Asis, Janine Marie Cortes, Maria Elaine Mendoza, Cheli Marie Santos, Micah Jill Silo, Marie Kyla

Louise

July 19, 2011 BIO 20 LAB -TFIJ

PRIMARY PRODUCTS OF PHOTOSYNTHESIS I. Carbohydrates in plants Procedure: 1. Cut up a leaf which has been exposed to bright light and crush it well with a little water. Filter the liquid into a test tube about one third filled. Add about 3 ml of Fehlings solution and boil ituntil the solution forms precipitate. Make sure that the opening of the test tube is away from you or from your classmates while boiling.
2. Get 5 test tubes and mix the indicated amount of substances in it. In the first test tube, put

1 ml of molasses and 2 ml of water. In second test tube, put a few crystals of cane sugar and 3ml of water. In the third test tube, put mashed raisins and put in 3 ml of water. And in the fourth test tube, put 1 ml of corn syrup and 2 ml of water. Shake the test tubes until the material are mixed well. In the fifth test tube, put 3 ml of distilled water. In all the test tubes, add 3 ml of Fehlings solution and boil until the solutions form precipitate. Label the test tubes according to the material used.
3. Make thin sections of banana, potato, cassava,and kamote. Mount in water in a slide and

examine with a low power objective under the microscope. Draw and compare the starch grains. Then place a drop of IKI solution on the slide at the edge of the cover glass. Watch the solution as it works under and note the effect as it comes in contact with the starch grains. Record your results.

A. Results As tabulated in Table 1.1, it was observed that the blue color of the reagent turned bright red, dark red, or brick red in some solutions and retained its color in others. The solution of the mango leaf turned bright red, the solution of cane sugar turned brick red, and the solutions of molasses, raisin, and corn syrup turned dark red. On the other hand, the solution of distilled water retained its bluish color.

Table 1.1Reaction of Plant Materials with Fehlings Solution. Legend: + = bright red, ++ = brick red, +++ = dark red, - = no precipitate Test Reagent Positive Result Mango Leaf Fehlings Solution + Molasses Fehlings Solution +++ Cane Sugar Fehlings Solution ++ Raisin Fehlings Solution +++ Corn Syrup Fehlings Solution +++ Distilled Water Fehlings Solution -

Fig. 1.1.1 Mango leaf extract solution reacted Fig. 1.1.1Molasses solution reacted with with Fehlings Solution and heated. Fehlings Solution and heated.

Fig. 1.1.3Cane Sugar solution reacted with Fehlings Solution and heated.

Fig. 1.1.4Raisin solution reacted with Fehlings Solution and heated

Fig. 1.1.5Corn syrup solution reacted with Fehlings Solution and heated.

Fig. 1.1.6Distilled water solution reacted withFehlings Solution and heated. 2

It is shown in Table 1.2that all of the thin sections of various plant materials were stained dark blue when the IKI solution is placed and exhibited different shapes. Banana exhibited an oval shape, potato exhibited a circular shape, cassava exhibited a rectangular shape, and sweet yam or kamote exhibited an irregular shape. Table 1.2Reaction of Plant Materials with IKI Solution. Legend: + = positively reacted with IKI Solution. Test Reagent Positive Results Shape Banana IKI Solution + oval Potato IKI Solution + circular Cassava IKI Solution + rectangular Kamote IKI Solution + irregular

Fig. 1.2.1 Thin section of banana reacted withFig. 1.2.2Thin sections of potato reacted with IKI IKI Solution and observed under the microscope.Solution and observed under the microscope.

Fig. 1.2.3 Thin section of cassava reacted with Fig. 1.2.4 Thin sectionof kamote reacted with IKI IKI Solution and observed under the microscope. Solution and observed under the microscope.

B. Discussion The test which was made to get the figures in Table 1.1 is called the Fehlings test. This test uses Fehlings solution which is made initially as two separate solutions known as Fehlings A and Fehlings B. Fehlings A is a copper (II) sulfate [CuSO 4] which is a blue aqueous solution. On the other hand, Fehlings B is a clear solution of aqueous potassium sodium tartrate, a double salt also known as Seignettes salt or Rochelle salt, and a strong alkali commonly known as sodium hydroxide [NaOH]. The final mixture of Fehlings solution is the equal mixture of the said solutions resulting to a royal blue color. 3

This test is used to indicate the presence of sugar in a material. If the solution, after being heated, formed red brick precipitate, it means that the material contains sugar. The formation of red brick precipitate occurs because the sugar reduces copper (II) ions to Copper (I). In the experiment, itcan be observed that the plant materials such as mango leaf, molasses, cane sugar, raisin, and corn syrup exhibited positive results when reacted to Fehlings Solution. As mentioned, precipitate formation occurs when there is sugar present to react with the Fehlings solution. It can be noticed that the color intensity of the resulting precipitate from each reactionvaries. Difference in sugar content may account for this difference in intensity.From our results, it can be said that cane sugar(brick red), has greater sugar content than mango leaf (light red), and molasses, raisin, and corn syrup (dark red) has higher sugar content than cane sugar. On the other hand, the reaction with distilled water solution did not exhibit any change in color. This reaction served as the negative control indicating the absence of sugar thus making the experiment valid. Sugar are present I plants because it is combined with carbohydrates to produce starch. It is also the source of energy of plants. On the other hand, figures in Table 1.2 are obtained using IKI Solution. The IKI solution is composed of 5% iodine, 10% potassium iodide in 85% distilled water. This solution reacts with starch making it an indicator of the presence of the said component. The reaction between the two produces a dark purple or black color. In the experiment, banana, potato, cassava, and kamote showed equal positive results when reacted to IKI solution. This result is expected because starch is found in all plants since it acts as energy storage for it. The dark region which is also needed to be observed hasdissimilar shapes in the different plant materials.Since the IKI solution only stains regions containing starch, and the starch pigment is contained inside the vacuole, it implies that this dark regionis the central vacuole of the plant. The shape of the bananas vacuole is oval, potatos vacuole is circular, cassavas vacuole is rectangular, and kamotes vacuole is irregularly shaped. II. Proteins Procedure: 1. Soak a small amount of mongo, soya, and corn seeds in water overnight. Ground these materials separately with a mortar and pestle. Add water and let stand for 30 minutes. Filter these materials. Put 3 ml of the filtrate to separate test tubes. To each test tube, carefully add 1 ml of concentrated nitric acid and boil it in a water bath for five minutes. A white precipitation forms which upon heating turns yellow and finally dissolves, giving rise to a yellow solution. Cool the solution and add 8-10 ml of 10% NaOH to make the solution alkaline. The yellow color deepens to orange. Record your results. 4

A. Results It is can be seen in Table 2.1 that the solutions which are reacted to HNO3 turned into different shades of yellow after being heated. The mongo seed solution became yellow orange, the soya solution became yellow, and corn solution became light yellow. However, the water solution remained to its clear color. Table 2.1Reaction of Plant Materials with HNO3 + NaOH (10%). Legend: + = light yellow, + + = yellow, +++ = yellow-orange, - = clear Test Reagent Positive Results Mongo HNO3+ NaOH (10%) +++ Soya HNO3+ NaOH (10%) ++ Corn HNO3+ NaOH (10%) + Water HNO3+ NaOH (10%) -

Fig.2.1.1 Mongo solution reacted with nitric acid Fig.2.1.2Soya solution reacted with nitric acidand and added 10% sodium nitrate after heating. added 10% sodium nitrate after heating. MISSING IMAGE

Fig.2.1.3Corn solution reacted with nitric acid Fig.2.1.4Water solution reacted with nitric acid and added 10% sodium nitrate after heating. and added 10% sodium nitrate after heating.

B. Discussion

The test used in this experiment is called Xanthoproteic test. This test is used to indicate the presence of proteins in plant materials. If proteins, which contain its building block amino acid with aromatic rings, are present, the mixture turns yellow. If a strong base is added the color turns orange.This color changes when reacted to HNO3 + NaOH (10%) are caused by nitrated aromatic rings in the protein. When the solutions of mongo, soya, corn, and water are reacted to nitric acid and added sodium hydroxide to the solutions when cooled after being heated, it indicated positive results. As mentioned, there will be changes that occur when a protein containing material is reacted to HNO3 + NaOH (10%). It can also be observed that different color intensities vary with different plant material. Difference in protein content can be accounted to the different color intensities. From the obtained results, it can be said that soya (yellow) has higher protein content than corn (light yellow) and mongo (yellow-orange) has higher protein content than soya. However, water did not exhibit any change in color which indicates absence of protein content. Making it the negative control of the experiment made the experiment valid. III. Lipids and Fatty Acids Procedure: 1. Get a 10x10 filter paper. Rub a peanut seed on one corner of the unglazed paper, coconut meat on another corner, cocoa powder on another corner, and a drop of vegetable oil on the fourth corner. In the center, put a drop of water. Hold the paper over a flame for a few seconds, but dont burn the paper. 2. Chop up a handful of grass cuttings or leaves using a mortar and pestle. Put the leaves into a flask with about 25ml of 85% acetone. Shake the flask and allow to stand until the color has passed into the acetone. When the acetone becomes deep green, view the solution by reflected light. A characteristic blood-red fluorescence is visible. 3. Cut a strip of filter paper. The length of the paper should exceed 25 ml test tube while the width should be less than the diameter of the test tube. Drop a spot of the solution about 1cm from the bottom of the strip of filter paper. Dry and put another drop on the same spot. Repeat the procedure for about three times. Completely dry the filter paper.
4. In the meantime, put a test tube on the stand and pour 2 ml of acetone. Cover the test tube

with a stopper. When the sides of the test tube are dry, carefully insert the strip of filter paper until the lower edge reaches the acetone. Make sure the level of the acetone does not reach the drop of chlorophyll solution. Stopper the test tube. Do not disturb the set-up and allow the solution to spread upward the filter paper. Observe the colors that appear as the acetone rises up the filter paper strip. Record your observations.

A. Results 6

It can be seen in the table below that the observed spot where the vegetable oil was dropped is the area which is the most translucent, followed by peanut, cocoa, and corn which are more translucent, next by coconut which is quite translucent. The area where the corn and water is rubbed and dropped respectively didnt show any signs of translucence. Table 3.1 Test for Fatty Acids in Various Seeds.Legend:+ = quite translucent,++ = translucent, +++ = most translucent, - = not translucent Positive result Corn Peanut ++ Coconut + Cocoa ++ Vegetable Oil +++ Water -

Fig 3.1.1 Rubbed or Droped corn, peanut, coconut, cocoa, vegetable oil, and water in different corners of filter paper and heated. (First Trial)

Fig 3.1.2 Rubbed or Droped corn, peanut, coconut, cocoa, vegetable oil, and water in different corners of filter paper and heated. (Second Trial)

According to the table below, the distance traveled by the bright yellow, light yellow, blue-green, and olive green pigments are 8.5 cm, 3.9 cm, 2.4 cm, and 1.5 cm respectively. In addition, the computed Rf factors for these pigments are 0.83, 0.38, 0.24, and 0.15 respectively. Table 3.2Distances travelled and calculated Rf factor for each pigment in the paper chromatography experiment performed using Hibiscus rosa-sinensis (Gumamela) leaves. Pigment Bright yellow Light yellow Blue-green Olive green Distance from lower end of filter paper (cm) 8.5 3.9 2.4 1.5
Solvent Front: 10.2 cm Bright yellow: 8.5 cm

Rf factor 0.83 0.38 0.24 0.15

Light yellow: 3.9 cm Blue-green: 2.4 cm Olive green: 1.5 cmfig.3.2.1 Figure 3.2.1 Gumamela pigments separated using paper chromatography.

B. Discussion The test used in this experiment tabulated in Table 3.1 is what you call the Grease Spot test which indicates the lipid and fatty acid contents of various seeds. A grease spot often looks like a wet spot. If it is a grease spot, the spot will spread and remain. If it a wet spot, it will dry out and disappear. This method of detecting lipid contents is done for centuries. Lipids that made the grease spots are derived from glycerol and sphingosine produces translucent spot on fabrics. Also, oil has higher evaporative cooling than water thus making the water evaporate when heated. When the peanut, coconut, cocoa, are rubbed and vegetable oil is dropped in the unglazed paper, it created a grease spot which according to the above statement indicates the presence of lipids and fatty acids. On the other hand the corn and the water disappeared and dry out which indicates the absence of oil. But according to the researches, oil grains have high oil content. Maybe, malpractices are done during the experimentation thus making the result incorrect. With water as the negative control, the experiment is considered valid. On the other hand, the procedure used to gain the results in Table 3.2 is called chromatography. Chromatography is the science which studies the separation of molecules based on differences in their structure and/or composition. Applied in the botanical perspective, it is 8

most commonly used to separate and identify different pigments in plants. Paper chromatography uses paper as the stationary phase (a solid or a liquid supported on a solid) and a liquid solvent as the mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase (capillary action) and carries the mixture or blot it passes through. Different components of the mixture travel at different rates. Chromatography is applied in various fields of science including biotechnology and pharmacy. In the paper chromatography activity performed using Hibiscus rosa-sinensis (gumamela) leaves as test specimen, filter paper as stationary phase and acetone as mobile phase, 4 colors were seen evidently. The colors: olive green, blue-green, light yellow, and bright yellow were arranged from the bottommost to topmost order respectively in the strip of filter paper. As shown in Fig. 3.2.1, the solvent front reached a distance of 10.2 cm while the pigments bright yellow, light yellow, blue green and olive green reached the distances 8.5 cm, 3.9 cm, 2.4 cm and 1.5 cm respectively. These distances aided in the computation of the Rf factor of each of the pigments.The Rf factor is calculated by getting the ratio of the distance travelled by the pigment and the distance travelled by the solvent front. According to the Table 3.2, the bright yellow, light yellow, blue-green, and olive green pigments have the Rf factors 0.83, 0.38, 0.24 and 0.15 respectively. Filter paper is made from trees which are composed of cellulose fibers. Cellulose is a hydrophilic (polar) polymer of glucose. Acetone is a colorless, volatile, polar protic organic solvent. Using the former as stationary phase and the latter as mobile phase in paper chromatography is efficient since both are polar and capable of adhering with each other. When the solution of the pigments is applied on the paper, the pigment molecules adsorb onto the cellulose fibers. When the bottom end of the filter paper is immersed into the acetone solvent, the solvent is then absorbed and moves up the paper through capillary action. As the solvent goes up the paper, it carries along with it some of the pigments present in the solution. The colors on the strip of filter paper correspond to different pigments present in Hibiscus rosa-sinensis. The bright yellow pigment is carotenoid, the light yellow pigment is xanthophyll, the blue-green pigment is chlorophyll-a, and the olive green pigment is chlorophyll-b. It was observed that the pigments separated at different rates and also travelled different distances. These differences are caused by several factors namely molecular weight, solubility of the pigment in the solvent and the affinity (attraction) for the paper. The greater the molecular weight of the compound, the shorter distance the pigment will travel and vice versa. The more soluble the pigment is in the solvent, the farther it will move up the paper and vice versa. Lastly, the greater the affinity of the pigment for the paper, the slower and nearer it will travel up the paper and vice versa. In the experiment performed, the carotene pigment reached the farthest distance among the other pigments. This is because this pigment has the smallest molecular weight, is most soluble in acetone and least attracted to the filter paper. The chlorophyll-b pigment, on the other hand, had the shortest travel distance; meaning it has the greatest molecular weight, is least soluble in the acetone solvent and has the greatest affinity for paper in the experiment. The computation of the Rf factor just gives the comparison of how the distances travelled by this pigments vary. A pigments retardation factor tells you about its molecular weight, solubility in the solvent and the affinity for the paper. 9

Guide Questions: 1. The following are some common drugs or pharmaceutical products which are used or prescribed by doctors. Indicate what is asked for. Medicine / Drug Dextrose Main ingredient present Monosaccharide (Glucose) Fiber Psyllium Test reagent Benedicts Reagent, Fehlings Solution Test for Phenolphthalein Test for Diphenylamine, Tollens Reagent Function of drug Primary source of energy and a metabolic intermediate. Taken to induce bowel movements or to loosen the stool. Help soothe and protect the skin when it becomes irritated; treats symptoms associated with poison ivy infection and chicken pox. Glucose is a primary source of energy for the brain. Treats skin problems, burns, sunburns, skin disorders, skin cuts, and abrasions. Helps remove freckles, thin the blood and discourage clotting; helpful against heartburn and indigestion. Acts to hydrolyze peptides into amino acids; catalyzes the hydrolysis of peptide bonds. Aids in digestion, stomach cramps, nausea, anxiety and vomiting; refreshes the body.

Laxatives

Calamine Lotion

Zinc oxide (ZnO) and 0.5% Ferric oxide (Fe2O3)

Caramel Castor Oil

Sugar (Glucose) Ricin oleic acid; Oil

Benedicts Reagent, Fehlings solution Schiffs Reagent Test

Papain

Protein, Latex

Carmine Fibrin , Ellman's Reagent, Xanthroproteic Test

Trypsin

Protein

Biuret Test, Ellmans Reagent

Mint

Mint substance

Mandelin Reagent, Marquis Reagent

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2. Why have plants been used as source of many pharmaceutical products? How is this related to photosynthesis? Plants show considerable effectiveness, are socially accepted, and economically viable. It is used as source of many pharmaceutical products because people have a future medicine bank to discover: there are millions of plants with flowers, most of which have not been investigated and which principles could be decisive in the treatment of diseases and the components of plants have synergic effect: they all interact simultaneously. Also, plants have preventive character regarding the appearance of diseases. It is related to photosynthesis by the principle of energy transformation in photosynthesis. Light has very high energy content, and when it is absorbed by a substance this energy is converted to other forms. When the energy ends up in the wrong place, it can cause serious damage to living organisms. Because plants and other photosynthetic species have been dealing with light for eons, they have had to develop photoprotective mechanisms to limit light damage. Learning about the causes of light- induced tissue damage and the details of the natural photoprotective mechanisms can help us find ways to adapt these processes for the benefit of humanity in areas far removed from photosynthesis itself. For example, the mechanism by which sunlight absorbed by photosynthetic chlorophyll causes tissue damage in plants has been harnessed for medical purposes. Substances related to chlorophyll localize naturally in cancerous tumor tissue. Illumination of the tumors with light then leads to photochemical damage which can kill the tumor while leaving surrounding tissue unharmed. Another medical application involves using similar chlorophyll relatives to localize in tumor tissue, and thus act as dyes which clearly delineate the boundary between cancerous and healthy tissue.

3.

Give the importance of using chemical tests in determining plant constituents.

Plant constituents, as the word implies are the individual chemicals from which plants are made. These constituents are organic in nature and synthesized in plants by the activity of individual cells. The process by which these complex organic chemical constituents are formed, utilizing simple substances and enzymes are known as biosynthesis. Chemical tests in determining plant constituents are important to derive pharmaceutical and medicinal plant sources. The medicinal value of plant depends on the nature of plant constituents present in it, which is known as active principal or active constituent. The chemical constituents present in plants that do not possess any definite therapeutic value are known as inactive constituents. The formation of different active and inactive constituents of plants involves various metabolic pathways.

References: Elementary Botany Laboratory manual by the Committee on Biology chemistry.msu.edu 800mainstreet.com chemguide.co.uk Britannica Encyclopedia Online 11

docshare.com library.thinkquest.org

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