Lab #1: Paper Chromatography
To separate a mixture using paper chromatography.
Vocabulary Words:
• Chromatography _______________________________________
• Homogeneous Mixture __________________________________
• Stationary Phase _______________________________________
• Mobile Phase __________________________________________
• Analyte _______________________________________________
• Solvent _______________________________________________
• Constituent ____________________________________________
There are 3 primary colors: Red, Blue & Yellow. Most colors are produced by mixing two or
more primary colors together.
Ex. Green color is produced by mixing blue & yellow.
In paper chromatography, small samples of analytes are spotted onto chromatography paper,
which serves as stationary phase. A liquid mobile phase is wicked up the stationary phase by
capillary action, causing the analytes to move upward. The distance traveled by each analyte
depends on its affinity for the mobile phase. The higher the affinity the farther it will travel. On
the other hand, if the particular analyte has a higher affinity for the stationary phase than to
the mobile phase, then it will stick to the stationary phase and not travel as far. The ration of
the distance traveled by an ink “spot” (analyte) to the distance traveled by the solvent (mobile
phase) is called retention factor, Rf.
$*%+(,(! -. /&%,.$(
Rf = !"#$%&'(
!"#$%&'( $*%+(,(! -. 01,+(&$
•
•
•
•
•
•
•
•
Black ink
Filter Paper
Metric Ruler
Clear Tape
Pencil
Isopropyl Alcohol
DI Water
250 ml Beaker
•
•
Timer
Camera to Record
1. Obtain Filter Paper and 250 ml Beaker
2. Measure and cut 12 cm of Filter Paper
3. Measure 1 cm from the base of the Filter Paper and make a straight line across using a pencil.
x
x
x
x
pg. 1
Lab #1: Paper Chromatography
4. Obtain a pencil and a tape. Attach the filter paper to the pencil such that when the pencil is rested
on the rim of the beaker, the filter paper barely touches the bottom of the beaker.
x
x
x
x
x
x
x
x
5. Add 5 mL of isopropyl alcohol to 250 mL of beaker.
6. Use the assigned 4 black markers to make the spots on the filter paper.
(Warning: The ink should be at least 3 mm from the edge of the filter paper, since liquids travel faster on the edges of a
paper. Can you think of why placing ink on the edge of the paper will cause an experimental error and unsuccessful
separation of colors).
x
x
x
x
7. REST the pencil-filter paper setup on the rim of the beaker with isopropyl alcohol.
5 mL of
Isopropyl
Alcohol
x
x
x
x
8. Observe and Record !
pg. 2
Lab #1: Paper Chromatography
Data Table 1: Assigned Black Inks
Black Ink
Assigned Black
Ink #
Black Ink Information
1.
2.
3.
4.
Data Table 2: Observations
Black Ink
Assigned
Black Ink
#
Observations in
1 min.
Observations in
5 min.
Observations in
15 min.
Observations in
25 min.
1.
2.
3.
4.
(Keep in mind that a few variations of the experimental procedure might be observed).
Example #1.
(Note: did not use pencil)
Example #2.
(Note: Then only used 3 Different Black Inks)
pg. 3
Lab #1: Paper Chromatography
(Note: You must calculate retention factor for each primary color of each black ink).
Use the following example to complete your calculations for each of the black inks assigned to your
group:
$*%+(,(! -. /&%,.$(
Rf = !"#$%&'(
!"#$%&'( $*%+(,(! -. 01,+(&$
1) Black Ink # _____
Distance that Mobile
Phase Traveled (Solvent)
6 cm
5 cm
Distance Blue Dye
Traveled
Distance Pink Dye
Traveled
2 cm
0 cm
Original position of the
black ink
Rf (BlueDye) =
2.4 '5.
6.4 '5
= 0.83
Rf (PinkDye) =
7.4 '5.
6.4 '5
= 0.33
Rf (YellowDye) =
8
6.4 '5
=
Repeat this Calculation for all 4 Black Ink Results.
2) Black Ink # ______
3) Black Ink # ______
4) Black Ink # ______
Mandatory
Watch:
Khan Academy
Calculating Retention Factor
for T.L.C.
pg. 4
Lab #1: Paper Chromatography
Answer questions # 1-13
1. Restate and clarify the purpose of this lab in your own words including “what will be analyzed,
the technique that will be used and what is it that we were trying to accomplish. 1-2 sentences.
2. What is analyte? (definition in your own words)
3. Which substance/substances are considered analyte(s) in this lab? (Temperature, beaker, filter
paper etc.)
4. How many analytes did you use in this lab?
5. Identify this(these) analyte(s).
6. In your own words, write the summery of the process of paper chromatography. (How did you
conduct this experiment?)
7. Identify each item of the lab as stationary phase, mobile phase, solvent and analyte.
8. Was your experiment successful?
9. Identify possible source of errors.
a. ___________________________
b. ___________________________
c. ___________________________
10. Report your results (your final observation for each analyte).
Analyte
Name or Analyte
Number
1.
2.
3.
4.
pg. 5
Lab #1: Paper Chromatography
11. Report all the values of Rf and explain what they mean in terms of degree affinity towards
stationary or mobile phase. (use your knowledge of paper chromatography technique. See the
background info provided for this lab).
Black Ink
#
Dye Color
Rf Values
Explain
1.
2.
3.
4.
12. Explain why Rf does not have units such as cm, m, km, etc.
(Hint: the answer lies in Rf formula)
13. Do all blue dyes separated from the 4 analytes tested have the same Rf values? What does this
tell you about the molecular shapes of those blue dyes?
14. Are these analytes consider to be Homogeneous or Heterogeneous mixtures? Explain.
pg. 6