This document describes how to perform paper chromatography to separate mixtures of colored ink dyes. Paper chromatography works by using solvents to differentially migrate the dye mixtures across a stationary filter paper phase based on differences in their polarities. The experiment demonstrated separation of 6 colored ink dyes (black, red, green, blue, purple, pink) across solutions with varying concentrations of isopropanol solvent. Observation of how far each dye migrated showed they separated into distinct components due to differences in solubility related to their chemical structures and polarities.

1. Clarinda clare linus
Diploma In Medical Sciences – January 2011

2.  At the end of this laboratory activity, the
students should be able to:
 Be introduced to the principles and
terminology of chromatography and
demonstrate separation of the dyes in
colored pens with paper chromatography.

3.  Paper chromatography is an analytical chemistry
technique for separating and identifying color
mixtures.
 In paper chromatography, substances are
distributed between stationary phase and a
mobile phase. Stationary phase is usually a piece
of filter paper and mobile phase is the colors that
travels up the stationary phase.
 Components of the samples will separate readily
according to how strongly they absorb on the
stationary phase vs. how readily they dissolve in
the mobile phase.

4.  6 beakers of jars
 6 covers of lids
 Distilled water
 Isopropanol
 Graduated cylinder
 6 strips of filter paper
 Different colors of water color pens
 Pencil
 Ruler
 Scissors
 tape

5.  Prepare 15 ml of isopropanol solutions in
labeled beakers
0%, 5%, 10%, 20%, 50% and 100%
 Cut 6 strips of filter paper
- Draw a line 1cm above the bottom edge of
the strip with the pencil
- Label each strip with its corresponding
solution
- Place a spot from each pen on your starting
line

6.  Place each strip in each beaker
- Cover the beakers
- Let the strips absorb the solutions about 2
cm from the top of the strips
- Remove strips and let them dry
 Observe the colors separated in low
concentrations of isopropanol and high
concentrations of propanol.
 Record data.

8. Distilled water 5% alcohol

9. 10% alcohol 20% alcohol

10. 50% alchohol 100% alcohol

11.  1st – black
 2nd – red
 3rd – green
 4th – blue
 5th – purple
 6th - pink

12.  According to the conducted experiment, the
results are as follows:
 Distilled water : Colors does not rise up to
stationary phase, dissolve
 Alcoholic solution: Colors rise up to stationary
phase and spread its colors
1. Black = purple and yellow color
2. Red = bright red and white
3. Green = blue and yellow
4. Blue = bright blue
5. Purple = bright purple and pink
6. Pink = Bright pink and white

13.  When a colored sample is placed on a filter
paper, the colors separate from the sample
by placing one of the paper in a low-leveled
solvent.
 The solvent diffuses up to the filter paper,
dissolving the various molecules in the
sample according to polarities of the
molecules and the solvent.
 If the sample contains more than one color, it
means that it have more than one kind of
molecule.

14.  Because of the different chemical structures
of each kind of molecules, the chances are
very high that each molecule will have at least
a slightly different polarity, giving each
molecule a different solubility in the solvent.
 The unequeal solubilities cause the various
color molecules to leave solution at different
places as the solvent continues to move up
the paper.

15.  Hence, the more soluble is a molecule, the
higher it will migrate up the paper.

16.  Chromatography has many uses. It is
commonly used in laboratories to isolate new
compounds, analyze subtle differences
between different environmental samples,
and even in the sequencing of DNA.

17.  To perform chromatography, one need two
things; a matrix and a color indicator. A
matrix is simply the materials to which a
sample is applied, the material is often
porous, action as filter paper that affects the
rate of flow of a sample. Special beads with
different pore sizes are commonly used to
separate proteins in biochemistry labs.

18.  The matrix may interact with a sample, and
depending on the chemical properties of
both, and is commonly used to analyze
mixtures of sugars.

19.  The color indicator may be the compound
itself. Some organic compounds are bright
yellow or orange, which makes them easy on
identifying on a chromatogram. Others need
to react with another chemicals to become
colored.
 Dyes specific for particular chemical
substances properties can be used to identify
samples. For examples, rhodamine dye can
be applied to visualize fats and oils.