Paper Chromatography
Chromatography is a method of separating and identifying
various components in a mixture, which are present in small
trace quantities. There are several types of chromatography,
one of which is paper chromatography. This uses a stationary
phase (paper) and a mobile phase (solvent). As the solvent
wicks up the paper by capillary action, an equilibrium is
established for each chemical in the mixture.
Part of the time the particular pigment is moving in the
solvent, while at other times it is adsorbed into the paper.
The pigments separate in the sample due to polarity; the
paper is more polar than the solvent so nonpolar substances
will spend more time in the nonpolar mobile phase and will
travel further up the paper
Spinach Chromatography
Lab Objective
1. Separate pigments from
spinach using paper
chromatography
2. Calculate Rf values of
separated pigments
*Rf = distance substance travels
distance solvent front travels
Materials
• Chromatography paper strips
• Petroleum Ether: Acetone
Solution
• 1 vial spinach leaf extract
• Capillary pipet
• Parafilm squares
• MSDS
• Transparent Tape
• Metric rulers
• Graduated Cylinder (100mL)
• Graphite pencil
Pre-lab Questions
• What is
chromatography?
• Calculate the Rf of a
substance that traveled
5.2 cm on
chromatography paper.
The solvent front
traveled 8.1 cm.
• Does each spot on the
developed
chromatography paper
represent one
substance?
Procedures
1.
2.
3.
4.
5.
Prepare a piece of
chromatography paper by
measuring 2cm from bottom of
paper and drawing a line with
pencil
At the other end, attach a piece
of tape long enough to drop the
paper to the bottom of the
graduated cylinder and attach
the top of the tape to the
cylinder mouth
At center station, touch the
capillary pipet with pigment to
center of line; allow the spot to
dry
Pour 5mL petroleum ether:
acetone solution into 100mL
graduated cylinder
Cover the top of cylinder with
Parafilm square
6. Place chromatography paper
inside cylinder so that the paper
extends into the solvent
7. Wait 10 minutes, then remove
the paper from the cylinder, and
mark the location of the solvent
front with pencil. Trace pigment
outlines with pencil after solvent
evaporates
8. In your notes, draw a life-size
picture of the developed
chromatography paper, labeling the
locations and colors of the spots.
9. Measure the distance each spot
and the solvent front traveled from
the pencil line; calculate the Rf
values for each pigment.
Follow-up Questions
• Find the distance traveled
by:
–
–
–
–
–
Solvent front _______cm
Carotene ________cm
Xanthophyll _______cm
Chlorophyll a ______cm
Chlorophyll b_______cm
• Calculate Rf for each
pigment:
Carotene ________
Xanthophyll _________
Chlorophyll a ________
Chlorophyll b ________
• Which pigment is
most polar? Which is
least?