In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into

its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the mobile phase,
which carries it through a system (a column, a capillary tube, a plate, or a sheet) on which a material
called the stationary phase is fixed. Because the different constituents of the mixture tend to have
different affinities for the stationary phase and are retained for different lengths of time depending
on their interactions with its surface sites, the constituents travel at different apparent velocities in
the mobile fluid, causing them to separate. The separation is based on the differential partitioning
between the mobile and the stationary phases. Subtle differences in a compound's partition
coefficient result in differential retention on the stationary phase and thus affect the separation.[1]

Chromatography may be preparative or analytical. The purpose of preparative chromatography is to

separate the components of a mixture for later use, and is thus a form of purification.[2][3] This
process is associated with higher costs due to its mode of production.[4][5] Analytical
chromatography is done normally with smaller amounts of material and is for establishing the
presence or measuring the relative proportions of analytes in a mixture. The two types are not
mutually exclusive.

Chromatography was first devised at the University of Kazan by the Italian-born Russian scientist
Mikhail Tsvet in 1900.[8][9] He developed the technique and coined the term chromatography in the
first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll,
carotenes, and xanthophylls. Since these components separate in bands of different colors (green,
orange, and yellow, respectively) they directly inspired the name of the technique. New types of
chromatography developed during the 1930s and 1940s made the technique useful for many
separation processes.

Aim

In this experiment using paper chromatography, mixtures of food colours extracted from ‘Smarties’
will be separated and compared with a set of standard food dyes by observing the movement of the
individual extracted colours up a sheet of paper using an aqueous salt solution.

Objectives

Paper chromatography is an analytical method used to separate coloured chemicals or substances.

It is now primarily used as a teaching tool, having been replaced in the laboratory by other
chromatography methods such as thin-layer chromatography (TLC).

REQUIREMENTS
1. Large glass jam jar with cover (eg flat piece of plastic or glass)
2. 6 sharpened wooden sticks (ie toothpicks)
3. Hair dryer or warm oven
4. Ruler
5. Stapler
6. Scissors
7. Sellotape
8. Paper clips
9. Cotton thread
10. Lollipop sticks
11. Pencil
 12. Sheet of chromatography paper, or large filter paper
13. 0.1%(w/v) aqueous sodium chloride solution (mobile phase)
14. Distilled water
15. Pack of assorted food colours (red, yellow, green and blue)
16. Small test tubes
17. Labels
18. 2 tubes of ‘Smarties’ (mixed colours)

Procedure

1) Sort the ‘Smarties’ into piles of the same colour. Tip one of the piles into a
small clean test tube and to the ‘Smarties’ add a small quantity of cold distilled
water (approx 1cm3). Agitate the test tube and its contents for 2 minutes until
the colour appears in the aqueous phase. Decant the coloured solution into a
clean dry test tube. Label the tube and its contents with name of the
‘Smarties’ colour.

Repeat the extraction process for each pile of ‘Smarties’.

2) Measure the height of the glass jar into which the chromatography will be
carried out. From the large sheet of chromatography paper, cut a strip, which
is 20mm less than the height of the jar.

3)Now form this strip into a loose cylindrical shape so that the diameter of the
cylinder is approximately 20mm less than the internal diameter of the glass
jar. Do not staple the paper at this stage but flatten it out, on a clean dry
surface, and rule a pencil line across the paper so that it is 10mm from the
base of cylinder shape when formed.

4)This is the line onto which the colours are spotted.

5) Onto the pencil line and 10mm from the left edge of the paper, using a clean
wooden stick, spot a small drop of the ‘Smartie’ extract. If the colour is not
intense allow the spot to dry and repeat the spotting until colour is visible.

6) Then spot, using a clean wooden stick along the pencil line, 10mm apart,
the following:
1. red standard
2. yellow standard
3. ‘Smartie’ extract (same extract as spotted at the edge)
 4. green standard
5. blue standard
6. ‘Smartie’ extract (same extract as spotted at the edge)
7. yellow standard
8. red standard

Continue spotting until there is no space left along the pencil line. Dry the
spots thoroughly using a hair dryer or warm oven.

7) When the spots are dry, form the cylinder and staple the top and bottom of
the vertical edges of the paper to form a cylinder with the ‘Smartie’ extracts
and standard colours at the bottom. Do not allow the sides of the paper to
overlap but leave a small (say 2 mm) gap up the side of the cylinder.

8)Pour a small volume of the aqueous sodium chloride solution (mobile

phase) into the glass jar to a depth of 5mm and carefully lower the cylinder
into the liquid so that the pencil line and spots are above the level on the
liquid.

9)Quickly slide on the lid and allow the liquid to move up the paper
cylinder. Do not move the jar and its contents during the chromatography.

Observation

. In the case of paper chromatography, the stationary phase is the filter paper, and
the mobile phase is the solvent4. The more soluble the molecules, the further they
are carried up the paper

Result

The paper chromatography did show that smartie colors could be separated
into various colors. The smartie gets its color from a mixture of various
colored inks blended together. The first color of ink to appear on the filter
paper was yellow followed by pink, red, purple then blue. The colors
separated the way they did because of the differences in their molecular
characteristics, specifically, their solubility in water and their rate of
absorption by the paper.
Precautions

• Some standard colours may contain more than one component.

• If components are not visible after chromatography, the experiment will
have to be repeated using a greater loading of standard colour or
‘Smartie’ extract along the pencil line. The original aqueous extract may
have to be concentrated by evaporation of water by heating over a
flame.
• If the coloured spots move slightly sideways across the filter paper,
there is probably a paper overlap in the cylinder or touching of the
paper against the walls of the glass jar.
• If the mobile phase does not rise high up the paper, a greater volume
may be required in the bottom of the jar. In this case stop the
experiment when the aqueous phase stops moving up the paper.

Conclusion
The paper chromatography experiment is a fascinating technique used for
separating and identifying components within a mixture. Let’s delve into the
conclusion based on the purpose and summary of this experiment:
1. Purpose and Summary:
o In chemistry, one common challenge is separating mixtures into their
pure constituents. Most natural materials, like seawater, air, oil, and
coal, are mixtures. To study them chemically, we need to separate
them into individual, pure substances.
o Paper chromatography is a method that exploits the different
interactions between dissolved mixture components and a fixed solid
(usually special chromatography paper). By comparing how various
materials move on the paper, we can separate and identify them.
2. Findings from Paper Chromatography Experiments:
o The experiment typically involves dissolving the mixture (if not already
in liquid form) and applying a small drop of the solution to porous
chromatography paper.
o Different components create tiny spots on the paper, which can be
placed side by side.
o The spotted paper is then exposed to a special solvent, allowing the
components to migrate at different rates.
o The result is distinct separation of the mixture’s components, each
forming a unique band or spot on the paper.
3. Conclusions from Various Reports:
 o In one experiment, black ink was separated into distinct colors using
water as the solvent. Bands of yellow, black, and purple were observed.
o Isopropyl alcohol led to noticeable changes, resulting in bands of
yellow, green, black, and purple1.
o Another report emphasized the importance of paper chromatography
in identifying unknown amino acids and other relevant
mixtures. Adhesion and cohesion play crucial roles in the separation
process2.
o Yet another experiment confirmed that black ink could indeed be
separated into various colors, with yellow appearing first, followed by
pink, red, purple, and blue3.

In summary, paper chromatography serves as a valuable tool for scientists and

researchers, enabling them to unravel the complexities of mixtures and uncover
hidden details about their components