Experiment 7:
RECRYSTALLIZATION,
MELTING POINT, and
HPLC ANALYSIS of ANALGESICS
Objectives

To learn the techniques of recrystallization
and melting point analysis.

To purify aspirin and acetaminophen
samples isolated by extraction using
recrystallization.

To evaluate the purity of samples by
melting point and HPLC analysis.
BEFORE COMING TO LAB…

Watch the following videos:

Recrystallization
http://www.wonderhowto.com/how-to/video/how-to-illustrate-recrystallization-inorganic-chemistry-271753/

Melting Point Analysis
http://www.wonderhowto.com/how-to/video/how-to-measure-melting-points-in-thechemistry-lab-259806/view/
EFFECT OF TEMPERATURE ON
SOLUBILITY
Solubility vs Temperature
Solubility (g/L)
30
25
20
15
10
5
0
0
LOW TEMPS: the
substance is much less
soluble, however some of
the substance will be
dissolved.
20
40
Temperature (ºC)
60
80
HIGH TEMPS: the
substance is much
more soluble.
ACTIVE INGREDIENTS IN
GOODY’S POWDERS
O
O
H
H 3C
O H
O
N
CH
3
O
OH
A s p irin
A ce tylsa licylic a cid
4 -a ce ta m id o p h e n o l
M F: C9H8O 4
M W : 1 8 0 .2 g /m o l
m p : 1 3 8 -1 4 0
A c e ta m in o p h e n
o
M F : C 8H 9N O 2
C
M W : 1 5 1 .2 g /m o l
o
H a za rd s : T o xic , Irrita n t
m p 1 6 9 -1 7 2 C
S o lu b ility in H 2 0 :
H a z a rd s: T o xic, Irrita n t
o
1 g in 1 0 0 m L a t 3 7 C
o
1 g in 3 0 0 m L 2 1 C
o
1 g in 4 0 0 m L 1 5 C
o
~ 1 g in 5 5 0 m L a t 0 C
S o lu b ility in H 2 0
o
1 g in 7 0 m L a t 3 1 C
o
1 g in 1 5 0 m L a t 2 1 C
PERCENT RECOVERY
• Percent recovery: an indication of how much of the active
ingredient you were able to recover after the purification
method.
• HPLC Area %: the percent of the active ingredient present in
the sample submitted for analysis.
• The percent recovery of compound B is calculated by:
% R e co ve ry o f B =
M a ss o f B re co ve re d
M a ss o f B use d initia lly
x 100
OVERVIEW—ASPIRIN

Reweigh aspirin + filter papers from last lab.

Subtract out filter paper weights to get ACTUAL RECOVERY OF ASPIRIN.

Prepare 2 melting point capillaries of the EXTRACTION
ASPIRIN.

Dissolve aspirin in hot ethanol, then hot water, then few
drops of hot ethanol.

Cool to room temp, then in ice for 15 minutes.

Suction filter.

Prepare HPLC sample and submit for analysis.

Secure small filter paper + aspirin in larger filter paper
and submit.

No final weight or melting point sample until next lab!
OVERVIEW—ACETAMINOPHEN

Transfer acetone/acetaminophen solution from
vial to beaker and evaporate acetone completely.

Dissolve acetaminophen in hot water (drop-wise).

Cool to room temp, then in ice for 15 minutes.

Suction filter.

Prepare HPLC sample and submit for analysis.

Secure small filter paper + aspirin in larger filter
paper and submit.

No final weight or melting point sample until next
lab!
Table 7.1:
Experimental Results
Aspirin
Acetaminophen
* Theoretical recovery after extraction (g)
• Same # as
Table 6.1
• Same # as
Table 6.1
*Actual recovery after extraction (g)
• Same # as
Table 6.1
• Same # as
Table 6.1
*% Recovery after extraction
• Same # as
Table 6.1
• Same # as
Table 6.1
 Same as…
 Same as…
• Obtain weight
during next lab
• Obtain weight
during next lab
Actual
Theoretical
Actual
Theoretical
Theoretical recovery after recrystallization (g)
Actual recovery after recrystallization (g)
% Recovery after recrystallization
• % Recovery is ≠ HPLC Area %
X 100
X 100
OVERVIEW—
MELTING POINT ANALYSIS

Prepare a melting point capillary containing the
provided impure aspirin.

Perform a melting point analysis on this along with
the EXTRACTION ASPIRIN prepared above.

Begin MelTemp on setting of 4. Back down temp
slightly as thermometer approaches 20o C from the
expected melting point (lit value).

Record melting range (Ti-Tf).
EXPERIMENTAL PROCEDURE
(Melting Point)

Dip the open end of a mp capillary tube into
the sample to be tested.

Invert the capillary tube and tap it on the
bench top to pack the sample into the
closed end.

Insert the capillary tube into one of the slots
in the heating block of the Mel-Temp
melting point apparatus.
EXPERIMENTAL PROCEDURE
(Melting Point)
• Turn the Mel-Temp power on and
adjust the temp control to the desired
rate of heating.
• In order to obtain an accurate mp, it
is necessary to heat SLOWLY (rate of 23oC/min).
Thermometer
Sample slot
Eyepiece
• SOURCES OF ERROR:
• Heating too fast = inaccurate mp (hard to
see 1st crystal melt!)
• Too much sample = broadened mp range
(takes a long time to melt entire sample!)
Power
Temp control
Table 7.2:
Melting Point Results
Aspirin
Acetaminophen
This lab!
---
Impure Aspirin preparation
Ti-Tf
This lab!
---
Extraction sample melting range (oC)
Ti-Tf
Next lab!
Next lab!
Ti-Tf
Ti-Tf
139-140
169-172
Recrystallized sample melting range (oC)
Literature melting point value (oC)
Table 7.3
Recrystallization HPLC Results
HPLC retention times (min) and area %
Sample
Compound
Standards
Rt
(min)
Aspirin
Sample
Chromatogram
Rt
(min)
area %
Acetaminophen
Sample
Chromatogram
Rt
(min)
area %
Aspirin
Acetaminophen
•Samples containing a single compound are considered PURE samples, while those
containing both compounds are considered MIXED (IMPURE) samples.
• Be sure to attach both sample chromatograms to final lab report!
• ANY HPLC SAMPLE CONTAINING VISIBLE SOLID WILL BE DISCARDED. Student
will receive a penalty for not providing a sample.
SAFETY CONCERNS
• Ethanol and reagent acetone are both extremely
flammable. Keep away from open flames, and use
extreme caution when applying heat!
WASTE MANAGEMENT

Place all liquid waste from recrystallization in
container labeled “LIQUID ORGANIC WASTE”.

Used melting point capillaries should be thrown
in the broken glass box, NOT the trashcan!

Any student leaving the melting point capillary tubes in
the MelTemp apparatus will receive a technique grade
penalty!
IN LAB QUESTIONS…
(The following questions should be answered in laboratory notebook.)
The UV detector on the HPLC instrument detects
compounds that absorb UV light in the range of 254 nm.
Compounds containing multiple bonds and aromatic rings
can typically be detected at this wavelength. Which of the
following compounds would you expect to be detected
and appear in the HPLC chromatogram of your sample?

O
O
H
N
O
O
OH
O
OH
H
H
O
O
OH
IN LAB QUESTIONS…
(The following questions should be answered in laboratory notebook.)

In some instances, a sample will appear to be pure
based on HPLC analysis; however the experimental
melting point will be lower than expected. Give one
possible source of this inconsistency and explain why
it occurs.