Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
4. Caffeine Isolation.
Wash the remaining ether layer in the separatory funnel by mixing with 10
mL of saturated NaCl.
Completely separate the layers and transfer the ether layer to a 50-mL
Erlenmeyer flask containing two spatula tips of anhydrous Na2SO4. Mix for
15 seconds.
Transfer the ether by carefully decanting it into a pre-weighed 25-mL
Erlenmeyer flask. Carefully remove the ether inside the fume hood using a
gentle stream of compressed air, as demonstrated by the instructor. This
should yield the pure neutral compound (caffeine) as a solid material.
Cover the flask with a piece of paper towel. Store inside the lab drawer for
subsequent weighing and TLC assay (Week 2).
5. Aspirin Isolation.
To the biphosphate extract (acid beaker), add enough 6M HCl to make the
solution acidic (this is usually 3-4 mL of acid). Verify this by using pH
paper.
Add 2 mL saturated NaCl. Mix.
Add the mixture to a separatory funnel. Add 10 mL of ether and extract as
before. Allow the layers to separate. Drain off the lower aqueous layer into a
beaker. Transfer the ether layer to a 50-mL Erlenmeyer flask containing
enough anhydrous Na2SO4 to cover the flask bottom. Mix for 30 seconds.
Return the aqueous layer to the funnel. Add another 10-mL portion of ether.
Repeat the above extraction, isolation and transfer process.
Make sure the combined ether extract is completely dried by noting that the
drying agent is no longer clumpy. If this is the case, add more drying agent
until clumping ceases.
Carefully decant the dried ether to a pre-weighed 50-mL beaker. Remove the
ether using a stream of compressed air. Allow the solid to air dry until the
following lab period (Week 2)
6. Acetaminophen Isolation.
To the hydroxide extract (phenol beaker) add enough 6 M HCl (this will
be at least 6-8 mL) until the extract is acidic . Verify this with pH paper.
Add 2 mL saturated NaCl. Mix.
Perform the extraction, isolation and drying procedure noted above for
aspirin. The acetaminophen can be isolated in a pre-weighed 30-mL beaker
and allowed to air dry until the following lab period (Week 2).
B. TLC: Pure Analgesic Compounds and Excedrin Components
Procedure:
Obtain a 2 x 5 cm TLC plate. Lightly draw a pencil line 1 cm from each
end of the plate. Lightly draw 2 hatch (x) marks, evenly spaced across
from the bottom (sample spotting) line. Label these marks S (standard)
and E (extract).
You will be provided with a stock solution containing a mixture of the pure
analgesic compounds. Using a separate micro-capillary pipette, deposit a
tiny amount (two 1-second applications, as demonstrated by the instructor)
of the standard on the S mark . On the E mark, spot four 1-second
applications of the ether solution of the soluble components of the Excedrin
tablet. View the undeveloped plate under the UV lamp to make sure that
there is enough of each material to see, but that the spots are not too large.
In a TLC developing chamber containing about 1 mL of a solution of 1:2
hexane:ethyl acetate with 1% acetic acid, carefully place the TLC plate.
Cover the chamber with the plastic cap and allow the solvent to ascend the
plate until it has reached the top pencil (solvent) line.
Remove the TLC plate and wait for 30 seconds to allow the solvent to
evaporate inside the fume hood.
Use the UV light to visualize your spots. Use a pencil to lightly trace the
location and size of the spots. Record a sketch (or photoimage) of your TLC
plate in your laboratory notebook. Calculate the Rf value of each spot (round
off to two decimal places). Dispose of the TLC plate according to the waste
disposal instructions.
Waste Disposal:
TLC solvent. Measure and record the volume of this solvent and place it in
the hazardous waste container.
Place the spotting micro-capillary pipettes in the designated waste glass
receptacle.
Place the TLC plates in the chemical paper waste container.
Note: the mortar and pestle must be cleaned as follows: (1) initially with
soap solution to remove inorganic residue; and, (2) finally with acetone to
remove organic residue and water. The acetone volume is recorded and
noted on the waste tag.
1. Tablet Preparation. Crush one Excedrin tablet using a mortar and pestle. To
the mortar, add 20 mL of diethyl ether and stir for 1 minute. Allow the solid binder
to settle (~ 1 minute). Carefully transfer the ether into a 50-mL beaker using a glass
pipette.
2. Column Preparation.
To the beaker containing the ether solution of the Excedrin components, add
a small amount of silica gel (approximately 0.5 mL, measured in a reaction
tube). Mix the contents for ~1 minute. In the fume hood, completely remove
the ether solvent using a gentle stream of compressed air, leaving a white
powder. Note: The solid MUST be broken up to a fine powder using a
spatula and exposed to the air stream for ~ 3 minutes.
From the Stockroom, obtain a glass chromatography column that has a frit
on the bottom. Attach the plastic conical funnel, using a short length of
rubber tubing, to the top of the column, and align the column vertically on a
ring stand using a clamp.
Add 3.0 g of silica gel to the column with gentle tapping, then enough sand
(~250 mg) to provide a cap that is 2-3 mm thick. Add the solids using
creased weighing paper, as demonstrated by the Instructor.
Add the powdered silica gel / Excedrin mixture from the beaker to the top of
the column, using a creased weighing paper.
In small covered beakers, obtain 15 mL of each of the following solvents:
1:1 hexane / ethyl acetate, 1:2 hexane / ethyl acetate, and acetone.
3. Column Elution.
Obtain six 10 mL Erlenmeyer flasks to collect fractions. Label them
Fraction 1 Fraction 6 and tare (pre-weigh) them before you start the
elution.
Detach the plastic conical funnel from the column.
Using a glass (Pasteur) pipette, add 1:1 hexane / ethyl acetate to the column
in increments of ~1 mL until eluate begins to come off the column, then fill
the column to the top with this solvent. Important: Do not allow the solvent
top to reach the solid packing, as this may create air pockets in the packing
bed which will result in poor separation efficiency. Do not allow the elution
solvent to travel beyond ~1 from the column top. If the packing settles and
develops air pockets during normal elution, use a wooden stick to release
the air and press the solid packing together.
Collect the first 10-15 drops of the eluate in a waste beaker, then begin
collecting two 5-mL fractions in the Fraction 1 and Fraction 2 flasks.
Caution: To prevent formation of solid on the column tip due to solvent
evaporation, place the column tip halfway into the collecting flask. Allow
the initial waste eluate to evaporate inside the fume hood.
At this point, change the elution solvent to 1:2 hexane / ethyl acetate and
collect two additional 5-mL fractions in Fraction 3 and Fraction 4
flasks.
Finally, change the eluent to acetone and collect the final 5- mL fractions in
the Fraction 5 and Fraction 6 flasks.
4. TLC Analysis of Fractions: TLC solvent 1:2 hexane / ethyl acetate with
1% acetic acid (provided for you in the hood read the bottle labels carefully!).
Prepare two 2 x 5 cm TLC plates as done previously for the pure analgesic
compounds and tablet. On the first plate, place three x marks on the
spotting line and label them 1,2, and 3. On the second plate, place three x
marks and label them 4, 5, and 6. These numbers correspond to the
chromatography column fraction numbers. Two 1-second applications of
each fraction sample to the plates should suffice.
Before you develop the plates, examine them under the UV light to see if
you have spotted enough sample on them.
Place each plate carefully into the TLC developing chamber a 100-mL
beaker to which 1-2 mL of TLC solvent has been added. Cover the chamber
immediately with aluminum foil and allow the solvent to reach the top
(solvent) line.
Remove the plates from the chamber and allow them to dry 30 seconds
inside the fume hood before examining them under the UV light.
Record a sketch (or photoimage) of each TLC plate in your laboratory
notebook, and calculate the corresponding Rf values (to two decimal places).
5. Isolation of Solids.
After spotting a specific elution fraction on the thin-layer chromatographic
plate, evaporate the solvent in the hood using a gentle stream of air, as
demonstrated by your instructor. Cover the flask with a piece of paper towel
and place inside the drawer to dry.
Next week (Week 3): you will determine the weight of the solid obtained for
all fractions. From the combined total weight of the eluted solids, you will
determine the % recovery of your total initial solids (aspirin, acetaminophen,
and caffeine), based on the amount of the three compounds present in the
original tablet.
Waste Disposal:
Used capillary and Pasteur pipettes are placed in the waste glass container.
TLC solvent volume must be measured , recorded on the waste slip, and
placed into the waste container.
Any excess solvent (hexane, ethyl acetate, acetone) is measured and
recorded on the waste slip and then placed in the hazardous waste container.
To clean the elution column, run compressed air through the top of the
column to dry the packing (~ 2 minutes). Then, run air through the tip end to
blow the silica gel packing into the waste bucket inside the waste fume
hood. Record the amount (~ 3.0 g) on the waste tag. Rinse the column with
warm tap water followed by an acetone (~ 5 mL) rinse; return the column to
the Stockroom.
Report:
Complete the report sheet for this experiment. Be sure to include the separate sheet
containing the sketched / photoimaged TLC plates and corresponding Rf value
calculations.