Pigment Separation Chromatography Lab

Plant Separation Technique:  Paper Chromatography

Lesson Objectives:  Students will explain the technique used to separate a mixture of pigments in spinach leaves.  Students will calculate the reference flow values for each of the separated pigments and use these values to identify the pigments present in spinach leavesStudents will explain the adaptive advantage to a plant that contains more than one pigment.  

chrom1        chromot2      chromo3

Tasks:  You will perform a solvent chromatography technique to see how many and what types of pigments are in a green spinach leaf.

1.  Download the chromatography activity word document.  It contains the questions to this activity.

2.  Here we will learn how to calculate the Reference flow values.  The Rf values indicate which pigment is the most soluble in the solvent used in this lab.  The solubilities of the pigments can then be compared.  The higher the Rf value, the higher the pigments solubility.  Soluble pigments travel the furthest up the paper chromatography strip.  The formula is the following: 

chromot 8

Click on image above to try an online chromatography lab and quiz.

a.  Using a ruler, draw a line across the origin line (where the plant mixture blot is).  Then draw a line across the top of what YOU estimate is where most of the pigment traveled.  Make sure you also draw a line where the solvent traveled to (in most cases this will be the top of the chromatography strip).  Use the diagram below to help you with your calculations.

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b.  Using the formula above (Rf =  ), calculate the Rf values for all the pigments you've identified by colour on your paper chromatography strip.  Distance pigment traveled divided by distance the solvent traveled.