Experiment # 3: Separation of Analgesic Tablet Components by

Two-Base Extraction and Column Chromatography; Purity

Analysis by Melting Point and Thin-Layer Chromatography
__________________________________________________________
Pre-Lab Mohrig Readings (Techniques): (1) pp. 147-149 (Acid-Base
Chemistry); (2) pp. 142-145, 161-162 (Extraction); (3) 163-169 (Drying Organic
Liquids); (4) 270-286 (Column Chromatography); (5) 256 269 (Thin-Layer
Chromatography); (6) 212 220 (Melting Points)
Experimental Objectives / Background
This is a three-week laboratory sequence involving the isolation of the three active
components of a commercial analgesic, such as Excedrin or its generic equivalent.
The components aspirin, acetaminophen, and caffeine- will be isolated by two
different methods: two-base extraction (Week 1) and column chromatography
(CC) (Week 2). The purity of the separated compounds will be assessed using thinlayer chromatography (TLC) and melting point ranges. A single tablet of extrastrength Excedrin contains three active compounds: aspirin a carboxylic acid
(250 mg), acetaminophen a phenol (250 mg), and caffeine (65 mg). In addition,
Excedrin contains a starchy material that is used to bind the active components
together. The three components differ in their acidity, with aspirin being the most
acidic (pKa 3.5), acetaminophen second (pKa 9.9), while caffeine is effectively not
acidic. The differences in acidity and corresponding polarity can produce
separation using two-base extraction and TLC. Note: Because of the unique
physical and chemical properties of each analgesic ingredient, quantitative
separation by either of the two extraction methods is not optimal. Therefore, the
main objective of the experiment is to determine which of the two extraction
methods (two-base extraction / column chromatography) provides the better
outcome according to the current protocol.
Safety Precautions
Diethyl Ether: Highly flammable; vapors have anesthetic effects when
inhaled.
KOH: Can cause severe skin irritation and eye damage.
K2HPO4: Can cause mild skin irritation.
Saturated NaCl: Mild skin irritant.
Na2SO4: Mild skin and nasal irritant.

 Hexane: Highly flammable; skin irritant; harmful if inhaled.

Ethyl Acetate: Mild inhalation hazard; skin irritant.
Silica Gel / Alumina: Dust particles are moderate nasal and skin irritant.

Part A (Week 1): Separation by Two-Base Extraction / TLC of Tablet

Components
A. Two-Base Extraction. This method employs liquid-liquid extraction, using
aqueous base solutions of different strengths to selectively extract the acidic
components aspirin and acetaminophen.
Procedures:
1. Tablet Preparation. Crush one Excedrin tablet into a fine powder using a
mortar and pestle (obtained from Stockroom).
To the mortar, add 25 mL of diethyl ether and stir for ~ 1 minute using the
pestle. While the three active components will dissolve, the starch binder
will not. Allow ~ 1 minute for the solid binder to settle.
To remove the binder and isolate the ether, carefully extract the ether from
the mortar using a glass pipette and transfer to a 50-mL Erlenmeyer flask.
Immediately use this extract for TLC analysis (described on the next page).
Note: At this point in time, one member of the student pair should complete
the TLC assay while the other member continues the current extraction
procedure.
Once the ether layer has been spotted on the TLC plate, the ether extract is
transferred into a separatory funnel. Use a plastic funnel for this transfer to
minimize spillage / loss of solvent.
2. Aspirin Extraction. To the separatory funnel, add 5 mL of 1 M K2HPO4.
Perform the extraction using the technique demonstrated by the instructor.
Allow the layers to separate, and completely drain the lower aqueous layer
into a 30-mL beaker and set aside. Label this beaker acid.
Repeat the aqueous extraction as described above using a fresh 5-mL portion
of 1 M K2HPO4. Add the aqueous extract to the acid beaker.

3. Acetaminophen Extraction. To the ether layer remaining in the separatory

funnel, perform two aqueous extractions using 5 mL of 1 M KOH for each
extraction. For each extraction, add 2 mL saturated NaCl along with the KOH to
minimize emulsification during extraction. Label the combined KOH extracts in
the beaker as phenol.

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.

Part B (Week 2): TLC Purity of Aspirin and Acetaminophen; Column

Chromatography
A. TLC Purity of Aspirin / Acetaminophen
Procedure: Note: One student from the group can perform this procedure while
the other student begins the column chromatography procedure.
Obtain and record the mass values for the dried isolated solids from Week 1
(aspirin, acetaminophen, caffeine). Based on the amounts of each compound
in the original tablet, calculate the percent recovery.
Place a small portion of each solid sample into a small glass vial and add
enough ethanol (3-5 drops) to dissolve the solid.
As described for Week 1, prepare and spot a TLC plate with each of the
dissolved solids along with a spot of a mixture of the three analgesic
components. Allow the plate to develop using the same TLC solvent as
before.
Upon UV analysis, calculate the corresponding Rf values for each observed
spot. Provide a sketch or photoimage of this TLC plate.
B. Column Chromatography (CC) / TLC Assay.
Procedure:

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.