Category: Handouts

Paper Chromatography Lab: Separating the Components of Black Ink

Introduction to Chromatography in the Science Classroom

Chromatography is a fundamental technique used in chemistry and biology to separate mixtures into their individual components. In this experiment, we explore how paper chromatography can be used to analyze and isolate the different pigments found in black ink.

The process relies on the movement of a solvent (in this case, water) through a medium (filter paper). As the solvent travels up the paper, it carries along the dissolved components of the mixture. Each substance moves at a different rate based on its molecular characteristics—such as size, solubility, and affinity for the paper. This separation allows for a visual and measurable representation of what the mixture is composed of.

Chromatography is widely used in scientific research for chemical analysis, substance purification, and compound identification. It also plays a role in detecting changes in chemical properties due to biological or chemical reactions. For students and educators, it’s a hands-on way to visualize how mixtures behave at the molecular level.

Hypothesis

Paper chromatography can be used to separate the individual pigments that make up black ink.

Materials

  • Filter paper strip
  • Test tube
  • Rubber stopper
  • Straightened paper clip (with hook)
  • Black felt-tip marker
  • Pencil
  • Metric ruler
  • Tap water (used as solvent)
  • Eraser
  • Computer (for data analysis or reporting)

Procedure: How to Perform Paper Chromatography

  1. Prepare the apparatus: Straighten a paper clip and add a hook at one end. Insert the other end into the center of the rubber stopper.
  2. Attach the filter paper: Hang a strip of filter paper from the hook. Ensure it hangs freely and doesn’t touch the sides of the test tube.
  3. Draw baseline: Remove the strip and draw a 5-mm-wide black band approximately 25 mm from the bottom using the black felt-tip pen.
  4. Mark solvent limit: Draw a pencil line 10 cm above the ink band. This will be the upper limit for the solvent’s movement.
  5. Add the solvent: Pour 2 mL of water into the test tube. Reinsert the filter paper so the bottom touches the water, but the black band remains above it.
  6. Observe separation: As the solvent moves up the paper, watch the ink pigments begin to separate.
  7. Remove and measure: When the solvent reaches the pencil line, remove the strip. Let it dry and measure how far each color moved.
  8. Record your data: Use a ruler to measure the distance traveled by each pigment and the solvent. Calculate the Rf value (retention factor) for each pigment using the formula:Rf = Distance traveled by pigment / Distance traveled by solvent

Results: Pigment Separation Data

ColorDistance Traveled (mm)Solvent Distance (mm)Rf Value
Yellow70 mm111 mm0.63
Pink82 mm111 mm0.74
Red101 mm111 mm0.91
Purple110 mm111 mm0.99
Blue111 mm111 mm1.00

Analysis & Discussion

  1. How many pigments were separated from the black ink?
    Five: yellow, pink, red, purple, and blue.
  2. What solvent was used?
    Tap water.
  3. What was the first visible color during the separation?
    Orange or yellow appeared first, indicating it traveled the fastest.
  4. In what order did the pigments separate (top to bottom)?
    Blue, purple, red, pink, and yellow.
  5. How far did the solvent travel?
    111 mm.
  6. What does this tell us about black ink?
    Black ink is not a pure substance—it’s a mixture of various colored pigments.
  7. Why did the pigments separate?
    Differences in molecular size, solubility, and paper affinity cause each pigment to move at a unique rate.
  8. Why did some pigments travel farther than others?
    Less absorbent pigments that are more soluble in the solvent moved farther up the paper.

Error Analysis

Some possible sources of error in this paper chromatography experiment include:

  • Measurement inaccuracies: Small errors in reading distances can affect Rf values.
  • Solvent volume inconsistencies: Too little or too much solvent may alter the pigment spread.
  • Filter paper length: A longer strip might have shown additional colors or more precise separations.

Conclusion

The results supported the hypothesis: paper chromatography successfully separated black ink into multiple pigments. The experiment demonstrated how different molecular characteristics—such as solubility and affinity to the filter paper—affect the movement of each component in a mixture. This hands-on experiment reinforces the idea that seemingly simple substances can contain complex chemical compositions.

Chromatography remains an essential tool in both educational and professional laboratory settings. From forensic science to biochemistry, the ability to separate and analyze chemical mixtures is foundational to understanding the materials around us.

Scientific Method Notes: Questions, Evidence, and Scientific Inquiry

Biology Foundations and Lab Skills Teaching Resources · High school biology; AP extensions where noted.

Biologists investigate questions using observations, comparisons, experiments, models, and existing data. A classroom sequence can help organize work, but research does not always follow one fixed list of steps. Scientists revisit ideas when evidence, methods, or questions change.

Learning goals and prerequisites

Students will distinguish an observation, hypothesis, prediction, and conclusion; identify what evidence could challenge an explanation; and recognize both experimental and observational research. No prior laboratory experience is required. Materials: paper and the example below.

Terms with a biological example

  • Observation: a recorded feature or measurement. “Seedlings beside the window are taller” describes a pattern; it does not yet explain its cause.
  • Question: a focused problem that evidence could help answer. “How does daily light duration affect seedling height after ten days?” is more testable than “Which plant is best?”
  • Hypothesis: a proposed explanation. For example, differences in light exposure may contribute to the height difference. Other explanations remain possible.
  • Prediction: an expected observation under specified conditions if an explanation is useful. State what would be measured, when, and compared with what. A predicted direction requires a biological reason; less light does not always mean a shorter seedling.
  • Evidence: observations analyzed in relation to the question, with attention to uncertainty and alternative explanations.
  • Conclusion: a statement explaining what the evidence supports and where the inference stops.

Plan a fair comparison

In a simple experiment, change the selected factor and keep other relevant conditions comparable. Define the response before collecting data. Use independent experimental units, repeat the comparison where practical, and assign treatments fairly. Measuring one plant ten times improves information about that plant; it does not create ten independent plants.

In an observational study, a researcher may measure variables without assigning a treatment. A relationship between two variables alone does not establish that one caused the other. Confounding factors and the sampling method matter.

Work through the example

  1. List two possible explanations for the window-side pattern.
  2. Choose one question and define how height will be measured.
  3. Describe a comparison that separates light duration from water supply as far as the classroom setup allows.
  4. Explain why several plants under each condition are more useful than a single plant per condition.
  5. Write a result that would make you reconsider your explanation.

Check understanding

Must a hypothesis be correct? No. It must be open to examination with evidence. An unsupported prediction can still lead to useful learning. Does a hypothesis turn into a law after enough experiments? No. Hypotheses, theories, and laws serve different roles; they are not levels on a ladder. Must all biology involve a controlled experiment? No. Field observations and comparisons can also provide evidence.

Teaching note: Ask students to justify revisions rather than reward a predicted result. For AP extension, discuss random assignment, independent replication, and the difference between evidence for a pattern and evidence for a mechanism.

Related teaching resources

Sources and further reading