CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING

ACTIVITY I PLANT PIGMENT CHROMATOGRAPHY PART I IN THIS
BIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017
CHROMATOGRAPHY LAB BIOLOGY LAB 10 OBJECTIVE TO USE THE

CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS STUDENTS’ SHEET INTRODUCTION THE
CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS TECHNICAL & TEACHING NOTES
CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING

CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING


Chromatography of photosynthetic pigments

Learning outcomes


Introduction

These learning outcomes aim to cover subject knowledge requirements for this part of A-level Biology in the specifications from OCR, AQA, Edexcel and Eduqas. You may need to adapt this information for your specification.

This information can be used to produce learning resources, revision materials, quizzes etc.

CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING

Students should be able to:

  1. State the precise location of the pigments involved in photosynthesis within the leaf

The pigments involved in photosynthesis are embedded in the thylakoid membrane in chloroplasts.


Chloroplasts have an internal membrane system of flattened sacs called thylakoids, stacked together to form grana, with grana joined by lamellae (extended sacs). These thylakoids, grana and lamellae are made of this thylakoid membrane.


  1. Name 5 photosynthetic pigments (or types of pigment) found in leaves

Chlorophyll a

Chlorophyll b

Carotenes (a category of carotenoids)

Xanthophylls (a category of carotenoids)

Pheophytins (a and b)


(Note: these molecules are not explicitly named in the specifications but are useful for interpreting the chromatograms.)


  1. Outline the role of the leaf pigments in photosynthesis

These photosynthetic pigments are involved in the “capturing” of light energy and its conversion to chemical energy.


The primary photosynthetic pigment is chlorophyll which uses energy from light to excite electrons. The carotenoids are accessory pigments that “channel” more captured light energy to the chlorophyll. The pheophytins are electron carriers that are part of the electron transport chain where the excited electrons are passed from one molecule to the next in a series of chemical reactions.


  1. Explain how the terms “light harvesting systems”, “photosystems” and “reaction centres” are related

The capturing of light energy to excite electrons is performed by a complex arrangement of molecules and so rather than use the name of a particular molecule these phrases are often used.


Photosystem is the term given to the whole arrangement of molecules. In green plants there are two different photosystems (termed photosystem I and photosystem II) each composed of a different set of molecules.


Each photosystem has a reaction centre where the light energy is used to excite electrons that are then passed down the electron transport chain. Surrounding the reaction centre is a light harvesting system that “channels” more light energy to the reaction centre than the reaction centre can capture on its own.


(Note: not all specifications use these terms – but an awareness that there are many different molecules involved in the capturing of light energy is important for the context of this chromatography)


  1. Explain why many plants have a variety of photosynthetic pigments

Different photosynthetic pigments absorb light best at different wavelengths. By having more photosynthetic pigments plants can absorb light energy over a wider range of wavelengths. This allows plants to capture more energy for the same sunlight intensity.


  1. Describe how to conduct chromatography to separate pigments from a leaf and explain the importance of each step

Step

Importance

  1. Tear up a sample of plant leaves into small pieces, place in a mortar with a pinch of sand, and grind with a pestle

To break open plant cells and chloroplasts.

  1. Add solvent and continue to grind with a pestle

To allow the photosynthetic pigments to dissolve in the solvent.

  1. Gently draw a pencil line 1cm from the base of the chromatography paper and repeatedly spot a small quantity of the leaf extract onto the centre of the line, allowing the spot to dry between each application

The pencil line marks the starting position of the pigments. It is in pencil so that the line doesn’t move with the solvent and won’t mask the results of the chromatography. The spot is made with small quantities through repeated spotting and drying in order to build up a concentrated, but still small, spot.

  1. Suspend the chromatography plate/paper from a bung in the glass vial with 1cm depth of solvent in the bottom. Ensure that the chromatography plate dips into the solvent but the spot of leaf extract remains above the surface of the solvent

The solvent is use to draw the photosynthetic pigments up through the chromatography plate. The spot needs to be above the surface of the solvent so that the pigments travel up the plate and don’t just dissolve out into the solvent in the vial.

  1. Allow the solvent to run up the chromatography plate until the solvent front is near the bung then remove the plate, mark the location of the solvent front, and allow to dry

Identification of pigments by chromatography relies on identifying how far each pigment has travelled as a proportion of how far the solvent itself has travelled. The solvent evaporates quickly and so the extent to which it has travelled up the plate needs to be marked whilst it can be seen. Drying ensures that the movement of solvent and pigments stops.

  1. The resultant chromatography plate is called a chromatogram which can then be photographed or the location of each separated pigment can be marked

To allow the distance each pigment has travelled to be measured and for its Rf value to be calculated.


  1. List 3 characteristics of the solute (in this case a pigment) that influences how far it travels during chromatography, and for each describe the effect it has

Factor

Explanation

Solubility in the solvent (mobile/liquid phase)

The more soluble the pigment is in the solvent the further it will travel

How it interacts with the plate used (stationary/solid phase)

Molecules that interact more strongly with the plate used will not travel as far

Size of the pigment molecule

The smaller the molecule the further it will travel

(Note: the size of the molecule is much less of a factor than the other two)



  1. Describe how to calculate the Rf value for a particular substance


Rf value = distance moved by the substance from its original location

distance moved by the solvent from the same location


CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING

Note: usually the distance moved by the substance is measured using the centre of the pigment spot’s final location







Note: Each chemical (in this case, pigment) has its own Rf value. So in this diagram there are 8 different pigments each with their own Rf value.


CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING





Distance moved by the solvent from the same location





CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING


Distance moved by the substance from its original location






CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING CHROMATOGRAPHY OF PHOTOSYNTHETIC PIGMENTS LEARNING OUTCOMES INTRODUCTION THESE LEARNING






  1. Explain how a chromatogram can be used to identify an unknown substance


Calculate the Rf value for the substance and compare this to the Rf values for known substances (specific to that solvent and that type of chromatography plate). The unknown substance should be one of the substances with the same Rf value.

However more than one substance can have the same Rf value for a particular solvent and chromatography paper and so it’s possible that multiple chromatograms will need to be run with different solvents (and/or chromatography paper) in order to find out the exact identity of the unknown substance. (other characteristics of the chemical may also help identification – e.g. the colour of photosynthetic pigments).

Science & Plants for Schools: www.saps.org.uk

Chromatography of leaf pigments – Learning outcomes: p. 3

Resource 1347, Version 1.1 (revised 2019)


Chromatography_Key
DEVELOPMENT OF LIQUID CHROMATOGRAPHYMASS SPECTROMETRIC PROCEDURES FOR THE ANALYSIS
EXPERIMENT 3 SEPARATION OF SPINACH PIGMENTS BY COLUMN CHROMATOGRAPHY


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