EXPERIMENT 1: CHROMATOGRAPHY -- SEPARATION OF A DYE MIXTURE and

EXPERIMENT 3 PART A: RESOLUTION OF ()--PHENYLETHYLAMINE, PART A

During this session, you will complete experiment 1 and prepare the crystals for experiment 3, which is to be
completed in a future lab session.

i. CHROMATOGRAPHY - SEPARATION OF A DYE MIXTURE

Introduction
Chromatography (from the Greek chroma, meaning colour, and graphein, meaning to write) is
a technique frequently employed by chemists and biochemists to separate the components of a
mixture. In its original application the method was used for the separation of coloured substances,
but colour is not required for compounds to be separated by this method. Colourless compounds
may be rendered visible either by the use of ultra-violet (UV) light or by chemical means. A number
of separation techniques using the principles of chromatography have been developed, some of
which are specific to a class of material (e.g. proteins, polysaccharides, lipids, organic polymers,
etc.).
All techniques depend upon the differential distribution of various components (analytes) of a
mixture between two phases, a mobile phase (liquid, gas or supercritical fluid) and a stationary
phase (solid or liquid adsorbent). Usually (and in this experiment), the stationary phase is a highsurface-area solid and the mobile phase is a liquid solvent. In that context, the differential
distribution usually (but not always) depends on the relative strengths of the intermolecular
interactions in a three-way competition between analyte, solvent and adsorbent: If the interactions
of the analyte with the stationary phase are much stronger than with the solvent and stronger than
those between the stationary phase and the solvent, then the analyte will tend to move slowly up
the plate. If the interactions of the analyte with the stationary phase are much weaker than they are
with the solvent or weaker than those between the stationary phase and the solvent, then the
analyte will tend to move quickly up the plate. The differences in mobility between analytes in a
sample mixture then depend on how differently the analytes interact with both solvent and
stationary phase. The choice of solvent is critical: Some solvents will out-compete both analytes for
the surface interactions, and both analytes will travel quickly, with little net separation. Some
solvents will fail to compete with either analyte for the surface, and neither analyte will move much,
with again little net separation.
The stationary phase may be polar or apolar. In this experiment, the surfaces of the stationary
phases will be polar, with both charged groups and neutral ones, and with hydrogen-bonding and

other dipolar interactions at play. Hence, both charged and neutral polar analytes will interact
strongly with such surfaces, and solvents that will adequately compete will need to be polar if the
most polar analyte is strongly polar. Solvents that have too little polarity will not compete and afford
little mobility, while those that are too polar will compete too successfully and afford too much
mobility. Similarly, the solvent will need to be less polar if the most polar analyte is less polar. An
ideal separation occurs when the solvent polarity is such that it outcompetes one analyte but not
the other. In practice, however, it is difficult to predict with any exactness just how polar the mobile
phase needs to be, and both experience and trial-and-error will help determine the appropriate
solvent for a given analyte.
Thin layer chromatography, frequently referred to as TLC, is largely an analytical technique to
separate nanomole to micromole quantities of analytes. It is used for determining the purity of
materials, for preliminary identification purposes and for determining the best solvent system to use
for later separations of mixtures on a larger, preparative scale for instance by column
chromatography. There is also a preparative version of TLC (called Preparative TLC) that uses
much thicker and wider layers to separate larger (milligram-scale) quantities of material. Paper
chromatography is a kind of thin-layer chromatography appropriate for analytes that can interact
with cellulose.
Analytical TLC plates are prepared as follows: A film of approximately 0.3 mm thickness of
alumina, silica gel, or other surface-active adsorbent material containing a small amount of binding
material (usually calcium sulphate) and optionally a fluorescent dye is applied as a slurry to one
side of a clean glass plate, aluminum sheet or plastic film. The slurry is then dried either at room
temperature or by heating. In this laboratory, we will use pre-prepared TLC plates available
commercially. Microliter-sized samples of mixtures to be analyzed are applied as solutions, placing
a small spot near the bottom of the plate or film using a glass capillary. The solvent is allowed to
air-dry, then the TLC plate is placed in an upright position in a chamber containing a small volume
of developing solvent (eluent). As the solvent ascends the thin layer by capillary action,
components of the mixture become separated and appear as distinct coloured spots (if the mixture
contains coloured substances). This is called a chromatogram. If the analytes are not coloured, UV
light can be used to visualize the migrated spots so long as the adsorbent contains a fluorescent
dye and the analytes can quench the fluorescence. Otherwise, a number of reagents can be used
to reveal spots by inducing a chemical reaction that produces a coloured product. Unlike in paper
chromatography, the thin material layer on the surface of a TLC plate can be treated with even very
corrosive reagents.

By marking the solvent front and measuring the distance travelled by each component, the
retention factors (Rf values) for individual compounds may be determined. In addition to being rapid
(from start to finish is usually a matter of minutes), the method is very sensitive using very little
material.
Column chromatography is a preparative technique used for separating mixtures of two or
more components, once an appropriate solvent has been determined using TLC. This technique
uses gravity or pressure (instead of capillary action) to move analytes downward along a vertical
column packed with a finely divided adsorbent already wet with a solvent (and thus free of air
pockets). The solvent used to prepare the column is often less polar than the one(s) used to eluent
the analytes. An analyte mixture is introduced as a solution at the top of the column, allowed to
penetrate the adsorbent, then fresh solvent is added continuously to the top of the column to carry
the analyte down and through the adsorbent. Each component of the mixture, in descending
through the column packing, partitions itself repeatedly between the adsorbent (the stationary
phase) and the solvent (the mobile phase). If the solvent is well chosen, the distribution coefficient
with respect to these two phases is different for each compound comprising the mixture, and,
therefore, each travels through the adsorbent at a different rate. Components of the mixture thus
become separated within a short time. If the analytes are coloured substances, the compounds
appear in the column packing as distinct, coloured bands. Continued addition of solvent to the
column finally elutes each compound as a coloured solution out the lower end and into a receiving
flask. A more polar solvent may be required to elute the more polar analyte(s) into separate
receiving flasks. The recovery of each compound as a pure substance then is possible by simply
evaporating the solvent.
Columns are usually prepared just before use, as in this experiment.

Pre-Lab Assignment
(must be submitted at the beginning of the lab)
1. Draw the chemical structures of sodium fluorescein and methylene blue dyes.
2. Which of these dyes is more polar and why?

Experimental
1. Thin Layer Chromatography
The mixture to be analyzed by TLC is a solution containing 0.2% sodium fluorescein dye and
0.2% methylene blue in 95% ethanol. Obtain two pre-coated thin-layer alumina plates. Use a
pencil and draw a line across the chalky side of the plate about cm from the shorter edge of
the plate. DO NOT draw with such force as to remove the alumina from the plate (this prevents
the dyes from flowing upwards). Draw a small quantity of the dye mixture into a micropipette
and lightly touch it to the plate on the line previously drawn. Keep the spot sizes to less than 1
mm in diameter. Repeat, spotting solutions of pure methylene blue solution and pure fluorescein
dye solution on the left and right sides of the dye mixture, parallel with the original spot as
shown below. These additional spots on the side will serve as controls.
Carefully place the TLC plate in the developing chamber (a 150 mL beaker) and allow the
back of the plate to lean against the side of the beaker at a slight angle from the vertical. Add
95% ethanol to the beaker such that the solvent level is just below the level of the spots when
the plate is immersed in the chamber. Cover the beaker with a watch glass to prevent solvent
evaporation from the plate. It is important that the level of the solvent not reach the level of the
sample spots, otherwise the sample will simply be dissolved off the plate instead of being
carried up by the rising solvent.

solvent front

baseline

fluorescein dye

dye mixture

methylene blue

Spotting TLC Plate

Rf =

b
=
a

distance traveled by spot

distance traveled by solvent

Determining Rf value

Once the solvent front has reached 9/10 of the height of the plate, remove it from the
chamber and immediately mark the solvent front with a pencil. After letting the solvent dry,

measure the Rf values for methylene blue and fluorescein. Note that the methylene blue may
contain a small amount of a pale purple impurity. If this additional purple spot appears on your TLC
plate, calculate its Rf value as well. Sketch the finished TLC plate to include with your results on the
Report Sheet (see Appendix).
Repeat the entire procedure using 65% ethanol as the eluting solvent.

2. Column Chromatography
The system to be separated is the same dye mixture used for the TLC exercise. To prepare the
column, clamp it in a vertical position with its Teflon stopcock closed. Insert a plug of cotton into the
bottom of the column with a long glass rod, using enough cotton to form a mat 1 cm thick. With the
aid of a plastic funnel, pour a layer of clean sand (about 1 cm in depth) on top of the cotton. In a 50
mL beaker, prepare a slurry of alumina (6 g) in ethanol (15 mL) and add to column. This aids in
the formation of an evenly packed column. Wash down the column walls with ethanol and add a 1
cm layer of sand to the top of the alumina.
solvent
sand

Always keep solvent level above

alumina

cotton plug

burette clamp

sand
stopcock

Chromatography Column

Open the stopcock and allow the solvent to drain just to the top of the upper layer of sand. At
no time should the solvent level be allowed to drain below this level; as this will produce entrapped
air bubbles, which forms channels that hinder separation. With the level of the eluent about 0.2 cm
above the sand, carefully add 0.5 mL of the dye mixture using a pipette. In order to maintain the
proper flow rate of the liquid at 1 drop per second from the column, attach one end of a rubber tube
to the air pump and the other end to the top of the column. Slowly open the stopcock. Introduce a
steady stream of air through the column. Run the mobile phase down and then carefully add fresh
95% ethanol at the top of the column using the squeeze bottle. Proceed to collect the blue dye
eluent. This is repeated until the eluent appears colourless. Now wash the column with 0.1 M

aqueous sodium hydroxide solution until no more yellow dye remains in the tube and collect the
eluent in another beaker. Record your observations.
ii. RESOLUTION OF ()--PHENYLETHYLAMINE, PART A
Experimental
Dissolve 12.0 g of (+)-tartaric acid in 165 mL of methanol in a 250-mL Erlenmeyer flask by
heating the unstoppered mixture on a steam bath. Remove the flask from the steam bath and
slowly add 10.0 mL of racemic ()--phenylethylamine (also called ()--methylbenzylamine) to the
warm solution (~ 1 mL each addition) while swirling the flask to mix well. [CAUTION: Salt formation
is exothermic and rapid addition of the amine will cause the solution to boil over and splash
upwards, possibly onto your face and hands.]
The desired salt will crystallize slowly as large clear prisms. The first crystals obtained are often
fine white needles instead of prisms; these needles do not provide optically pure amine and must
be avoided. To ensure that the desired prisms form, add a few seed crystals of the correct
composition. Cork and label the flask and set it aside until your scheduled day to complete
Experiment 3, Part B (see page 22).