complete the following worksheet in this organic chemistry activity regarding extractions. total of 7 questions
ENZYME ACTIVITY
Enzymes are an important class of molecules in cells and organisms. Without
enzymes, essential chemical reactions required for a cell's survival are not possible.
Virtually all enzymes in living cells are proteins although some classes of RNA
molecules (ribozymes) perform critical enzymatic roles.
Like all chemical reactions, some metabolic reactions are spontaneous (having a
negative ∆G) and some are non-spontaneous (having a positive ∆G). Regardless of
the ∆G value, all chemical reactions, including all metabolic reactions, require an initial
input of energy to start the reaction. This “starting” energy is called the ACTIVATION
ENERGY. The activation energy for most metabolic reactions is prohibitively high. This
is due to the specific orientation the reactants must adopt in order to produce the
desired reaction (termed an EFFECTIVE COLLISION). Enzymes increase the number
of effective collisions. Each enzyme’s 3-D structure attracts and binds the reacting
molecules greatly increasing the number of effective collisions and the formation of
enzyme/substrate complexes. Without enzymes, even metabolic reactions with a
overall negative ∆G will not occur under physiological conditions.
Like all proteins, an enzyme's 3-D structure is critical to its function. An enzyme's
functional 3-D structure is called its NATIVE CONFIGURATION. The domain on an
enzyme's 3-D structure where the reactants bind is called the ACTIVE SITE. The
reactants in an enzyme-mediated reaction are designated as the SUBSTRATE(S) of
that enzyme. Some enzymes can process thousands of substrate molecules per
second. Since enzymes are a type of catalyst they are not destroyed or permanently
altered while performing their function. Therefore, a few molecules of enzyme may be
all that is necessary to catalyze thousands of reactions.
Enzyme activity can be affected by the number of available enzyme molecules
(i.e., the concentration of the enzyme) and physical factors that disturb the native
configuration of the enzyme or impact the formation of an enzyme/substrate complex.
Any disturbance of the native configuration will impact the active site, reducing the
efficacy of the enzyme. When an enzyme loses its native configuration, it is
DENATURED, and the process is called DENATURATION. Environmental factors can
cause denaturation by disrupting the hydrogen and ionic bonds that are the basis of an
enzyme’s secondary, tertiary and, in multi-subunit enzymes, quaternary structures.
Increased energy in a system causes an increase in intramolecular atomic vibrations.
Too much vibration will disrupt these relatively weak bonds and cause denaturation.
Denaturation caused by increased energy is sometimes reversible (the enzyme can
RENATURE) provided the energy increase was not too extreme and the cooling
process is gradual. Changes in H+ concentrations (i.e., changes in pH) can also cause
denaturation by ionizing amino groups (-NH2 ˆ NH3
+
) or hydrogenating ionized alcohol
groups (-Oˆ -OH) in the amino acid backbone and R-groups, thereby disturbing ionic
and hydrogen bonds that stabilize the enzyme’s secondary and tertiary structures.
23Apr19 5:33 pm Enzyme Activity - 1
In this lab exercise you will examine the effect of several factors on two enzymemediated reactions. One experiment will collect objective qualitative data and the other
using objective quantitative data. An objective qualitative analysis may be limited to a
“yes” or “no” answer or may allow for a modest amount of degree assessment. In the
latter case, degree assessment in objective qualitative data must adhere to an
acceptable scale. For example, pH paper, which changes colors in response to specific
[H+] includes a color scale for assessing the results. In this lab, the objective
qualitative analytical technique uses a standard scale (see the Lab portion of the
website) and another scale that allows the conversion of objective qualitative results to
a quantitative scale.
STUDENT LEARNING OBJECTIVES
The following STUDENT LEARNING OBJECTIVES are designed to provide
quantifiable data on Student Learning. They do not represent all the learning
objectives for this Lab Activity. Students will be provided with additional objectives in
the Lab Report Instructions and Test Review for this Lab Activity.
Student Learning Outcomes
- Students will collect and analyze objective qualitative data on the effect of pH on
Invertase activity. - Students will formulate general conclusions about enzyme activity and structure
based on qualitative data. - Students will analyze objective quantitative data on the effect of Temperature on
Amylase activity - Students will formulate general conclusions about enzyme activity and structure
based on quantitative data.
23Apr19 5:33 pm Enzyme Activity - 2
Note - Since the Benedict’s Test
is a Qualitative Assay, it might
be necessary to “create”
intermediate numbers
(e.g., 2.5, 0.5, 1.7, etc.) on the
scale. In the experiment below
you will “create” intermediate
values that correspond to the
data all the pH groups collect.
PART 1 - A QUALITATIVE ANALYSIS OF ENZYME ACTIVITY
In this exercise you will examine the activity of the enzyme, Invertase. All
enzyme names end with -ase. Invertase hydrolyzes sucrose into glucose and fructose.
You will monitor the appearance of glucose and fructose as an indication of Invertase’s
level of activity (i.e., Invertase’s reaction rate). If the enzyme is working, then some or
all the sucrose present will be converted to glucose and fructose. Therefore, the
amount of glucose and fructose produced is directly related to the level of Invertase
activity (assuming no contamination by glucose and fructose from another source).
The assay you will use to detect the appearance of glucose and fructose is called
the Benedict's Test. The Benedict's Test detects the presence of reducing agents.
Reducing agents readily give up electrons to other atoms or molecules, thereby
reducing those atoms or molecules. Glucose and fructose are a type of reducing agent
called reducing sugars. The Benedict's Test does not detect glucose and fructose
directly, it detects an activity (reduction) that the two molecules perform. The test
cannot distinguish between different reducing agents. It only detects the presence of
any reducing agent. So, the Benedict's test does not assay for Invertase activity
directly. The assay detects Invertase activity indirectly by detecting a characteristic of
the products of the enzyme's activity.
The Benedict's test uses Cu2+
as an electron acceptor. Cu2+
is blue in solution. The
reduction to Cu1+ causes a color change. It is this color change that you will use to
monitor Invertase activity. The color change can vary from Brownish Green to Orange
to Red. Since sucrose is not a reducing sugar, if the solution remains Blue, then the
sucrose was not hydrolyzed to glucose and fructose . The degree of color change is
directly related to the amount of glucose & fructose present and, therefore, the level of
activity of the Invertase enzyme. For the purposes of this lab, we will use the common
scale below to convert the observed color changes to objective numerical data. The
conversion scale is provided below:
Qualitative Result Quantitative Scale
Blue 0
Brownish-Green 1
Orange 2
Red 3
23Apr19 5:33 pm Enzyme Activity - 3
Determining the Effects of pH on Invertase Activity - A Qualitative Study
(50 minutes)
Now that you know what to expect from the Benedict's Test, you will use it to assay for
Invertase activity.
i Monitor your time carefully, this experiment including data collection and brief analysis is
allotted 50 minutes - Each group will perform one CONTROL and one EXPERIMENTAL.
- Each group will be assigned one pH solution as their EXPERIMENTAL condition.
All groups will also do a CONTROL at pH=4.4. See the Table 1 for your group’s
experimental condition. - You will need the following items to perform your experiments:
• 6 glass test tubes
• 1 test tube rack
• Labeling tape and a sharpie OR a grease pencil
• Disposable plastic pipettes. - You will use the following reagents. Regents are in bottles with pumps. Pumps
are labeled as delivering 1ml per pump.
• pH buffer solutions
• Enzyme (Invertase)
• Substrates (sucrose + water)
• Benedict’s reagent - The flowgram on the next page shows the steps for each experiment.
- Use the disposable plastic pipettes to transfer liquids.
- Your instructor will provide instructions on adding specific reagents (pH
solutions, enzyme, substrate and Benedict’s solution) to your test tubes
02Dec19 12:25 pm Enzyme Activity - 4
FLOWGRAM FOR TESTING THE EFFECT OF pH ON INVERTASE ACTIVITY
VERY IMPORTANT- GENTLY SWIRL TUBES AFTER ADDING SOLUTIONS to
ensure even mixing
02Dec19 12:25 pm Enzyme Activity - 5
See Page 3 for the
suggested Quantitative
values for each
Benedict’s test result.
Create intermediate
values as necessary to
fully express the
qualitative Benedict’s
test results.
After you have completed your assigned assays have your instructor review your
results. All the results will be gathered together and used to complete Table 1 below.
The summary data will be provided on the Canvas
TABLE 1: Invertase ACTIVITY AT DIFFERENT pH's
Group
Number
pH solution Qualitative
Benedict's
Test Results
“Created”
Benedict's Test
Quantitative
Scale
1 1
2 2
3 3
ALL:
CONTROL 4.4
4 5
5 7
6 8
7 9
8 10
9 11
10 12
02Dec19 12:25 pm Enzyme Activity - 6
PART 2: A QUANTITATIVE ANALYSIS OF ENZYME ACTIVITY
In this exercise you will use a different enzyme to gather objective quantitative data
on enzyme activity. The enzyme you will use is called AMYLASE. Amylase is a
catabolic enzyme that hydrolyzes the sugar AMYLOSE, a constituent of the larger
carbohydrate starch, into the disaccharide maltose. The assay you will use relies on
the fact that iodine binds with amylose (starch) to produce a blue/black color. In a
solution of amylose and iodine, there is a direct relationship between the amount of
color and the amount of amylose present. Since maltose does not bind iodine as the
amylose is hydrolyzed by amylase, the iodine in the solution has nothing to bind to and
the blue/black color fades. As with the Benedict's Test, this assay indirectly measures
enzyme activity. However, instead of measuring the appearance of the product
resulting from the enzyme's activity as the Benedict's Test does, the Iodine Test
measures the disappearance of the substrate of the enzyme, amylose.
To quantify your data, you will use a SPECTROPHOTOMETER to measure the loss
of color. Spectrophotometers measure the amount of light transmitted or absorbed by a
liquid sample. You will gather data on the amount of light transmitted by a sample. This
data is called the Transmittance or %T. The %T value indicates the percentage of a
particular light wavelength passing through the specimen. The larger the %T, the more
light is passing through a specimen. Transparent liquids have higher %T values than
opaque liquids. Therefore, as the amylose is hydrolyzed by amylase the solution
will become more transparent and the %T value will increase. After you collect
your data, you will enter it into a computer program that will produce a graph of the
data. The data will be posted on Canvas.
SOLUTIONS PROVIDED FOR THIS EXPERIMENT - Plain Substrate: 1% (v/v) solution of amylose. For use in calibrating the
spectrophotometer. MUST BE mixed via pipetting (use a disposable pipette) just
prior to each use to ensure the mixture is uniform. - Iodine: 1% solution
- Stock Enzyme: (w/v) solution of amylase. Your instructor will provide
the amylase concentration for your experiment.
ASSAY PRINCIPLE: - Amylose (starch) and Iodine form a Blue-Black complex that has a low %T.
- Amylase digests Amylose (starch) into smaller sugars, notably Maltose.
- Maltose DOES NOT BIND TO IODINE.
- Therefore, as Amylase digests the Starch, the blue/black color will fade and the %T
will rise. HOWEVER, the iodine will remain part of the solution.
23Apr19 5:33 pm Enzyme Activity - 7
THE FOLLOWING EQUIPMENT WILL BE NEEDED FOR THIS EXPERIMENT
Provided at each lab table to be shared among all table groups:
! 1 bottle of 1% amylose (starch) solution (substrate)
! 1 bottle of 1% iodine in smoked glass bottle - iodine degrades in light. These
bottles are designed to minimize degradation
! 1 bottle of amylase (enzyme)
Each group will gather the following items:
G Clean, dry disposable pipettes, start with 6, more as needed
G Square spectrophotometer cuvettes as needed, start with 10, more as needed
G Spectrophotometer cuvette rack (cuvettes are square)
G Micropipettors of various size + tips (in Orange Biotech toolboxes)
G Blue-capped 15ml falcon conical tubes, start with 4, more as needed
G Spectrophotometer (on your lab table)
INITIAL SPECTROPHOTOMETER CALIBRATION:
(20 minutes) - Your instructor has completed the following steps for each spectrophotometer prior
to lab.
• Basic calibration and Dark Zeroing of Spectrophotometer
• Set the Spectrophotometer to read %Transmittance (%T)
• Set the wavelength (500nm) for today's experiment
• Data screen is FROZEN - Your instructor will review the parts of the spectrophotometer with you before
you proceed with next steps. - Secure 2 spectrophotometer cuvettes.
• For simplicity sake, spectrophotometer tubes will be referred to as “cuvettes” and
the spectrophotometer will be referred to as the “spec” for the balance of this
protocol.
• Always handle the cuvettes by the top. Bodily fluids from your fingers will affect
the light transmitting characteristics of the spec tube. Lens paper has been
provided to wipe off cuvettes if they are accidently soiled. - Using a clean, dry disposable pipette, mix the substrate,1% amylose, by pipetting.
Always mix the substrate prior use. The substrate is a suspension not a solution.
Never use the substrate without first mixing by inversion or pipetting. - Transfer 2500µl of substrate into a cuvette. This is your “Blanking Cuvette” you will
use it throughout the experiment.
23Apr19 5:33 pm Enzyme Activity - 8
Initial Spectrophotometer Calibration:(30 minutes) - You will now “blank the spectrophotometer”. This step “tells” the spec what
materials to ignore in determining the transmittance of the solution. In this case, the
spec is “told” to ignore the transmittance characteristics of the amylose solution and
the spec tube. - Mix amylose suspension in the Blanking Cuvette by pipetting and insert it into the
sample chamber and close the lid. - UNFREEZE THE DATA SCREEN then Press the Blanking Button. Wait until the %T
reads 100% - IMMEDIATELY Press ENTER (5 ) to freeze the data screen
- Remove the Blanking Cuvette from the sample chamber and store it in your cuvette
rack. - Use the Blanking Cuvette to “re-blank” your spec before each experimental
run. Since the 1% amylose is a suspension, be sure to mix the solution in the
cuvette by inversion or pipetting prior to each use. - You must blank the spec before every reading to ensure proper calibration
and increase the consistency of %T readings.
Instructions continue on the next page
23Apr19 5:33 pm Enzyme Activity - 9
Instructions for preparing a Calibrated Substrate tube
(30 minutes)
The starch and iodine suspension is unstable at best. Expect that %T readings
for specific amounts of Iodine added to substrate to vary.
Purpose: Determine the amount of iodine needed to reach a range of 14%T to
17%T. - Secure 10 cuvettes and a rack.
- Place 5 of the cuvettes in the rack (set the other 5 aside for later use as needed)
- Add 2500µl of 1% amylose to each cuvette in the rack.
- Add 1% iodine to each cuvette as listed below and mix by pipetting.
• Cuvette #1 - 150µl
• Cuvette #2 - 175µl
• Cuvette #3 - 200µl
• Cuvette #4 - 225µl
• Cuvette #5 - 250µl - Determine the %T for each cuvette+iodine solution FOR EACH SAMPLE:
• BLANK the spec. previously described (use your Blanking Cuvette)
• MIX BY INVERSION OR PIPETTING BEFORE testing a sample
• Insert a cuvette in the sample chamber, close the lid, press ENTER (5 ) to
unfreeze the data screen.
• AS SOON as the %T value changes, IMMEDIATELY Press ENTER (5 ) to
freeze the data screen.
• Record the %T in the data table on the next page - Review your %T data.
• Did you find an amount of Iodine that yielded a %T between 14% & 17%? If you
did, draw a circle around this amount. You will use this amount to create a
calibrated substrate tube for the experiment.
• If you did not find an amount of Iodine that yielded a %T between 14% & 17%
then continue trying different amounts of Iodine until you find the appropriate
volume. The volumes you try will depend on your initial data set. If you are
unsure how to proceed, ASK YOUR INSTRUCTOR
DO NOT SPEND MORE THAN 20 MINUTES ON THIS EXERCISE
TEST A MAXIMUM OF 10 IODINE VOLUMES.
ASK YOUR INSTRUCTOR IMMEDIATELY IF YOU CANNOT FIND A SUITABLE
IODINE VOLUME AFTER 10 TRIALS
23Apr19 5:33 pm Enzyme Activity - 10
Data Table: Determining amount of Iodine needed to reach target % T range
Cuvette # µl of Iodine %T
1 150
2 175
3 200
4 225
5 250
6
7
8
9
10
DO NOT SPEND MORE THAN 20 MINUTES ON THIS EXERCISE
TEST A MAXIMUM OF 10 IODINE VOLUMES.
ASK YOUR INSTRUCTOR IMMEDIATELY IF YOU CANNOT FIND A SUITABLE
IODINE VOLUME AFTER 10 TRIALS
Lab Exercise Continues on the Next Page
23Apr19 5:33 pm Enzyme Activity - 11
PART 2. Determining the Effect of Temperature on Enzyme Activity: A Quantitative Study
Before proceeding, go back to the first page of this exercise and re-read the
material on enzyme function.
In this study you will determine the effect of kinetic energy on enzyme activity. We
will use temperature as a measure of the amount of kinetic energy in a system.
Kinetic energy is directly related to the amount of molecular motion within a system.
As kinetic energy increases reaction rates first rise, as effective collisions and
enzyme/substrate complexes increase, but then reaction rates fall as the effective
collisions become to energetic for the formation of enzyme/substrate complexes. .
As the temperature approaches 1000
C the enzyme structure is denatured due to
increases in intramolecular vibrations that destabilize the non-covalent bonds that
maintain the enzyme’s secondary & tertiary structure.
You will conduct experiments to determine the effect of temperature (as a measure
of kinetic energy) on amylase activity. Each group will be assigned one of five
temperatures (0o
C, 25o
C, 40o
C, 55o
C, and 100o
C)
Experimental Procedures: (1 hour and 10 minutes) - Secure a blue-capped 15ml Falcon tube.
- Transfer 2000µl of Amylase into the Falcon tube. Replace the lid but don’t
tighten completely. - Transfer 2500µl of 1% starch into a clean cuvette. This is the cuvette you will
calibrate for your experiment. You will also need your Blanking cuvette. - For the 250
C (room temperature) experiment, the experiment will be on the lab
desktop. No incubation is needed. - For the 00
C, 400
C, 550
C, 1000
C experiments, THE FALCON TUBE
(containing the Amylase) WILL BE PLACED IN THE WATER BATH.
DO NOT HEAT THE CUVETTE WITH THE SUBSTRATE. - For the 00
C, 400
C, 550
C, 1000
C , allow all the Amylase solutions to equilibrate to
the designated temperatures for 15-20 minutes. - Place your Falcon tube with Amylase in the bath for your group’s experimental
temperature. Note the time. Incubate for at least 15 minutes
i For 1000
C, the water bath must be at a rolling boiling and stay that way for
full 20 minutes.
02Dec19 12:25 pm Enzyme Activity - 12
DURING THE 15' INCUBATION, follow the instructions below. It is OK if the samples
remain in the baths longer than 15'. If you are doing 250
C, no incubation is needed so
proceed with the steps below including the Experimental Run.
i Re-blank your spec. Don’t forget to freeze the data screen when the blanking
is complete (%T reads 100%). Leave the screen frozen.
i Prepare a Calibrated Substrate cuvette using the volume of Iodine you
determined earlier. Confirm the %T is between 14% & 17%.
• Remember: Press ENTER (5 ) to unfreeze the data screen. AS SOON as the
%T value changes, IMMEDIATELY Press ENTER (5 ) to freeze the data
screen.
• Due to the instability of the starch/iodine complex, you may need to do some
adjustments of the Iodine volume.
• DO NOT SPEND MORE THAN 10 MINUTES ADJUSTING IODINE
VOLUMES. Choose the volume that is closest to the desired range and
proceed to the Experimental Run.
• Leave the calibrated substrate tube in the spectrophotometer and the
data screen frozen. Record the %T in the data table under Time 0.
EXPERIMENTAL RUN - Read all the instructions before beginning.
i Designate group members as follows to perform the following tasks
• Start time and call out 30" time periods
• Data recording (see data table on the next page)
• Adding enzyme, mixing by pipetting between 30" time periods and calling out
%T readings.
i Using a micropipettor, remove 700µl of enzyme from your Falcon tube. IF
YOUR TUBE IS IN A TEMPERATURE BATH, DO NOT REMOVE THE TUBE
FROM THE BATH.
i IMMEDIATELY, add the enzyme to your calibrated substrate tube in the spec
AND START THE TIMER. Using a clean disposable pipette, mix the solution by
gentle pipetting. The timer will run continuously until 5:00 is reached or the %T
is 100% or above.
i At 30" unfreeze the screen. As soon as the %T changes, IMMEDIATELY
PRESS ENTER (5 ) to freeze the data screen & record the %T. Continue to
take %T readings every 30". Carefully mix the solution between 30" readings.
Stop your run after 5:00 or when the %T is 100% or above.
i Take your data to your instructor for review. DO NOT RE-RUN AN ASSAY
UNTIL YOU HAVE SHOWN YOUR DATA TO THE INSTRUCTOR.
23Apr19 5:33 pm Enzyme Activity - 13
EFFECT OF TEMPERATURE ON AMYLASE ACTIVITY
TIME Enter your group’s Temperature below
Run 1 Run 2
if needed
Run 3
if needed
%T @Time ‘0' is the calibrated %T value between 14% & 17%
Initial %T
- 30 sec
- 1 min
- 1 min 30 sec
- 2 min
- 2 min 30 sec
- 3 min
- 3 min 30 sec
- 4 min
- 4 min 30 sec
- 5 min
i Depending on your data set, you may need to re-run your assay. There is sufficient
enzyme in your Falcon tube for several assays.
i If your data set is accepted, clean up your area. Cuvettes, Falcon tubes, and
disposable pipettes go in the trash. Place your equipment back in your kits and
return the kits to the cabinet.
23Apr19 5:33 pm Enzyme Activity - 14
Review Questions:
The data from today’s experiments will be posted on Canvas. Use that data to answer
these questions.
Effect of pH on Invertase Activity
Review the material on page 1 of the exercise regarding the effect of pH on enzyme
structure and function.
Using appropriate terms (secondary & tertiary structure, and denaturation) and the data
from the pH experiment, describe what is happening to the structure of Invertase as the
pH values change.
02Dec19 2:10 pm Enzyme Activity - 15
Review Questions (con’t)
Effect of Temperature of Amylase Activity
Review the material on page one of this exercise and the introductory paragraph for the
Temperature experiment regarding the effect of energy on enzyme structure and
function. Review the material on enzyme function in the lecture outline.
Using appropriate terms (molecular motion, molecular collisions, effective collisions,
enzyme/substrate complexes, secondary & tertiary structure, and denaturation) and the
data from the temperature experiment describe what is happening to the structure of
amylase as the temperature rises from 00
C to1000
C
02Dec19 2:10 pm Enzyme Activity - 16
1
Liquid-liquid Extraction Online Activity
Separation and Identification of an Unknown Cutting Agent
One of the primary skills that an organic chemist must develop is the ability to purify a mixture
of compounds using a liquid-liquid extraction. The goal of this lab is to review the concepts of liquidliquid extraction introduced in CHEM 241L and to expand on what you previously learned by testing
the solubility of colored dyes in an organic solvent and aqueous solutions. Parts A and B of this lab use
colored dyes for ease of visualizing solubility. You will make conclusions about each dye’s likely
structure (charged or neutral?) when dissolved in an organic solvent or an aqueous solution of different
pH values. Remember that neutral compounds will likely dissolve in the organic layer and charged
compounds are much more soluble in an aqueous solution. After testing for solubility of each dye in
an immiscible mixture of hexane (organic layer) and aqueous solutions of pH 2,7,14, you will devise a
method to separate mixtures of two of the dyes. As a real-world example of this technique, in Part C,
you will apply what you have learned to isolate a simulated street drug (MDPV, ‘bath salts’ – a designer
drug that has hallucinogenic effects) from common cutting agents (an agent used to dilute a pure illicit
drug before sale).
Figure 1: The structures of the dyes.
Part A: Solubility Testing with Colored Dyes
Watch the following video: https://youtu.be/wduziLVPUfY
Answers the following questions:
- Describe the procedure in part A.
2 - Summarize the results in table format. Draw the structure of the dye in either the organic or
aqueous layer. Carefully show a neutral or charged structure as would be expected in each layer.
If nothing is in the organic layer or aqueous layer for a particular test, put an X in that box.
Sudan Blue
Hexanes and Water Hexanes and Aqueous NaOH Hexanes and Aqueous HCl
Organic Layer Organic Layer Organic Layer
Aqueous Layer Aqueous Layer Aqueous Layer
Sudan Orange
Hexanes and Water Hexanes and Aqueous NaOH Hexanes and Aqueous HCl
Organic Layer Organic Layer Organic Layer
Aqueous Layer Aqueous Layer Aqueous Layer
3
Congo Red
Hexanes and Water Hexanes and Aqueous NaOH Hexanes and Aqueous HCl
Organic Layer Organic Layer Organic Layer
Aqueous Layer Aqueous Layer Aqueous Layer - Which dye was protonated/deprotonated from the original structure? Show the chemical
reaction that took place.
4
Part B: Separation of a Mixture of the Dyes
Watch the following video: https://youtu.be/PTE9_WCdkHw - Based on the solubility tests for these molecules in part A, describe how you would separate
each of the following mixtures, predicting which dye will be partitioned into which layer (organic
or aqueous), and determine what would be the optimal pH (acidic, neutral, or basic) for the
aqueous layer.
Mixture A: Sudan Blue and Congo Red
Mixture B: Sudan Blue and Sudan Orange
Fill in the table shown below to record a successful separation method, indicating in each column which
aqueous solvent was chosen. It is okay to just write the name of the dye instead of the structure in this
table.
Mixture A Mixture B
Extraction Layer List Dye and Describe Aqueous Layer List Dye and Describe Aqueous Layer
Hexanes
Aqueous Layer
(Indicate if
Acidic, Neutral,
or Basic)
5
Part C: Simulated Analysis of Illicit Drug Samples
Watch this video to review the principles of liquid-liquid Extraction:
https://www.youtube.com/watch?v=5mugRn5erNM
For part C, you will apply what you’ve learned about liquid-liquid extractive separations to the analysis
of a simulated 4:1 mixture of methylenedioxypyrovalerone (MDPV, aka ‘Bath Salts’) and an unknown
cutting agent. A cutting agent is another chemical that is added to cut or dilute an illicit drug. Since the
HCl salt of MDPV is a white solid, any other non-toxic white solid could potentially be used as the cutting
agent. Likely candidates are readily available over the counter drugs. The goal of this experiment is to
determine the identity of the cutting agent by TLC, use your extraction skills to generate a plan to
separate the cutting agent from the drug, and prove that your amine is now pure via 1
H NMR
spectroscopy.
In this experiment we will not be using the HCl salt of MDPV but instead we will use the HCl salt of 3-
(dimethylamino)-1-phenylpropan-1-one as a “simulated MDPV”. The sample that is provided to
students initially will contain 80% of the “simulated MDPV” HCl salt (and 20% cutting agent), because
the HCl salt is an easy to handle white solid whereas neutral MDPV is a viscous liquid. After extraction,
you will need to isolate the neutral MDPV in order to obtain a 1
H NMR.
Figure 2: The chemical structures of MDPV and the HCl salt of MDPV
Figure 3: The chemical structure of what will be used as ‘simulated MDPV’ (provided as HCl salt)
6
Figure 4: The chemical structures of the possible cutting agents for this lab
The first goal will be to identify the cutting agent by TLC with standards for comparison. For TLC, a 1:1
mixture of ethyl acetate and hexanes was found to show separation of the components of the mixture. - Given this TLC plate, determine the identity of the cutting agent:
- Now use your knowledge of liquid-liquid extractive separations to determine an extraction
procedure which would isolate/separate the cutting agent from the MDPV. Assume the mixture
dissolves in dichloromethane (DCM). Don’t worry about measuring a % recovery or detailing
the exact amounts of the solvent or aqueous solutions used.
7 - The 1
H NMR spectrum that the student obtained is shown below. Determine if the purified
“simulated MDPV” a.k.a 3-(dimethylamino)-1-phenylpropan-1-one was pure. Explain.