Experiment 1. Thin
in Layer Chromatography (TLC) and
Column Chromatography.
References:
Vogel, A Textbook of Practical Organic Chemistry
Fieser, Organic Experiments
Landgrebe, Theory and Practice in the Organic Laboratory
Silverstein and Basler, Spectrometric Identification of Organic Compounds
Eaton, Laboratory Investigations in Organic Chemistry
INTRODUCTION:
Organic chemists use a variety of techniques to prepare and analyze organic compounds.
Synthetic chemists make new molecular structures by performing reactio
reactions,
ns, for example interinter
converting functional groups and making new arrangements of carbon chains and rings. You will
perform some reactions later in this course, but for now let's focus on purification and
characterization. After each chemical reaction is ccomplete,
omplete, the desired product is often present as
a mixture with solvents, reagents and undesired by
by-products.
products. The chemist uses techniques, such
as chromatography (this lab), distillation (Exp. 2), extraction (Exp. 3), and crystallization (Exp.
3) to obtain the pure product. Chromatography is by far the most favoured technique in the
research lab today.
BACKGROUND:
General Principles of Chromatography
Chromatography is a technique which is widely used to separate a mixture of substances into its
component parts.
The term chromatography encompasses a number of different techniques which, although they
will be discussed separately, are all based on common principles. Chromatography may be
divided broadly into three kinds: adsorption, partition and ion-exchange,, and the simplest type
of these is adsorption chromatography.
1) Adsorption Chromatography
In this technique, the substance under investigation is adsorbed onto a solid support (the
stationary phase) and separation of a mixture into the component pparts
arts is achieved by elution
with solvents of different polarity. Adsorption chromatography may be carried out in two ways,
by column, or thin layer techniques. Both techniques will be used in this experiment and they
will be discussed separately.
Column Chromatography
In column chromatography a finely divided adsorbent such as silica or alumina is
placed in a glass column, supported at the bottom by a wad of glass wool, as shown
to the left.
A layer of sand is placed over the top of the adsorbent, and the whole column is wetted with the
solvent to be used. A solution of the substance to be purified in this solvent is then applied
evenly to the top of the column, and this solution is allowed to pass down into the column so that
the dissolved solid is adsorbed at the top of the column. The column is then eluted by passing
down a number of solvents of increasing polarity. In this way, weakly adsorbed substances will
pass rapidly through the column while the more strongly adsorbed substances will pass through
at a slower rate. By eluting with a series of solvents of increasing polarity it is therefore possible
to separate the components of a mixture and to elute them, one after the other, from the solid
adsorbent.
Choice of Adsorbent
The order in which the compounds are eluted will depend on how strongly they are adsorbed on
the surface of the stationary phase. A few adsorbents used in column chromatography, with
different binding abilities are Cellulose, Calcium oxide, Silica gel (oxides of silicon), and
alumina (aluminum oxide). Alumina unless specially pretreated is slightly basic and hence
strongly adsorbs acidic substances or materials capable of forming hydrogen bonds to the basic
oxygen atoms of the alumina. Compounds without the ability to form hydrogen bonds but with
substantial dipole moments will be somewhat less strongly adsorbed due to electronic
interactions between their dipoles and those of the alumina. Compounds with neither acidic
hydrogens nor dipole moments are only very weakly adsorbed due to dipole induced dipole
interactions. The complete order for the strength of all these bonding interactions is generally the
following: Salt Formation > Coordination Complexes > Hydrogen Bonding > Dipole-Dipole
Interactions > Van der Waals. Some examples of these bonding interactions with alumina are
shown:
Choice of Solvent
The choice of solvents used to elute the various components of the mixture from the column will
depend upon the components in the mixture. For a very weakly adsorbed component a very nonpolar solvent such as petroleum ether or benzene would be used. For more strongly adsorbed
components, a more polar solvent such as ether might be used. For very strongly adsorbed
components, a very polar solvent such as ethanol or even acetic acid might be required to
displace the material from the column. A list of common solvents in order of increasing eluting
power follows:
increasing
eluting
Petroleum Ether
Carbon Tetrachloride
Cyclohexane
Carbon Disulfide
Benzene
Toluene
Methylene Chloride
Chloroform
(non-polar)
increasing
polarity
power
Diethyl Ether
Ethyl Acetate
Acetone
Ethanol
Methanol
Water
Acetic Acid
(polar)
Thin layer Chromatography
Thin layer chromatography (TLC) is another type of adsorption chromatography. In fact, TLC
can be considered simply column chromatography in reverse,, with the solvent ascending the
adsorbent, rather than descending. Here, the solid adsorbent, again usually alumina or silica gel,
ge
is spread out in a thin layer over a glass, metal or plastic sheet, and the substance under
examination is placed on this layer in the form of a spot. A suitable solvent is then allowed to run
up the sheet by capillary action, as shown below:
Under an established set of conditions (solvent system, adsorbent, etc.), a given compound
always travels a fixed distance relative to the distance traveled by the solvent front. This ratio is
known as the Rf value and is given as a decimal fraction:
Rf = y / x
where,
x = distance (in cm) from origin to solvent front
y = distance (in cm) from origin to the centre of the sample spot (if too much sample had been
applied and the spot has a faint tail, then its 'centre of density' is estimated).
During the elution with the solvent, the sample will partition itself between the stationary phase
(the adsorbent layer) and the moving phase (the solvent) so that the distance which the sample
moves up the plate is characteristic of that substance and will differ from one substance to the
next. A mixture of substances will thus give rise to a series of spots, one corresponding to each
component. This technique is extremely useful for analysis on a micro scale and for the
purification of small quantities (usually less than 00.1 g) of material.
In preparative work, a substance is recovered from the plate after development by removing the
particular region of the adsorbent layer containing that substance from the plate, followed by the
removal of the substance from the adsorben
adsorbentt layer by extraction with a suitable solvent.
2) Partition Chromatography
The second general type of chromatography is partition chromatography,, consisting of gasliquid chromatography, liquid-liquid
liquid chromatography and paper chromatography (which is an
application of liquid-liquid
liquid chromatography). In gas chromatography (Exp 4), the moving phase
is a stream of an inert gas, such as nitrogen or argon. The sample under investigation is
volatilized and the vapour swept through a column of the stationary phas
phasee by the carrier gas.
Partition of the sample between the stationary and moving phases will occur. However, for liquid
chromatography,, the system is eluted with a moving liquid leading to separation of the
components of a mixture by the partitioning of the
these
se components between the stationary and
moving liquid phases so that the components will move at differing rates through the column.
Paper chromatography is analogous to thin layer chromatography except that in this case, the
support material consists of a sheet of specially prepared paper, and the stationary phase is
considered to be water adsorbed on the paper. Elution with a solvent, measurement of the Rf
value and preparative work is carried out in the same way as for thin layer chromatography.
3) Ion-Exchange
Exchange Chromatography
The third type of chromatography, ion-exchange chromatography,, is of somewhat more limited
application in the chemistry lab, but is widely used in biochemistry. Here, the solid support
consists of a resin which can have either bas
basic
ic or acidic properties, and mixtures of acidic or
basic substances can be separated using these resins by eluting with buffers of different pH's.
Part A: Thin Layer Chromatography of Analgesic Drugs
In this experiment, TLC will be used to examine the composition of various analgesic (pain
relieving) drugs. The best known of these is aspirin, but several other chemically similar
compounds are (or were) also used as analgesics. Among these are phenacetin, salicylamide and
acetaminophen. Caffeine is some
sometimes
times added to these formulations to overcome drowsiness. A
few other compounds such as N--cinnamylephedrine
cinnamylephedrine (cinnamedrine) and diphenylpyrilene are
included for other therapeutic effects, such as antispasmodic or slight sedative action. In addition
to the active ingredients, the tablets of these drugs contain starch, lactose, and other substances
that act as binders and permit rapid solution, and sometimes also inorganic bases. The objective
of this experiment is to identify an unknown drug tablet by a TLC comparison with standard
compounds.
Part B: The Separation of Fluorene and Fluorenone by Column
Chromatography
Fluorenone can be produced by oxidizing fluorene with a strong oxidizing agent, like sodium
dichromate (Na2Cr2O7). The mechanism is rather complex, but the result is a doubly bonded
oxygen in the place of two hydrogens. You will not perform the oxidation that gives fluorenone,
but you will be separating a mixture of fluorene and flurenone (which would result from the
oxidation reaction).
In Part B of this experiment you will use a Pasteur pipette to prepare a chromatographic column
and separate 5 mg samples of fluorene and fluorenone. You will also use thin layer
chromatography to analyze the 1 mL fractions collected during the elution of the compounds to
determine the degree of separation and the purity of the compounds.
PRE-LAB PREPARATION:
Read the experimental procedure so that you are prepared for the lab and you understand the
safety and disposal information for the chemicals you are using in this experiment.
1.
Unknown
Acetaminophen
Aspirin
Ibuprofen
Caffeine
Unknown
Unknow
n
Based on the three TLC plates shown above, and the table of reference compounds in the
experimental procedure (pg 16), what would be the identity of the unknown drug?
2. What would be the result of the following errors in TLC technique:
a) Too much sample applied? b) Solvent pool in developing jar too deep?
c) Allowing the solvent front to proceed off the top of the plate?
3. Draw out the structures of fluorene and fluorenone. Based on their structures (using alumina
as the absorbent), which compound would you expect to elute first (fluorene or fluorenone)?
Which would have the higher Rf value (using silica as the absorbent)? Explain your reasoning
for both questions.
EXPERIMENTAL PROCEDURE:
Part A: Thin Layer Chromatography of Analgesic Drugs
This will take about 70 minutes to complete: do not start too late. DO INDIVIUALLY.
Safety Data for Solvents used in Part A.
Solvent/Solution
Mol. Wt.
(g/mol)
Safety Data
Acetic Acid
60.05
Irritating to eyes and skin. Harmful if swallowed.
Ethyl Acetate
88.10
Irritating to eyes. Highly flammable.
Hexane
86.17
Highly flammable. Irritating to eyes. Harmful by inhalation.
Methanol
32.04
Highly Flammable. Toxic by inhalation, in contact with skin
and if swallowed.
Toluene
92.14
Highly Flammable. Harmful by inhalation.
1. Preparation of the Developing Chamber
Using the chromatography jar provided, place 3-4 mL of the solvent mixture (which is also
provided to you) in the chromatography jar, cover tightly and allow to stand 5 - 10 minutes
before using. CHROMATOGRAPHY JARS SHOULD BE FILLED IN THE FUMEHOOD
AND STAY IN THERE UNTIL DISPOSAL TIME (Note: Dispose of solvent in the “Organic
Waste” container). The developing solvent system that was provided is a mixture of:
74 mL ethyl acetate
25 mL hexane
1 mL acetic acid
*When finished with the lab empty the jar into the waste, DO NOT WASH JAR with water,
simply leave upside down where you obtained it
2. Preparation of the Sample
a) Obtain an unknown sample from your TA
b) Write down the number of your unknown sample, then remove the label from your vial and
stick it onto your 10 mL erlenmeyer flask.
c) Dissolve your unknown in 4 mL of methanol-toluene (1:1) in your 10 mL Erlenmeyer flask.
[NOTE: some insoluble material will not dissolve. This is normal.]
FYI: The reference standards have been prepared at a strength of 25 mg/mL.
3. Application of the Samples
a) Obtain 2 of the commercially prepared fluorescent Silica Gel TLC plates provided.
b) To know where your spots started, draw LIGHTLY in pencil a horizontal line 1 cm from the
bottom of the TLC plate. If you push too hard, you will crack the silica.
c) You will have five solutions (4 reference compounds and 1 unknown) to examine. They
should be spotted on the coated side (not smooth) on the line 1 cm from one end of the sheet,
equally spaced apart, with the outer two spots about 0.75 cm from the edge of the sheet. The
unknown should be placed in the centre, with one reference compound on each side. You should
use two TLC plates. You should use one plate to run your unknown plus two of the reference
samples, and the other plate to run the unknown plus the remaining two reference samples.
To apply a sample, touch one end of an applicator capillary tube to the solution, and then gently
and quickly touch the Silica Gel plate at the proper spot. It is important to touch the plate very
lightly and not to gouge a hole in the absorbent on the plate. It is also important to touch the plate
very briefly so that the entire contents of the capillary tube is not transfer to the plate. Use the
other end of the capillary tube or a fresh capillary tube for each sample. The sample spots should
not be larger than 2 mm diameter.
d) BEFORE elution, look at the TLC plates under the ultraviolet lamp (see step 5) to make sure
some compound is present.
Solvent Front
Reference
Unknown
Reference
1cm
TLC Before Elution
TLC After Elution (example)
4. Development of the Chromatogram
When each plate has been prepared, place the plate, spotted end down, in the developing jar.
Make sure that the solvent pool begins below the spots. To develop both plates at once, have
them sitting back to back (shiny sides without sample) in the developing jar. Cover tightly, do
not disturb (do not touch the developing jar until elution is complete, you will be able to see the
solvent rise up on the silica), and allow about 5 - 10 minutes for the solvent to rise to within
about 1 cm from the top of the plate - do not allow the solvent to run all the way to the top of the
TLC plate! Remove the TLC plate(s) and immediately mark the solvent front lightly in pencil (a
single horizontal line across). Allow the TLC plates to dry (IN THE FUMEHOOD!).
5. Visualization
The colourless compounds are visualized by illumination of the plate with an ultraviolet lamp.
Many substances, particularly aromatic compounds, will show a bright fluorescence, which may
have a characteristic colour. The thin layer plates used contain a trace of fluorescent dye.
Compounds which are fluorescent show up as bright spots on a light background; any others
appear as a dark spot since they quench the fluorescence of the background dye. Circle the spots
lightly in pencil, and note any distinctive colours.
CAUTION: DO NOT LOOK DIRECTLY AT THE UV LAMP!
6. Comparison of the Unknown with Reference Standards
(a) Calculate the Rf values of the reference compounds and the components of the unknown.
Only once do you need to show a full set of calculations.
(b) Draw the chromatogram (developed TLC plate) to scale in your lab notebook; identify and
label (ingredients) the spots in the chromatogram, including as many of the spots in the unknown
as possible. Also label the solvent front!
(c) From the number, position and appearance of the spots in the unknown, and the composition
of the possible unknowns, identify your unknown analgesic. You should use the table below to
aid the identification of your unknown.
ie: If Midol is your unknown, you will see 2 spots, that match up with the reference compounds
Acetaminophen and Caffeine
Drug
(Brand Name)
Possible
Unknowns
Ingredients
Acetaminophen
Aspirin
Caffeine
Ibuprofen
200 mg
Advil
Anacin
Excedrin
250 mg
Midol
500 mg
Tylenol
325 mg
400 mg
32 mg
250 mg
65 mg
60 mg
325 mg
Bayer
The reference compounds are: Acetaminophen [4-acetamidophenol], Aspirin [acetylsalicylic
acid],
Caffeine, Ibuprofen [2-(4-Isobutylphenyl)-propionic Acid]
Part B: The Separation of Fluorene and Fluorenone by Column
Chromatography
Begin Part B after you have begun Part A. Work in PAIRS for Part B only.
Safety and Disposal Data for Compounds used in Part B.
Compound
Mol. Wt. (g/mol)
Safety and Disposal Data
Alumina
101.96
May cause irritation. May be harmful if inhaled.
Avoid all contact. Wash thoroughly after use.
Dispose in empty pail in Waste Fumehood.
Diethyl Ether
74.12
Irritant. Extremely flammable. May form
explosive peroxides. Dispose in Organic Waste.
Fluorene
166.22
Irritant. Dispose in Organic Waste.
Fluorenone
180.20
Irritant. Dispose in Organic Waste.
Ligroin
(Petroleum Ether)
---
Extremely flammable. Dispose in Organic Waste.
1. Preparing the Column
a) You will be provided with a `long' Pasteur pipette that has a small bend in the
closed tip.
b) Fill the pipette at lea
least half-full with pure Ligroin (petroleum ether) NOT the 4:1
Ligroin:ether solution. Place a small wad of glass wool into the bottom of the pipette
(3mm glass rod is ideal to push the glass wool down to the bottom or another long
pipette),
), pour in some san
sand
d (0.3 cm). Tap the column to try and remove all air
bubbles.
c) Pack the `column' with alumina (5 cm), using a micro spatula, by slowly adding
the alumina to the solvent in the column and tapping the column gently. Be sure to
keep the solvent level above the level of the alumina. Note: The tapping promotes
even settling and mixing in the column, and frees air bubbles. Try and keep the
pipette vertical while doing this so the alumina settles evenly.
d) Then pour sand (0.3 cm) on top of the alumina and clam
clamp
p the pipette carefully with a
microclamp to the scaffolding at the back of your fumehood. It is important that the column be
mounted as close to vertical as possible your column is ready for use.
Microclamp
2. Separation and Collection of Fluorene and Fluorenone
NOTE: Do not break the pipette tip until you have everything ready!
DO NOT let the column run dry!
a) Take 1 mL of the fluorene/fluorenone solution provided, which has been dissolved in
pet-ether.
ether. Break off the tip of the `colum
`column'
n' (hold the pipette near the bend when breaking) with a
piece of paper towel and when the Ligroin just about disappears through the sand add the
solution of fluorene/fluorenone to the top of the column drop wise until the sample has been
loaded on the column.
b) When the final volume of fluorene/fluorenone solution disappears through the sand at the top,
add elution solvent (4:1 Ligroin/ether) to the top of the column drop wise (about 10 drops), then
fill the column to the top. (Do not let the solvent lev
level
el drop below the sand at the top of the
column. Channels will develop in the alumina resulting in a poor separation of the compounds.)
You will likely need to add more elution solvent while you are collecting, have a small amount
ready BEFORE you break the
he column tip!
c) Collect at least five (5) one mL fractions in test tubes. If the second compound has not
appeared by fraction 5 (light yellow colour) collect 2 - 3 more 1 mL fractions.
3. Analysis of the Fractions
a) Spot the fractions on Silica Gel T
TLC
LC strips along with the reference fluorene and fluorenone.
b) Develop the strips in a chromatography jar containing a 4:1 mixture of pet-ether/ether
ether/ether.
c) Visualize the chromatograms with the ultraviolet lamp to determine which fractions are pure
fluorene and which are pure fluorenone. Draw the chromatogram in your lab notebook
(identifying and labeling the spots in the chromatogram). Calculation of Rf values is not needed,
but may aid you in identifying the correct spots.
e) Based on your TLC data for Part B, record which compound was eluted first. Explain your
reasoning.
Note: Since this is a problem based lab, you will be required to include a
conclusion of your findings for both parts.