Laboratory Activity 3: Separation of Ink Pigments Using Paper Chromatography
Objective:
To separate and identify the different pigments in ink using paper chromatography.
Principle:
Paper chromatography is a separation technique based on the differences in solubility and affinity of
substances in a solvent. A mobile phase (solvent) moves through a stationary phase (chromatography
paper), carrying different components at different rates based on their polarity and solubility.
The retention factor (Rf) value for each pigment is calculated as:
where:
A higher Rf means the substance is more soluble in the solvent.
A lower Rf means the substance has more attraction to the paper.
Materials and Equipment:
Filter paper or chromatography paper
Black ink pen (water-soluble)
Ruler and pencil
Beaker or glass jar (100 mL)
Wooden stick or pencil (to hold the paper)
Binder clip or tape
Water or ethanol (as solvents)
Dropper or pipette
Procedure: (Pen Ink)
Step 1: Prepare the Chromatography Paper
1. Cut a strip of filter paper (about 10 cm x 3 cm).
2. Draw a light pencil line (about 2 cm from the bottom) – this is the baseline.
3. Place a small ink dot in the center of the baseline using the black pen.
Step 2: Set Up the Experiment
4. Pour about 1 cm of solvent (water or ethanol) into the beaker.
5. Attach the top of the paper strip to a pencil using a binder clip or tape.
6. Suspend the paper so that the bottom edge just touches the solvent, ensuring the ink spot does
not touch the solvent directly.
Step 3: Allow the Chromatography to Run
7. Leave the setup undisturbed for 10–15 minutes as the solvent moves up the paper.
8. Once the solvent reaches about ¾ of the paper’s length, remove the strip and mark the solvent
front with a pencil.
9. Let the paper dry completely.
Step 4: Analyze the Results
10. Measure and record:
o Distance traveled by each pigment (from the baseline to the pigment spot).
o Distance traveled by the solvent front.
11. Calculate the Rf value for each pigment:
Procedure: (Leaves)
1. Cut the leaves into small pieces with scissors.
2. Put 10 grams of leaves into a mason jar or drinking glass.
3. Add four teaspoon of isopropyl alcohol (70% or higher).
4. Crush the leaves into the alcohol using the blunt end of a wooden spoon for about five minutes,
until the solution is dark.
5. Let the solutions sit for 30 minutes in a dark place indoors.
6. Transfer the crushed leaf solution into a resealable plastic bag. Do this for each leaf color
separately. Then close each bag but leave one end open. Squeeze the leaves and collect the
colored liquid in a small heat-resistant container.
7. Pour some water into a pot and heat it up on the stove. Then switch off the stove and place the
three heat-resistant containers with your leaf extracts into the hot water. Let them sit in the hot
water for about 20–30 minutes, or until the isopropyl alcohol has mostly evaporated. As the
alcohol evaporates, your extracts should become thicker when stirred with a fork.
a. Concentrating the extracts should not require more than 30 minutes of evaporating off
the isopropyl alcohol, although the extracts will become more concentrated the longer the alcohol
is allowed to evaporate off.
b. Stir each color thoroughly to blend and loosen any bits of dried up pigment from the
side of the bowl. Be sure to use a clean fork for each color so you do not mix them!
8. Measure and record:
o Distance traveled by each pigment (from the baseline to the pigment spot).
o Distance traveled by the solvent front.
9. Calculate the Rf value for each pigment:
Data Table:
A. Pen Ink
Rf
Pigment Color Distance Traveled by Pigment (cm) Distance Traveled by Solvent (cm)
Value
B. Leaves
Rf
Pigment Color Distance Traveled by Pigment (cm) Distance Traveled by Solvent (cm)
Value
Discussion Questions:
1. How many pigments were separated from the black ink?
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
2. Which pigment traveled the farthest? What does this indicate about its solubility?
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
3. How does the choice of solvent affect separation?
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
4. What are some real-world applications of chromatography (e.g., forensic analysis, food testing)?
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
