Separation of Pigments from the extract of Spinach leaves by paper chromatography

● Introduction:

Paper chromatography is the process of separating pigments, it is an analytical process. In this process, samples are delivered in two phases, a stationary phase and a mobile phase. The stationary phase is almost always a piece of high-quality filter paper. The mobile phase, on the other hand, is an increasing solution up to the stationary phase, supporting the samples with it. As the mobile phase moves over the sample over the stationary phase, the components of the sample gradually separate from each other. This in-house experience will show how denaturing actually works.

● Objective: The objective is to test the efficacy of the chromatography paper, to separate pigments from an extract of spinach leaves, as well the components that affect the final result.

● Scientific question: What will be the actual efficiency of the chromatography paper will differ depending on the amount of spinach leaf extract added to it ?.

● Hypothesis: The result will appear on the paper W. Depending on how many drops of spinach extract you put with the different substances, it is necessary to make several tests.

Theoretical background:

Photosynthesis occurs inside plant cells, in small cells called chloroplasts. Chloroplasts contain a green colored substance called chlorophyll. Chlorophyll absorbs the light energy necessary for photosynthesis. Plants and algae can photosynthesize only in the presence of light. On the other hand, take the photosynthetic pigments; “Most photosynthetic pigments, such as xanthophyll and alpha and beta-chlorophyll, have parts of the molecule that have an affinity for one solvent and others that have an affinity for another. Because of this, these pigments move according to the parts in which they "roll" in the still water phase, and the greater the ratio they cause, the slower they move. As a result, each pigment usually moves at a certain speed, forming discrete bands on the paper. Almost always, the stationary phase is water.

● Variables

-Control Variable: The quantity of Isopropyl alcohol- water mixture -Independent Variable: In the w. paper, the concentration of the spinach leaf extract.

-Dependent variable: The appearance of the different pigment spots.

● Materials

- Chromatographic chamber with lid

- Extract of spinach leaves

- Isopropyl alcohol- water mixture

- Whatman filter paper strip

- Fine capillary tube

- Thread

- Scale

- Pencil

Experimental procedure:

1. Pick up a strip of Whatman filter paper and draw a line with a pencil drawn over 2 cm from one end.

2. Make another longitudinal line from the center of the paper, and name the intersection point of the two lines as P.

3. Pick up the spinach leaf extract using a capillary tube. 4. Place a drop of leaf extract on point P and allow it to air dry. 5. Place another drop on the same stain and blot again, so that the stain is rich in the mixture.

6. Grab a piece of string and attach the filter paper to it. 7. Hang the filter paper vertically in the chromatographic chamber containing the solvent,that is a mixture of isopropyl alcohol and distilled water.

8. Hold the chamber undisturbed for some time. Observe the solvent rising along with the leaf extract .

9. When the solvent has risen, you will notice different spots of the pigments, such as: carotene, xanthophyll, chlorophyll a and chlorophyll b, from the spinach leaf extract on the filter paper.

10. Take the filter paper out of the chamber and mark with a pencil the distance the solvent has risen on the paper,this is called the solvent front.

11. Wipe the filter paper dry and put pencil marks in the center of the pigment spots.

12. Determine the distance of the solvent

...