Determination of an Unknown by Extraction, Crystallization, Thin Layer
Chromatography, Melting Point, and Spectroscopy.
Megan Yap
California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, CA 93405, United States
Abstract: Over the course of several weeks an unknown sample was analyzed and its components were
identified. This was accomplished using the lab techniques of extraction, crystallization, IR and 13C NMR
spectroscopy, melting point analysis, and thin-layer chromatography. During the extraction experiment, the
acidic and neutral components were isolated from each other. The neutral component was identified through IR
and 13C NMR spectroscopy. The acidic component was purified during crystallization and identified during
melting point analysis. Finally, TLC was used to confirm the identity of the unknowns. The acidic component
was identified as p-toluic acid and the neutral component was identified as isobutyrophenone.
 Introduction
An unknown organic compound containing both an
organic acid and a neutral organic component was
analyzed. Chemically active extraction was
performed on the unknown to separate the two
components. Extraction is a separation process in
which a solvent is introduced that is intended to
retrieve the desired material. An example of
extraction is the process of preparing coffee; water is
used as the extracting solvent in order to retrieve the
desired material (caffeine). An example of washing
is the process of washing clothes; water is used as
the washing solvent but in this case, its purpose it to
remove the undesired material (dirt, grease, ect.).
Crystallization is the most common technique for
purifying organic solids. A suitable solvent was
found for crystallization of the unknown acidic
component and the acidic component was purified
by crystallization. Solvents are chosen based a
certain criteria. The best solvents dissolve a small
amount of solid at room temperature, dissolve a large
amount when heated, do not react with solid, have
high boiling points, and relatively volatile.
IR and 13C NMR spectroscopy are used to identify
functional groups and derive structural information
on the isolated neutral component a molecule from
the formula give; C10H12O.
Melting point analysis was performed to identify the
unknown acidic component. The technique called
“mixture melting point” was performed on mixtures
of the unknown and two to three different acids that
had melting points close to that of the unknown.
Thin-layer chromatography was performed on the
neutral component, the acidic component to see if
you separated your compound correctly.
 Results
The reaction that was occurring in the initial
extraction lab is as follows:
Chemically Active Extraction. The percent
recovery for the neutral component was 82.19%. The
percent recover for the acidic component was
67.92%. The percent recovery could have been
negatively affected as a result of human error by not
fully extracting the basic compound during the
extraction steps.
Table 1. Percent Recovery of Neutral Compound
Percent Recovery
Extraction
Neutral
82.19%
Acid
67.92%
Crystallization. At room temperature, the solute in
the tube containing isopropyl acetate, heptane, and
toluene dissolved. All tubes were heated in a hot
water bath. Upon heating, the tubes isopropanol,
heptane, toluene, isopropyl acetate, and 50:50
ethanol/water dissolved the solute. The tubes were
cooled to room temperature and only 50:50
ethanol/water fully crystallized. Since the unknown
did not dissolve in 50;50 ethanol water at room
temperature, but did dissolve upon heating and
crystallize upon cooling, 50:50 ethanol/water was
chosen as the best solute for crystallization of the
unknown. The percent recovery for the unknown
crystallization was 69.81%. This percentage could
have been affected by the loss of a small amount of
sample during the boiling step.
Table 2. Percent Recovery for Crystallization
Percent
Crystallization
Recovery
Unknown
69.81%
Table 3. Solvents Used in Crystallization
Solvent
Room
H20 Bath
Ice Bath
Temp.
()
H20
Solid
Solid
Did not
crystallize
Isopropanol
Solid
Dissolved
Did not
crystallize
Heptane
Precipitate Dissolved
Did not
crystallize
Toluene
Precipitate Dissolved
Did not
crystallize
Isopropyl
Dissolved Dissolved
Did not
Acetate
crystallize
MethylcycloSolid
Solid
Did not
hexane
crystallize
50:50
Solid
Dissolved Crystallize
Ethanol/Water
13C
IR and
NMR Spectroscopy. The unknown has
5 units of unsaturation, which was determined from
the molecular formula C10H12O. This suggests that
the unknown contains a benzene ring. The IR graph
shows a peak at 699.29, which suggests the benzene
ring has a mono substituted pattern. The IR graph
also has peaks at 2973.98 and 2932.42, which means
it has C-H bonds, and a peak at 1680.34, which
means the C=O bond is conjugated.
The 13C NMR graph showed peaks at 18.245 and
34.239. These peaks indicate that the unknown
contains CH2 and CH3 bonds. The 13C NMR graph
also showed 4 peaks (1s, 1m, 2 t) in the C=C region
(from 100-150) which indicates a mono substituted
benzene ring. The IR graph indicates mono as well
so the unknown must contain a mono-substituted
benzene ring. Lastly, there is a peak on the 13C NMR
graph at 202.274, which indicates that the unknown
contains a ketone and since there is no peak on the
IR graph between 2700 and 2800, the unknown must
contain a ketone, not an aldehyde. All this
information combined indicates that the unknown
neutral component is isobutyrophenone. The SDBS
website confirmed the identity of the unknown. The
refractive index temperature correction was 1.5136
and after temperature correction, was 1.5176. This
matches the refractive index of isobutyrophenone,
which has a refractive index of n20 = 1.517.
Table 4. IR Spectrum Results
IR (cm-1)
Possibilities
2973.98
C-H bonds
2932.42
C-H bonds
1680.34
C=O bond.
Conjugated
699.29
Mono?
Table 5. 13C NMR Results
13
C NMR
a. 202.274
b. 135.518
(Small)
c. 131.885
(Medium)
d. 127.864 (Tall)
e. 127.499 (Tall)
f. 34.239
g. 18.245
Possibilities
Ketone
Benzene
Benzene
Benzene
Benzene
C-H
C-H
Table 6. Rf Values of Different Solvents
Rf
Methanol Ethyl
25%
Values
acetate
ethyl
acetate/
75%
heptane
Acidic
0.72
0.58
0.17
Neutral
0.75
0.67
0.52
Heptane
0
0.11
Figure 1. Isobutyrophenone.
Melting Point. The melting point of the unknown
acid compound was between 177C-182C. The
mixture of unknown and p-aminobenzoic acid
melted between 132C-140C. These temperatures
were nowhere near the unknown melting point.
However, the mixture of unknown and p-toluic
acid melted right in the range of the unknown’s
melting point (173C-178C). Based on these
results, the unknown acid was determined to be ptoluic acid.
Figure 2. P-toluic acid.
Thin Layer Chromatography (TLC). The further
the unknown travelled, the greater the Rf value is,
and the more polar the compound is in relation to the
TLC plate. The most significant chromatography
reading for the identification of the unknown came
from the 25% ethyl acetate/ 75% heptane plate. This
TLC plate had the best separation between the
neutral and acidic components. The Rf value for the
acidic component was 0.17 and the Rf value for the
neutral component was 0.52. The unknowns on the
methanol plate all moved very far because methanol
is a very polar solvent. The unknowns on the heptane
plate didn’t travel at all because heptane is nonpolar.
 Experimental
Chemically Active Extraction. The general
procedure was followed using ethyl acetate, NaOH
and MgSO4. The unknown number 145 was obtained
and a sample was weighed by difference. The weight
of the sample was 3.71 g. The majority of the
unknown sample was transferred to a 125 mL
Erlenmeyer flask and dissolved in 40 mL ethyl
acetate. The dissolved solution was then transferred
to a separatory funnel and an additional 10 mL ethyl
acetate solution. The 10 mL rinse and an additional
20 mL of 5% aqueous NaOH was also added to the
separatory funnel. The basic aqueous layer was
drained into a 250 mL beaker. The extraction was
repeated twice more with 20 mL of 5% aqueous
NaOH.
Approximately 30 mL 10% aqueous HCl solution
was added to the basic extraction and the solution
turned acidic. The resulting mixture was chilled in an
ice bath. Vacuum filtration was performed to collect
the solid from the now acidic solution.
The ethyl acetate solution remaining in the
separatory funnel was washed with 15 mL deionized
water. The water was drained into a 50 mL
Erlenmeyer flask and the ethyl acetate solution was
poured into a clean, dry 125 mL Erlenmeyer flask.
The ethyl acetate solution was dried with MgSO4,
and the dried solution was filtered through fluted
filter paper into a clean and dry 100 mL roundbottomed flask that weighed 61.78 g. The solvent
was removed using the rotary evaporator. Traces of
solvent were removed using a high vacuum line. The
percent recovery for the neutral component was
82.19%. The percent recover for the acidic
component was 67.92%.
Crystallization. A little amount of the unknown
acidic component was placed in each of seven
separate tubes. Each tube contained 10 drops of a
different solvent. Water, isopropanol, heptane, and
toluene were the first solvents used. These solvents
did not crystallized at room temperature. The second
round of solvents used were isopropyl acetate,
methylcyclohexane, and 50:50 ethanol/water. The
50:50 ethanol/water test tube was placed in an ice
water bath and the unknown crystallized. The
unknown acid was placed in a 25 mL Erlenmeyer
flask. Just enough 50:50 ethanol/water was added to
cover the unknown. A 50 mL Erlenmeyer flask with
25-30 mL of 50:50 ethanol/water was prepared. The
solvent in both flasks was brought to a gentle boil on
a hot plate. The boiling 50:50 ethanol/water was
added to the unknown a few mL at a time, until the
solid dissolved. The solution was cooled to room
temperature, and crystals formed. The solution was
placed in an ice-water bath to cool even further. The
crystals were then removed by vacuum filtration.
The percent recovery for the unknown crystallization
was 69.81%.
IR and 13C NMR Spectroscopy. IR and 13C NMR
spectroscopy was performed on the isolated neutral
component and the graphs were analyzed to identify
the neutral component. The important peaks in my
IR spectroscopy were at: 2973.98 cm-1, 2932.41 cm1
, 1680.34 cm-1, and 699.29 cm-1.
The peaks on the 13C NMR graph were at:
202.274ppm, 135.518ppm, 131.865ppm,
127.865ppm, 127.499ppm, 34.239ppm, and
18.245ppm.The refractive index before temperature
correction was 1.5136 and after temperature
correction was 1.5176.
Melting Point Analysis. Melting point analysis was
performed on the unknown acidic component. A
crude melting point of the unknown was taken to get
a rough idea of the melting point range 178C187C. The melting point of the unknown was
repeated for a more precise value 177C-182C. The
two acids that had melting points closest to the
unknown’s melting point (p-toluic acid and paminobenzoic acid) were chosen as the best
unknowns to be used in the “mixture melting point”
experiment. A 50:50 mixture of each of these two
acids and the unknown was prepared and the melting
points of these two mixtures was determined. The
mixture of the unknown and p-aminobenzoic acid
melted between 132C-140C. The mixture of the
unknown and p-toluic acid melted between 173C178C, and p-toluic acid was determined to be the
unknown.
Thin Layer Chromatography (TLC). Thin-layer
chromatography was performed on the neutral
component, the acidic component, and the original
unknown to confirm the identities that were
determined by melting point analysis lab and the IR
and 13C NMR spectroscopy lab. Three separate
samples were prepared: 0.01 g of the acidic
component was diluted in 10 mL of acetone, 0.01 g
of the original unknown was diluted in 10 mL of
acetone, and 1 mL of the neutral component was
diluted in 10 mL of acetone. Each plate was placed
in a capped jar, filled a 10 mL deep with it’s
respective solvent. The solvents traveled up the
plates at different rates, and when each solvent
reached the top line in the plate, that plate was
removed from the jar. The spots showed ultravioletactive compounds. See Table 6 for the Rf values of
the four different solvents.