Last Update: 4 November 2017 Part II

M 12

Paper Chromatography
Definition: Chromatography is a laboratory technique that is used to separates and to identify components
within a mixture by using the differential affinities of the components for a mobile medium and for a
stationary adsorbing medium through which they pass.

Principle: Chromatography is based on differential migration. The solutes in a mobile phase go through a
stationary phase. Solutes with a greater affinity for the mobile phase will spend more time in this phase than
the solutes that prefer the stationary phase. As the solutes move through the stationary phase they separate.
This is mainly based on the following properties:
Capillary Action the movement of liquid within the spaces of a porous material due to the forces
of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its
attraction to itself is stronger than the force of gravity.
Solubility the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into
solvents that have similar properties. (Like dissolves like) This allows different solutes to be
separated by different combinations of solvents.

Separation of components depends on both their solubility in the mobile phase and their differential
affinity to the mobile phase and the stationary phase.

Purpose of use:
Analyze examine a mixture, its components, and their relations to one another
Identify determine the identity of a mixture or components based on known components
Purify separate components in order to isolate one of interest for further study
Quantify determine the amount of the a mixture and/or the components present in the sample

How it works
In all chromatography there is a mobile phase and
a stationary phase. The stationary phase is the
phase that doesn't move and the mobile phase is the
phase that does move. The mobile phase moves
through the stationary phase picking up the
compounds to be tested. As the mobile phase
continues to travel through the stationary phase it
takes the compounds with it. At different points in
the stationary phase the different components of the
compound are going to be absorbed and are going
to stop moving with the mobile phase. This is how
the results of any chromatography are gotten, from
the point at which the different components of the

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compound stop moving and separate from the other components.
In paper and thin-layer chromatography the mobile phase is the solvent. The stationary phase in paper
chromatography is the strip or piece of paper that is placed in the solvent. In thin-layer chromatography the
stationary phase is the thin-layer cell. Both these kinds of chromatography use capillary action to move the
solvent through the stationary phase.
In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is
immersed in a solvent, and the solvent moves up the paper by capillary action. Components of the mixture
are carried along with the solvent up the paper to varying degrees, depending on the compound's preference
to be adsorbed onto the paper versus being carried along with the solvent. The paper is composed of
cellulose to which polar water molecules are adsorbed, while the solvent is less polar, usually consisting of a
mixture of water and an organic liquid. The paper is called the stationary phase while the solvent is referred
to as the mobile phase. Performing a chromatographic experiment is basically a three step process:
1) application of the sample,
2) "developing" the chromatogram by allowing the mobile phase to move up the paper, and
3) calculating Rf values and making conclusions.

What is the Retention Factor, Rf ?

The retention factor, Rf, is a quantitative indication of how far a particular compound travels in a particular
solvent. The Rf value is a good indicator of whether an unknown compound and a known compound are
similar, if not identical. If the Rf value for the unknown compound is close or the same as the Rf value for
the known compound then the two compounds are most likely similar or identical.
The retention factor, Rf, is defined as:
Rf = distance the solute (D1) moves divided by the distance traveled by the solvent
front (D2)
Rf = D1 / D2
where D1 = distance that color traveled, measured from center of the band of color
to the point where the food color was applied
and D2 = total distance that solvent traveled
Preparation of solvent
A solvent is a liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. The most
common solvent in everyday life is water. Most other commonly-used solvents are organic (carbon-
containing) chemicals. These are called organic solvents. Solvents usually have a low boiling point and
evaporate easily or can be removed by distillation, thereby leaving the dissolved substance behind. Solvents
should therefore not react chemically with the dissolved compounds they have to be inert. Solvents can
also be used to extract soluble compounds from a mixture, the most common example is the brewing of
coffee or tea with hot water. Solvents are usually clear and colorless liquids and many have a characteristic
odor. The concentration of a solution is the amount of compound that is dissolved in a certain volume of
solvent. The solubility is the maximal amount of compound that is soluble in a certain volume of solvent at
a specified temperature.
e.g. Chromatographic solvent contained 4:1:5 n-butanol : acetic acid : water mixture as a mobile
phase. This is generally used for monosaccharide oligosaccharide separation
Common uses for organic solvents are in dry cleaning (e.g. tetrachloroethylene), as paint thinners (e.g.
toluene, turpentine), as nail polish removers and glue solvents (acetone, methyl acetate, ethyl acetate), in
spot removers (e.g. hexane, petrol ether), in detergents (citrus terpenes), in perfumes (ethanol), and in
chemical syntheses.

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Polarity, solubility, and miscibility
Solvents and solutes can be broadly classified into polar (hydrophilic) and non-polar (lipophilic). The
polarity can be measured as the dielectric constant or the dipole moment of a compound. The polarity of a
solvent determines what type of compounds it is able to dissolve and with what other solvents or liquid
compounds it is miscible. As a rule of thumb, polar solvents dissolve polar compounds best and non-polar
solvents dissolve non-polar compounds best: "like dissolves like". Strongly polar compounds like inorganic
salts (e.g. table salt) or sugars (e.g. sucrose) dissolve only in very polar solvents like water, while strongly
non-polar compounds like oils or waxes dissolve only in very non-polar organic solvents like hexane.
Similarly, water and hexane (or vinegar and salad oil) are not miscible with each other and will quickly
separate into two layers even after being shaken well.
Protic and aprotic solvents
Polar solvents can be further subdivided into polar protic solvents and polar aprotic solvents. A polar protic
solvent is one that contains an O-H or N-H bond. A polar aprotic solvent is one that does not contain an O-H
or N-H bond. Water (H-O-H), ethanol (CH3-CH2-OH), or acetic acid (CH3-C(=O)OH) are representative
polar protic solvents. A polar aprotic solvent is acetone (CH3-C(=O)-CH3). In chemical reactions the use of
polar protic solvents favors the SN1 reaction mechanism, while polar aprotic solvents favor the SN2 reaction
mechanism.
Boiling point
Another important property of solvents is boiling point. This also determines the speed of evaporation.
Small amounts of low-boiling solvents like diethyl ether, dichloromethane, or acetone will evaporate in
seconds at room temperature, while high-boiling solvents like water or dimethyl sulfoxide need higher
temperatures, an air flow, or the application of vacuum for fast evaporation.
Density
Most organic solvents have a lower density than water, which means they are lighter and will form a
separate layer on top of water. An important exception: many halogenated solvents like dichloromethane or
chloroform will sink to the bottom of a container, leaving water as the top layer. This is important to
remember when partitioning compounds between solvents and water in a separatory funnel during chemical
syntheses.
Chemical interactions
A solvent will create various weak chemical interactions with the solute to solubilize the solute. The most
usual of these interactions are the relatively weak van der Waals interactions (induced dipole interactions),
the stronger dipole-dipole interactions, and even the strongest interaction, hydrogen bonds (interaction
between O-H or N-H hydrogens with adjacent O or N atoms).
General precautions
Avoiding the generation of solvent vapors by working in a fume hood, local exhaust ventilation
(LEV), or a well ventilated area
Keeping the storage containers tightly closed
Never using open flames near flammable solvents, use of electrical heating instead
Never flushing flammable solvents down the drain to avoid explosions and fires
Avoiding the inhalation of solvent vapors
Avoiding contact of the solvent with the skin many solvents are easily absorbed through the skin.
They also tend to dry the skin and may cause sores and wounds.

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Properties table of common solvents
The solvents are grouped into non-polar, polar aprotic, and polar protic solvents and ordered by increasing
polarity. The polarity is given as the dielectric constant. The density of nonpolar solvents that are heavier
than water is bolded.

Solvent Chemical Formula Boiling point Dielectric constant Density

Non-Polar Solvents

Hexane CH3-CH2-CH2-CH2-CH2-CH3 69 C 2.0 0.655 g/ml

Benzene C6H6 80 C 2.3 0.879 g/ml

Toluene C6H5-CH3 111 C 2.4 0.867 g/ml

Diethyl ether CH3CH2-O-CH2-CH3 35 C 4.3 0.713 g/ml

Chloroform CHCl3 61 C 4.8 1.498 g/ml

Ethyl acetate CH3-C(=O)-O-CH2-CH3 77 C 6.0 0.894 g/ml

Dichloromethane CH2Cl2 40 C 9.1 1.326 g/ml

Polar Aprotic Solvents

1,4-Dioxane /-CH2-CH2-O-CH2-CH2-O-\ 101 C 2.3 1.033 g/ml

Tetrahydrofuran (THF) /-CH2-CH2-O-CH2-CH2-\ 66 C 7.5 0.886 g/ml

Acetone CH3-C(=O)-CH3 56 C 21 0.786 g/ml

Acetonitrile (MeCN) CH3-CN 82 C 37 0.786 g/ml

Dimethylformamide (DMF) H-C(=O)N(CH3)2 153 C 38 0.944 g/ml

Dimethyl sulfoxide (DMSO) CH3-S(=O)-CH3 189 C 47 1.092 g/ml

Polar Protic Solvents

Acetic acid CH3-C(=O)OH 118 C 6.2 1.049 g/ml

n-Butanol CH3-CH2-CH2-CH2-OH 118 C 18 0.810 g/ml

Isopropanol CH3-CH(-OH)-CH3 82 C 18 0.785 g/ml

n-Propanol CH3-CH2-CH2-OH 97 C 20 0.803 g/ml

Ethanol CH3-CH2-OH 79 C 24 0.789 g/ml

Methanol CH3-OH 65 C 33 0.791 g/ml

Formic acid H-C(=O)OH 100 C 58 1.21 g/ml

Water H-O-H 100 C 80 1.000 g/ml

Partition coefficient (LogP)

A partition coefficient or distribution coefficient is a measure of differential solubility of a compound in

two solvents. The logarithmic ratio of the concentrations of the solute in the solvent is called log P
(sometimes LogP). The best known of these partition coefficients is the one based on the solvents octanol
and water. The octanol-water partition coefficient is a measure of the hydrophobicity and hydrophilicity of a
substance. In the context of drug-like substances, hydrophobicity is related to absorption, bioavailability,
hydrophobic drug-receptor interactions, metabolism and toxicity. In the field of hydrogeology, the octanol
water partition coefficient, or Kow, is used to predict and model the migration of dissolved hydrophobic
organic compounds in soil and groundwater.
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Definition of Partition Coefficient P and Distribution Coefficient D

The partition coefficient is the ratio of the concentration of the compound in octanol to the concentration
of the compound in water. The distribution coefficient is the ratio of the sum of the concentrations of all
species of the compound in octanol to the sum of the concentrations of all species of the compound in water.
Based on acid dissociation reactions, we can introduce the concept of a partition coefficient for cationic and
anionic species and for neutral species. The following gives the definition of partition and distribution
coefficients for ionized and unionized species.

The partition and distribution coefficients for multiprotic compounds are defined in much the same way as
for monoprotic compounds, using the following formulas.

The Different Types of Chromatography

There are four main types of chromatography. These are Liquid Chromatography, Gas Chromatography,
Thin-Layer Chromatography and Paper Chromatography.

Liquid Chromatography is used in the world to test water samples to look for pollution in lakes and rivers.
It is used to analyze metal ions and organic compounds in solutions. Liquid chromatography uses liquids
which may incorporate hydrophilic, insoluble molecules.

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Gas Chromatography is used in airports to detect bombs and is used is forensics in many different ways. It
is used to analyze fibers on a persons body and also analyze blood found at a crime scene. In gas
chromatography helium is used to move a gaseous mixture through a column of absorbent material.

Thin-layer Chromatography uses an absorbent material on flat glass or plastic plates. This is a simple and
rapid method to check the purity of an organic compound. It is used to detect pesticide or insecticide
residues in food. Thin-layer chromatography is also used in forensics to analyze the dye composition of
fibers.

Paper Chromatography is one of the most common types of chromatography. It uses a strip of paper as the
stationary phase. Capillary action is used to pull the solvents up through the paper and separate the solutes.

The table below summarizes the information from above.

Type of Chromatography Applications in the Real Why and What is it

World
Liquid Chromatography test water samples to look Used to analyze metal ions and organic
for pollution, compounds in solutions. It uses liquids
which may incorporate hydrophilic,
insoluble molecules.
Gas Chromatography detect bombs in airports, Used to analyze volatile gases. Helium is
identify and quantify such used to move the gaseous mixture through a
drugs as alcohol, used in column of absorbent material.
forensics to compare fibers
found on a victim
Thin-Layer detecting pesticide or Uses an absorbent material on flat glass
insecticide residues in food, plates. This is a simple and rapid method to
Chromatography also used in forensics to check the purity of the organic compound.
analyze the dye composition
of fibers
Paper Chromatography separating amino acids and The most common type of chromatography.
anions, RNA fingerprinting, The paper is the stationary phase. This uses
separating and testing capillary action to pull the solutes up
histamines, antibiotics through the paper and separate the solutes.

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