LAB 7 :PLANT PHYSIOLOGY ( paper chromatography )

LAB 7 :PLANT PHYSIOLOGY ( paper chromatography )

INTRODUCTION:

Plants consists of two categories of pigment that used for photosynthesis which are  primary and accessory pigment. Chlorophyll is a primary pigment in organelle chloroplast with two types of chlorophylls which are chlorophyll a and chlorophyll b. The chlorophyll are green  pigment that absorb blue, red, orange and yellow but reflect green light as light contains different colours of different spectrum. Besides, accessory pigment are red, yellow or orange that absorb all the other colours. Paper chromatography is a porous paper that used to separate the plant pigments from  plant using hydrophobic solvent. Different molecules from a mixture have different solubility. With the aid of the solvent, the rate of solubility of the molecule can be known from “ molecular race track” that are created. Some compounds are hydrophobic that do not like to dissolve in water (water fearing) and some compounds are hydrophilic that like to dissolve in water (water loving). Chromatography is a method of separating and isolating molecules based on their level of hydrophobic or hydrophilic properties. Different plants pigments will move at different rates through the piece of paper as the liquid solvent is absorbed upward. From the band or distance of different movement of pigment in the paper chromatography, the solubility of pigment can  be known.

OBJECTIVE:

• To separate the plants pigments that are present in leaves using chromatographic technique.
• To determine the Retention factor (Rf ) of different pigment based on distance travelled  by pigment carried by solvent in paper chromatography.

MATERIALS:

• pin                             
• chloroplast extract
• hair dryer                   
• microbiurette
• cork                           
• test tube
• solvent                       
• paper strip

METHODOLOGY:

1.         Used the microbiurette / pin head as the dropper, the chloroplast extract was dropped on the prepared chromatography paper ( see figure 1 )
2.           Dropped the extract at about 1.0 cm from the pointing end of the paper. Dryed the drop with a hair dryer and repeated the process for 3-4 times until one small dot of thick pigment available.
3.       Attached the paper strip at the cork stopper using a pin. Placed the strip vertically and straight into the test tube which contained solvent.
4.       Let the solvent moves and removed the paper before the solvent front reaches the top of your chromatography paper.
5.       Marked with pencil the last range of the solvent.

RESULTS:

 R_{f  =  Distance travelled by compound}
            distance travelled by solvent


1. Identify each pigment obtain from the experiment, and record your result in the table.

2. Calculate the Rf value of each pigment observed.

Leaf A (Green spinach)

Pigments	Colour	Rf
Pigment 1	Light Green (chlorophyll b)	2.0/10.4=0.19
Pigment 2	Light Yellow (carotenoids)	4.0/10.4=0.38

Leaf B (Red spinach)

Pigments/ colour	Colour	Rf
Pigment 1	Blue Green (chlorophyll a)	2.9/10.2=0.28
Pigment 2	Light Yellow (carotenoids)	4.5/10.2=0.44

Leaf C (Bougainvillea leaf)

Pigments/ colour	Colour	Rf
Pigment 1	Blue Green (chlorophyll a)	3.0/8.4=0.36
Pigment 2	Light Green (chlorophyll b)	2.3/8.4=0.27
Pigment 3	Light Yellow (carotenoids)	4.8/8.4=0.57

DISCUSSIONS:

1.      Why the developing solvent mixture is prepared fresh before use?

•       the developing solvent mixture comprises of a pure solvent but more often it is a mixture of two or more solvents in specified proportions. in case solvents are mixed and stored for long periods there could be loss of volatile component which will alter the maxing proportions.

2.      Why is it important to keep the dyes spots (leaf extract) above the solvent level?

• it is important to keep the dye spots above the solvent level because if the dye spots of submerged in the solvent, then the spots would dissolve into the solvent preventing them from separating out and no measurement observation could be made.

3.      Why is it necessary to cover the test tube during the paper development

• during the chromatogram developing chamber is covered. this is essential as the environment inside the chamber should remain saturated with the solvent vapour. developing times can vary from about an hour to several hours and a sarurated environment prevents losses due to evaporation.

4.      Why it is important to stop the chromatogram before the solvent front reaches the top of your chromatography paper?

•       it is important to stop it because you cannot determine the Rf value unless you can measure the distance from the start to the front of solvent, you have to be able to see where if stops event if you let it go as high you want

5.      Why is it important to mark the solvent level on the chromatography paper when you remove it from the test tube?

•       it is important to mark the solvent level on the chromotography paper when you remove it from it from the petri dish because so the point at which the solvent stopped could be noted in case the solvent kept advancing when removed.

6.      Which of the pigments migrated the farthest and why the separation of pigments occur as it did? How does paper chromatography work?

•      carotene (orange) because it was the most soluble in the solvent. paper chromatograph as the layer of absorbent is known as the stationary phase. after the sample has been applied on the plate, a solvent or solvent mixture( known as the mobile phase) is drawn up the plate via capillary action. because different analytes ascend the plate at different rates separation is achieved.

7.      Explain what would happen to your chromatogram if you let it run too long?

•      therefore we should not be able to calculate the Rf values without a measured solvent front.

CONCLUSION:

If pigments are separated, then Rf values can be determined. Rf value was determined by distance travelled by compound divide by distance travelled by solvent. thus, from Rf value, we can determined the affinity of the solute to the solvent. greater Rf value, greater affinity of solute to the solvent.

Besides, the paper chromatography did show that from a dot of chloroplast extract could be separated into various colours of pigment. the chloroplast contain mixture of various pigment together. the first colour of pigment to appear on the filter paper was pale yellow. the colour separated because of the differences in their molecular characteristics, specifically, their solubility in water and their rate of absorption by the paper. the most soluble and readily absorbed chloroplast was the pale yellow green in colour . the least soluble and least absorbable of chloroplast pigment was blue in colour.

REFERENCE:

1. The Editors of Encyclopaedia Britannica. (2018, June 1). Paper chromatography. Retrieved from https://www.britannica.com/science/paper-chromatography

2. ochemistree. (2013, June 14). Calculating Rf Values. [Video File] Retrieved from https://www.youtube.com/watch?v=VtkwBLTd0rA