Exp No. 3 Chem 31.1

Group Name: Chem 31.1 hx Group 1

Date PeRformed: 02 / 10 / 15 Date Submitted: 02 / 17 / 15

Experiment no. 3 CHROMATOGRAPHY OF PLANT PIGMENTS 1 Alindogan, Marianette, 1Asuncion Arnel, 1Avisado Adrienne, 1Bantola Dianne 1 College of Science University of the Philippines, Baguio ABSTRACT Chromatography is a method used to separate mixtures of compounds that exists in a mixture. There are different types of chromatography used in laboratories such as Thin-layer chromatography and Paper chromatography. In this experiment paper chromatography was used in order to acknowledge the different pigments present in the spinach leaves. Leaves possess different types of pigments in their cytoplasm which aid in their photosynthesis. Some of these pigments are chlorophyll which gives its green color, carotenoids that gives orange, red and yellow color to the plant and many more. This experiment aims to distinguish the pigments present in spinach leaves by obtaining its extract through maceration and addition of acetone. A solvent was prepared by mixing acetone, diethyl ether and petroleum ether. This will serve as the solvent that would be a medium for the pigments to travel depending on their polarity. The extract was gathered through capillary reaction and placed in a specialized filter paper and was suspended on the prepared solvent where the distance traveled by the solvent and the solute are gathered. The results were then recorded where different pigments were observed, whichever travelled the farthest, the nearest or whether it travelled at all, these data were then gathered and used to measure the distance travelled by the solute against the distance travelled by the solvent, which is called Retention Factor.

I.

INTRODUCTION Paper chromatography is a technique which separates dried liquid sample with a liquid solvent (mobile phase) and a paper strip (stationary phase). A mobile phase is a gas or liquid that carries the components while the stationary phase is the part of the apparatus that does not move with the sample. Paper chromatography involves principles which show their characteristics. One of which is the ability to move of a liquid within the spaces of a porous material due to the forces of adhesion, cohesion and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity hence this is called capillary action. In addition to this is solubility which is the degree to which a material (solute) dissolves into a solvent. This also includes the ability of solutes to dissolve into solvents having similar properties and this allow different solutes to be separated by different combinations of solvents. Paper chromatography is important in making small-scale separations and identifications. In addition to this, paper chromatography is important in forensic chemistry for identifying complex mixtures of chemical compounds in the crime scenes. This will help to identify the unknown

sample and the source of the colors that were used and can totally provide evidence. Also, included in this is the calculation of the retention factors or retardation factors which gives the quantitative measure of the properties of a component’s mixture. This is calculated by having the formula distance travelled by solute divided by distance travelled by solvent. In this experiment, spinach leaves are used as a source of pigments which will be used in paper chromatography. These pigments were separated by this technique and its retention factor was also calculated using the data obtained. The objectives of this experiment is to separate pigments from the leaves of spinach then to identify the different pigments present in spinach leaves and lastly to compute the approximate rates of flow (Rf values) of each of the component pigments of the leaves. The Rf value of each color can be identified by the movement of any spot found in the paper.

II. RESULTS AND DISCUSSION The pigments found in plants vary in their respective polarities. As such, dissolving a plant extract with a certain solvent will cause these pigments to dissolve at different rates. By using this property to our advantage, the separation of pigments present in a plant is made possible. The common photosynthetic pigments found in the plant leaf include chlorophyll a, chlorophyll b, xanthophylls, and carotene. Chlorophyll a is characterized by its deep green color. Chlorophyll b on the other hand appears as a lighter, yellowish-green. Xanthophylls are yellow in color, and carotene is orange. It is possible to predict the results based on the set up of the experiment, particularly, the difference in polarities between the reagents to be used. According to plant anatomy textbooks, the photosynthetic pigments can be arranged according to increasing polarity in the following order: xanthophyll, chlorophyll a, and chlorophyll b. Carotene is a nonpolar pigment, and is therefore considered the least polar among the pigments. Next, we have to take into account the nature of the solvent/s to be used – petroleum ether, diethyl ether, and acetone; which are nonpolar. The composition of Whatman paper, cellulose, is a polar substance. With this information, we apply the rule of “like dissolves like.” The most nonpolar substance, carotene, easily dissolves with the nonpolar solvent and therefore dissolves first. As such, it should rise at almost the same rate as with the solvent. The polar pigments would then follow suit, as they are not soluble with the solvent. The least polar of the remaining three pigments, xanthophyll, would rise behind carotene. Chlorophyll a would move next; and the most polar pigment, chlorophyll b, would be the last to rise. The separation of the pigments can thus be predicted in the following sequence, with the white gaps representing possible varying intervals between the pigments:

Fig. 1. Predicted outcome of the separation of pigments

Fig. 2. Experimental outcome of the separation of pigments Only two colors that appeared on the surface of the Whatman paper and these are the colors of dark shade of green and the other one is yellowish green. Therefore the latter shows the presence of chlorophyll b while the darker shade of green indicates the presence of chlorophyll a. These two pigments are dominant in the leaf as they are responsible for the leaf’s photosynthetic abilities. As predicted, chlorophyll a appears higher than chlorophyll b, which indicates the more polar nature of the latter

Fig. 3. Chemical composition of Chlorophyll a and b The longer distance travelled by the chlorophyll a (green) indicates that it is less polar than chlorophyll b (light green), because the higher polarity of chlorophyll b causes it to dissolve last in the presence of a nonpolar solvent. This causes the chlorophyll a to rise first, followed by the more polar chlorophyll. Measuring the distance travelled by each pigment spot, the following results are obtained:

oreDistance Travelled by Solute Distance travelled by Solvent

A B C

RTF Value

A B C

Mean

Yellow Green 45.5mm 47.5mm 47mm

Green 52.5mm 51mm 51.5mm

54mm 0.84 0.88 0.87 0.86

0.97 0.94 0.95 0.95

Before obtaining the results, the beaker filled with petroleum ether, diethyl ether and acetone was sealed to make sure that the atmosphere inside the beaker is saturated with solvent vapour. This is done to stop the solvent from evaporating as it rises up on the paper. And when it is done, the saturated vapour concentration inside the beaker will cause the substances present on the extract to appear during the experiment. The Whatman filter paper was lowered below the

beaker touching only the very end of the tip. And for this to possibly happen, there are several factors that affect the movement of the solute contained on the pigment and these factors include the porosity of the chromatography paper or the Whatman filter paper, the solubility of the solvent and also the molecular size o the solute. The Whatman filter paper is made of cellulose from plants which are polar in nature. Materials made of cellulose, as defined by its function on everyday life, is porous and most importantly absorbent. The solvent moves and diffuses on the paper upward; dissolving the molecules it ran into. And this event depends on the polarities of the molecules and the solvent. For the solute to rise together with the solvent, its solubility should be determined to explain this occurrence. Their unequal solubilities caused the variety of color pigment molecules to rise at different places as the solvent continues to travel upward of the paper. And as solubility depends on the polarities of substance, it will further affect the results during the experiment, if a chemical is non-polar, it will not dissolve on polar solvent, same for a polar molecule with a non-polar solvent. Due to capillary action, it allows the separation of compounds due to their attraction or affinity of the compounds for the mobile phase (the solvent) versus the stationary phase (the Whatman paper). To add some more, insoluble compounds that were attracted to the stationary phase stay in place while the other way around travel upward together with the the solvent. As the solution moves upward the paper, like soluble pigments will travel along with the solvent until the bonds break between the solvent and solute becoming so weak and will later on break the attraction and imprint itself on a certain height up in the paper. .

The solvents used in the experiment are petroleum ether, diethyl ether and acetone which all possessed the properties of having both polar and non-polar characteristics. This is for the molecules on the extract especially the pigments that are extracted to efficiently go with the flow travelled by the solvent as it rose upward on the paper without minding their properties prior to their polarity. Chlorophyll pigments, a and b, are said to be polar in nature according to some literatures. And this explained why they were able to rise together with the solvents, wherein this all follow the principle of “like dissolves like”. The above explanations synthesized the process involving the paper chromatography, which is defined as a method used to separate very small quantities of substances from each other and through this the identity of separated substances can be determined. But this can be furthermore explained and proven through the help of finding the so-called retention factors. Based on Table 1 above, the data results are used to calculate the retention factor which is the ratio of the distance the spot move above the origin to the distance travelled by the solvent. On this experiment, finding the retention factors of the data can be useful on identifying the substance identity rather than by just observing the chromatogram. The distance travelled by the solvent reached 54 mm and for the results in three trials to be united, the mean of the retention factors were computed separately for the two pigments. Chlorophyll a has reached the farthest distance of 0.95 as shown on the mean from its obtained retention factors while chlorophyll b travelled a distance of 0.86 on the other hand. Computing for the R f is important because molecules bond in different ways to the surface of the Whatman filter paper and these differences caused the molecules to move slower or faster making it possible to separate them. And when the products are separated, it is easier to identify them using R f tables. And because of these retention factors, is how the explanations are also constructed, wherein the compound with a larger Rf was considered less polar as it interacts less strongly with the polar Whatman filter paper and vice-versa. So it can be said that chlorophyll a is less polar than chlorophyll b. It also helps in the comparison of molecular size of the pigment molecules because according to some literatures, retention factors in inversely proportional to molecular size, so it can also be implied that chlorophyll a is smaller in size.

III. CONCLUSION The paper chromatography is an effective technique widely used in order to separate very small quantities of substances from each other. In this way, the identities of the separated substances in the spinach leaves are determined. The fllter paper showed dark shade of green and yellow green colors rising until they reached a point. This is because of the capillary action taking place in the points made in the filter paper. These green colors are because of the pigment chlorophyll a and chlorophyll b which is found on leaves and which makes them appear shades of green. This pigment is maximized for photosynthesis and for a wider range of absorption of light. This pigment exhibits absorption in which their molecular characteristics speed up their absorption process in the filter paper. Also, the colors were absorbed at different rates through a mat of cellulose fibers which is the Whattman filter paper. Rf values were computed for each color absorbed by the Whattman filter paper. For the dark green color, the following Rf values are 0.97, 0.94 and 0.95. On the other hand, the Rf values for the yellow green color are 0.84, 0.88 and 0.87. This shows that there is a large Rf value for the dark color green than the yellow color green. This implies that chlorophyll a is more mobile compared to chlorophyll b and travelled farther than the base line compared to the former one. The objectives of this experiment were achieved successfully and the data results obtained were all studied and observed carefully throughout the experiment.

IV. POST LAB QUESTIONS 1. How many separate bands of color appeared in the chromatogram? Answer: There were 2 bands of colors that appeared in the chromatogram. These were color green and yellow green. The figure is shown below:

Green band Yellow green band

2. In what order did the bands of color appear in the chromatogram? Answer: The green band travelled farther than that of the yellow band. The green band travelled an average of 46.67 millimeters while the yellow band travelled an average of 51.67 millimeters.

3. Compute the approximate Rf values of each pigment using the following formula:

Rf 

dis tan ce travelled by solute distan ce travelled by solvent

YELLOW GREEN BANDS

 dis tan ce travelled by solute 45.5mm Rf    0.84 dis tan ce travelled by solvent 54mm A.



Rf 

dis tan ce travelled by solute 47.5mm   0.88 dis tan ce travelled by solvent 54mm

Rf 

distan ce travelled by solute 47mm   0.87 dis tan ce travelled by solvent 54mm

B.

 C.





Rf(green)average 

0.84  0.88  0.87  0.86 3

GREEN BANDS

Rf 

dis tan ce travelled by solute 52.5mm   0.97 distan ce travelled by solvent 54mm

Rf 

distan ce travelled by solute 51mm   0.94 dis tan ce travelled by solvent 54mm

Rf 

dis tan ce travelled by solute 51.5mm   0.95 distan ce travelled by solvent 54mm

A.



B.

 C.





Rf(green )average 

0.97  0.94  0.95  0.95 3

4. Submit the chromatogram together with your report.

V. REFERENCES Paper Chromatography.Retrieved from www.darngoodsolutions.com/stogascience/scienceworks/intro/TQCHROM.html Clark, J. (2007). Paper Chromatography. Retrieved from http://www.chemguide.co.uk/analysis/chromatography/paper.html Paper Chromatography Science Background. (n.d.). Retrieved from http://www.ihmc.us/groups/voluntology/wiki/291f0/Paper_Chromatography_Science_Background. html TLC - Retention Factor (Rf). (n.d.). Retrieved from http://www.ce.gxnu.edu.cn/organic/net_course/content/tlc/Retention_Factor.htm Retention Factors. (n.d.). Retrieved from http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/chrom/wback3.htm St. Rosemary Educational Institution (2015). Chromatography Lab Answers. Retrieved from http://schoolworkhelper.net/chromatography-lab-answers/. Goh, D. (2014). PIGMENT SEPARATION USING PAPER CHROMATOGRAPHY. Retrieved from https://prezi.com/afsngpazf40x/pigment-separation-using-paper-chromatography/ Skyline College Chemistry 210 Laboratory Manual. (2010). Separation of compounds by paper chromatography. Retrieved from https://prezi.com/afsngpazf40x/pigment-separation-using-paperchromatography/ Liam, M. (2009). Why is retention factor or Rf important? Retrieved from https://answers.yahoo.com/question/index?qid=20080921000150AAfNMxi Anonymous. Experiment 3: Paper Chromatography: A Technique of Separation and Identification. PDF file, Retrieved February 15, 2015. Anonymous. Paper Chromatography. PDF file, Retrieved February 15, 2015. Beck, C. B. (2005). An introduction to plant structure and development: Plant anatomy for the twenty-first century. Cambridge, UK: Cambridge University Press. Pavia, D. L., Lampman, G. L., Kriz, G. S., & Engel, R. G. (1999). Introduction to Organic Laboratory Techniques: A Microscale Approach. Isolation of Chlorophyll and Carotenoid Pigments from Spinach, 3. Retrieved from http://voh.chem.ucla.edu/vohtar/spring03/classes/14CL/pdf/14clisol.pdf

Quach, H. T., Steeper, R. L., & Griffin, G. W. (n.d.). “An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach”, Chem. Educ. 2004, 81, 385-7.