CHM 112 Lab Procedure 2:

THIN-LAYER CHROMATOGRAPHY
Objectives:
(a) to investigate the separation of mixtures with thin-layer-chromatography and
calculate Rf values
(b) to demonstrate the presence of at least five colored compounds in extract of
spinach leaves
(c) to determine the presence of vanillin and/or ethyl vanillin in pure vanilla extract
and imitation vanilla flavoring
(d) to determine the composition of various over-the-counter analgesics

Introduction:

Since the investigation of chemically pure materials has provided the basis for
understanding chemistry, it is critical that chemists be able to separate mixtures of
substances as they are often found in nature. Biochemistry relies heavily upon methods
of separation of pure chemical substances from the very complex mixtures present in
living materials. A great deal of effort has gone into developing analytical methods that
can be used to separate mixtures into their component pure substances. Often the
science of chemistry advances as the techniques of separation are improved.

Chromatography is a very important separation technique, and one type of

chromatography is thin-layer chromatography (abbreviated TLC). You will be using
TLC in this procedure to separate several different types of mixtures.

All forms of chromatography depend upon the distribution (or separation) of solute
particles between a moving phase (a gas or liquid; you'll be using a liquid moving phase
in this procedure) and the stationary phase (a liquid or solid; in this procedure you will
be using a solid stationary phase. The solid phase is solid silica particles that have been
applied to a plastic or aluminum support plate). When solute particles in a mixture
have a different size or charge, they have a different affinity for the stationary phase
and will therefore move at different rates through the stationary phase. Those with low
affinity will have weak interactions with the stationary phase and will therefore spend
most of the time in the moving phase. They will move quickly through the stationary
material. Those solutes that have a strong affinity for the stationary phase will adhere
more to the stationary phase and will move slowly through the stationary phase.

In TLC, the stationary phase consists of a thin layer of adsorbent (usually silica gel
consisting of partially hydrated SiO2 – a type of sand) combined with a small amount of
gypsum (CaSO4.H2O) as a binder. A backing, either glass, plastic or aluminum supports
this adsorbent. The mixture of compounds to be separated is dissolved in a volatile
solvent and a small spot is carefully spotted close to one edge. This is called the origin
line. This spot should be as small in diameter as possible, approximately 1-2 mm in

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diameter. This prevents the developed spots from streaking and tailing. When the
sample spots are thoroughly dry, the sheet (called the chromatogram) is placed in a
chamber (a jar or tank) with a shallow layer of developing solvent at the bottom.
The edge of the chromatogram with the spots applied will sit down into the developing
solvent, which must not be so deep as to submerge the origin line, or the applied spots
will wash out into the solvent tank. The developing solvent rises up the adsorbent layer
in a front, drawn up by capillary action. As the solvent passes the origin line, the
sample compounds begin their migration up the chromatogram. When the solvent
front has reached almost the top of the chromatogram (which takes different amounts
of time depending upon the separation), the chromatogram is removed from the tank
and allowed to dry. Then the migrated spots must be located and identified.

If the compounds being separated have visible colors, the spots are easy to find (you
will notice this in the spinach juice separation). But because most organic compounds
are colorless (such as the vanillin and the analgesic drugs), another means of spot
detection must be employed. One method is to mix a fluorescent compound (such as
ZnS or CdS) into the adsorbent, so that the whole sheet will fluoresce under UV light.
But the spots of separated material will quench this fluorescence, and spots will appear
as dark, non-fluorescent spots against a fluorescent background when the sheet is
viewed under UV light. Then these dark spots can be marked with a pencil. Other
times, the compounds will themselves fluoresce, so that these will appear as brightly
fluorescent spots against the faint fluorescence of the background chromatogram, and
these spots can also be marked with a pencil. Finally, the chromatogram can be
stained with a reagent that can react with some of the spots and make them visible.
Iodine vapor works well for this purpose (you will use it in this procedure for both the
vanillin and the analgesic drugs).

Because each chromatogram will be developed under slightly different conditions

(slightly more or less solvent, slightly different temperatures, differing size and
concentration of the origin line applications), the general practice in TLC is to place
known and unknown compounds on the same chromatogram. The unknowns can then
be easily identified by comparison with the known samples that have been run under
the exact same conditions on the same chromatogram. A few characteristics that
remain consistent from run to run can be used to compare different chromatograms.
The generally accepted method for making such comparisons is to compute the Rf
value (meaning "Relationship to the Front") of the spots. This is easily calculated by:

Eq. 1:
Rf = distance traveled by the spot from the origin line (cm)
distance traveled by the solvent front (cm)

(Note that a more polar compound will more strongly adsorb to the polar
stationary phase and will have a lower Rf value)

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The means, therefore, available to identify the components of a TLC mixture are:

• Migration identical to that of a standard or reference (known) compound

• Intrinsic fluorescence like that of a reference compound
• Staining behavior like that of a reference compound (Staining characteristics may be
concentration-related. Therefore, a spot of lower concentration may have a lighter
color than a spot of higher concentration)
• The Rf value that is identical to that of a reference compound. This Rf value may be
obtained from direct measurement or obtained from literature.

These criteria must be met in order to identify an unknown spot by TLC!

In this procedure, you will doing separations of several different mixtures –

(a) the colored components of spinach,
(b) the components of vanilla and imitation vanilla flavoring and
(c) solutions of over-the counter analgesic (pain-relieving) drugs.

Part A: Separation of Plant Pigments

The characteristic green color of all leaves comes from chlorophylls, which are
responsible for the absorption of solar energy that maintains all life on the planet. The
structures of chlorophyll a and b are shown in Figure 6.1. Look at these structures –
the arrangement of alternating single and double bonds (which, you may remember, is
an aromatic arrangement) in these chlorophylls serves to provide the optimal
arrangement of electrons to interact with and absorb visible light.

Figure 5.1: Chlorophylls a and b

HC CH2 CH3 H O
C
CH2CH3
N N
Chlorophyll b
Mg

N N
H
CH3

O H3C
H
C
H2 H
C H 2C
O O
O C
C20H39
O CH3

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 The zeaxanthin molecule, shown in Firgure 5.2, is a second category of plant pigments,
known as the xanthophylls. They generally have yellow colors and assist the
chlorophylls in absorbing light.

Figure 5.2: a representative xanthophyll

CH3
H3C OH
CH3 CH3 CH3

CH3 CH3 CH3 CH3

HO
CH3

Zeaxanthin -- a xanthophyll

The third type of pigment, the pink-orange carotenoids, resembles the xanthophylls in
structure (see Figure 5.3) – they only lack the two -OH groups at either end of the
molecule that the xanthophylls have. But the carotenoids have the added function in
that they can be converted into vitamin A (Figure 5.4), so the carotenoids are of great
nutritional significance. Notice that vitamin A is almost the same as the β-carotene
molecule split in half with an -OH added at the split point.

Figure 5.3: β-carotene – a representative carotenoid

CH3
H3C
CH3 CH3 CH3

CH3 CH3 CH3 CH3

CH3

beta-carotene

Figure 5.4 – Vitamin A

CH3 CH3 CH3
CH2OH

CH3
CH3

Vitamin A
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Part B: Separation of Vanillin and Ethyl Vanillin

In this portion of the procedure, you will be analyzing pure vanilla extract and imitation
vanilla flavoring for the presence of vanillin and ethyl vanillin, both of which may be
components of vanilla beans.
O O H
H
C C

OCH3 OCH2CH3

OH OH

Figure 5-5: Figure 5-6:

vanillin ethyl vanillin
Vanilla extract is an alcohol and water solution of the substances in vanilla beans.
Imitation vanilla also contains some of the same compounds, but it is made from lignin,
a waste product of the wood pulp industry. Both solutions, vanilla extract and imitation
vanilla flavoring, contain the same molecules – vanillin and ethyl vanillin – but natural
vanilla costs about twice as much to produce as imitation vanilla. It is not surprising
that natural vanilla extract and imitation vanilla flavoring contain the same compounds;
chemical logic knows that vanillin and ethyl vanillin from vanilla beans are exactly the
same molecules as the vanillin and ethyl vanillin that come from a vat of wood pulp
(although the wood pulp thing seems a little less appetizing, doesn't it?). The
difference between pure vanilla and artificial vanilla flavoring is what isn't there – the
beans have many additional, different molecules that work together to make the flavor
of pure vanilla what it is (vanillin and ethyl vanillin are the MAIN components). The
artificial flavoring has just the major molecules that are responsible for the flavor.

One interesting fact: chemical testing can tell the difference between the two
flavorings. The test concerns measuring carbon isotopes in the two solutions. The
ratio of carbon-12 to carbon-13 in vanilla made from vanilla beans is different from the
ratio in flavoring made from lignin. So, it can be determined if the solution in the bottle
is actually what the label says it is. Then you can be sure that you ARE getting exactly
what you pay for! A triumph for chemistry!

Looking at the structures of vanillin and ethyl vanillin presented here, the one that has
the most non-polar bonds (the most number of non-polar C-H and C-C bonds) would

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be considered to be the more non-polar molecule of the pair. Which has the lowest Rf
value – the more polar compound or the more non-polar compound?

Part C: Separation of Analgesic Drugs

You will be using TLC to separate and identify the components of some familiar
mixtures of organic compounds – over-the-counter analgesic drugs. Several of these
compounds have related structures, but are different enough so that they can be
separated.

Aspirin is acetylsalicylic acid – made from the esterification reaction of salicylic acid
with acetic anhydride. It was first used in ancient times, when the Greek physician
Hippocrates had his patients chew willow bark to ease pain and reduce fever. It was
later discovered that willow bark contains salicin, a derivative of the molecule we know
as aspirin today. Aspirin is an analgesic (pain reliever) as well as an antipyretic (fever
reducer). Its action in both cases, analgesic and antipyretic, is due to the ability of
aspirin to inhibit prostaglandins, which are chemicals in the body involved in sending
pain messages to the brain. Any analgesic doesn't really stop the ache or pain; an
analgesic just keeps the brain from hearing about the pain. Aspirin also can inhibit the
clotting of blood, so sometimes it is prescribed for lowering the risk of heart attack and
stroke (cerebral vascular accidents) by lowering the formation of blood clots that can
dislodge and plug arteries or veins in the heart and brain.

Acetaminophen is structurally related to aspirin, and it is an analgesic and an

antipyretic, but does not reduce inflammation like aspirin does. It also does not
promote gastrointestinal upset like aspirin does, so it can be taken in larger doses over
longer periods of time.

Caffeine is often found in preparations of over-the-counter analgesics, but there is

little evidence that caffeine enhances the effects of aspirin. Caffeine is a mild stimulant.

Salicylamide is also structurally related to the aspirin, and is sometimes used in

combination with aspirin.

Ibuprofen is also an analgesic (but not used in this procedure). It only bears a slight
structural resemblance to aspirin, but may be superior to aspirin in the reduction of
inflammation. It can also reduce fever and relieves mild pain, but does not appear to
be better than aspirin for these effects.

For the questions following the lab procedure, you will be required to draw the
structures of some analgesic compounds. These structures may be found in any
number of textbooks or Websites (one site to try is www.chemfinder.com), in a
Physician's Desk Reference (PDR), or in a Merck Index (this is NOT the same as a Merck
Manual. The library has both books, so be sure to get the Merck INDEX!)

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Procedure:

Work in pairs for Parts A and B, but if your instructor assigns you an
individual unknown for Part C, you must perform part C individually.

Your instructor will direct you whether or not to turn in any of the
chromatograms with the lab report. It may be best NOT to discard them
until you have been instructed on this.

When calculating Rf values: since you are making measurements utilizing 3

significant figures (your distances will be measured to the nearest 0.01 cm),
your calculated Rf values should also contain 3 significant figures.

Three important rules to ensure success for ANY thin-layer-chromatography portion in

the procedure:

1) DO NOT TOUCH THE PLATE WITH YOUR FINGERS – OILS, GREASE AND OTHER
SUBSTANCES FROM YOUR FINGERS MAY LEAVE RESIDUE ON THE PLATE AND
MAKE THE SPOTS RUN IN AN INCORRECT MANNER. HANDLE THE PLATES BY
THEIR EDGES; IF YOUR FINGERS TOUCH A LITTLE ON THE EDGES, THAT IS OK.

2) MARK YOUR ORIGIN LINE LIGHTLY WITH A PENCIL ON THE CHROMATOGRAM

BEFORE YOU PLACE THE CHROMATOGRAM INTO THE CHAMBER AND LIGHTLY
MARK THE SOLVENT FRONT IMMEDIATELY AFTER REMOVING THE DEVELOPED
CHROMATOGRAM FROM THE TANK. IF YOU DO NOT MARK THE SOLVENT FRONT,
THE CHROMATOGRAPHY WILL HAVE TO BE DONE OVER.

3) AFTER YOU HAVE PLACE THE CHROMATOGRAM IN THE DEVELOPING JAR, DO

NOT MOVE THE JAR! ANY SPLASHING SOLVENT COULD RUIN THE
CHROMATOGRAM AND THE PROCEDURE WILL HAVE TO BE DONE OVER. IF YOU
HAVE A QUESTION ABOUT THE DEVELOPMENT OF YOUR CHROMATOGRAM, ASK
YOUR INSTRUCTOR TO COME TO YOUR BENCH. DON’T TAKE THE JAR AND
CHROMATOGRAM TO THE INSTRUCTOR.

Part A: TLC of spinach juice

Use the 80% petroleum ether/20% acetone solvent for this section.

1. Obtain one 2.5 x 7.5-cm silica gel thin layer chromatography plate from your
instructor. Handle the plate by the edges only. With a pencil and ruler, lightly mark
a line about 0.7 cm from the narrow edge of the plate. Using a capillary tube, pipet
a small spot of spinach juice onto the center of the marked line, keeping the spot as
small as possible, approximately 1-2 mm in diameter.
2. While the spot is drying, prepare the chromatography chamber. Place enough of
the solvent (80% petroleum ether/20% acetone solvent) into the bottom of the

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 chromatography so that the depth of solvent is enough to cover the bottom of the
chamber, but not enough to touch the pigment spot.

3. Insert the spotted, dried plate into the prepared chamber and cover the chamber.
Watch the progress of the solvent front on the plate. Do not move the jar during
development. When the solvent front is about 0.5-1.0 cm from the top of the plate,
remove the plate from the chamber and QUICKLY mark the solvent front level with a
pencil line across the top of the chromatogram before the solvent evaporates. After
marking the solvent front, allow the chromatogram to dry. Place the used solvent in
the proper waste container. Although not required, you may clean the chamber
with a small amount of acetone and dry (place the acetone used for this cleaning in
the proper acetone waste container – not down the sink!).

4. Make a drawing of this chromatogram in your laboratory notebook, indicating the

proper color and shape of each spot. Measure the distance from the origin line to
the solvent front. Measure all distances in centimeters and measure to the
nearest 0.01 cm (two decimal places). Then measure the distance from the origin
line to the center of each spot (again, to the nearest 0.01 cm). Write these
distances on the drawing in your notebook. Using these measured distances,
calculate the Rf of these spots (to 3 significant figures).

5. You will see quite a few spots (between 7-15 spots), but (looking at the
chromatogram from the solvent front down to the origin line) the important ones
are:
a) a spot of bright orange-yellow pigment (the carotenes)
b) a gray spot (pheophytin – a decomposition product of chlorophyll). A second
(more pale) gray spot may be seen under it. This is another pheophytin.
c) another yellow spot (the xanthophylls). This is a pale gray-yellow spot and
may be just below the second gray spot.
d) one or two bluish-green spots (close together) that are both chlorophyll a
e) one or two yellowish-green spots (close together) that are both chlorophyll b

6. You will also look at the chromatogram under long-wave UV light and observe the
red fluorescence of the chlorophyll spots.

7. After observation of the spots, you will calculate the Rf value of each of these
compounds.

Part B: TLC of Vanillin and Ethyl Vanillin

Use the 50% ethyl acetate /50% cyclohexane solvent for this section.

1. Obtain two 2.5 x 7.5-cm silica gel thin layer chromatography plates from your
instructor. Handle the plate by the edges only. With a pencil and ruler, lightly mark

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 a line about 0.7 cm from the narrow edge of the plate. Also, lightly mark an
identification mark on each plate, whether it is plate #1 or plate #2 (#1 = the
vanilla extract plate; #2 = the imitation vanilla plate).
2. On each plate, place a spot of 0.1% alcoholic solution of vanillin (reference solution)
about 1/3 the distance from the left edge of the plate, keeping the spot as small as
possible, approximately 1-2 mm in diameter. Let the spot dry repeat the spotting
procedure with a 0.1% alcoholic solution of ethyl vanillin (reference solution) about
1/3 the distance from the right edge of the plate.

3. In the center of the origin line of plate #1, repeat the spotting procedure using pure
vanilla extract (test solution).

4. In the center of the origin line of plate #2, repeat the spotting procedure using
imitation vanilla flavoring (test solution) as the center spot.

5. To reprise the samples on each plate:

Plate #1

• • •
Vanillin reference Vanilla extract test Ethyl vanillin reference

Plate #2

• • •
Vanillin reference Imitation vanilla flavoring test Ethyl vanillin reference

6. Dry the plates in an 80 °C oven for 2-3 minutes (especially if the weather is humid).
The chromatograms will be developed in a 50%/50% mixture of ethyl
acetate/cyclohexane.

7. While the plates are drying in the oven, prepare the chromatography chamber.
Place enough of the solvent (50% ethylacetate/50% cyclohexane) into the bottom
of the chromatography so that the depth of solvent is enough to cover the bottom
of the chamber, but not enough to touch the applied spots.

8. Insert the spotted, dried plates into the prepared chamber and cover the chamber.
Do not move the jar during development. With careful arrangement of plates, you
should be able to fit both plates into the same chromatography chamber. If not,
use two prepared chromatography chambers, and place one plate into each
chamber. Watch the progress of the solvent front on the plate. When the solvent
front is about 0.5-1.0 cm from the top of the plate, remove the plate form the

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 chamber and QUICKLY mark the solvent front level with a pencil line across the top
of the chromatogram before the solvent evaporates. After marking the solvent
front, allow the chromatogram to dry. Place the used solvent in the proper waste
container. Although not required, you may clean the chamber with a small amount
of acetone and dry (place the acetone used for this cleaning in the proper acetone
waste container – not down the sink!).

9. Place the chromatograms into a jar with a few iodine crystals, cover the jar and let
stand until brownish-yellow spots develop. Remove the plates and outline the spots
with a pencil.

10. Make a drawing of this chromatogram in your laboratory notebook, indicating the
proper color and shape of each spot. Measure the distance from the origin line to
the solvent front. Measure all distances in centimeters and measure to the
nearest 0.01 cm (two decimal places). Then measure the distance from the origin
line to the center of each spot (again, to the nearest 0.01 cm). Write these
distances on the drawing in your notebook. Using these measured distances,
calculate the Rf of these spots (to 3 significant figures).

11. Decide the presence of vanillin and/or ethyl vanillin in each test solution. Look at
the structures of vanillin and ethyl vanillin and determine the relationship between
polarity and Rf of the vanillin and ethyl vanillin.

• Any other spots on the chromatogram that do not have the same Rf as vanillin or
ethyl vanillin (the two references) are additives and will not be identified in this
procedure.
• These are likely additives to the vanilla solution. In this procedure, the only
spots to be characterized are vanillin and/or ethyl vanillin.

Part C: TLC of Analgesic Drugs

You may be assigned an individual unknown for this section. You should
therefore perform this section individually.
Use the ethyl acetate + 0.5% acetic acid solvent for this section.

1. Obtain one 7 x 10-cm fluorescent silica gel thin layer chromatography plate from
your instructor. Handle the plate by the edges only. With a pencil and ruler, lightly
mark a line about 1 cm from the narrow edge of the plate (not the wide edge of
the plate).

2. Divide the plate into six equal portions. Six solutions will be spotted on each plate
(keeping the spots as small as possible, approximately 1-2 mm in diameter) – a
solution of each of four standards/reference solutions (acetaminophen, aspirin,
caffeine, salicylamide) will be applied to the plate, as well as a combined solution of

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 all four standards/references together, so it can be determined how the compounds
run when together in solution. You will also spot your assigned unknown on the
plate. Apply each of the six solutions once; then spot your unknown a second time,
allowing the spot to dry before placing the other spot on top of it. It might be
easiest to apply the solutions in alphabetical order, to prevent confusion.

3. To reprise the samples on the plate:

Plate

• • • • • •
Acetaminophen Aspirin Caffeine Salicylamide Unknown Combined
(apply twice) Reference

4. Dry the plates in an 80 °C oven for 2-3 minutes (especially if the weather is humid).
The chromatograms will be developed in a mixture of ethyl acetate/acetic acid.

5. While the plates are drying in the oven, prepare the chromatography chamber. You
will use your 400-mL beaker as a development chamber. Place enough of the
solvent (ethylacetate/acetic acid) into the bottom of the chromatography so that the
depth of solvent is enough to cover the bottom of the chamber, but not enough to
touch the applied spots.

6. Insert the spotted, dried plate into the prepared chamber and cover the chamber
with a plastic sandwich bag. Do not move the jar during development. Watch the
progress of the solvent front on the plate. When the solvent front is about 0.5-1.0
cm from the top of the plate, remove the plate from the chamber and QUICKLY
mark the solvent front level with a pencil line across the top of the chromatogram
before the solvent evaporates. After marking the solvent front, allow the
chromatogram to dry. Place the used solvent in the proper waste container. Clean
the chamber (a 400-mL beaker) with soap and water. Rinse the beaker with
deionized water as a final rinse.

7. Visualize the plate under short-wave UV light, and mark any spots (either
fluorescent or dark spots) that are seen with a pencil on your chromatogram. Then
place the chromatogram into a jar with a few iodine crystals, cover the jar and let
stand until brownish-yellow spots develop. Remove the plates and outline the spots
with a pencil.

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8. Notice the placement of the compounds:

Compound Visualization
Acetaminophen Approximately 3/4 of the way up the chromatogram from the
origin line; can be visualized as a dark spot under short- wave UV
light, and has a VERY strong brown/yellow color in the I2 vapors
Aspirin Just slightly above acetaminophen; will be visualized as a dark
(quenched) spot under short-wave UV light. May have a "tail" off
the spot. Aspirin will not be seen (or only slightly seen) with iodine
crystals
Caffeine Approximately 1/4 of the way up the chromatogram from the
origin line; can be visualized as a brown spot in the iodine jar
Salicylamide Just slightly above acetaminophen; will be visualized as a brown
spot in the iodine jar. Salicylamide will be seen under the short-
wave UV light as a deep blue/purple fluorescence
Aspirin and salicylamide run at about the same Rf value; the way they are told apart
is by the visualization process. If both compounds are present, both visualization
steps will show the presence of the proper compounds.

9. Make a drawing of this chromatogram in your laboratory notebook, indicating the

proper color and shape of each spot. Measure the distance from the origin line to
the solvent front. Measure all distances in centimeters and measure to the
nearest 0.01 cm (two decimal places). Then measure the distance from the origin
line to the center of each spot (again, to the nearest 0.01 cm). Write these
distances on the drawing in your notebook. Using these measured distances,
calculate the Rf of these spots.

10. Determine the components (there are three lines in the report sheet; if your
unknown only contains one or two components you will not need all three lines) and
brand name of the unknown solution you have been assigned from Table 5-1.

11. Save your chromatogram for this section unless your instructor tells you that you do
NOT need to turn it in with your lab report. If you are to turn it in with your lab
report, staple it to the front of the completed lab report when you turn it in.

Table 5-1
Brand name Component ingredients
Anacin Aspirin and caffeine
Bufferin Aspirin
Excedrin Acetaminophen, aspirin and caffeine
Tylenol Acetaminophen
B. C. Powder Aspirin, caffeine and salicylamide

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Lab partner(s) ________________________ Notebook points/initials __________

Date ______________________ Name ___________________________

REPORT FORLAB PROCEDURE-2:

THIN-LAYER CHROMATOGRAPHY

Part A: Separation of Plant pigments

Illustrate separation by drawing the relative location, color, and size of ALL the
spots on your chromatogram below. Also draw the solvent front and label each spot.
Your chromatogram may show more than the seven spots requested here. Only these
seven need to be identified – in the table as well as the chromatogram.

Origin line →

Solvent front distance from origin line _______________cm

Compound Color Distance from Rf value

origin line (cm)
Carotenes

Pheophytin

Xanthophyll

Chlorophyll a

Chlorophyll a

Chlorophyll b

Chlorophyll b

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Part B: TLC of vanillin and ethyl vanillin
Slide #1: Slide #2:

Vanillin reference – Rf value

Ethyl vanillin reference – Rf

value
Pure vanilla extract – Spot
#1 – Rf value
Pure vanilla extract – Spot
#2 – Rf value
Imitation vanilla extract –
Spot #1 – Rf value
Imitation vanilla extract –
Spot #2 – Rf value

Which compound(s) are present in the pure vanilla? In the imitation vanilla? Are these
results what you would expect if you compared the content labels of each product with
your results here? (If you get a chance, check this out at a grocery store)

What is the relationship between the polarity of vanillin and ethyl vanillin and the Rf
values you determined here?

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Part C: TLC of Analgesic Drugs

Appearance of Developed Chromatogram:

ACET ASP CAFF SAL UNK REF

COMPOUND DISTANCE DISTANCE

MIGRATED BY MIGRATED BY Rf OF COMPOUND
COMPOUND (cm) SOLVENT FRONT
(cm)
ACETAMINOPHEN
ASPIRIN
CAFFEINE
SALICYLAMIDE
UNKNOWN:
LIST ALL SPOTS
THAT APPEAR

Assigned unknown: _________________

Identify all constituent compounds in unknown: ______________
______________
______________
Brand name of unknown (From table 5-1):________________________________

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Questions about TLC:

1. Define the term over-the-counter.

2. What size sample spots will you place on the origin line of your chromatograms?

3. What would happen if the solvent layer in your development chamber is too deep,
so that the origin lines of your chromatograms are submerged in it?

4. What are the two methods you will use to visualize the spots on your developed
chromatograms?

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5. Using an appropriate source, draw the structural formula of the following
compounds in the spaces provided above each name.

a) aspirin b) ibuprofen

c) acetaminophen d) caffeine

e) salicylamide

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