Abstract
Chromatographic techniques are used to separate different components in a mixture based on their solubility in the mobile phase and adsorption on the stationary phase. The most soluble components move further with the mobile the components that adsorb more on the stationary phase move a shorter distance. The main component of green leaves is chlorophyll. However more information can be obtained on the content of leaf extracts by separating a homogenate made from a leaf sample. In this experimental report, spinach leaves were used to obtain a homogenate that was separated on paper chromatography to obtain the various leaf pigments. Isopropanol was used as the solvent (mobile phase) Three main components were separated from the leaf sample. The lowest on the chromatograph paper was chlorophyll b, characterized by its blue-green colour. The second component on the paper was chlorophyll a which has a green colour. The last component was carotenes which have a yellow colour on the paper. Paper chromatography separates compounds based on their polarity. Using a non-polar solvent like isopropanol, the plant extract separates in the order chlorophyll b, chlorophyll a then carotenes, with carotenes being the least polar of the three. Therefore the three plant extract components that were separated by paper chromatography were chlorophyll a, chlorophyll b and carotenes.
Introduction
All life on earth depends on the process of photosynthesis. Photosynthesis is a process that plants use to convert carbon dioxide (CO2) and water (H2O) in the presence of light energy into carbohydrates (sugars) . Photosynthesis requires the presence of a substance that absorbs light of particular wavelength and this substance is called chlorophyll pigments . Its colour depends upon the color of light that it reflects. For instance, a green leaf appears green because it reflects the green light and absorbs all other colors. There are two major types: chlorophyll a, which is light green color and chlorophyll b, which is green to dark green colour . In addition to chlorophyll, the leaves of many plants also contain one or more additional pigments, including carotenes which are yellow to orange, and xanthophyll, which are yellow . These pigments in plant cells can be separated from each other by a technique known as chromatography. Chromatography is a common technique used for separating and identifying substances in a mixture, based upon their solubility in a solvent and adsorption on the stationary phase . Chromatography usually involves the use of two phases, one moving phase called mobile phase and the other stationary phase. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it . Components of the sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase. The most well-known type of chromatography is paper chromatography in which separation is established from the movement of molecules on a piece of chromatography paper by capillary action. In this experiment paper chromatography was used to separate photosynthetic pigments extracted with isopropanol (solvent) from frozen chopped spinach. Studies have shown that frozen spinach leaves contain chlorophyll a and b and carotene as major pigments and less abundant pigments such as xanthophyll . According to a previous study it has been indicated that carotenes are soluble in polar solvents while, xanthophyll are less soluble than carotene and chlorophyll are the least soluble .
Following chromatography we expect to see at least two distinct pigment bands/spots namely: i) chlorophyll and ii) carotene and depending on the type of spinach we might have other pigments such as xanthophyll (yellow). In this process, various pigments will separate along the chromatography paper depending on polarity of the pigments. The most polar will be in the lowest band on the paper, while the least polar will be in the uppermost band . The identity of separated components can be determined calculating Rf value, which is defined as.
The purpose of this investigation is to separate the pigments in a plant extract by paper chromatography and to determine the identity of the pigments using ratio factor (Rf).
Procedure
Prior to starting the lab experiment some plant material was to be prepared especially if chopped frozen spinach were not to be used. However, in this experiment frozen chopped spinach was used; therefore no preparation was required other than just taking it out of the freezer. A strip off the chromatography paper, 1cm shorter than the test tube was cut. Thereafter, water bath was prepared by placing a mug into a pot of water one-third full and heated to a temperature just below boiling. Then the frozen chopped spinach was added into a test tube to a depth 1.5 cm. A small plastic pipette was used to add about the same volume of isopropanol in the test tube. The spinach was crushed using a large pipette and the test tube placed into the mug of water bath. The test tube was left in the water bath for 30 minutes and the spinach leafs repeatedly crushed with the large pipette. Once the solution became distinctly green the heat was turned off. Using a pencil a line was made on the chromatography paper 1.5cm from the end and a tiny drop of the solution added on the mark. The paper was allowed to dry for 2 minutes and the process repeated 10 times to create a distinct green dot. Subsequently, about 1cm of isopropanol was added to a new test tube using a small pipette. Thereafter the chromatography paper with the distinct green dot of spinach homogenate was placed in the test tube 1 cm above the solvent (isopropanol) and the test tube sealed. The chromatography strip was left in the test tube for 60 minutes to allow separation of the pigments. After 60 minutes the extraction was complete (see Fig. 1 in result section) and Rf value was calculated to identify the pigments.
Results
At the end of the experiment chromatography paper had three distinct spots namely:
i) blue- green
ii) yellow-green and
iii) Yellow.
Discussion
This clearly agrees with the hypothesis that we expect to see at least two distinct bands with a possible additional band. However the additional band is dependent on the type of spinach and the resolution of the chromatographic technique; in this experiment the third spot/ band was not clearly visible because paper chromatography does not give a very good resolution. Paper chromatography separates compounds based on their polarity. The main visible spots on the paper chromatograph green, yellow and blue spots. The green spot is the most prominent one and it mainly contains chlorophylls (a and b). The yellow spot represents the carotenes. Pigments are carried along at different rates because they have different solubility in the solvent and because they are attracted differently to the fibres in the paper through the formation of intermolecular bonds, such as hydrogen bonds. Therefore carotenes travelled a longer distance on the paper more than the chlorophylls and this is evident with the high Rf value of the carotenes (0.97) compared to that of the chlorophylls (0.62 and 0.83).
Chlorophylls are both polar compounds. Chlorophyll a is blue green in colour and is responsible for the blue colour that remained around the base line where the sample was spotted. Chlorophyll b is green in colour and is the represented in the middle of the chromatograph paper. The difference in polarity between chlorophyll a and b is because chlorophyll a has an methyl (-CH3) group on the same position in chlorophyll b which has an aldehyde group (-CHO). Since –CHO is polar, this makes chlorophyll b slightly more polar than chlorophyll a . On the other hand, carotenes are non-polar and yellow in colour.
Isopropanol was used as the mobile phase in the chromatograph system. This is a non-polar solvent and it will only dissolve hydrophobic compounds. Therefore, during chromatography, the non-polar carotenes dissolve in the mobile phase and move along with the isopropanol more than they remain (adsorb) on the stationary phase (paper) resulting to high Rf values. Chlorophylls are polar and they are therefore adsorbed to the polar stationary phase (paper) longer than they travel in the solvent. Chlorophylls therefore had low Rf values relative to the carotenes. Chlorophyll b is slightly more polar than chlorophyll a and will therefore have a smaller Rf value and its blue colour characterizes it on the baseline.
The stationary phase paper is made of cellulose. Cellulose is a homopolymer of glucose and it has hydroxyl groups (–OH) on its surface which form a thin layer of water molecules. The paper is therefore hydrophilic and it will retain polar compounds, preventing them from moving along with the mobile phase. This results to polar compounds having small Rf values.
Conclusion
The purpose of this investigation was to separate the pigments in a plant extract by paper chromatography and to determine the identity of the pigments using ratio factor (Rf). This technique yielded a good overview of the components of a spinach leaf which include chlorophyll a, chlorophyll b and carotenes. The three pigments were identified based on their Rf values. These three components are coloured and easily differentiated from each other without having to stain the paper with any reagent. Chlorophyll a is green; chlorophyll b is blue green while carotenes are yellow. These pigments were observed although the two chlorophylls were not very distinct because paper chromatography does not give a very good resolution. However it gives an idea of the possible constituent pigments in the spinach leaves. Thin layer chromatography is be a better chromatographic method for this experiment as it yields a higher resolution and only uses a small amount of sample.
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