Separating the Components of a MixtureIntroduction: The natural world around us is an abundant source of new and interesting organic
compounds. Many pharmaceutical compounds including aspirin (an anti-inflammatory agent derived from
salicin), morphine (a compound used to treat moderate to severe pain), and quinine (an anti-malarial drug)
were originally isolated or derived from natural sources (Figure 1).1
Figure 1: Medicinally important natural products
When obtaining and testing therapeutic agents from naturals sources, the separation of compounds of
interest from the many other organic compounds which are present in a crude sample is very challenging.
One way compounds can be readily separated is by taking advantage of the acidic and/or basic properties
of the compound. For instance, a neutral carboxylic acid is often soluble in organic solvents such as ethyl
acetate but is insoluble in water. Treatment with an appropriate base will deprotonate the carboxylic acid,
yielding a water soluble carboxylate that is insoluble in ethyl acetate. For instance, benzoic acid is insoluble
in water (0.3 g per 100 g of water) but is soluble in ethyl acetate. Upon deprotonation with hydroxide the
product, sodium benzoate, is now soluble in water (66 g per 100 g of water, see Figure 2) but insoluble in
Figure 2: Acid/base chemistry and solubility of benzoic acid
In contrast, a compound that contains a basic site can be protonated under acidic conditions. When the
basic compound is not protonated, it will be organic soluble. Upon protonation, the conjugate acid of the
basic molecule will be positively charged and will therefore be soluble in aqueous solution (see Figure 3).
“A Historical Overview of Natural Products in Drug Discovery” Daniel A. Dias, Sylvia Urban and Ute Roessner, 2012, Metabolites,
2, 303 – 336.
Figure 3: Acid/base chemistry and solubility of aniline
Neutral organics (compounds that will not be protonated or deprotonated with typical Brönsted acids and
bases such as sodium hydroxide or hydrochloric acid) will still be neutral after treatment by an acid or base.
Thus, the neutral compound will always be insoluble in water but soluble in organic solvents. The
differences in solubilities of the neutral species and the ions provides an efficient method for separation of
compounds in a mixture.
Description of the Experiment:
The goal of this experiment is to separate, isolate and characterize the two components of an acid/neutral
mixture and a base/neutral mixture. In the first experiment, we will work with a larger quantity of material.
In the second experiment we will instead work at a scale often referred to as “microscale”. The basic
concepts and techniques are the same, the only difference is that we use slightly different glassware.
1 M sodium hydroxide
6 M sodium hydroxide
benzoic acid (122 °C)
2-chlorobenzoic acid (142 °C)
3-methoxybenzoic acid (103 – 108 °C)
1 M hydrochloric acid
6 M hydrochloric acid
Acetanilide (114 °C)
Phenacetin (134 °C)
1,4-dimethoxybenzene (54 – 56 °C)
4-chloroaniline (69.5 °C)
Ethyl 4-aminobenzoate (88 – 90 °C)
para-toluidine (44 °C)
1. Create a table of important physical properties for reagents and possible unknowns.
2. Write out the acid/base reaction that will occur when you treat 2-chlorobenzoic acid with 1 M
sodium hydroxide. Do the same to indicate what happens when you treat 5-chloroanline with 1 M
3. Prepare a flow chart of the steps that you will take in today’s experiment.
4. Look up the Safety Data Sheet (SDS) for each of the unknowns that you may be using today.
Indicate any safety concerns in your laboratory notebook.
5. Consider the following questions:
a. After you extract your organic layer with 20 mL of 1 M sodium hydroxide:
– Which layer is the organic layer and which layer is the aqueous layer? How can you
predict? How can you make sure?
– Which layer will the neutral compound be in and which layer will contain the unknown
b. Why do we wash the organic layer two times with 20 mL of 1 M aqueous sodium hydroxide
solution each time rather than just once with 40 mL?
125 mL Separatory Funnel
100 mL round bottom flask
– Ethyl acetate is a flammable solvent.
– Aqueous solutions of sodium hydroxide and hydrochloric acid are corrosive. Appropriate PPE
(Personal Protective Equipment) must be worn at all times
– Aqueous solutions can be poured down the drain with plenty of water.
– Ethyl acetate should be disposed of in the non-halogenated waste container.
– Solids should be disposed of in the solids waste container.
A. Separation of an unknown carboxylic acid from an unknown neutral compound: Large Scale
Dissolving your solids: Weigh approximately 2 g of your assigned unknown, being sure to record which
unknown you were assigned and the actual weight of the material in your laboratory notebook.
Transfer the solid to a 125 mL Erlenmeyer flask. Add 50 mL of ethyl acetate and swirl the solution until
all of the solids have dissolved.
Washing with Aqueous Base
Transfer the solution of your unknown to a 125 mL separatory funnel (place the funnel in a ring stand
before doing this and make sure that the stopper is closed.) and wash the solution 2 x 20 mL with
aqueous 1 M sodium hydroxide. Be sure to label your each container as to whether it is the aqueous or
Isolating the unknown neutral compound
Transfer the organic layer to a 100 mL round bottom flask. Remove the ethyl acetate on the rotary
evaporator and recrystallize your unknown from water. Obtain a weight for the isolated compound,
determine the % recovery (assuming that 50% of the unknown was the neutral compound) and obtain
the melting point of the material. Use this information to determine the identity of the material.
Isolating the unknown acid
Acidify the aqueous solution to pH 1 – 2 by the careful addition, dropwise, of 6 M aqueous hydrochloric
acid. Isolate the precipitate by vacuum filtration and continue pulling a vacuum until the solid is dry.
Determine the weight, % recovery and melting point of the material. Which unknown acid did you
B. Separation of an unknown aryl amine from an unknown neutral compound: small scale
Dissolving your solids
Weigh approximately 0.2 g of your unknown, being sure to record which unknown you were assigned
and the actual weight of the material in your laboratory notebook. Transfer the solid to a 10 mL
Erlenmeyer flask, add 5 mL of ethyl acetate, and swirl the solution until all of the solids have dissolved.
Washing with aqueous acid
Transfer your solution to a 15 mL centrifuge tube (NOTE: it works well to set the centrifuge tube in a 100
mL beaker so that it doesn’t tip) and wash the solution twice with 2 mL with 1 M aqueous hydrochloric
acid. Be sure to label each container as to whether it is the aqueous or organic layer!
Isolating the unknown neutral compound
Transfer the organic layer to a pre-weighed 10 mL Erlenmeyer flask and remove the solvent by gently
warming the solution under a stream of nitrogen gas. Obtain a weight for the isolated compound,
determine the % recovery (assuming that 50% of the unknown was the neutral compound) and
determine the melting point of the material (which unknown neutral is it?).
Isolating the unknown acid
Neutralize the aqueous solution by the dropwise addition of 6 M aqueous sodium hydroxide solution to
pH 10 (litmus paper). You should observe the formation of a solid precipitate (note: to maximize your %
recovery you can cool the solution in an ice bath). Isolate the precipitate by vacuum filtration. After the
compound is dry determine the weight, % recovery and melting point of the material. Which unknown
amine did you have?
Lab Report Conclusions
– Be sure to clearly indicate the identity of each of your unknowns and the data that you used to
come to each conclusion.