Column Chromatography (CC)

1. Introduction to chromatography

Chromatography is perhaps the most important technique used by organic chemists to

separate the components of a mixture. This technique involves the distribution of the different
compounds or ions in the mixture between two phases, one of which is stationary and the
other moving.

Chromatography works on much the same principle as solvent extraction. In extraction, the
components of a mixture are distributed between two solvents according to their relative
solubilities in the two solvents. In chromatography, the separation of a mixture is achieved by
distribution of the mixture between two phases, the stationary and the moving phases. The
separation process in chromatography depends on differences in how strongly the components
of the mixture are adsorbed to the stationary phase and how soluble they are in the moving
phase. These differences depend primarily on the relative polarities of the components in the
mixture.

There are many types of chromatographic techniques, ranging from thin-layer

chromatography (TLC) and column chromatography (CC), which are relatively simple and
inexpensive, to high-performance liquid chromatography (HPLC), which is very sophisticated
and expensive.

Various types of chromatography are possible, depending on the nature of the two phases
involved: solid–liquid (column, thin-layer, and paper), liquid–liquid (high performance
liquid), and gas–liquid (vapor-phase) chromatographic methods are common.

2. Column chromatography (CC)

All chromatography works on much the same principle as solvent extraction. Basically, the
methods of CC depend on the differential solubilities or adsorptivities of the substances to be
separated relative to the two phases between which they are to be partitioned. Here, column
chromatography, a solid–liquid method, is considered.

2.1. Adsorbents

CC is a technique based on both adsorptivity and solubility. It is a solid–liquid phase-

partitioning technique. The solid may be almost any material that does not dissolve in the
associated liquid phase; the solids used most commonly are silica gel (SiO2 xH2O), and
alumina (Al2O3 · xH2O). These compounds are used in their powdered or finely ground
forms.

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2.2. Interactions

If powdered or finely ground alumina (or silica gel) is added to a solution containing an
organic compound, some of the organic compound will adsorb onto or adhere to the fine
particles of alumina. Many kinds of intermolecular forces cause organic molecules to bind to
alumina/ silica gel. These forces vary in strength according to their type. Nonpolar
compounds bind to the silica gel using only van der Waals forces. These are weak forces, and
nonpolar molecules do not bind strongly unless they have extremely high molecular weights.
The most important interactions are those typical of polar organic compounds. Either these
forces are of the dipole–dipole type or they involve some direct interaction (coordination,
hydrogen bonding, or salt formation). Similar interactions occur with alumina. The strengths
of such interactions vary in the following approximate order:

Salt formation > coordination > hydrogen bonding > dipole–dipole > van der Waals

The more polar the functional group, the stringer the bond to alumina (or silica gel).

Strength of interaction varies among compounds. For instance, a strongly basic amine would
bind more strongly than a weakly basic one (by coordination). In fact, strong bases and strong
acids often interact so strongly that they dissolve alumina to some extent. You can use the
following rule of thumb. A similar rule holds for solubility. Polar solvents dissolve polar
compounds more effectively than nonpolar solvents do; nonpolar compounds are dissolved
best by nonpolar solvents. Thus, the extent to which any given solvent can wash an adsorbed
compound from alumina depends almost directly on the relative polarity of the solvent. For
example, although a ketone adsorbed on alumina might not be removed by hexane, it might be
removed completely by chloroform. For any adsorbed material, a kind of distribution
equilibrium can be envisioned between the adsorbent material and the solvent.

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The distribution equilibrium is dynamic, with molecules constantly adsorbing from the
solution and desorbing into it. The average number of molecules remaining adsorbed on the
solid particles at equilibrium depends both on the particular molecule (RX) involved and the
dissolving power of the solvent with which the adsorbent must compete.

2.3 Principle of CC Separation

The dynamic equilibrium and the variations in the

extent to which different compounds adsorb on
alumina or silica gel underlie a versatile and
ingenious method for separating mixtures of
organic compounds. In this method, the mixture of
compounds to be separated is introduced onto the
top of a cylindrical glass column packed or filled
with fine alumina particles (stationary solid phase).
The adsorbent is continuously washed by a flow of
solvent (moving phase) passing through the Figure 3. A chromatographic column
column.

Initially, the components of the mixture adsorb onto the alumina particles at the top of the
column. The continuous flow of solvent through the column elutes, or washes, the solutes off
the alumina and sweeps them down the column. The solutes (or materials to be separated) are
called eluates or elutants, and the solvents are called eluents. As the solutes pass down the
column to fresh alumina, new equilibria are established among the adsorbent, the solutes, and
the solvent. The constant equilibration means that different compounds will move down at
differing rates depending on their relative affinity for the adsorbent on the one hand, and for
the solvent on the other. Because the number of alumina particles is large, because they are

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closely packed, and because fresh solvent is being added continuously, the number of
equilibrations between adsorbent and solvent that the solutes experience is enormous.

As the components of the mixture are separated, they begin to form moving bands (or zones),
with each band containing a single component. If the column is long enough and the other
parameters (column diameter, adsorbent, solvent, and flow rate) are correctly chosen, the
bands separate from one another, leaving gaps of pure solvent in between. As each band
(solvent and solute) passes out from the bottom of the column, it can be collected before the
next band arrives. If the parameters mentioned are poorly chosen, the various bands either
overlap or coincide, in which case either a poor separation or no separation is the result. A
successful chromatographic separation is illustrated in Figure 4.

Figure 4. Sequence of steps in a chromatographic separation

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2.4. Parameters affecting separation

The versatility of CC results from the many factors that can be adjusted. These include:

(1) Adsorbent chosen

(2) Polarity of the solvents chosen

(3) Size of the column (both length and diameter) relative to the amount of material to be
chromatographed

(4) Rate of elution (or flow)

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Thin-layer chromatography (TLC)

TLC is a very important technique for the rapid separation and qualitative analysis of small
amounts of material. It is ideally suited for the analysis of mixtures and reaction products in
both macroscale and microscale experiments. The technique is closely related to column
chromatography. In fact, TLC can be considered CC in reverse, with the solvent ascending
(moving up) the adsorbent rather than descending (going or coming đown).

1. Principles of TLC

Like CC, TLC is a solid–liquid partitioning technique. The principles of TLC are similar to
those of CC. The primary difference is that the moving phase in CC travels downward the
adsorbent, whereas in TLC the solvent ascends (moves up) the plate. The most typical
backing is a plastic material, but other materials are also used. A thin layer of the adsorbent is
spread onto the plate and allowed to dry. A coated and dried plate is called a thin-layer plate.
When a thin-layer plate is placed upright in a vessel that contains a shallow layer of solvent,
the solvent ascends the layer of adsorbent on the plate by capillary action (Figure 1).

Figure 1. A development chamber with a TLC plate undergoing development

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In TLC, the sample is applied to the plate (as a small spot near the base of the plate using a
small capillary pipet) before the solvent is allowed to ascend the adsorbent layer. When the
filled pipet touches the plate, capillary action delivers its contents to the plate and a small
spot is formed. As the solvent ascends the plate, the sample is partitioned between the moving
liquid phase and the stationary solid phase. During this process (developing/running the TLC
plate). In development, the various components in the applied mixture are separated. The
separation is based on the many equilibrations the solutes experience between the moving and
the stationary phases.

As in CC, the least polar substances advance faster than the most polar substances. A
separation results from the differences in the rates at which the individual components of the
mixture advance upward on the plate. When many substances are present in a mixture, each
has its own characteristic solubility and adsorptivity properties, depending on the functional
groups in its structure. In general, the stationary phase is strongly polar and strongly binds
polar substances. The moving liquid phase is usually less polar than the adsorbent and most
easily dissolves substances that are less polar or even nonpolar. Thus, the most polar
substances travel slowly upward, or not at all, and nonpolar substances travel more rapidly.

When the thin-layer plate has been developed, it is removed from the developing tank and
allowed to dry until it is free of solvent. If the mixture that was originally spotted on the plate
was separated, there will be a vertical series of spots on the plate. Each spot corresponds to a
separate component or compound from the original mixture.

2. The Rf value

TLC conditions include:

(1) Solvent system

(2) Adsorbent

(3) Thickness of the adsorbent layer

(4) Relative amount of material spotted

Under an established set of such conditions, a given compound always travels a fixed distance
relative to the distance the solvent front travels. This ratio of the distance the compound
travels to the distance the solvent front travels is called the Rf value. The symbol Rf stands for
“retardation factor,” or “ratio to front,” and it is expressed as a decimal fraction:

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3. Visualization methods

If the mixture components are colored substances, various spots will be clearly visible after
development. More often, however, the “spots” will not be visible because they correspond to
colorless substances. If spots are not apparent, they can be made visible only if a visualization
method is used. Three common techniques to visualize/ detect spots on a TLC plate:

(1) Put the TLC plate under UV light: some organic compounds illuminate or fluoresce
under short-wave UV light

(2) Stain the TLC plate with iodine: iodine vapor forms brown/yellow complexes with
organic compounds

(3) Spray the TLC plate with sulfuric acid and then heat the plate: permanent charged spots
are produced

4. Applications of TLC in organic chemistry

In organic chemistry, TLC can be used in the following applications:

(1) To establish that two compounds are identical

(2) To determine the number of components in a mixture

(3) To determine the appropriate solvent for a column-chromatographic separation

(4) To monitor a column-chromatographic separation

(5) To check the effectiveness of a separation achieved on a column, by

crystallization/extraction

(6) To monitor the progress of a reaction

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Homework questions

Chromatography

1) What is chromatography technique used for?

2) What kind of distribution does chromatography technique involve?
3) How is chromatographic separation achieved?
4) What kind of differences does the separation process in chromatography depend on?

Column chromatography (CC)

1) What is the technique of CC used for?

2) What are the pieces of apparatus needed to run a chromatographic column?

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 3) What are the two phases of a chromatographic column?
4) What is the separation technique of CC based on?
5) What are the two factors that CC depends on?
6) What are the parameters affecting separation in CC?
7) What are the two most common adsorbents used in CC?
Thin-layer chromatography (TLC)
8) What is the technique of TLC used for?
9) What causes separation in TLC technique?
10) What action/ force that drives the travel of the mobile phase over the surface of the
stationary phase in TLC?
11) What is the main similarity between CC and TLC?
12) What is the main difference between CC and TLC?
13) What does Rf stand for and what does it mean?
14) What are the three common techniques to visualize/ detect spots on a TLC plate?
15) What are the six applications of TLC in organic chemistry?

(From Pavia, D. L. 2011. A small-scale Approach to organic laboratory techniques. Belmont,

CA: Brooks/Cole Cengage Learning)