Western Kentucky University Melting point determination and Thin Layer Chromatography Lab

Table of physical constants (5 points): (tabulate name, structure, molar mass, density (liquids only), melting point, boiling point, and safety hazards for all chemicals mentioned in the procedure)

Observations:(Include pictures of TLC plates)

Tabulated data (10 points): (construct a table including name of compound or over-the-counter drug, distance spot traveled, distance solvent traveled, calculated Rf values for each spot visualized, and color change in iodine chamber)

Answer the questions below (5 points each):

MELTING POINT DETERMINATION and THIN LAYER CHROMATOGRAPHY
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Student learning objectives – What you will learn from this experiment
• Use solid-liquid extraction to obtain a solution of analgesic drugs from common medicines
• Perform thin layer chromatography
• Calculate R values
• Predict relative R values based on molecular structure
Accurately measure melting point ranges
Experiment
• Strategy
In this experiment, you will extract analgesic
drugs from four over-the-counter medicines by solid
liquid extraction, and you will compare the behavior of each extracted drug with pure samples using
thin
layer chromatography. The four over-the-counter medicines are generic Excedrin migraine,
No-Doz. Tylenol, and Bayer. The pure analgesic compounds obtained from a chemical supplier are
caffeine, acetaminophen, and aspirin (acetylsalicylic acid).
You will also measure the melting points of pure and impure compounds. A pure compound should
melt over a narrow temperature
range, while impurities usually cause the melting point range to
begin at a lower temperature and a broadder range of temperatures. To obtain the melting point
range, you record the temperature at which the first crystals begin to melt (solid to liquid phase
transition) and the temperature at which the last crystal melts, e.g.: “m.p. = 124-126*c.
Procedure
Part A – Prepare solutions from four over-the-counter medicines
• Create a powder of each of the four over-the-counter medicines by crushing tablets separately
with mortar and pestle. Avoid cross-contamination between medicines and be sure to remove
any colored coating or lettering before crushing your tablets. Transfer the powder obtained from
each tablet into separate test tubes.
• Add approximately 2 mL of ethanoldichloromethane (1:1 v/v) to each test tube and swirl for
two minutes. Allow the mixture to settle for several minutes before use.
Part B – Prepare TLC plates and developing jar
Add eluent to the developing jar until a height of approximately 0.5 cm is reached. The eluent
used for this experiment is 1:2 hexane:ethyl acetate with 5% acetic acid.
· Seal the jar and allow the atmosphere in the jar to equilibrate with solvent vapors. Leave this
Obtain 2 TLC plates. Using a pencil. gently draw a line about 1 – 2 cm from the bottom of each
TLC plate. Be careful to avoid scratching the thin coating on the plate. Use a regular (non-
mechanical) pencil to prevent scratching. Do not use a pen or marker.
. Gently create four equally spaced marks on the line of each TLC plate. The 1 and 4 marks
should
be located at least as far from the edge of the plate as the other marks are from each
other. These marks are where you will add spots of your solutions.
Part C-Plate #1 – Pure compounds obtained from a chemical supplier
Your TA will provide you with ethyl acetate solutions of chemical grade caffeine,
acetaminophen, and aspirin. Spot each solution onto a separate lane on your TLC plate. Spot
a solution containing the mixture of all three pure compounds (given to you by your TA) onto
the 4h lane.
Record the location of each compound in your notebook. Allow each spot to dry completely
before developing.
. Gently place TLC plate #1 into the developing jar without splashing, promptly seal the jar, and
allow the solvent to move up the plate until it reaches approximately 1 cm from the top. Do not
disturb the developing jar while the plate is inside.
• Open that jar, remove the plate, and promptly
mark the position of the solvent front in pencil.
Note that the solvent front will quickly recede due to evaporation, so mark the location of the
solvent front immediately upon removing the plate from the jar.
Part D-Plate #2 – Drugs extracted from over-the-counter medicines
• Spot each solution that you extracted from an over-the-counter medicine (in test tubes from
Part A) onto a separate lane on your TLC plate. Record the location of each medicine in your
notebook. Allow each spot to dry completely before developing.
MELTING POINT DETERMINATION and THIN LAYER CHROMATOGRAPHY
. Gently place TLC plate #2 into the developing jar without splashing, promptly seal the jar, and
allow the solvent to move up the plate until it reaches approximately 1 cm from the top. Do not
disturb the developing jar while the plate is inside.
• Remove the plate from the jar and promptly mark the position of the solvent front in pencil.
Note that the solvent front will quickly recede due to evaporation, so mark the location of the
Solvent front immediately upon removing the plate from the jar.
Part E-Visualize spots and record Re values
. After the solvent has completely evaporated from each developed plate, visualize both TLC
plates by placing them under the UV lamp and gently circling each spot with a pencil.
After visualizing spots with the UV lamp.place each TLC plate into the iodine developing
chamber. Circle any new spots
that may appear, and mark
which spots reacted with the iodine
(changed color). Record all of this information in your notebook.
• Calculate R values for each
spot on each TLC plate and record these values.
Part F- Prepare melting point capillaries
Your TA will provide vials containing solid samples of pure acetaminophen and aspirin
(acetylsalicylic acid) for you to measure melting point Fila capillary tube to approximately 2-3
mm by holding the vial in one hand and the rounded side of the capillary tube between your
thumb and forefinger. Carefully insert the open end of the capillary tube into the powdered
sample. Tap the tube gently on the edge of the vial before taking it out to knock off any excess
sample on the outside of the tube. Then drop the capillary tube (round side down) into a long,
straight packing tube on a hard surface. The bouncing of the capillary tube will efficiently pack
your crystals.
Dispense a small amount of pure acetylsalicylic acid into a mortar (bowl) by holding the vial of
aspirin over the mortar, titing the vial, and gently tapping the side of the vial with a finger of the
same hand that is holding it. Obtain enough material for 2-3 melting point experiments. Then
add a similar amount of pure acetaminophen into the mortar. Any volumetric ratio in the range
of 90:10 – 10.90 should be adequate for this experiment.
Use the pestle to grind and mix the powders in roughly equal proportions. Do not slam the
mortar and pestle together; keep the pestle in contact with the mortar and move it around to
grind the powder (think of scrubbing something to clean it). Load a melting point capillary with
the combined mocture.
Part G-Measure melting points
Use the melting point apparatus to determine the melting points of all three samples
simultaneously. Think about the order in which you expect them to melt (use your table of
physical constants). Start at a low heat setting, observe the temperature for several minutes to
familiarize yourself with the apparatus, then adjust the setting so that the temperature rises at
a reasonable rate to about 10° C below the expected melting point range. (a reasonable rate
is approximately 1 every 5 seconds…rapidly enough so that time is not wasted but slowly
enough so that the temperature does not increase past the expected melting point range.) The
setting is then adjusted to achieve the desired temperature rise of 1/minute near the melting
point of the sample (this heating rate will seem very slow). Note the settings and temperatures
for your reference in future experiments.
Run additional trials to increase your confidence in your results if time permits.
• Special safety concerns
Keep al open vessels containing liquids in the fume hood.
There
will be many hot surfaces in the lab. Keep the Mel-Temps away from the flammable solvents
used for the TLC developing jars.
The melting point capillary tubes are potential sharps hazards.
MELTING POINT DETERMINATION and THIN LAYER CHROMATOGRAPHY
• Student notes
Use the chemical structure of each compound on your table of physical constants to make
predictions about what you expect to see before running each plate. Which compounds are the
most polar? Sketch the expected result in your notebook based on the relative polarity of each
compound, and compare your expectations with the actual results. This habit of predicting,
observing, and comparing will improve your understanding of chromatography. This habit will also
help you identify abnormal results.
The height of the sample in the melting point capillary tube should be about 2-3 mm.
• Prep/TA notes
Salvent for all spotting solutions is ethyl acetate.
Eluent is 1:2 hexane:ethyl acetate with 5% acetic acid.
0.5 L of 1:2 hexane:ethyl acetate with 5% (25 mL) acetic acid (i.e., 158 mL hexane + 317 mL
ethyl acetate + 25 mL glacial acetic acid).
Acetylsalicylic acid, acetaminophen, and caffeine spotting solutions are 2.5 g analgesic / 100 mL
ethyl acetate
The spotting solution for the mixture contains 2.5 g of each analgesic / 100 mL ethyl acetate.
Questions to test your understanding that will help you with your report and exams:
• Tabulate distance spot traveled, distance solvent traveled, and calculated R values for each
component (each spot visualized). Some lanes may have more than one component. Which spots
changed color in the iodine chamber?
. Compare your two TLC plates. Do the Rr values match your expectations for each drug? Do the R
values change going from the medicine extracts to the chemical-grade samples? Should they change?
• Do your TLC results match the expected contents for each over-the-counter drug? Explain your answer
using your tabulated TLC data.
Explain any anomalies that you observed in your TLC plates (e.g.streaking or large spots).
How does the melting point of the mixture in Part G compare with the melting points in Part F? Are
these differences expected? Why?
• Why is it important that spots are completely dry before developing the TLC plate? What might happen
if you developed your TLC with wet spots ?
• Why is it important to report melting points as ranges instead of a single temperature? How could you
use melting point to identify an unknown compound?
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Acetylsalicylic acid
L 134-1380
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