CHEM 231: Organic Form and Function
Laboratory 1
CSI Meadville: Preparative purification of a mixture
Specific Practical Goals




to
to
to
to
effect physical separation by leveraging chemical properties
master the technique of aqueous extraction using a separatory funnel
separate a solid from liquid using vacuum filtration
analyze samples using TLC and HPLC
The Premise
Benzocaine, or ethyl p-aminobenzoate (4), is a widely used inexpensive local anæsthetic. As part of a
homicide investigation, your forensics lab receives a 2 gram sample of impure benzocaine seized from
a basement laboratory. The sample is believed to contain significant amounts of benzoic acid (1),
most probably the result of low-quality starting materials. Your job is to use the chemical properties
of benzocaine and benzoic acid to separate the two components in high purity and recovery (yield), to
estimate the purity of the isolated compounds using analytical tools (balance, TLC, HPLC), and to
determine the percent composition of the original sample.
O
O
OH
pK a = 4.2
1 , benzoic acid
O
2, benzoate
O
O
O
H 3N
3, ethyl p-ammoniobenzoate
pK a = 4.8
O
H 2N
4 , ethyl p-aminobenzoate
Scheme 1. Proton transfer reactions of benzoic acid and ethyl p-aminobenzoate
Week 1
Overview of Extraction
The basis of extractive techniques is the "like dissolves like" rule. Water typically dissolves inorganic
salts (such as lithium chloride) and other charged species, while solvents (ethyl acetate, methylene
chloride, diethyl ether, etc.) dissolve neutral organic molecules. It is important to remember that this
method of separation relies on partitioning—that is, the preferential dissolution of a compound into
one solvent over another. For example, 2-pentanol is somewhat soluble in water (ca. 17 g / 100 mL
H2O), but infinitely soluble in diethyl ether. Thus, 2-pentanol can be preferentially partitioned into
ether.
This technique is usually carried out using two immiscible solvents—that is, that do not mix with each
other—one of which is almost always water. Typical organic solvents include ethyl acetate, hexane,
chloroform, methylene chloride, and diethyl ether. All of these form crisp delineations between
phases.
The two layers are commonly referred to as the aqueous phase and the organic phase. It is important
to keep track of these phases, as their positions are solvent dependent. For example, diethyl ether is
lighter than water, so the organic phase will rest on top in the separatory funnel, whereas methylene
chloride is heavier than water and so will sink to the bottom (Figure 1). Many are the students who
have realized only too late that they have discarded the wrong layer from an extraction.
methylene
chloride
extraction
H2O
diethyl
ether
extraction
aqueous layer
CH2 Cl2
Et2O
organic layer
H2O
organic layer
aqueous layer
Figure 1. Two possibilities for aqueous extraction
By way of vocabulary, actually two operations are encountered in the separatory funnel. When
components are removed from an organic layer by shaking with an aqueous solution, the organic
phase is said to be washed (e.g., “The combined ether extracts were washed with aqueous sodium
bicarbonate solution.”). On the other hand, when components are removed from water by treatment
with an organic solvent, the aqueous phase is said to be extracted (e.g., “The aqueous layer was
extracted with three portions of ethyl acetate.”). Thus, aqueous layers are extracted, and organic
layers are washed—although these two terms are sometimes (erroneously) used interchangeably.
Usually low-boiling organic solvents are used for extractions because they are easily removed by
evaporation, leaving behind the organic compound of interest. Many of these, however, carry over a
considerable amount of residual water from the aqueous washes. Ethyl acetate and diethyl ether both
dissolve large quantities of water (3.3% and 1.2%, respectively). Therefore, it is advantageous to
wash these organic layers with brine (saturated NaCl solution) at the end of the extraction sequence—
the brine draws out the dissolved water through an osmotic-like effect. For methylene chloride and
chloroform a brine wash is unnecessary, since the solubility of water in these solvents is quite low.
Once freed from the bulk of residual water, the organic layer is dried over a desiccant, such as sodium
sulfate, calcium chloride, or magnesium sulfate, and then decanted or filtered before evaporation.
Review of Acid-base Chemistry
You will recall from freshman chemistry that the pKa of an acid corresponds to the pH at which it is
exactly half ionized. Therefore, if hydrofluoric acid (HF) has a pKa of 3.2, and you make an aqueous
solution of HF at pH 3.2, then the solution will consist of exactly 50% HF and 50% F-. Also bear in
mind that the pH (and pKa) scale is logarithmic, so that by changing the pH by one unit, we change
this balance tenfold. In other words. an HF solution at pH 4.2 would consist of 90% F-, and one at pH
5.2 would be 99% F-. Thus, pH can be a powerful tool for modifying the charge properties of a
compound.
If this feels a bit rusty, you should brush off the freshman chemistry text and review the section on
acid-base chemistry, particularly the passages dealing with the acidity constant.
Safety Considerations
You must abide by all laboratory safety rules
Exposure routes for methylene chloride are transdermal and inhalation. Avoid contact with skin and
always work in a fume hood.
Concentrated hydrochloric acid is corrosive to skin, eyes, and clothing. Handle with extreme caution!
Procedural Overview
Important Note: You will be evaluated on the amount of compound you recover. Therefore, make
sure all transfers are quantitative. If you are unsure how to do this, ask your instructor. Likewise,
purity counts, so make sure all glassware is clean before using.
As soon as you receive the sample, record its weight and ID number in your notebook. Completely
dissolve the solid in 20 mL of methylene chloride, and save two drops in a centrifuge vial for a TLC
sample. Transfer the remaining solution to a separatory funnel1 and partition using an equal amount
of 3M sodium hydroxide. Separate the phases into two Erlenmeyer flasks, taking care to keep track of
which flask contains which phase (aqueous or organic).
Extract the organic phase with another portion of 3M sodium hydroxide and separate the phases again
into two Erlenmeyer flasks. Dry the organic phase with sodium sulfate2 and remove the solvent using
rotary evaporation.3 Store the flask until next laboratory period according to your instructor’s
directions.
Notes:
1
always use a ring stand for a sep funnel—never use a clamp for holding the funnel.
2
make sure the sodium sulfate container is immediately and tightly resealed!
3
remember to tare the round-bottom flask before you transfer the solution to it.
Week 2
Overview of Chromatography
Chromatography represents the most versatile separation technique readily available to the organic
chemist. Conceptually, the technique is very simple—there are only two components: a stationary
phase (usually silica or cellulose) and a mobile phase (usually a solvent system). Any two compounds
usually have different partitioning characteristics between the stationary and mobile phases. Since
the mobile phase is moving (thus the name), then the more time a compound spends in that phase,
the farther it will travel.
Chromatographic techniques fall into one of two categories: analytical and preparative. Analytical
techniques are used to follow the course of reactions and determine purity of products. These
methods include gas chromatography (GC), high-performance liquid chromatography (HPLC), and
thin-layer chromatography (TLC). Sample sizes for these procedures are usually quite small, from
microgram to milligram quantities. In some cases, the chromatograph is coupled to another analytical
instrument, such as a mass spectrometer or NMR spectrometer, so that the components that elute can
be easily identified.
Preparative methods are used to purify and isolate compounds for characterization or further use. The
most common techniques in this category are preparative HPLC, preparative TLC, and column
chromatography. One popular modification of column chromatography is the flash column, which
operates at medium pressures (about 10 psi) and provides very rapid separation (ca. 5 min elution
times). Column chromatography is suitable for sample sizes ranging from a few milligrams to several
grams.
Collectively, these methods fall under the category of LC (liquid chromatography), meaning the mobile
phase is some liquid solvent system. Occasionally, this is a single solvent, but more often than not it
is a binary mixture of solvents with different polarities. The advantage of the latter is that the bulk
polarity can be modulated by varying the ratio of the two solvents.
For example, consider a typical TLC plate (Figure 1) developed in a 1:1 mixture of ethyl acetate and
hexane, which exhibits two well-separated components. The spots can be characterized by their Rf
value, which is defined as the distance traveled from the origin divided by the distance traveled by the
mobile phase. Generally speaking, the slower moving component (Rf 0.29) is either larger, more
polar, or both. If we wanted a larger Rf value, we could boost the solvent polarity by increasing the
proportion of ethyl acetate in the mobile phase. Conversely, more hexane would result in lowerrunning spots.
50% EtOAc
in hexane
solvent front
c
b

Rf2 = b/c (0.69)

Rf1 = a/c (0.29)
a
baseline (origin)
Figure 1. A typical thin-layer chromatography (TLC) plate
Sometimes one or more of the components interact so strongly with the silica gel that departure from
the origin comes only with great difficulty. One answer to this problem is to derivatize the silica with
non-polar substituents, such as long-chain aliphatic residues. This creates a situation known as a
reverse phase system. In normal phase chromatography, the stationary phase (i.e., silica) is more
polar than the mobile phase; in reverse phase, the stationary phase is less polar than the mobile
phase. The derivatized silica is not only much less polar itself, but it also allows for the use of very
polar solvents, such as water and methanol. The reversed phase results in some counterintuitive
outcomes. For example, more polar components actually elute faster, and a more polar solvent
system results in an increased retention time (i.e., lower Rf value).
These parameters are
summarized in Table 1. In this laboratory, you will be using both types: the TLC plates are normal
phase and the HPLC column is reverse-phase.
parameter
normal phase
reverse phase
typical stationary phase
underivatized silica gel
C8-hydrocarbon derivatized silica
representative mobile phase
ethyl acetate / hexane mixture
acetonitrile / water mixture
more polar components:
have lower Rf values
have higher Rf values
increasing solvent polarity:
increases Rf values
decreases Rf values
Table 1. Comparison of normal and reverse-phase chromatography
Safety Considerations
You must abide by all laboratory safety rules
Exposure routes for methylene chloride are transdermal and inhalation. Avoid contact with skin and
always work in a fume hood.
Do not look directly into the UV lamp! Protect skin from prolonged exposure.
Procedural Overview
Important Note: You will need to bring a ruler and pencil to lab.
Add 1 mL of acetonitrile to the centrifuge tube containing the sample from last week. Then, after
weighing the isolated solids, prepare analytical samples of each (1-5 mg in 1 mL acetonitrile) and
analyze using TLC and HPLC according to the procedure below.
TLC Procedure
a.
Spot the two analytical samples from last week on a single TLC plate
b.
Develop the plate in a solvent of methylene chloride containing 3% methanol and 0.5% acetic acid
c.
Visualize the developed plate with a UV lamp and calculate all Rf values
HPLC Operating Procedure
a.
Make sure you are operating in the proper software window (Instrument 1 is on the left)
b.
Ensure that the system is available—a green "Ready" box will show in the upper left quadrant
c.
On the top menu, go to RunControl  Sample Info; enter your name under Operator Name
d.
Clean a 50 L Hamilton syringe (handle with utmost care!) by drawing methanol into it to full
capacity; purge into a waste container; repeat
e.
With injector in “Load” position (counterclockwise), flush out sample loop with methanol 4 or 5
times using a full syringe
f.
Load sample into injector using at least 3 full injections, avoiding any air bubbles in the syringe;
leave the syringe in the injector port after the last injection
g.
Turn injector to “Inject” position (clockwise), leaving the syringe in the injector; software will set a
zero point on the trace
h.
Do not make any hardware adjustments while a run is in progress
i.
When the sample has finished, your trace will automatically print out