Chromatography and Spectrophotometry Lab
Purpose: To extract and separate pigments (there are 4) from a spinach leaf and analyze them
separately for transmission and absorption of light at wavelengths from 380 to 720 nm.
Groups: Each lab station will prepare 2 chromatographs and analyze one pigment. Class data for all
pigments will be pooled for tabulation and graphing.
Materials:
Spinach leaf
Chromatography paper
Scissors
Solvent (9:1, hexane:acetone)
Chromatography tube w/ stopper
Spectrophotometer with 20 nm bandwidth ("Spec 20")
Spectrophotometer tubes (perfectly clean)
A US quarter and/or an ID card
Procedures: Day to day procedures may vary
Chromatography
Day one:
Practice the placement of pigment on the chromatography paper with short pieces of paper.
NOTE: This lab takes 4 days in total because you must first learn the techniques involved. If your
chromatograph is not good, you will not be able to produce good data. So we practice first.
There are several ways to do this, the "quarter" (coin) method and the "student ID" method. I
would suggest you practice both.
Day two:
Cut 2 strips of chromatography paper 2-3 cm longer than the tube, and cut a point on the end.
Roll a line of pigment (plant juice) 1 cm above the bevel.
Set it in solvent midway up the bevel.
Allow 10 min for chromatography to happen.
Staple 2 chromatographs to a piece of plain white paper and present to your teacher.
Spectrophotometry
Day three:
Catch up on theory, and practive spectrophotometry with colored water. I will demonstrate
proper technique.
Day four:
Carefully separate the four pigment bands with scissors.
Collect all of each pigment in small, labeled beakers.
Add solvent to beakers, swirl for 2 min to remove pigments.
Analyze transmission from 380-720 nm for your assigned pigment.
Subtract transmission from 100% to get absorption.
Get data for other pigments from other lab groups in your period. Make sure to credit them.
Tabulate data for all four pigments on the table provided.
Afterward: Graph results…%Absorption as a function of wavelength….transpose color spectrum
onto wavelength scale