to a piece of paper to dry over the week. When dry remove the filter paper and weigh the solid.
p-Anisic acid: Place the beaker containing “bicarbonate extract” in the ice bath and add 6 M HCl
slowly while stirring with a glass rod. Foaming will occur as CO2 is formed. Add acid slowly until the
aqueous phase is acidic (pH approximately 3 with pH paper). This will require some patience. If the
acid is added too fast the foam will overflow. Cool in the ice bath for 10 minutes with occasional
stirring. Filter on the Buechner funnel rising with ice water. Allow air to pull through the funnel for 2-3
minutes. Remove the solid and filter paper to a piece of paper to dry over the week. When dry remove
the filter paper and weigh the solid.
p-Phenylphenol: Place the flask containing “NaOH extract” in the ice bath and add 6 M HCl slowly
while swirling to mix. Some foaming may also occur. Add acid until the aqueous phase is acidic. Cool
in the ice bath for 10 minutes and filter on the Buechner funnel rinsing with ice water. Remove the solid
and filter paper to a piece of paper to dry over the week. When dry remove the filter paper and weigh
the solid.
Biphenyl: [This may be done the following week] Pour the ether from the separatory funnel into a 50
mL Erlenmeyer flask and dry with 1-2 g sodium sulfate. After 5 minutes with occasional swirling
gravity filter the solution through a cotton-plugged short step funnel into a clean and dry tared 50 mL
round bottom flask . It is important to tare the round bottomed flask because your recovered weight of
biphenyl is based on that. Always use the same balance when taring a flask and weighing it later. Rinse
the flask and sodium sulfate with several small portions of ether (~5 mL) . Evaporate the ether on the
rotary evaporator. Leave the flask open over the week to allow the residual ether to evaporate, then
weigh the flask. Scrape out as much of the biphenyl as possible. Rinse the flask with acetone several
times into the waste container. Then wash the flask using the ultrasonic bath and soap/test tube brush.
Submit the purified four products in labeled vials after the chromatography has been completed.
Thin-Layer Chromatography
Background
Chromatography is an important laboratory method for
separating organic compounds for purification or analysis.
Although originally developed for separating colored
substances of plant origin (chromatography actually means
color-writing), the technique may be applied to the separation
of colorless substances. Chromatography is often the most
effective method for separating compounds which, in many
cases (e.g., small quantities), cannot be separated by the
traditional techniques of distillation and recrystallization. In
fact, chromatography, in a broad sense, is not only important in
organic chemistry but also in many related chemical sciences
such as biochemistry, medicinal chemistry, clinical chemistry,
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forensic chemistry, etc. There are many types of chromatographic methods, each based on a similar
principle, but differing in equipment design.
Principles
Column and thin layer chromatography are types of liquid-solid adsorption chromatography in which
separation depends on the selective adsorption of compounds between the solid phase (stationary phase
or absorbent) and the liquid phase (mobile phase or solvent). A separation of two components of a
mixture by thin layer chromatography is shown on the next page.
A dilute solution, containing substances A and B, is applied to a thin-layer chromatographic plate
using a capillary tube. The term "thin-layer" refers to a thin layer of silica gel deposited on a glass or
plastic support. The photograph on the previous page illustrates this. Silica gel coated glass plates can
be purchased in 20 x 20 centimeter squares which can be cut into smaller sized plates using a diamond
glass cutter. A pencil line is drawn on the silica gel surface and the compound, dissolved in an
appropriate solvent, is spotted on the line. After the solvent evaporates the plate is placed in a tank
containing the developing solvent. The four photographs below illustrate this sequence
Capillary action draws the solvent up the plate through the thin layer of silica gel: the stationary phase.
As the solvent passes over A and B, those molecules which are less strongly adsorbed to the stationary
phase will travel at a faster rate and separate from the more strongly held molecules. At time T>0
compound A moves faster than compound B. As the solvent continues to move (T>O, T>>O), a
separation is achieved. After removing the plate and evaporating the developing solvent the
components are visualized by one or more methods. The distance a compound travels up the plate is a
physical characteristic of that compound.
TLC provides a method for identification of compounds by comparison with standards. It can also be
used to estimate the impurities in a product, to monitor the rate of a reaction and thereby determine
when it is finished. It is used to determine the proper solvent, adsorbent, etc., for a separation using
A
A
B
B
A and B
time = 0
time > 0
time >> 0
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column chromatography, a large-scale version of thin layer chromatography. Thin layer
chromatography is used to determine the number of components in a mixture and to make
identifications with reference to known standards. Very small amounts of material can be separated by
this method; the limit is the ability to detect the components. When applied to the separation of small
quantities, thin layer chromatography becomes a quantitative analytical method.
Adsorbents
The most commonly used adsorbents for column and thin layer chromatography are alumina (Al2O3)
and silica gel (SiO2) although others (charcoal, magnesium carbonate, sugar, and starch) have been
used. Alumina and silica gel are very polar and adsorb polar compounds to a greater extent than nonpolar compounds. This relationship is shown in the following order below:
Polarity of Functional Groups
As the polarity of a functional
group increases so will its
adsorption to the solid phase;
more polar groups adsorb more
strongly to the adsorbent, lowering
Rf's
saturated hydrocarbons
least polar
unsaturated hydrocarbons
ethers
halides
esters
ketones, aldehydes
amines
alcohols
carboxylic acids
most polar
Solvents
The extent of adsorption of a given compound on the adsorbent also depends on the polarity of the
solvent (mobile phase). If a solvent of improper polarity is used, the compound may be adsorbed too
weakly and move too fast, or it may be adsorbed too strongly and not move at all. In general a strongly
polar organic compound will require a polar solvent to minimize the extent of adsorption and carry the
compound up the plate. Mixtures of solvents are frequently used to fine-tune the solvent polarity. In
column chromatography the solvent can be gradually changed during the course of the separation.
Ultimately, the best choice of solvent is determined by experimentation. The following partial list of
solvents and their dielectric constants (measure of polarity) is useful for selecting a possible solvent
system.
Solvents
water
methanol
ethanol
acetone
methylene chloride
ethyl acetate
diethyl ether
toluene
hexane
Dielectric Constant
80
33
24
21
9
6
4
2.4
1.9
80
Increasing
polarity
General Procedure to Obtain a Thin Layer Chromatogram
NOTE: Test tubes or vials used to prepare solutions for TLC must be completely dry. The TLC
developing chamber must be dry. Pipets must be dry. When doing TLC do not let water contact the
TLC plate. TLC = no water.
Dissolve approximately 1-5 mg of a sample in 5-10 drops of
identification of samples
a volatile solvent (ethyl acetate, methylene chloride, etc.) in
1
2 3
a test tube or vial. Obtain a silica gel plate (handle by its
solvent front
edges only) and draw a light pencil line 6-8 mm from the
bottom of the plate across the silica gel surface being careful
not to scratch through the surface. This is the starting line
6.6 cm
where the spots will be applied, called the origin. Mark light
lines where you will apply the spots and add markers to
show the identity of the samples. Using a fine capillary tube
carefully apply a spot of each solution 1-2 mm in diameter
6-8 mm
on the line. Several (3-6) spots can be placed on the same
plate with very little separation between spots, provided that
the size of the spots is kept to a minimum. Pour 4-6 mL of the eluting solvent into a 150 mL beaker .
Carefully place the plate in the tank allowing it to rest on the side of the beaker. Cover with a watch
glass. It is very important that the level of the solvent in the tank not come up to the level of the spots
on the TLC plate. If this should happen, the spots will merely be washed into the eluting solvent and
defeat the purpose of the thin layer technique. Allow the solvent to climb to within approximately 1 cm
of the top of the plate. This is called developing the TLC plate. The leading edge of the solvent is
called the front or solvent front. Remove the plate and quickly mark the solvent front with a pencil
before the solvent evaporates. Allow the solvent to evaporate for a short while in the hood and then
visualize the spots. If TLC is done over several hours it may be necessary to add fresh solvent to the
tank since it is continually evaporating.
Visualization
Most organic compounds are not colored. The spots on TLC
plates must be visualized to determine their positions. Some
colorless compounds fluoresce and can seen by irradiating
the plate with long wavelength ultraviolet light. In this lab
plates that contain a fluorescent material mixed with the
silica gel will be used. Most organic compounds quench the
fluorescence of these plates and appear as dark spots against
a fluorescing background. A short wavelength ultraviolet
lamp is used for this purpose. Since the radiation used is in
the near ultraviolet you should not keep your hands in the
box with the TLC plate for more than a few seconds. Spots
are circled with a pencil to preserve their location. It should be noted that some compounds quench the
fluorescence much better than others. This can give misleading results when interpreting a TLC plate.
Molecules which contain aromatic rings show up especially well while molecules with no unsaturation
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or only isolated pi bonds (for example, simple esters, aldehydes ketones, etc.) may be nearly invisible
on the plate under ultraviolet light
The plate may also be placed in a tank containing iodine crystals or silica gel
coated with iodine as the photograph shows. Materials that absorb iodine
appear as brown spots. The spot for the compound anthracene can be seen
with iodine adsorbed on it. This is not a very sensitive method but is
convenient for some compounds. This method often works for those
molecules invisible under ultraviolet light. Another visualization method is to
spray or dip the plate with a reagent that reacts with the compound, producing
a color.
Calculation of Rf
The distance the spot moves from the point of application (the origin) relative to the distance the
solvent moved from that same point (the origin), under specified conditions (adsorbent, solvent, and
temperature) is a physical constant of the compound. This value is the Rf and is defined as:
Rf = distance traveled by sample/distance traveled by solvent
Due to limitations in determining the centers of spots, Rf values are only calculated to two significant
figures at most. Some materials, such as carboxylic acids and amines, give elongated spots so that the
center is just an estimate. Tentative identifications can be made from Rf data. More definitive
identifications are only made if a standard is available. If two samples are the same compound only one
spot will be seen on a thin-layer plate when they are spotted at the same location. Likewise, if two
samples are not the same, two spots will be seen, assuming they are separable on the plate, which is not
always the case. This is shown below where A and B are identical but C and D are not. The central spot
on each plate is made by applying solutions of A and B or C and D to the same place on the plate.
If A and B are identical
If C and D are not identical
The Experiment:
TLC Analysis
of Bthe Components
A A+B B
A A+B
C C+D of
D the Four-Component
C C+D D
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Mixture
Obtain three TLC plates. Handle them carefully and only by their edges. Remember, they are cut from
a larger glass sheet and their edges are sharp. Develop all plates with hexanes/ ethyl acetate (7:3) and
visualize them under ultraviolet light. Circle the spots with pencil. You may also spray the plates with
reagents if suggested by your instructor.
l. Plate One
On plate one, spot the samples that were saved during the extraction procedure, (1) “mixture”, (2) “after
HCl”, (3) “after bicarbonate”, (4) “after NaOH”. It will be necessary to add 5-10 drops of ether or ethyl
acetate to each sample since the ether more than likely has evaporated. This plate will show how
effective each stage of the extraction was in removing each component from the original mixture. You
may follow your flow chart for interpreting the plate. If the spots are too large it may be necessary to
redo the TLC with more dilute solutions. This also applies to other plates. Remember to rinse capillaries
well. Contamination will give erroneous results.
2. Plate two
On this plate, spot solutions prepared from your isolated components, (1) lidocaine, (2) 4-anisic
acid, (3) p-phenylphenol, and (4) biphenyl. The solutions are prepared by dissolving a small portion
of each material in a solvent such as ethyl acetate. This will allow you to estimate the purity of each
isolated component.
3. Plate three
Spot authentic/pure samples of (1) lidocaine, (2) p-anisic acid, (3) p-phenylphenol, and (4) biphenyl
that will be provided for you as solutions. From this plate you will be able to identify the components of
the mixture.
A note about TLC interpretation: Because lidocaine and p-anisic acid may react with themselves in an
acid base reaction the spots may not be round and well-resolved compared to neutral compounds such
as biphenyl.
Submit the products in labeled vials.
The report should contain:
1.
2.
3.
4.
5.
6.
7.
Title
Purpose
Structures of materials
The procedure (reference and outline)
Flow Chart containing reagents and structures.
Observations
Data (given in the following order)
83
Grams of Recovered Components
p-anisic acid
biphenyl
p-phenylphenol
lidocaine
TLC developing solvent _______________________
Plate 1
Extraction Progress
1
2
3
4
Plate 2
Isolated Compounds
5
6
7
Plate 3
Standards
8
5
6
7
8
1 = mixture
5 = lidocaine
5 = lidocaine
2 = after HCl
6 = p-anisic acid
6 = p-anisic acid
3 = after bicarbonate
4 = after NaOH
7 = p-phenylphenol
7 = p-phenylphenol
8 = biphenyl
8 = biphenyl
Rf's
lidocaine
p-anisic acid
biphenyl
8. Results (A detailed interpretation of the TLC plates)
9. Conclusions
ASA Questions:
84
p-phenylphenol
1.
Complete the reactions and add arrows to show how bonds are formed and broken.
OCH3
1 M HCl
N
CH3
OH
O
NaOH (aq)
OH
2.
Write a flow chart for the separation of 4-nitroaniline from naphthalene. Use structures to show
how the compounds exist at each step.
3.
The product of the following reaction is a liquid. What steps should you follow in the
laboratory to isolate and purify it?
O
HCl (aq)
O Na
in water
4.
Circle those compounds that form water soluble salts (quantitatively) when added to a
saturated aqueous sodium bicarbonate solution.
OH
NH2
OH
CO2H
OH
Cl
5.
Ethanol cannot be used as the organic layer in extractions where an aqueous layer is
involved. Why?
6.
Which compound is more polar? Compound A, Rf = 0.76; compound B, Rf = 0.56.
7.
What criteria should you use to determine what solvent to use in preparing a sample for TLC
analysis?
8.
What solvents could be added to increase and decrease the polarity of ethyl acetate?
9.
In this experiment the chromatography plates are coated with a thin film of:
(a) silica gel
10.
(b) aluminum oxide (c) magnesium sulfate
(d) talc
What is the structure of
(a) p-anisic acid (b) biphenyl (c) p-phenylphenol (d) diethyl ether (e) sodium bicarbonate
85