Thin Layer Chromatography
INTRODUCTION:
Sandra, a child of six, was racked with chills, vomiting and in
convulsions when her mother found her. Sandra had been at a friend's house, had gotten into the
medicine chest and had swallowed a large number of `headache pills'. The symptoms Sandra
was experiencing is not uncommon for those suffering from a drug overdose. Fortunately for
Sandra, her parents were able to determine from the open empty bottle the drug the child had
taken and, with this information, the doctor was able to take proper corrective action in time to
prevent permanent damage.
For a doctor to take proper action in cases of drug overdoses, it is of vital importance the doctor
know precisely what drug the individual ingested. One method to rapidly determine drug
identification is through chromatography of a portion of the victim's blood. In the experiment
you will be performing today, the brand name of an unknown analgesic will be determined by
identifying the components in it by thin layer chromatography.
CHROMATOGRAPHY: the general principle
There are many forms of chromatography used to separate mixtures. All forms involve three
things:
1.
a stationary (or nonmobile) phase
2.
a mobile (or moving) phase
3.
an analyte (the compound or mixture being analyzed)
Chromatography separates the components in a mixture so they can be separately identified.
Separation is achieved by placing a portion of the mixture on the stationary phase and allowing
the mobile phase to pass through. Depending on the properties of the compounds in the mixture,
some will be more attracted to the stationary phase and not move (or move very slowly) others
will be more attracted to the mobile phase and will move very quickly.
TLC, page 2 of 8
THIN LAYER CHROMATOGRAPHY
In thin layer chromatography, or TLC, a small portion of dissolved material is applied as a small
dot near the bottom edge of a plastic or glass plate coated with a thin layer of adsorbent. (The
adsorbent constitutes the stationary phase). The plate is placed in a closed container containing a
small amount of solvent. Capillary action pulls the solvent (the mobile phase) slowly up the
plate like water being soaked up by a sponge.
The compounds in the sample on the TLC plate (the analyte)
can do two things as the solvent moves up the plate:
If a compound is attracted to the
If a compound is not attracted to the
coating it sticks and does not move up coating it will not stick and it does move
the plate:
up the plate:
Actually, most compounds will be partially attracted to both the coating and the solvent.
Compounds more attracted to the coating move up the plate slowly, while those more attracted to
the solvent travel more quickly and separation is achieved.
What features cause some compounds to prefer the stationary phase to the mobile phase? A
major factor is polarity of the bonds. The coating is composed of aluminum oxide (Al2O3).
The aluminum - oxygen bonds are very polar. The solvent is usually a nonpolar or very
moderately polar organic solvent, in this experiment ethyl acetate is used (formula C4H8O2). In
general, the more polar bonds a compound has then the more attracted it is to the very polar
aluminum oxide and the more slowly it moves up the plate.
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The structures of the compounds you will be analyzing are shown below. The bonds possessing
the important polarity are those bonds between the very electro- negative atom O and C or O
and H, and between N and C, or N and H. It is primarily these bonds that determine the overall
polarity of these compounds. Remember that it is the difference in electronegativity of the two
atoms in the bond that makes a bond polar. With this in mind, carbon-carbon bonds are not polar
at all, while carbon-hydrogen bonds are only very slightly polar so both of these types can be
ignored.
O
H3C
O
N
C
C
N
H
CH3
H
N
C
C
N
C
H
H
C
C
H
C
C
C
C
C
N
CH3
C
O
H
ACETAMINOPHEN
CAFFEIN
H
C
C
H
CH3
H
C
H
H
O
C
N
C
H
O
H
SALICYLAMIDE
H
H
H
H
C
C
C
C
H
O
C
C
O
O
H
C
O
CH3
ACETYLSALICYLIC ACID
TLC, page 4 of 8
ANALGESICS
The best known and most widely used analgesic is acetylsalicylic acid (known as aspirin). Other
commonly used analgesics are those shown previously. Caffeine is not an analgesic, but is
sometimes added for its stimulating effects. Caffeine increases the body's sensitivity to carbon
dioxide causing an increase in both the rate and depth of respiration.
The analgesics given above work to inhibit pain by preventing transmission of pain impulses
from the hypothalamus gland (located in the base of the brain) to the cerebral cortex. These
analgesics also reduce fever by the following modes of action:
1.
causing blood vessels to dilate (enlarge) so more blood can be cooled when it
reaches the skin;
2.
stimulating diaphoresis (perspiration) so the skin cools blood even more
efficiently;
3.
by inhibiting the production of certain prostaglandins which normally function to
increase body temperature.
When viruses or bacteria invade the body, prostaglandins are activated to INCREASE body
temperature. Because the invading organisms are more susceptible to high temperatures than
your own uninfected cells, this increase in temperature serves as one of the body's methods of
ridding itself of invading organisms. Thus, while taking an analgesic drug serves to reduce
symptoms associated with infectious disease, it does NOTHING to attack the invading organisms
and may, in fact, be counterproductive!
Aspirin, and to some extent salicylamide, also exhibit an anti-inflammatory action that is
especially useful to people suffering from arthritis. Aspirin is able to reduce swelling and relieve
the pain. Some analgesics tablets include other compounds for therapeutic effects such as
antihistamines, decongestants and sedatives. In addition to the active ingredients, the tablets
must also contain substances such as starch that act as binders to hold the tablets together.
TLC, page 5 of 8
EXPERIMENTAL
In this experiment you will obtain as an unknown, a well-known analgesic tablet. The object is
to identify the drug by comparison with several known compounds. The tablet will be one of
those listed below.
DRUG
INGREDIENTS
(BRAND NAME)
ANACIN
acetylsalicylic acid, caffeine
EXCEDRIN
acetylsalicylic acid, caffeine,
acetaminophen, salicylamide
VANQUISH
acetylsalicylic acid, caffeine,
acetaminophen
TYLENOL
acetaminophen
PROCEDURE
 Capillaries for applying samples can be made by heating and turning a glass tube until the
glass softens and then pulling it in opposite directions. The instructor will demonstrate this
for you. Be careful, hot glass looks just like cool glass. Break off six short pieces of the
small capillary you just made.
 Obtain from the stockroom a TLC plate. Don't touch the surface of the plate, or finger prints
will appear on the plate. Lay the TLC plate on a page in your note book. With a pencil mark
on the page where the corners of the plate are. Also make a mark on the page about 1 cm up
from the bottom of the plate. Below the plate make five evenly spaced marks on the page
and number them 1-5.
 Obtain from the stockroom your unknown analgesic in a small test tube. A small amount of
solvent will be added, then crush the tablet. Don't worry if it doesn't all dissolve. Using a
clean capillary tube for each sample, apply one dot of the following in the order named on the
TLC plate (use the lines as a guide):
1. acetaminophen
2. acetylsalicylic acid
4. caffeine
5. salicylamide
3. unknown
TLC, page 6 of 8
 When all five samples have been applied, place the TLC plate spotted end down in a
developing jar and screw on the lid. Don't disturb the jar while the TLC plate is developing
or the samples may be ruined. About fifteen minutes are needed for the solvent in the jar to
be pulled up the plate. Keep you eye on the TLC plate, and when the solvent is about 1 cm
from the top, pull out the plate and quickly mark the solvent line on top of the plate. The
solvent will evaporate very easily so don't be slow. Allow the solvent to evaporate in the
fume hood.
 What do you see? Probably nothing. You need to visualize the spots on the plate. This is
done in two ways. First, shine an ultraviolet light on the plate and circle any spots you see
with a pencil. Record the colors and any other observations you can make about the spots.
ANALYSIS OF THE PLATE
In TLC, the compounds are compared by their RETENTION FACTORS (or Rf's). These are
determined by measuring the distance each sample moved and dividing by the distance the
solvent moved.
 Start at the origin (the place where the sample was applied) and measure the distance to the
CENTER of a spot. This is the sample distance.
 Now measure the distance from the origin to the line where the solvent reached (called the
solvent front). This is the solvent distance.
 Divide the sample distance by the solvent distance, this is the Rf for that spot.
 Calculate the Rf for each spot on your TLC plate.
 Using the Rf values for each spot and the observations you made when you visualized the
plate, determine which of the known standards are in your unknown analgesic.
 Using the listed ingredients given above, identify the brand name of the analgesic you
analyzed.
PRELAB
There is no prelab for this experiment. Be sure to write the procedure your
notebook!
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NAME and Drawer #___________________________
Lab Section:
Th am
Th pm
REPORT FOR THIN-LAYER CHROMATOGRAPHY
Follow the directions below and answer the questions as directed.
Your report will consist of the attached pages. Remove these pages and answer the
questions. Turn in your report with the TLC plate stapled to the upper left corner.
 Reproduce your TLC plate on the outline below. Label the samples with their
names.
 Report the Rf for each. Indicate what was seen for each spot using both
visualization techniques.
Sample name
visualization
1
2
3
4
5
1
2
3
4
5
1. List the ingredients
in your unknown:
2. What is the brand name of your unknown? _____________________
Rf
TLC, page 8 of 8
3. Rf values have no units, explain why. Show how you calculated the Rf
value for each component in your unknown.
4.
An aspirin tablet contains 300 mg of aspirin. An arthritic patient requires
0.90 grams of aspirin daily. How many tablets should you give the patient
each day? Show unit analysis.
5.
A patient requires 1500 mg of sodium bicarbonate (NaHCO3). How many
2.5 grain tablets of sodium bicarbonate should be administered to the
patient? ( 60 mg equals 1 grain )
Finally, write a conclusion for this experiment.