Chromatography

Question 1

1. What factors are involved in the separation of the pigments? (What principle is this technique based upon? Why does it work for separating pigments?)

Answer 1

The key method used in this lab for plant chromatography is capillary action, when the solvent is attracted to the inner surface of a tube using adhesion and gradually moves upward. Here, the solvent molecules are attracted to the surface of the thin strip of paper, causing the solvent molecules to travel upward. Using adhesion, the solvent molecules also carry the various pigment molecules with them. Cohesion happens in this capillary action as solvent molecules are attracted to each other. The solvent carries the pigment molecules at different rates because the various pigments are not equally attracted to the solvent or to the paper surface. This causes the solvent molecules to first leave behind the pigment molecules that are least soluble in the solvent (which are strongly attracted to the cellulose in paper), and as the solvent moves up, the solvent leaves behind the pigments that are more soluble in the solvent (which are not so strongly attracted to the cellulose in paper).

Question 2

2. Would you expect the Rfvalue of a pigment to be the same if a different solvent were used, such as water? Explain.

Answer 2

Yes, the Rf value would definitely be different. This is because various pigment molecules [solutes] are soluble in various solvents to varying degrees because of the various molecular structures. These structures may allow the solutes to form more or less hydrogen bonds with the solvent, thus affecting adhesion. For example, if water was used as the solvent in this lab, then Carotine might not migrate very far because Carotine might not adhere so well with the water because of structural reasons, thus forming less hydrogen bonds.

Question 3

3. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments?

Answer 3

Chlorophyll a is the primary and most important photosynthetic pigment in plants. In the reaction center of photosystems, a molecule of chlorophyll a is located. It participates directly in the light reactions. Chlorophyll b does not directly participate in the light reactions, but it widens the range of colors that a plant can use by transporting absorbed energy to chlorophyll a, which then uses the energy to do work. The energy in from of a photon will be used to excite and break an electron from chlorophyll a, which will eventually be used to make NADPH.

     There are also Carotenoids in the chloroplast, which appear and reflect yellow-orange. Some pass energy to chlorophyll a. Others are protective: they absorb and scatter excess light, which could damage the photosystem. During autumn, trees appear yellow-orange because of strong decrease in green chlorophyll and the increasing visibility of carotenoids. Xanthophyll and Carotine are carotenoids because they are creamy yellow and orange-yellow respectively.  

*Source of this answer is on the last page: *

Question 4

name all of the pigments in order.

Answer 4

chlorophyll b

chlorophyll a

xanthophyll

Carotine

Question 5

Diagram showing how to measure the migration of the pigments and solvent:

Answer 5

Note: when measuring the distance a band of pigment travelled, always measure from the bottom of the band to the place where the _spot _was initially located.

Question 6

how to caclulate Rf value

Answer 6

Rf = distance pigment migrated (mm) __\__ _

                                           Distance solvent front migrated (mm)