TCHEM 261 CAU Wk 2 Determine the Types of Hydrogen Atoms Lab Report

University of Washington – Tacoma
Organic Chemistry (TCHEM 261)
Separation of Spinach Components Lab
Student Version
INTRODUCTION
Spinach leaves, like other leaves, contain a variety of compounds, called pigments,
which contribute to the color of the leaves. 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. They also contain
pheophytins (similar in structure to chlorophylls, but grey in color).
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.
H2C
H3C
R
N
H3 C
N
CH3
Mg N
N
H3C
H3 C
O
O
H3 C
CH3
H3 C
H3 C
O
O
R = CH3 or CHO
H3 C
Chlorophyll
H3C CH3
CH3
CH3
H3C
CH3
CH3
β-carotene
CH3
H3C CH3
Figure 1 Structures of various visible light absorbing pigments found in spinach
In order to investigate the electronic absorption spectra of the pigments extracted from
spinach, the chlorophylls and carotenoids need to be separated. Thin-layer
chromatography (TLC) can be used to investigate the solvent system for separation of
the compounds. In thin-layer chromatography, the stationary phase is the adsorbent
(usually silica or alumina) coated on a sheet of glass, metal, or plastic. The sample is
applied as a spot near the bottom of the plate. The TLC plate is then placed in a
developing chamber containing a shallow layer of solvent, where the mobile phase
(solvent) slowly rises by capillary action.
Under a given set of conditions, a specific compound will travel a unique fixed distance
relative to the solvent front. Different compounds generally move at different rates. As a
result, if the sample is a mixture of compounds, it will separate into a series of spots at
varying distances up the plate (see Figure 2). TLC separation results are expressed in
terms of Rf (retention factor) values. The Rf is a ratio calculated by dividing the distance
traveled by the sample by the distance traveled by the solvent at the end of the
experiment.
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, and the solid stationary phase.
In this experiment, you will extract the pigments from spinach leaves and isolate the
chlorophylls and carotenoids using column chromatography. TLC will be used to
evaluate the purity of your fractions. Using electronic absorption spectroscopy, the
wavelengths of absorbance peaks for the chlorophyll and carotenoid pigments will be
identified.
PREPARATION
1. Read the Whole Lab, especially the post-lab write-up instructions.
2. Learn or Review any concepts or skills you are not confident about applying during
lab.
a. Column Chromatography, Nichols – Click on Chromatography tile, then on
“column chromatography” link and “2.4B: Microscale (Pipette) Columns or try this
link:
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_
Techniques_(Nichols)/02%3A_Chromatography/2.04%3A_Column_Chromatography
/2.4B%3A_Microscale_(Pipette)_Columns
3. Prepare your lab notebook as described below.
LAB NOTEBOOK Set-Up
• Title – Use the one given for this lab.
• Clerical Information – Your name, the name of your assigned lab partner for that lab,
and the date on which the in-lab work was started. Be sure to also enter the lab in your
Table of Contents.
• Objective – After reading the entire lab, identify and write down the objective of the lab.
The objective is the question or questions your data will hopefully allow you to answer.
• Hypothesis – For this lab, please write a hypothesis about which compound, chlorophyll
or β-carotene, will absorb elute fastest based on its structure.
•
Physical & Chemical Properties Table – Use the format below.
Chemical
Name
Use/Purpose Relevant Physical Properties
in THIS lab
xx
xx
xx
Hazards
Response to
hazards
•
Pre-lab questions – Do these on a separate sheet of paper, NOT in notebook.
1. For each of the following pairs of compounds, circle the one that will absorb at
the longest wavelength.
a)
b)
2. You are given a sample composed of three compounds, A-C with different polarities.
The sample is loaded onto an alumina column and eluted with 30% methanol/water. After
eluting with 10 mL of solvent and collecting 1 mL fractions, compounds A and B are found to be
in the 3rd and 6th fractions, respectively. Compound C is still in the column.
(a) Which compound is more polar, A or B?
(b) Is compound C more or less polar than compounds A & B?
(c) After eluting with another 10 mL of 30% methanol/water, compound C has still not
eluted. Describe how you would obtain compound C.
•
•
•
•
Experimental Plan – Use the Experimental directions below to write a plan for the work
you need to do in lab.
Data – Create spaces in your notebook to record all the data you will collect.
Have lab notebook checked and initialed
After preparing your lab notebook, please take the on-line pre-lab quiz to ensure
that you have a solid background for the investigation you will carry out in this lab.
Experimental Directions
Part I Extraction of the Pigments (Week 1)
1. Using a mortar and pestle, or a blender, grind up about 4 g of spinach in methanol. Use a
minimal amount, but it is important that all of the spinach comes in contact with
methanol (Lab staff does this.)
2. Add about 10 mL hexane to the spinach slop and stir thoroughly.
3. Pour off the liquid, squeezing as much juice out of the leaf residue as possible.
THE INSTRUCTOR OR LAB STAFF WILL DO UP TO HERE FOR YOU
4. You now need to remove the methanol. To do this, place the mixture in a separatory
funnel. Add about 10 mL of water, mix thoroughly, and drain the water layer only out of
the separatory funnel. Repeat this step a second time. (Since methanol is more soluble in
water than in hexane, it will come out with the water. It’s important to get all of the
methanol out, so make sure you remove all of the water layer, even if this means you
remove a little bit of the hexane layer.)
5. No matter how well you do step 4, there will now be a little water in with the hexane.
This needs to be removed. To do this, transfer your hexane layer to an Erlenmeyer flask
and add a pinch of anhydrous sodium sulfate. If this coagulates, add a little more. Keep
adding until some of the sodium sulfate still looks powdery after swirling it around in
your hexane mixture.
6. Remove the hexane mixture from the drying agent and heat it gently, in the hood, on a
hot plate. Keep heating until the total remaining volume is about 0.5 mL. (Sometimes
it’s easier just to evaporate to dryness and then add back about 0.5 mL hexane.)
7. Save a small amount of your initial mixture to run a TLC tests after column
chromatography.
Part II Column Chromatography
1. Obtain 10 mL of acetone and 10 mL of hexane in separate 100 mL beakers.
2. Assemble the pipet column. CAUTION: Alumina and Silica powders are harmful if
inhaled. Use only in the fume hood.
a) Place a small piece of cotton into the bottom of the pipet followed by 0.5 cm of sand
and enough alumina to fill the pipet about 2/3 full. Note: Weighing paper may be
used to help fill the column with silica.
b) Gently tap the side of the pipet for 1 minute to pack the alumina.
c) Add another 0.5 cm of sand to the top of the pipet.
3. Place the pipet column in a clamp and secure it to the ring stand. It must be vertical.
4. Obtain six test tubes to collect the column fractions and a 100 mL beaker for waste.
Important: Once you start the elution process, it cannot be stopped. You must go to
completion. Do not start after 3:30 pm. Read Step 8 carefully before continuing.
5. Prepare the column for chromatography:
a. Place the waste beaker under the pipet column and elute 2−3 mL of hexane through the
column. Note: If air bubbles or cracks appear in the column, discard and repeat Step 2.
b. Elute another 2−3 mL of hexane. Important: Do not let the column get dry during this
procedure. Replenish with solvent as needed.
c. Once the solvent has drained to just above the silica, pipet the green pigment solution
from Step I.7 evenly onto the column. Allow the solution to adsorb onto the silica.
d. A yellow band will appear and begin to separate from the green band. Continue adding
hexane.
e. Collect the yellow fraction in a clean test tube.
f. Once you have collected the yellow fraction, change the solvent by adding acetone.
g. Continue adding the solvent and collect the clear portion in the waste beaker.
h. The green band should be moving down the column. Collect this fraction in a clean test
tube. Note: Not all of the pigments will be removed from the column.
6. Save the fractions collected off the column.
Part III Thin-Layer Chromatography
1. Prepare a 2 development chambers: For each,
a. Place a piece of filter paper against the side of a 400 mL beaker. The filter paper will help
saturate the beaker with solvent vapors.
b. Add 5–10 mL of 30%:70% acetone/hexane mixture to one of the 400 mL beakers. Cover
with a watch glass.
c. Add 5-10 mL of either 100% hexane or 100% acetone or 100% methanol to the second
beaker. Try to use a different solution than the people around you, so that between
everyone you will have tested a variety of solvents. Cover the chamber with a watch
glass.
2. Obtain two TLC plates. Handle them carefully by the edges so that the adsorbent does not
flake off or pick up oils.
3. Prepare the TLC plates. Do the following on both plates as identically as possible.
d. Using a pencil (NOT an ink pen), lightly draw a line across the plate, approximately 1 cm
from the bottom. Across this line, mark the location indicating where the sample will be
spotted, making sure it is not too close to the edge of the plate (see Figure 2).
e. Take a spotting (open-ended capillary) tube and dip one end into one of the solutions
containing the spinach extracts. Capillary action will draw the liquid into the tube.
f. Lightly tap the tube on the mark on the TLC plate. Only a small amount of sample needs
to be delivered. The spot should be 1−2 mm in diameter. Let the spot dry. If it is not a
clearly visible green, again lightly tap the spotting tube on the TLC plate in the exact same
spot. Repeat until you see a distinctly green spot.
g. You should have a spot from your initial spinach mixture and from each of the fractions
eluted from the column which you think may contain material.
4. Place one TLC plate in each beaker and cover with the watch glasses. Make sure the plate is
not touching the filter paper. The solvent level must not be above the spots on the plate or
your sample will dissolve into the solvent.
5. When the solvent has risen to within 1-5 cm from the top of the plate, remove the plate from
the chamber and with a pencil, gently draw a line to mark the position of the solvent front.
6. After the plate has dried, observe the TLC plate and lightly outline the spots with the pencil.
7. Draw a picture of your developed TLC plate. Colored pencils are available. Calculate the Rf
value for each spot and record the values in a data table.
University of Washington – Tacoma
Organic Chemistry I (TCHEM 261)
Spinach Lab Post Lab Write-Up
POST LAB
RESULTS – ORGANIZE INTO TABLES
Part II. Column Chromatography
Number of fractions collected, volume of different fractions, time they were eluted, visual
appearance. A table might be good.
Part III Thin-Layer Chromatography
a) Drawing of TLC Plates
b) Show calculation of Rf values
c) Make a table reporting all Rf Values
Pigment color
Calculated Rf
Questions to Answer
1. Was column chromatography effective in separating different fractions from spinach? Be
sure to use your data to support your answer to this question.
2. Was our suggestion of a solvent system appropriate? Again, use data to support your
answer.
3. You wrote a hypothesis about which material, the chlorophyll or cartenoids, would elute
fastest. Was your hypothesis upheld by the data, contradicted by the data, or was the
experiment inconclusive?
4. Did Column Chromatography and TLC show similar relative movement of the different
compounds? Is this expected or surprising?
5. Can you suggest further experiments one might do to investigate the relationship between
conjugation and UV-Vis absorbance?
6. What applications might this have? You might consider the importance of sunlight
absorbance in plants, marine algae, or by solar cells for energy production. Don’t try to
cover everything; pick one of these or something else and just discuss the implications for
that one case. Use the introduction to provide background information about whatever
topic you chose to focus on, and come back to that same topic at the end of the
discussion.
University of Washington – Tacoma
Organic Chemistry I (TCHEM 261)
Compound ID Lab – Revised W23
StudentVersion
INTRODUCTION
Chemists often wish to determine the identity of an unknown compound. Traditionally
they did this by using a series of chemical tests, derivatizations, and determination of physical
properties. These tests are still useful, but chemists now also have available a powerful array of
spectroscopic tools: Mass Spectrometry (MS), Infrared Spectrometry (IR), and Nuclear Magnetic
Resonance Spectroscopy (NMR). UWT has both IR and 1H-NMR spectrometers available for
student and research use.
This lab has an urgent objective. An illegal dump was found in east Pierce County with
about 64 unlabeled drums filled with organic compounds. The Pierce County Environmental
Services Division has asked the UWT chemists to identify the unknown compounds in these
drums so that they can be safely reused or disposed. Each lab group needs to determine the
identity of two unknowns, one solid and one liquid. To accomplish this task, students will obtain
IR and 1H-NMR spectra of their unknowns. They will also determine melting and boiling points
and other physical constants such as density and refractive index. If desired, students may
request a mass spectrum and/or 13C-nmr spectrum. Reagents needed to carry out some
potentially helpful chemical tests will also be available, and students may perform these tests at
their discretion. If students are unable to identify their unknowns, elemental analysis can be
requested, but this will result in a deduction of points.
When a chemist is faced with identifying an unknown chemical compound, there is a
systemic approach often used. The following story about Harold and Erma and Unknown X
illustrates a common approach, but not the only possibility.
Sample of Steps in Identifying an Unknown.
Organic Chemistry students Harold and Erma investigate solid Unknown X. It is a solid,
so they take its melting point (m.p. = 60-62 oC). They next take an IR spectrum (shown in
Figure 1 below), as IR is good for determining the presence or absence of major functional
groups in a compound.
Figure 1: IR spectrum of Unknown Solid X.
Harold and Erma decide that the strong peak at 1700 suggests the presence of a carbonyl
group. The lack of an –OH peak above 3000 rules out a carboxylic acid. The weak peaks
above 3000 suggest the possibility of an aromatic group, while the strong signals just below
3000 indicate at least some aliphatic hydrogens. At this point, Harold and Erma suspect that
they have either an aldehyde, a ketone, or an ester which contains both aromatic and aliphatic
groups.
Harold and Erma then obtain the 1H-NMR spectrum shown below.
By studying this 1H- NMR, Harold & Erma determine that the compound contains 9 aromatic
and 3 aliphatic Hydrogens. A 13C-NMR tells them that the compound has 10 unique carbons.
From this information, they write 3 or 4 possible structures, and check melting points of the
possible structures. One matches. This proposed structure has a methyl group, so they run the
iodoform test and it is positive, confirming their proposed structure. They turn in this structure
and are thrilled to receive an “A” for this lab.
The moral of this example is “Be like Harold and Erma.” Use a logical, step-wise approach to
identifying your unknowns, chose your tests wisely, and be sure to consider ALL the data.
This is a Three-Week Lab for Winter 23
PREPARATION for Week 1
1. Read the Whole Lab. Note that you will be writing a complete lab report for this lab.
2. Learn or Review any concepts or skills you are not confident about applying during lab. Note:
We will be covering IR and NMR and Mass Spec in lecture; you do not have to teach
yourself the topic. This is, however, a great time to read ahead. You should also review
melting points and boiling points.
Melting points: Nichols 6.1D
Boiling point determination: Nichols 6.2A, Thiele Tube method
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_Technique
s_(Nichols)/06%3A_Miscellaneous_Techniques/6.02%3A_Boiling_Point/6.2B%3A_Step-byStep_Procedures_for_Boiling_Point_Determination .
IR: Smith Spectroscopy B
1
H-NMR: Smith Spectroscopy C
3. Prepare your lab notebook as described below.
LAB NOTEBOOK Set-Up Week 1
• Title – Use the one given for this lab.
• Clerical Information – Your name, the name of your assigned lab partner for that lab,
and the date on which the in-lab work was started. Be sure to also enter the lab in your
Table of Contents.
• Objective – After reading the entire lab, identify and write down the objective of the lab.
The objective is the question or questions your data will hopefully allow you to answer.
• Hypothesis – For Week 1 of this lab, please write a hypothesis about how useful melting
points and boiling points will be for helping to determine which functional groups your
compounds contain.
• Physical & Chemical Properties Table – You do not need to do any Material Data &
Safety/hazards table for this lab, as all of our compounds are unknown. HOWEVER,
remember that these compounds are organic, and thus likely to be flammable, toxic,
irritants, corrosive, and/or mutagenic. Wear gloves and perform transfers in the hood.
• Safety – List any particular safety issues associated with this lab.
• Pre-lab questions: None for Week 1
1. What is the objective of this lab?
•
•
Experimental Plan – Use the Experimental directions below to write a plan for the work
you need to do in Week 1’s lab session
Data – Create spaces in your notebook to record all the data you will collect.
PREPARATION for Week 2
LAB NOTEBOOK Set-Up Week 2
• Clerical data, especially date, name, and partner’s name.
• Hypothesis – For Week 2 of this lab, please write a hypothesis about how useful H-NMR
will be for helping to determine the types of hydrogen atoms present in your compounds.
• Pre-Lab Questions Week 2 – Do on separate sheet, not in Lab Notebook
1. During last week’s lab, Harold and Erma started by taking a melting point of their
solid, unknown X. However, when interpreting their data, is the melting or
boiling point of the unknown going to be the piece of data that they use first to
begin to identify the unknown? Explain.
2. What did the IR spectra tell you about your solid?
3. What did the IR spectra tell you about your liquid?
•
•
Experimental Plan – Use the Experimental directions below to write a plan for the work
you need to do in Week 1’s lab session
Data – Create spaces in your notebook to record all the data you will collect.
PREPARATION for Week 3
LAB NOTEBOOK Set-Up Week 3
• Clerical data, especially date, name, and partner’s name.
• Hypothesis – For Week 3 of this lab, please write a hypothesis about how useful Mass
Spec will be for helping to determine the types of functional groups present in your
compounds.
• Pre-Lab Questions Week 3 – Do on separate sheet, not in Lab Notebook
1. During last week’s lab, you obtained H-NMR spectra. Copy the following table
twice and fill it in once for your liquid unknown and again for your solid
unknown.
Chemical
Shift
Area
Relative Possible
Area
# of H
Observed
Splitting
# of
Neighbors
Possible types of H
2. What possible structures or structural fragments are suggested by your H-NMR?
3. Harold and Erma started by taking a melting point of their solid, unknown X.
However, when interpreting their data, is the melting or boiling point of the unknown
going to be the piece of data that they use first to begin to identify the unknown?
Explain.
Experimental Directions
The following steps do not need to be done in a particular order. Your instructor will
guide you in proceeding in a manner that will prevent long waits for a certain piece of
equipment.
WEEK 1
Melting Point of Solid Unknown
Determine the m.p. of your solid unknown using the Mel-Temp apparatus.
Boiling Point of Liquid Unknown
An apparatus for determining boiling point is shown in this link
https://www.google.com/search?q=boiling+point+apparatus&safe=active&biw=1413&bih
=620&tbm=isch&tbo=u&source=univ&sa=X&sqi=2&ved=0ahUKEwiHhd2L_4fLAhVB3W
MKHc_nAfkQsAQIGw&dpr=0.95#imgrc=fJYDX3FBOGQ37M%3A
Or this one.
https://www.google.com/search?q=boiling+point+apparatus&safe=active&biw=1413&bih
=620&tbm=isch&tbo=u&source=univ&sa=X&sqi=2&ved=0ahUKEwiHhd2L_4fLAhVB3W
MKHc_nAfkQsAQIGw&dpr=0.95#imgrc=tEAiUbIiemhfWM%3A
Place a small quantity of the unknown liquid in a little test tube. Invert the open end of a closed
end capillary tube in the test tube. Rubber band the test tube to a thermometer. Place the test tube
(but NOT the rubber band) in a cool oil bath. Heat until bubbles come out of the mouth of the
capillary tube. Turn off the heat and cool. Watch for the temperature at which liquid is sucked
back into the capillary tube. Record these two temperatures as the b.p. range.
IR Analysis
Run an IR on each of your unknowns. Dan or Dr. Meg will be available to assist you. Make
sure you clean the instrument after use. You should analyze the spectrum obtained from your
compound. Be sure to include the spectrum in your post lab submission.
WEEK 2
1
H-NMR Analysis
Prepared NMR samples will be available for each unknown. The lab staff made these by
dissolving enough of each sample in 0.5 mL of deuterated solvent to create a 0.5 M solution. A
drop of TMS was added to your sample. The instructor will show you how to run our NMR
spectrometer.
13
C-NMR will be provided.
WEEK 3
Mass spec will be provided.
Based on the information obtained from the IR and NMR, consider running any of the following
chemical tests. If you think you wish to do any of these tests, please talk to the instructor about
how to carry them out.
Additional Physical Tests
Density determination of a liquid
Refractive Index
Optical Rotation
Solubility
Fluorescence
Possible Chemical Tests
Bromine Test
Cold dilute KMnO4 test
Alcoholic Silver Nitrate Test
FeCl3 test
Tollen’s Test – aldehydes & some ketones
Iodoform Test – methyl ketones
pH
University of Washington – Tacoma
Organic Chemistry I (TCHEM 261)
Compound ID Post-Lab: Formal Lab Report
You will write a complete formal lab report for this lab. See general
directions posted on canvas in the scientific writing module. I also
suggest that you read the grading rubric both prior to writing your
report and again before you submit your final draft.
Compound ID Lab Report Rubric
Criteria
Evaluation
ABSTRACT
States purpose of work/
problem being investigated.
Hypothesis stated
Major methodology stated
Key result(s) stated
Major outcome given
INTRODUCTION
P1: Background & context
P2: Objective/ Hypothesis/
Proposed work
P3: Results & Implications
EXPERIMENTAL
The lab work for each of the following should be described in
sufficient but not excessive detail. This section must be written in
the past tense and passive voice.
Physical constant
determination
IR
NMR
Chemical tests
RESULTS
Measured Physical constants
given
IR – spectra attached with
labelled peaks.
H-NMR
– spectra attached.
Possible Awarded
Points
Points
10
2
2
2
2
2
12
4
4
4
10
2
2
2
2
2
18
2
4
8
– Table of signals with shift, splitting,
areas included – see examples.
MS, 13C-NMR, other tests
4
DISCUSSION
The discussion is your chance to make a strong argument for your proposed
identity of each of your compounds. Build an argument that will convince your
reader that the unknown is what you propose it is. Refer to data using Figure
and Table numbers so that your reader can look at the data you are
discussing. Be sure to comment on ALL the data you have for a compound –
don’t cherry-pick!
Solid compound ID
• Discusses information that can
be deduced from each piece of
data
• Builds a cohesive, logical
argument
• Discusses any anomalies
• Nothing overlooked
20
2
2
4
2
2
4
Liquid compound ID
•
•
•
•
•
•
Physical constants
IR
H-NMR
Other spec. if used
Chemical tests if app.
Conclusion
Conclusion and
Recommendations to the
Pierce County Environmental
Services Team
Mechanics
*Typed, double –spaced?
*Complete sentences?
*Understandable
grammar, punctuation &
spelling?
*References/citaitons
Quality
* Logic
* Connections, flow
* Effort
TOTAL
20
2
2
4
2
2
4
4
6
1
1
2
2
4
100
You will also receive a grade (25 pts) based on your in-lab work. This grade will include
notebook preparation, pre-lab quiz score, understanding of the tests you are running,
care and gentle treatment of our equipment, teamwork, tidiness & cleanliness, safe
handling and proper disposal of chemicals, and general attitude.