATERIALS
Part I Extraction
fresh spinach leaves
mortar and pestle
three 12 ´ 75 mm test tubes
test tube rack
disposable Pasteur pipets and bulb
one No. 0 stopper
hot plate
250 mL beaker
Temperature Probe or thermometer
sodium sulfate, Na2SO4, anhydrous
acetone
hexane
saturated aqueous sodium chloride
cotton plug
10 mL graduated cylinder
weighing paper
balance
boiling stone
Part II Column Chromatography
disposable Pasteur pipets and bulb
2
ring stand with utility clamp
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
six medium test tubes
test tube rack
cotton plug
sand
silica
spatula
acetone/ hexane solvent mixture
micro funnel
three 100 mL beakers
2.5 cm piece of cut rubber tubing
pinch clamp
weighing paper
Part III Measure the Absorbance
LabQuest or computer
LabQuest App or Logger Pro
SpectroVis Plus spectrophotometer
glass cuvettes with lids
disposable Pasteur pipets and bulb
acetone/ hexane solvent mixture
hexane
acetone
Kem-wipes
samples from Part II
TLC RESULTS FOR SPINACH EXTRACT
Previous TLC study of the Spinach extract with a very low polarity solvent system produces the
results below. Consider the relative Rf values of the target components and use this information
to rationalize why the procedure is performed as it is.
Adapted from Organic Chemistry with Vernier
3
PROCEDURE FOR COLUMN CHROMATOGRAPHY
Part I Extraction of pigments from Spinach leaves
1. Obtain and wear goggles and gloves. Conduct this experiment in front of student fume hood.
**to save time, see step 9 and prep hot water. Set-up outside of hood area to keep that
space clear for extraction.**
2. Weigh out 1.0 g of fresh spinach leaves. Record the mass to the nearest 0.01 g.
3. Tear the leaves into small pieces and place them in a mortar. Add 5 mL of acetone/hexane
mixture (as indicated by your instructor) and grind the mixture for 3–5 minutes.
4. Create a filter pipet with a cotton plug and a disposable Pasteur pipet. Filter the green extract
into a clean test tube. Try to avoid transferring the ground leaves.
5. Wash the extract in the test tube:
a. Add 2 mL of saturated aqueous sodium chloride solution to the test tube.
b. Place a piece of parafilm over the top (do not wrap it around), hold in place with thumb,
and shake the test tube.
c. Immediately vent the test tube by lifting the parafilm edge.
d. Repeat this process of shake and vent for 1-2 minutes.
6. Place the test tube in the test tube rack and allow the mixture to separate while prepping
drying agent column (Step 7).
7. Create a drying agent filled filter pipet: Obtain a disposable Pasteur pipet and add a cotton
plug. Fill the pipet half-way with solid sodium sulfate (anhydrous). Note: Use a micro funnel
or rolled weighing paper to fill the pipet.
8. The final traces of water are removed by treating the organic solution with the drying agent.
Draw up the yellow/green organic layer from your test tube with a disposable pipette and
pass it through the sodium sulfate plug into a clean test tube. Avoid removing the aqueous
layer when your draw up your organic layer.
9. Prepare a 70°C hot water bath in a 250 mL beaker. Monitor the temperature of the water bath
using a Temperature Probe or thermometer.
10. Carefully move the hotplate and bath in front of/under your fume hood. Add a glass bead to
the test tube to prevent the solution from bumping. Use a test tube holder and carefully warm
the tube with gentle agitation. Take care to not allow it to boil aggressively and flash/froth
out of the tube. Keep tube directed toward the back of your hood, not at a neighbor or self.
Evaporate the solvent to approximately 0.2–0.3 mL (about the depth of the glass bead.)
4
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
Part II Separation of pigments via Column Chromatography
19. Assemble the pipet column. CAUTION: Silica powder is harmful if inhaled. Use only in the
fume hood.
a. Place a small piece of cotton into the bottom of the pipet to support the separation
materials. Use a piece of aluminum wire to seat he plug gently into the tapered part of the
pipette. Do not pack too tightly; enough to prevent particles from passing it but not so
densely that liquid struggles to pass through.
b. Add approx. 0.5 cm of sand to support and provide a flat base for the stationary phase
c. Add enough silica to fill the pipet about 3/4 full. Note: Micro funnel or weighing paper
may be used to help fill the column with silica. See instructor for micro-funnel.
d. Gently tap the sides of the pipet for 1 minute to pack the silica.
e. Add another 0.5 cm of sand to the top of the pipet to maintain a flat surface for loading
the mixture onto the silica.
20. Use a micro clamp to secure the pipet column to a ring stand. Place the fingers of the clamp
in the middle of the silica column for easy viewing of the top and bottom. Adjust the height
of the column on the ring stand to allow the bottom to insert into the top of a small test tube
which can be easily swept from beneath it and replaced with another.
21. Obtain six test tubes to collect the column fractions and a 50/100 mL beaker for waste.
Important: Once you start the elution process, it cannot be stopped.
You must go to completion. Read Step 22-23 carefully before continuing.
22. Prepare the column for chromatography (work in front of/under student fume hood)
a. Place a waste beaker under the pipet column. This will catch the eluent which does not
contain the target molecules.
b. Obtain approx. 10 mL of the hexane in a 10 ml graduated cylinder. Set to back of your
student fume hood, not on open benchtop, for later use as the first eluting solvent (step h
below).
c. Obtain 8-10 mL of the acetone in a 10 ml graduated cylinder. This will be used first to
pack the column, and later as the second eluting solvent.
d. Carefully load a portion of acetone into the pipet to fill the space above the column. Use a
small bulb to apply pressure and push the solvent through the silica gel to force air from
the column. Only apply pressure; tilt bulb to side while still holding slight pressure to
prevent pulling air back up and ‘cracking’ the column. This process will provide a
consistent stationary phase for separation.
e. Repeat loading acetone and pushing with bulb until all air is removed from the column.
The solvent should be clean and can be recycled through the column during this process.
Note: If air bubbles or cracks appear in the column, tap to pack and continue the solvent
flush process.
f. Important: Do not let air move into the top of the column during this procedure.
Replenish with solvent as needed. When the column is prepared for loading with the
green extract fill with solvent to give you time to pull up the extract and get ready to load
it.
Adapted from Organic Chemistry with Vernier
5
23. Load and run the colum chromatography:
a. Let the solvent line drop until the meniscus is just above top sand layer, then use a
clean pipette to load the concentrated green spinach extract onto the top of the
column.
b. Allow the green solution to adsorb onto the silica. As the extract flows through
the sand to reach the silica get, drip acetone above it only as needed to not let the
sand get dry.
c. Do not let the column get dry during this procedure
d. Once the extract is properly loaded, switch to using pure hexane to elute the
yellow layer faster. Refer to the TLC results above and consider what target
compounds are in this yellow extract which is flowing the fastest with the nonpolar hexane solvent.
***Take a good picture of the loaded column to later insert into your report
e. A yellow band will appear and begin to separate from the green band. Allow the
colorless solvent to flow into a small beaker until the yellow band nears the
bottom. Do not let the column get dry during this procedure, continuing
flushing with pure hexane.
***Take a good picture of the eluting column to later insert into your report
f. To collect the yellow extract, replace the beaker with the first test tube. Collect
the entire yellow fraction into this test tube. WATCH THE SOLVENT LEVEL Do not let air get to the silica layer during this procedure.
g. Once you have collected the yellow fraction, place another small waste beaker
under the column and change the eluting solvent to pure acetone to begin
pushing the green layer through the column.
h. Continue adding the solvent, collect the colorless portion in the waste beaker. The
green band should be moving down the column.
***Take a good picture of the eluting column to later insert into your report
24. To collect the green extract moving with the acetone, replace the beaker with a clean test
tube. Collect all of the green portion. Do not let the column get dry during this procedure
Note: All of the pigments will NOT be removed from the column.
23. Place a beaker back under the column and allow it to drain/dry during next portion of
experiment.
6
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
Part IV Measure the Absorbance
24. Locate an available laptop/Spectrovis station. Connect the SpectroVis Plus
spectrophotometer to the USB port of LabQuest or a computer. Start the data-collection
program, then choose New from the File menu.
25. Calibrate the Spectrophotometer for the yellow fraction.
a. Prepare a blank for testing the yellow fraction by filling a glass cuvette with hexane and
cover with a cap or parafilm. Note: Always wipe the cuvette thoroughly to remove any
trace amounts of solvent before placing in the Spectrophotometer. The exterior of the
Spectrophotometer is not resistant to organic solvents!
b. Place the blank cuvette in the Spectrophotometer assuring that the proper sides are in the
light path.
c. Choose Calibrate from the Sensors menu of LabQuest or the Experiment menu of
Logger Pro.
d. When the warm-up period is complete, select Finish Calibration. Select OK.
26. Determine the wavelengths of maximum absorbance for the peaks in the yellow solution.
Note: If the sample is too concentrated, dilute with the appropriate solvent.
a. Empty the blank cuvette and fill the cuvette about 3/4 full with the yellow solution and
place it in the Spectrophotometer.
b. Start data collection. A full spectrum graph of the solution will be displayed. Stop data
collection.
***Take a good picture of the spectra and insert into your report.***
c. In Logger Pro, choose Analyze and then choose Examine. Move the cursor to identify the
wavelengths of maximum absorbance for each peak and prominent shoulders. Record
these values on your image neatly.
d. Store the run by choosing Store Latest Run from the Experiment menu in Logger Pro.
e. Remove the cuvette from the Spectrophotometer and dispose of the solution as directed.
27. Analyze the green fraction.
a. Recalibrate the Spectrophotometer with acetone by repeating Step 25.
b. Repeat Step 26 using the green solution to determine the wavelengths of maximum
absorbance. Note: If the sample is too concentrated, dilute with the appropriate solvent.
***Take a good picture of the spectra and insert into your report.***
28. Dispose of waste as directed by your instructor.
Adapted from Organic Chemistry with Vernier
7
DATA ANALYSIS
1. Column Chromatography Images:
a. Insert and label images of the loaded column, column while eluting the yellow
compounds, column while eluting the green compounds as indicated above.
b. Keep them in chronological order and label.
2. Absorbance Spectroscopy:
a. Consider the reference spectra below. Use this information to analyze your
results.
8
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
3. Insert large image of each pigment spectra collected in this experiment (or one spectra if
both sample lines are overlayed).
a. Clearly indicate the color of sample and wavelengths of absorbance maxima for
each peak or obvious shoulder.
b. Using the provided refence spectra in Q2 above, identify your samples as either
chlorophylls or carotenoids. Comment on the quality of your extracted samples.
4. Combine what you learned in your pre-lab about why something has a certain color based
on light absorption/transmission/reflection with what you observed in your absorption
spectra to fully explain why your samples are yellow or green. Refer to your notes taken
in pre-lab assignment and use that logic.
Adapted from Organic Chemistry with Vernier
9
CHEM 2010 Organic I
Laboratory
Extraction of Spinach Pigments and Analysis by
UV/Vis Electronic Absorption Spectroscopy
THEORY
Ultraviolet-visible spectroscopy is a useful tool for studying the electronic structure of
unsaturated molecules and their conjugation. The electronic absorption spectra can generally
reveal the degree of delocalization of the conjugated π system. The electronic transition between
bonding and anti-bonding orbitals in organic molecules are large and normally require higher
energy. As the number of π molecular orbitals increase in conjugated systems, the energy gaps
between the filled and unfilled orbitals decrease. Lower energy is needed to promote electrons
into an excited stated, resulting in molecules that can absorb in the visible region.
The pigments found in spinach are good examples of highly conjugated molecules that can
absorb in the visible region. Spinach contains green pigments known as chlorophylls and yellow
pigments known as carotenoids, both which are involved in the photosynthesis process. There
are several types of chlorophyll, chlorophyll a and b being the most common. The difference
between the two chlorophylls is that a methyl side-chain in chlorophyll a is substituted with a
-CHO group in chlorophyll b. Carotenoids are a class of hydrocarbons (carotenes) and their
oxygenated derivatives (xanthophylls). The yellow color due to the carotenoids is obscured by
the chlorophyll pigments. Structures of chlorophyll and β-carotene are shown in Figure 1.
H2 C
H 3C
R
N
H3 C
N
CH3
Mg N
N
H 3C
H 3C
O
O
H3 C
H3 C
CH3
O
H3 C
O
R = CH3 or CHO
H3 C
Chlorophyll
H3C CH3
CH3
CH3
H3 C
CH3
CH3
CH3
H3C CH3
β-carotene
Figure 1 Structures of various visible light absorbing pigments found in spinach
Adapted from Organic Chemistry with Vernier
1
In order to investigate the electronic absorption spectra of the pigments extracted from spinach,
the chlorophylls and carotenoids need to be separated.
Column chromatography is a purification technique used to isolate compounds from a mixture.
In column chromatography, the stationary phase is a solid adsorbent placed in a column and the
mobile phase is a solvent that is added to the top and flows down through the column. Separation
is achieved based on the polar and non-polar interactions among the compounds, the solvent (the
mobile phase), and the solid adsorbent (stationary phase).
In this experiment, you will extract the pigments from spinach leaves and isolate the chlorophylls
and carotenoids using column chromatography. Using electronic absorption spectroscopy, the
wavelengths of absorbance peaks for the chlorophyll and carotenoid pigments will be identified.
PRE-LAB READING AND VIDEOS FOR REVIEW
Ø Review each of the references above and take notes on technique and theory to be
included in this report.
General concept of Column
Microscale(Pipette) column chromatography
Spectrophotometry

Klein Textbook: Refer Chapter 16 covering conjugated pi systems with particular focus on the
characteristics of light absorption and the basis of color.
OBJECTIVES
In this experiment, you will
• Extract the pigments in spinach leaves.
• Isolate the green and yellow pigments using column chromatography.
• Identify the wavelengths of absorbance peaks for the extracted spinach pigments.
MATERIALS
Part I Extraction
fresh spinach leaves
mortar and pestle
three 12 ´ 75 mm test tubes
test tube rack
disposable Pasteur pipets and bulb
one No. 0 stopper
hot plate
250 mL beaker
Temperature Probe or thermometer
sodium sulfate, Na2SO4, anhydrous
acetone
hexane
saturated aqueous sodium chloride
cotton plug
10 mL graduated cylinder
weighing paper
balance
boiling stone
Part II Column Chromatography
disposable Pasteur pipets and bulb
2
ring stand with utility clamp
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
six medium test tubes
test tube rack
cotton plug
sand
silica
spatula
acetone/ hexane solvent mixture
micro funnel
three 100 mL beakers
2.5 cm piece of cut rubber tubing
pinch clamp
weighing paper
Part III Measure the Absorbance
LabQuest or computer
LabQuest App or Logger Pro
SpectroVis Plus spectrophotometer
glass cuvettes with lids
disposable Pasteur pipets and bulb
acetone/ hexane solvent mixture
hexane
acetone
Kem-wipes
samples from Part II
TLC RESULTS FOR SPINACH EXTRACT
Previous TLC study of the Spinach extract with a very low polarity solvent system produces the
results below. Consider the relative Rf values of the target components and use this information
to rationalize why the procedure is performed as it is.
Adapted from Organic Chemistry with Vernier
3
PROCEDURE FOR COLUMN CHROMATOGRAPHY
Part I Extraction of pigments from Spinach leaves
1. Obtain and wear goggles and gloves. Conduct this experiment in front of student fume hood.
**to save time, see step 9 and prep hot water. Set-up outside of hood area to keep that
space clear for extraction.**
2. Weigh out 1.0 g of fresh spinach leaves. Record the mass to the nearest 0.01 g. 1
00g
3. Tear the leaves into small pieces and place them in a mortar. Add 5 mL of acetone/hexane
mixture (as indicated by your instructor) and grind the mixture for 3–5 minutes.
4. Create a filter pipet with a cotton plug and a disposable Pasteur pipet. Filter the green extract
into a clean test tube. Try to avoid transferring the ground leaves.
5. Wash the extract in the test tube:
a. Add 2 mL of saturated aqueous sodium chloride solution to the test tube.
b. Place a piece of parafilm over the top (do not wrap it around), hold in place with thumb,
and shake the test tube.
c. Immediately vent the test tube by lifting the parafilm edge.
d. Repeat this process of shake and vent for 1-2 minutes.
6. Place the test tube in the test tube rack and allow the mixture to separate while prepping
drying agent column (Step 7).
7. Create a drying agent filled filter pipet: Obtain a disposable Pasteur pipet and add a cotton
plug. Fill the pipet half-way with solid sodium sulfate (anhydrous). Note: Use a micro funnel
or rolled weighing paper to fill the pipet.
8. The final traces of water are removed by treating the organic solution with the drying agent.
Draw up the yellow/green organic layer from your test tube with a disposable pipette and
pass it through the sodium sulfate plug into a clean test tube. Avoid removing the aqueous
layer when your draw up your organic layer.
9. Prepare a 70°C hot water bath in a 250 mL beaker. Monitor the temperature of the water bath
using a Temperature Probe or thermometer.
10. Carefully move the hotplate and bath in front of/under your fume hood. Add a glass bead to
the test tube to prevent the solution from bumping. Use a test tube holder and carefully warm
the tube with gentle agitation. Take care to not allow it to boil aggressively and flash/froth
out of the tube. Keep tube directed toward the back of your hood, not at a neighbor or self.
Evaporate the solvent to approximately 0.2–0.3 mL (about the depth of the glass bead.)
4
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
Part II Separation of pigments via Column Chromatography
19. Assemble the pipet column. CAUTION: Silica powder is harmful if inhaled. Use only in the
fume hood.
a. Place a small piece of cotton into the bottom of the pipet to support the separation
materials. Use a piece of aluminum wire to seat he plug gently into the tapered part of the
pipette. Do not pack too tightly; enough to prevent particles from passing it but not so
densely that liquid struggles to pass through.
b. Add approx. 0.5 cm of sand to support and provide a flat base for the stationary phase
c. Add enough silica to fill the pipet about 3/4 full. Note: Micro funnel or weighing paper
may be used to help fill the column with silica. See instructor for micro-funnel.
d. Gently tap the sides of the pipet for 1 minute to pack the silica.
e. Add another 0.5 cm of sand to the top of the pipet to maintain a flat surface for loading
the mixture onto the silica.
20. Use a micro clamp to secure the pipet column to a ring stand. Place the fingers of the clamp
in the middle of the silica column for easy viewing of the top and bottom. Adjust the height
of the column on the ring stand to allow the bottom to insert into the top of a small test tube
which can be easily swept from beneath it and replaced with another.
21. Obtain six test tubes to collect the column fractions and a 50/100 mL beaker for waste.
Important: Once you start the elution process, it cannot be stopped.
You must go to completion. Read Step 22-23 carefully before continuing.
22. Prepare the column for chromatography (work in front of/under student fume hood)
a. Place a waste beaker under the pipet column. This will catch the eluent which does not
contain the target molecules.
b. Obtain approx. 10 mL of the hexane in a 10 ml graduated cylinder. Set to back of your
student fume hood, not on open benchtop, for later use as the first eluting solvent (step h
below).
c. Obtain 8-10 mL of the acetone in a 10 ml graduated cylinder. This will be used first to
pack the column, and later as the second eluting solvent.
d. Carefully load a portion of acetone into the pipet to fill the space above the column. Use a
small bulb to apply pressure and push the solvent through the silica gel to force air from
the column. Only apply pressure; tilt bulb to side while still holding slight pressure to
prevent pulling air back up and ‘cracking’ the column. This process will provide a
consistent stationary phase for separation.
e. Repeat loading acetone and pushing with bulb until all air is removed from the column.
The solvent should be clean and can be recycled through the column during this process.
Note: If air bubbles or cracks appear in the column, tap to pack and continue the solvent
flush process.
f. Important: Do not let air move into the top of the column during this procedure.
Replenish with solvent as needed. When the column is prepared for loading with the
green extract fill with solvent to give you time to pull up the extract and get ready to load
it.
Adapted from Organic Chemistry with Vernier
5
23. Load and run the colum chromatography:
a. Let the solvent line drop until the meniscus is just above top sand layer, then use a
clean pipette to load the concentrated green spinach extract onto the top of the
column.
b. Allow the green solution to adsorb onto the silica. As the extract flows through
the sand to reach the silica get, drip acetone above it only as needed to not let the
sand get dry.
c. Do not let the column get dry during this procedure
d. Once the extract is properly loaded, switch to using pure hexane to elute the
yellow layer faster. Refer to the TLC results above and consider what target
compounds are in this yellow extract which is flowing the fastest with the nonpolar hexane solvent.
***Take a good picture of the loaded column to later insert into your report
e. A yellow band will appear and begin to separate from the green band. Allow the
colorless solvent to flow into a small beaker until the yellow band nears the
bottom. Do not let the column get dry during this procedure, continuing
flushing with pure hexane.
***Take a good picture of the eluting column to later insert into your report
f. To collect the yellow extract, replace the beaker with the first test tube. Collect
the entire yellow fraction into this test tube. WATCH THE SOLVENT LEVEL Do not let air get to the silica layer during this procedure.
g. Once you have collected the yellow fraction, place another small waste beaker
under the column and change the eluting solvent to pure acetone to begin
pushing the green layer through the column.
h. Continue adding the solvent, collect the colorless portion in the waste beaker. The
green band should be moving down the column.
***Take a good picture of the eluting column to later insert into your report
24. To collect the green extract moving with the acetone, replace the beaker with a clean test
tube. Collect all of the green portion. Do not let the column get dry during this procedure
Note: All of the pigments will NOT be removed from the column.
23. Place a beaker back under the column and allow it to drain/dry during next portion of
experiment.
6
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
Part IV Measure the Absorbance
24. Locate an available laptop/Spectrovis station. Connect the SpectroVis Plus
spectrophotometer to the USB port of LabQuest or a computer. Start the data-collection
program, then choose New from the File menu.
25. Calibrate the Spectrophotometer for the yellow fraction.
a. Prepare a blank for testing the yellow fraction by filling a glass cuvette with hexane and
cover with a cap or parafilm. Note: Always wipe the cuvette thoroughly to remove any
trace amounts of solvent before placing in the Spectrophotometer. The exterior of the
Spectrophotometer is not resistant to organic solvents!
b. Place the blank cuvette in the Spectrophotometer assuring that the proper sides are in the
light path.
c. Choose Calibrate from the Sensors menu of LabQuest or the Experiment menu of
Logger Pro.
d. When the warm-up period is complete, select Finish Calibration. Select OK.
26. Determine the wavelengths of maximum absorbance for the peaks in the yellow solution.
Note: If the sample is too concentrated, dilute with the appropriate solvent.
a. Empty the blank cuvette and fill the cuvette about 3/4 full with the yellow solution and
place it in the Spectrophotometer.
b. Start data collection. A full spectrum graph of the solution will be displayed. Stop data
collection.
***Take a good picture of the spectra and insert into your report.***
c. In Logger Pro, choose Analyze and then choose Examine. Move the cursor to identify the
wavelengths of maximum absorbance for each peak and prominent shoulders. Record
these values on your image neatly.
d. Store the run by choosing Store Latest Run from the Experiment menu in Logger Pro.
e. Remove the cuvette from the Spectrophotometer and dispose of the solution as directed.
27. Analyze the green fraction.
a. Recalibrate the Spectrophotometer with acetone by repeating Step 25.
b. Repeat Step 26 using the green solution to determine the wavelengths of maximum
absorbance. Note: If the sample is too concentrated, dilute with the appropriate solvent.
***Take a good picture of the spectra and insert into your report.***
28. Dispose of waste as directed by your instructor.
Adapted from Organic Chemistry with Vernier
7
DATA ANALYSIS
1. Column Chromatography Images:
a. Insert and label images of the loaded column, column while eluting the yellow
compounds, column while eluting the green compounds as indicated above.
b. Keep them in chronological order and label.
1 Clean
loaded
ow
Y
compounds
green
compounds
2. Absorbance Spectroscopy:
a. Consider the reference spectra below. Use this information to analyze your
results.
8
Adapted from Organic Chemistry with Vernier
Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy
3. Insert large image of each pigment spectra collected in this experiment (or one spectra if
both sample lines are overlayed).
a. Clearly indicate the color of sample and wavelengths of absorbance maxima for
each peak or obvious shoulder.
b. Using the provided refence spectra in Q2 above, identify your samples as either
chlorophylls or carotenoids. Comment on the quality of your extracted samples.
Blankcompoundgreenongraph
compoundblue
ongraph
enow
compound
green
redongraph
4. Combine what you learned in your pre-lab about why something has a certain color based
on light absorption/transmission/reflection with what you observed in your absorption
spectra to fully explain why your samples are yellow or green. Refer to your notes taken
in pre-lab assignment and use that logic.
Adapted from Organic Chemistry with Vernier
9
CHEM 2010
Laboratory
Aspirin Synthesis & Analysis
INTRODUCTION
Watch the video Organic Lab: Aspirin 1 – Theory and Prelab Discussion
Since 500 B.C., the bark and leaves of willow trees have been used as a pain killer. The
active component, salicylic acid (SA), can, however, cause stomach upset because its acidity
(pKa = 2.97) can be higher than the pH of the human stomach (pH ~ 4 after digestion is complete).
I
C
Figure 1. Salicylic Acid
Salicylic acid is a diprotic organic acid with two acidic functional groups: a carboxylic acid
and a phenol. (Acidic hydrogen atoms are blue.) In comparison, the monoprotic acetylsalicylic
acid (ASA, aspirin) is less acidic (pKa = 4.57). The reason: an ester has replaced the acidic phenol
in ASA.
Figure 2. Acetylsalicylic Acid (ASA)
The synthesis of ASA from salicylic acid results in the formation of an ester functional
group and, therefore, is called an esterification. The first step in this esterification is to create a
suspension of salicylic acid (a solid at room temperature) in an excess of acetic anhydride (a
liquid at room temperature). Acetic anhydride serves as both a reactant and a solvent. A
catalyst is required for this reaction. Phosphoric acid, H3PO4, donates a H+ which binds to the
reaction complex. As a catalyst, H+ is regenerated (not consumed) by the end of the reaction. As
1
CHEM 2010
Laboratory
the reaction proceeds, the solid salicylic acid disappears and the acetylsalicylic acid product
remains dissolved in the hot solution. Once all solid has disappeared (all the salicylic acid has
been consumed) the reaction is complete.
CH
e
C
e
catalyst
Clb
113C
500C
Figure 3. Formation of Acetylsalicylic Acid (ASA, a.k.a., Aspirin)
At this point the excess acetic anhydride must be hydrolyzed (split apart by the addition of
water) to acetic acid. Acetic anhydride is very reactive toward water, so the hydrolysis must be
done slowly – water should be added drop-wise.
Z
3C
CEI z
3C
e
Figure 4. Hydrolysis of Acetic Anhydride
More water is then added and the flask is placed in an ice bath to lower the solubility and
Precipitate/crystalize the ASA product. The product is then collected by filtration.
The reaction’s progress is monitored by TLC and the final product’s purity is analyzed 2 ways:
1) The melting point of the synthesized ASA product will be compared to the literature value.
2) Samples with unreacted salicylic acid complex with Fe3+ to create a purple complex in
aqueous solution. Pure ASA samples will remain colorless.
SAFETY PRECAUTIONS
Safety goggles, aprons, and gloves must be worn at all times in the laboratory. Acetic
anhydride (a liquid at room temperature) is violently reactive toward water, flammable,
corrosive, and can cause burns; add water slowly, prevent contact with eyes, skin, and clothing.
Securely cap acetic anhydride when transferring it from the supply hood to the hood where you
will work. Work with acetic anhydride in the fume hood only. The Fe3+ solution has been
acidified to pH < 2 with hydrochloric acid and sodium hydroxide is a strong base. Both are corrosive and can cause burns; prevent contact with eyes, skin and clothing. Wash affected areas thoroughly with cold water. Ethyl acetate is flammable and harmful by inhalation, ingestion, and when in contact with skin. Any container holding ethyl acetate should be capped when not 2 CHEM 2010 Laboratory in use to prevent evaporation of the solvents, as they are harmful when inhaled. The salicylic acid, acetylsalicylic acid, and aspirin tablets used may be contaminated through student activities and are NOT for internal use. Solutions containing ethyl acetate or Fe3+ must be placed in appropriate waste bottles and NEVER be poured down the drain. Report all spills, accidents, or injuries to your instructor. PRELAB 1) Review your previous studies on the techniques involved with TLC analysis, crystallization, and vacuum filtration. 2) Using a legitimate reference, record the literature melting point range for acetylsalicylic acid as well as the reference used: Literature value: ___________________________________ Literature reference: ________________________________________________ 3) Using a legitimate reference, record the literature density for acetic anhydride as well as the reference used: Literature value: ___________________________________ Literature reference: ________________________________________________ 4) Evaluate the theoretical yield of acetylsalicylic acid if you were to use exactly 1.50 grams of salicylic acid and 3.50 mL of acetic anhydride. Your calculations should clearly define the limiting reactant, excess reactant, and expected mass yield of aspirin. If you are unfamiliar with these calculations from general chemistry, review these videos on doing limiting reactant based theoretical yield calculations- General Chem – Stoichiometry Part 5 Limiting Reactant and General Chem – Stoichiometry Part 6 Limiting Reactant Practice 3 CHEM 2010 Work in Pairs Use time wisely Laboratory PROCEDURE Watch the video: Organic Lab: Aspirin 2- Synthesis and crystallization, and Crystal collection via vacuum filtration Follow along with the directions provided below. To illustrate you have paid attention to the video provide time stamps in the left margin at all major steps. experience Requiredonly toa limited that Part A. Synthesis & TLC Analysis. **Set-up the warm water bath and get the temperature regulated as you prepare your TLC plates so it is ready to use for the reaction!! TLC Preparation 1. Create a TLC plates with four spots: salicylic Acid (SA) reactant, acetylsalicylic acid (ASA), reaction Mixture before heating (Sample A) & reaction mixture after heating (Sample B) 2. Use a pencil to lightly mark the origin line and small dots where the sample spots will be made. 3. Take a small amount of the salicylic acid (SA) starting material and dissolve in ethyl acetate by adding the solvent drop-wise until it dissolves. Once dissolved, dilute with the same number of drops it took to dissolve it. Use this solution to load the SA onto the appropriate spot on our TLC plate. 4. Use the provided solutions of ASA in ethyl acetate solution to laod ASA onto the appropriate spot. 5. Set the plate aside where it is ready to be spotted but out of the way to not get disturbed as you proceed with the reaction. 6. Prepare one TLC developing chamber: Add the provided 1:1ethyl acetate/hexane solvent mixture into the TLC developing chamber. (Reviewing the TLC video is a very good idea to assure you do not forget any steps in this portion of the experiment.) Synthesis & TLC Sampling 7. Tare a clean and DRY 50 mL Erlenmeyer flask and add ~ 1.5 g salicylic acid, recording an accurate mass. 8. In the fume hood and wearing gloves, add 3.50 mL acetic anhydride using a 10 mL graduated cylinder and a long stem pipette. Use a piece of parafilm to cover the flask when out of a hood. 9. Add 5 drops conc. H3PO4 to the suspension. Use a piece of parafilm to cover the flask when out of a hood. 10. Before placing the flask in the warm bath, use a pipet (no bulb, just capillary action) to remove a small bit of the reaction solution, use a spotter to draw a small bit of this sample 4 CHEM 2010 Laboratory up by touching the tips of the pipette and spotter. Load this solution onto the TLC plate on the spot marked as Sample A. Clean the spotter thoroughly! 11. Add a small stir bar to your flask, support with a clamp/ring stand, and lower the flask into the 50°C water bath warm bath. Begin gently stirring and heating the mixture. Continue heating and stirring until all solid disappears (about 10 minutes). 12. During the 10 minute reaction time: use a beaker/dish to prepare an ice bath deep enough to chill the portion of the flask your solution uses but not deep enough to submerge the flask if it were to tip. Use a small beaker or flask to chill a few milliliters of distilled water for rinsing your crystals in the funnel later. 13. After all solid disappears from the reaction mixture, use a clean pipet to transfer another sample to your spotter and spot the plate as Sample B. Place the plate aside, you can develop it later, once the ASA product is isolated and drying in in the oven. 14. Remove the flask from the water bath and carefully add 15 drops distilled H2O. Wait one minute. If precipitation does not begin, add 15 more drops of H2O. If precipitation does not occur after 5 minutes, go on to the next step. 15. Once solid begins precipitating out of solution, add 15 mL distilled H2O. The flask and its contents may now be removed from the fume hood safely. Cool in ice bath for 10 minutes. 16. During the ice bath time: set up for vacuum filtration. 17. Collect the precipitate by vacuum filtration. Use small washings of chilled distilled H2O and a spatula to transfer your crystals to the Buchner funnel. Rinse with a few more milliliters of chilled water. 18. Dry the crystals for 15 minutes under vacuum and then place them on a labeled (initials with sharpie on bottom) pre-weighed watch glass in the oven for at least 10 minutes. You should check the temperature on the oven; if the temperature exceeds 65 °C, your product will decompose. 19. While waiting for your product to dry in the oven: place TLC plate into the developing chamber, develop, dry, and use UV lamp to visualize your plate. Takeagoodpictureofdry developed marked Insertbelow 20. Remove from the oven, allow time to cool to room temperature, and accurately record the mass. 21. Take an image of your product up close and in focus for your instructor to judge its quality. Note: Organic liquids should be discarded in the appropriately labeled containers in the hood Aqueous waste can be flushed down the sink with water, synthesized aspirin can be placed in the jar labeled “Aspirin-student prep”. Part B. Purity Analysis Melting Point of synthesized ASA (if you do not have 30 min of lab time remaining, do not begin this step. Proceed to the Fe+3 analysis). art 5 CHEM 2010 Laboratory 22. Be sure to crush and pulverize a small bit of your sample rather than try to cram flaky crystals into your mp tube. Be suretousecrystallinematerial notimpureprecipitate ifpossible 23. Turn on the Mel-Temp and adjust it as appropriate to increase the temperature quickly to within twenty degrees of the expected melting point. Fill a small capillary tube with 2-3 mm of solid. Determine the melting point range for your synthesized aspirin. 3+ Fe test 24. Place a small amount of your product in a small test tube. 25. Place ~1 mL distilled H2O into the test tube 26. Add 1 drop FeCl3(aq) solution to each test tube. 27. Record your observations. Take a goodpicturewithwhitebackground Insert below NOTE: Solutions should be discarded of in the hood labeled “Metal Containing Waste”. SYTHESIS DATA AND RESULTS Mass of salicylic acid (g) 1.502 Molar Mass of salicylic acid (g/mol) Moles of salicylic acid (mol) Vol. acetic anhydride (mL) 3.50 he Density of acetic anhydride (g/mL) Molar Mass of acetic anhydride (g/mol) Moles of acetic anhydride (mol) Molar Mass acetic anhydride (g/mol) Theoretical yield of acetylsalicylic acid (g) Mass of watchglass (g) 110.338 Mass of watchglass + aspirin (g) 111.762 Mass of aspirin (g) Percent yield of acetylsalicylic acid (g) Melting point range of synth. Aspirin (oC) 136.5 –c138.6 6 CHEM 2010 Laboratory TLC RESULTS A. Insert an image of the developed TLC plate. B. Determine the Rf for all of the components on your TLC plate. Clearlyindicateoriginpoint solventline andeachmeasurementusedtocalculateeach Rfvalue IRON (III) TEST RESULTS Insert an image of your aspirin sample solution treated with Fe+3 and explain their relevance. 7 CHEM 2010 Laboratory DATA ANALYSIS A. What are the similarities and differences in the TLC of the reaction mixture before heating and after completion? Are your results logical? What do these results suggest? B. Use the results from the TLCs, the melting points, and the Fe3+ tests to confirm if the product you synthesized is ASA, and comment on its purity level. Use specifics; no vague statements! Insert theimageofyourfinaldryproduct here 8