STEM103

Lab Worksheet 1: Paper Chromatography

Name: ______________________________________________ Date Performed:_________

Section:____________________________ Teacher: Ms. Lyka D. Lamoste____

Introduction

Chromatography is a way to look at complex mixtures by separating them into their

components. Criminal investigators use this technology to identify substances such as drugs,
blood, ink and other fluids. Different inks have different properties, such as how much they can
be dissolved in solvents. When you place chromatography paper into the solvent, the solvent
begins to move up the paper. As the solvent rises, it dissolves the ink on the paper and
separates the ink into its components. The farther the ink travels, the more it is attracted to the
solvent. A good understanding of solutions and mixtures and their components is essential for
environmental engineers. Most experiments and data gathering done for the purpose of
improving groundwater quality involve the preparation of solutions to monitor and test different
conditions for contaminated water.

Instruction:
I. You will be working as a group. Read Lab Mission attached in the Google Classroom.
Use the procedure here or the Lab Mission Procedure file as your guide. You can also
watch this video for further reference.
II. Make sure that the materials are complete before your group begins the activity. Be
aware of the safety precautions in conducting this activity.
III. Answer the pre-lab and post-lab questions below. Use blue text font color in answering
this worksheet.
IV. After completing the mission, you would need to submit a Scientific Report as a group.
You can also include pictures of the experimental setup and the results. Effort marks per
group should also be accomplished and attached with your report.
A. Pre-Lab Question:
1. What is the purpose of using paper chromatography, and how does it help in
separating different pigments present in a mixture?
● Chromatography is a method of physically separating mixtures into its
individual components. It is a common laboratory technique used to
identify unknown components in mixtures (Libretexts, 2022). By eye, we
cannot know if each ink color is a mixture or pure substance. Using
chromatography, the components in a sample will migrate along the filter
paper at different rates such that they become spread out and separated
from each other.
● Paper chromatography is an analytical technique used to separate and
analyze mixtures of soluble substances. It is a type of chromatography. In
a paper chromatography experiment, a small amount of a sample solution
is placed on a strip of chromatography paper. The chromatography paper
is then suspended in a solvent. As the solvent moves up the paper, the
individual components of the sample solution separate out in bands of
individual color.

2. Based on the evidence presented in the Lab Mission, what is your hypothesis?
The evidence provided suggests that there are three potential sources for the ink
blots on the bed sheet: black ink from my little brother's doodling, red ink from my
sister's new markers, and blue ink from my own marker use. Based on this
information, here are my (our) hypotheses for each ink color:

Black Ink - Hypothesis 1:

The black ink blot on the bedsheet is likely the result of my little brother's
doodling using black ink. I (We) hypothesize that the black ink used for doodling
contains a mixture of various color components. Conducting paper
chromatography on the black ink will reveal distinct bands of color, helping
identify the specific pigments present in the black ink.

Red Ink - Hypothesis 2:

The red ink blot on the bed sheet may be attributed to my sister's use of red
markers for bullet journaling. I (We) hypothesize that the red markers contain a
mixture of different pigments responsible for the red color. By employing paper
chromatography, we can separate and analyze the components in the red ink,
confirming the source of the red blot.

Blue Ink - Hypothesis 3:

Considering my usage of a blue marker the other night, the blue blot on the bed
sheet might be associated with my activities. I (We) hypothesize that the blue
marker ink comprises a mixture of pigments. Paper chromatography will allow for
the separation of these components, and analyzing the chromatogram will help
identify the specific pigments contributing to the blue color in the ink.
3. Why do we use ethyl alcohol as a solvent in this activity?
● Ethanol, or ethyl alcohol, is a popular solvent in paper chromatography
due to its:
A. Broad solubility - ethanol's capacity to dissolve both polar
and nonpolar compounds is crucial for chromatography, as
it enables the separation of diverse substances with
varying polarities in a mixture.
B. Quick evaporation - ethanol's rapid evaporation is
beneficial in chromatography as it prevents oversaturation
of the paper and ensures concentrated migration of
mixture components.
C. Low toxicity - ethanol is a safe choice for laboratory and
educational settings as it is less toxic compared to other
solvents, making it suitable for experiments involving
students or individuals without specialized chemical
handling training.
● These qualities make it well-suited for separating and identifying
components in a mixture during chromatography. Ethanol also enhances
capillary action, aiding the movement of the solvent and the effective
separation of mixture components on the paper.
B. Data and Observations: (make sure you use tables or graphs to show your data)

Interpretation of Chromatogram
● The Rf (Retention Factor) is the distance, D, traveled by the spot divided by the
distance traveled by the eluting solution, or Solvent Front, F (Libretexts, 2022).
Calculate the Rf value for each colored pigment using the formula below:
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑝𝑖𝑔𝑚𝑒𝑛𝑡𝑠 𝑖𝑛 𝑖𝑛𝑘)
Rf = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑒𝑡ℎ𝑎𝑛𝑜𝑙)

Distance Traveled (cm)

Ink Rf Value
Solve Components
Color
nt
front

Blue 4 Light Blue = 1.2 1.2 𝑐𝑚

= 0.3
4 𝑐𝑚

Dark Blue = 3.3 3.3 𝑐𝑚

= 0. 83
4 𝑐𝑚

Red 4 Light Pink = 1.1 1.1 𝑐𝑚

= 0.28
4 𝑐𝑚

Red to Pink = 3.3 3.3 𝑐𝑚

= 0.83
4 𝑐𝑚

Orange = 3.8 3.8 𝑐𝑚

= 0.95
4 𝑐𝑚

Black 3.5 Light Gray = 0.6 0.6 𝑐𝑚

= 0.17
3.5 𝑐𝑚

Black = 2.2 2.2 𝑐𝑚

= 0.63
3.5 𝑐𝑚

Brown = 3.3 3.3 𝑐𝑚

= 0.94
3.5 𝑐𝑚

Leaf 3.5 Pale Green = 1.9 1.9 𝑐𝑚

= 0.54
3.5 𝑐𝑚
Extract
Pale Yellow = 2.4 2.4 𝑐𝑚
= 0.69
3.5 𝑐𝑚
C. Conclusion

The paper chromatography experiment provided valuable insights into the composition
of various ink colors and a leaf extract. The use of ethanol as a solvent proved effective in
separating different pigments based on their solubility and polarity. We were able to determine
the Rf values—which represent the relative mobility of each pigment in the solvent—by
calculating the component distances traveled on the paper (distance traveled by
solute—pigments in ink) divided by the distance traveled by solvent (ethanol).
According to Libretexts (2020), Rf values and polarity are inversely related. The spot
with the highest Rf value is the least polar (fastest moving), and the spot with the lowest Rf value
is the most polar (slowest moving).

1. Blue Ink
I. Chromatogram Analysis:
➢ The blue ink chromatogram revealed two distinct components:
light blue and dark blue pigments.
➢ The light blue pigment traveled a distance of 1.2 cm, while the
dark blue pigment migrated to 3.3 cm.
II. Rf Values and Polarity:
➢ Rf values were calculated as follows:
Light Blue: 1.2 cm / 4 cm = 0.3
Dark Blue: 3.3 cm / 4 cm = 0.83
➢ Interpretation: The higher Rf value for dark blue indicates lower
polarity, suggesting it is less attracted to the stationary phase and
moves faster. In contrast, the lower Rf value for light blue signifies
higher polarity, resulting in a slower migration.
2. Red Ink
I. Chromatogram Analysis:
➢ The red ink chromatogram displayed a mixture of pigments,
including light pink, red to pink, and orange.
➢ Light pink traveled a distance of 1.1 cm, red to pink at 3.3 cm, and
orange at 3.8 cm.
II. Rf Values and Polarity:
➢ Rf values were calculated as follows:
Light Pink: 1.1 cm / 4 cm = 0.28
Red to Pink: 3.3 cm / 4 cm = 0.83
Orange: 3.8 cm / 4 cm = 0.95
➢ Interpretation: Higher Rf values (e.g., orange) suggest lower
polarity and faster migration, while lower Rf values (e.g., light pink)
indicates higher polarity and slower movement.
3. Black Ink
I. Chromatogram Analysis:
➢ The black ink chromatogram revealed components including light
gray, black, and brown.
 ➢ Light gray traveled a distance of 0.6 cm, black at 2.2 cm, and
brown at 3.3 cm.
II. Rf Values and Polarity:
➢ Rf values were calculated as follows:
Light Gray: 0.6 cm / 3.5 cm = 0.17
Black: 2.2 cm / 3.5 cm = 0.63
Brown: 3.3 cm / 3.5 cm = 0.94
➢ Interpretation: The higher Rf value for brown indicates lower
polarity and faster migration, while the lower Rf value for light gray
suggests higher polarity and slower movement.
4. Leaf Extract
I. Chromatogram Analysis:
➢ The leaf extract chromatogram displayed two distinct components:
pale green and pale yellow.
➢ Pale green traveled a distance of 1.9 cm, while pale yellow
migrated to 2.4 cm.
II. Rf Values and Polarity:
➢ Rf values were calculated as follows:
Pale Green: 1.9 cm / 3.5 cm = 0.54
Pale Yellow: 2.4 cm / 3.5 cm = 0.69
➢ Interpretation: The higher Rf value for pale yellow indicates lower
polarity and faster migration, while the lower Rf value for pale
green suggests higher polarity and slower movement.

Collectively, the experiment provided valuable hands-on experience in chromatographic

techniques and analysis, contributing to a deeper understanding of the principles behind paper
chromatography.

D. Post-Lab Questions for the Scientific Report

1. What are the uses of paper chromatography?

As highlighted by Mlblevins (2011), paper chromatography is a simple
chromatography technique which has many applications. Its main advantage is that it is
not very expensive to perform, and provides clear results. Given below are some
important uses of paper chromatography.
● Separating Colored Pigments - paper chromatography is an effective technique
for separating colored pigments from a mixture.
➔ Ex. Used in the separation of plant pigments.
● Obtaining Pure Compounds - paper chromatography is used to obtain pure
compounds from a mixture. This is done by cutting out and redissolving the
patterns formed by each constituent. Also, this technique can be effectively used
to remove impurities from chemical compounds.
➔ Ex. Used in the separation of proteins into homogenous groups for use in
medicine.
 ● Qualitative Analysis - paper chromatography is one of the methods of
qualitative analysis, to analyze or separate the different constituents of a mixture.
It is a useful tool for separating polar as well as nonpolar solutes. Pharmaceutical
companies use this technique to analyze the different compounds in drugs.
➔ Ex. Used in the testing of antibiotics and determining the pollutants in
water.
● Pathology and Forensic Science - paper chromatography is useful in the field
of forensic science, for investigation of crime. This is because this process can
be successfully carried out even with very small quantities of material. Samples
from crime scenes are collected to be analyzed and identified, using this
technique.
➔ Ex. Used in DNA and RNA fingerprinting. Pathological laboratories use
paper chromatography to detect the presence of alcohol or chemicals in
blood.
● Analyzing Complex Mixtures - paper chromatography is used to detect the
presence of, or identify certain organic compounds such as carbohydrates and
amino acids, from a complex mixture of organic compounds.
➔ Ex. Used in the separation of amino acids and anions.
2. What sort of errors did you experience when doing the experiment?
Several errors occurred during our lab experiment, affecting the accuracy of our
results.
● One major error was the use of a black whiteboard marker, as we were
unaware that the marker's ink would not react with the alcohol in the
chromatography process. In addition, inconsistencies in the measurement
of the filter paper occurred, leading to multiple cutting and measuring
attempts.
● Additionally, we faced challenges in extracting ink from leaves as the
quantity was limited. As a result, the ink obtained was quite pale, showing
weak migration patterns on the filter paper during chromatography.
● Moreover, due to time constraints, we were unable to complete the full set
of chromatography experiments for all four colors. This limitation resulted
in insufficient and potentially less accurate data.
These errors had a significant impact on the completeness and precision of our
experiment, emphasizing the necessity of careful preparation, accurate measurements,
and adequate time management in chromatographic analysis.

3. What other separation methods can you suggest to solve the mission?
I. Paper Electrophoresis. Brown and Kirk (1954) emphasized that paper
electrophoresis and paper chromatography should be regarded as
mutually supplementary methods for identifying inks. The indications
suggest that electrophoresis may ultimately prove considerably more
valuable than chromatography. This is because it appears to separate
and utilize constituents of the ink that do not separate well with
chromatography, as it has been applied up to the present.
 II. Chalk Chromatography. Helmenstine (2020) notes that while certain
chromatography methods require expensive lab equipment, others can be
conducted using common household items. For instance, chalk and
alcohol can be employed for chromatography to separate pigments in
food coloring or inks. Chalk chromatography is a straightforward
technique used to differentiate pigments in a dye or ink.
4. What colors are present in black ink?
In black ink, the colors that are present based on the paper chromatography
results are the following:
I. Light Gray Component:
● Distance Traveled: 0.6 cm
● Rf Value: 0.17
II. Black Component:
● Distance Traveled: 2.2 cm
● Rf Value: 0.63
III. Brown Component:
● Distance Traveled: 3.3 cm
● Rf Value: 0.94
The presence of these components suggests that black ink is not a simple,
single-color mixture. Instead, it comprises various pigments contributing to the observed colors,
including light gray, black, and brown. The Rf values provide insights into the relative mobility of
each component during chromatography, indicating their solubility and interaction with the
chromatography paper and solvent.

5. How might the ability to separate solutions into their components be important to
environmental engineers looking into water quality issues?
A thorough comprehension of solutions, mixtures, and their components is crucial for
environmental engineers engaged in addressing water quality issues. This knowledge is
particularly vital for tasks involving the preparation of solutions for monitoring and testing
groundwater contaminants, as well as for developing remediation processes to separate
pollutants from water solutions.
Environmental engineers heavily rely on experiments and data gathering to enhance
groundwater quality. The ability to separate solutions into their components is foundational to
these efforts, ensuring accurate analysis and effective remediation strategies.
Chromatography, as highlighted by Chromatography Today (2021), plays a pivotal role in
water quality analysis worldwide. This analytical technique enables researchers to trace and
analyze contaminants, trace metals, and potential pollutants like pharmaceuticals and solvents.
Chromatography's capability to detect these substances, even in minimal concentrations, is
instrumental in providing comprehensive insights into water composition.
The use of chromatography in water quality projects is instrumental in moving towards a
world where safe and clean water is accessible to everyone. Its contribution to identifying and
understanding contaminants supports ongoing efforts to improve water safety and accessibility
globally.
References

Brown, C., & Kirk, P. L. (1954). Paper Electrophoresis in the Identification of Writing Inks.
Comparison with Horizontal Paper Chromatography. The Journal of Criminal Law,
Criminology, and Police Science, 45(4), 473. https://doi.org/10.2307/1140040
Libretexts. (2022, October 15). 2: Paper chromatography of gel ink pens (Experiment).
Chemistry LibreTexts.
https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/Wet_Lab_Experi
ments/General_Chemistry_Labs/Online_Chemistry_Lab_Manual/Chem_9_Experiments/
02%3A_Paper_Chromatography_of_Gel_Ink_Pens_(Experiment)

Libretexts. (2020, August 5). 6.2: Thin Layer Chromatography (TLC). Chemistry LibreTexts.
https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/Wet_Lab_Experi
ments/Organic_Chemistry_Labs/Lab_I/06%3A_Exp_5-_A_and_B_TLC/6.02%3A_Thin_
Layer_Chromatography_(TLC)?fbclid=IwAR2BhyMs9S2XeEpBSYa7GAePioD-PamNYak
hPBJwmujjT-PGvawmvGbTGwE

Mlblevins. (2011, November 10). Paper chromatography uses. Science Struck.

https://sciencestruck.com/paper-chromatography-uses?fbclid=IwAR3iozSfugpcPM5lrj0cJ
1lpcgnHAzytobOqsx830-T9L51wWcvSnrEXsFY

Today, C. (n.d.). How Can Chromatography Help with Clean Water? Chromatography Today.
https://www.chromatographytoday.com/news/liquid-chromatography/65/breaking-news/h
ow-can-chromatography-help-with-clean-water/55693