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Title

The effect of temperature on beetroots cells membrane. To use beetroot to investigate the effect of temperature on cell membranes and relate the effect observed to the membranes structure and properties.

Objective : Introduction. Beetroot

Beetroot used for experiment. Pictures adapted from : testwithskewer.blogspot.com

Beetroot is one of many type of beets known as Beta Vulgaris. It can be used in cooking and drinking as it is one of the source of vitamins, minerals, nutrients and antioxidant as well including magnesium, potassium, sodium and vitamin C and betaine. Betaine in the cells serve as organic osmolytes which are synthesized or taken from the surroundings by the cells for protection against osmotic stress, drought and high temperature. It is also important for cardiovascular disease. The high amount of nitrate content in the beetroot help to lower the blood pressure and thus, help to prevent the cardiovascular diseases. the nitrate or known as dietary nitrate in our blood is believed to be the source of biological messenger nitric oxide. This biological messenger nitric oxide is used by the endothelium for signaling the smooth muscle to relax. Thus, vasodilation occurs and enhance the blood flow. Other than its benefits to health, beetroot, especially the juice is used for making dyes or the food colorant as it is known for no allergic side-effects. Besides it is cheap.but, beetroots colour will change when heated so it is mainly used in confectionary, ice creams, tomato paste, jam and jellies. Beetroot dye may also be used in ink.when the bulb is older, the colour is deep crimson and the flesh is softer.

Cell Surface Membrane and Properties.

Cell membrane is a structure of cell that functions as the barier separating the interior cell from the outsides surrounding. This structure selectively let substances in and out of the cell. It is made up of lipid molecules arranged side by side with their tails facing each other forming a phospolipid bilayer. tail

phospolipid bilayer. Adapted from :

http://en.wikipedia.org/wiki/Cell_membrane
This lipid bilayer has partial-permeability for ions and polar molecules as the arrangement of hydrophilic heads and hydrophobic tails prevent the solute such as amino acids, carbohydrates, protein and ions from diffuses through it. But simple diffusion occurs for substances like water (osmosid) and glucose which doesnt require extra helps from protein.

The model of cell membrane that is accepted widely is the Fluid Mosaic Model by S.J. Singer and Garth Nicolson. This model of cell membrane shows that the cell membrane is described as fluid as the composition of it that consist of lipids and protein that always move sideways or literally. This is why it is said to be more fluid-like than a solid.

The picture of Fluid Mosaic Model. picture adapted from vcebiology.edublogs.org

The simple structure of cell membrane consist of lipid bilayers, protein and cholesterol. Picture adapted from graphicshunt.com

The protein in the phospolipid bilayer is known as transport protein which helps the transportation af substances in and out of the cell. As we know, molecules of amino acid, carbohydrate, proteins and charged ions cannot pass through the lipid bilayer by simple diffusion. Thus, the carrier protein is used to transport these substance sometimes with help of Adenosine Triphospate (ATP).

Diagram of simple diffusion, passive transport and active transport through lipid bilayer with the help of carrier protein and ATP. Pictures adapted from : 12knights.pbworks.com

Colorimeter Colorimeter is a device used to measure the absorbency of substances. It is a light-sensitive device that simply measures how much colour is absorbed by objects or substances. With the help of colorimeter, we can analyse the concentration of that particular substances. This device works according to Beer Lamberts law that state, the absorption of light transmitted through a medium is directly proportional to the concentration of the medium. Colorimeter works by passing through a ray of light through the solution thus measures the amount of light. The more concentrated the object or solution, the less light passing through it as more light is absorbed by the solution. The difference can be seen between the light before passing through the solution and after it passed through it. The result or the absorbance do not have a specific unit. But it is called absobance unit or AU.

A type of colorimeter. Picture adapted from : analabsystem.tradeindia.com

(1) Wavelength selection, (2) Printer button, (3) Concentration factor adjustment, (4) UV mode selector (Deuterium lamp), (5) Readout, (6) Sample compartment, (7) Zero control (100% T), (8) Sensitivity switch

Parts of colorimeter. Picture and information adapted from :

http://en.wikipedia.org/wiki/Colorimeter_(chemistry)

REPORT OF PRACTICAL
Problem Statement : What is the effect of temperature on cell membrane and what is the relation between the result observed and the structure of cell membrane. Hypothesis :

When the beetroot cell surface membrane is exposed to high temperature, the membrane will break down causing the red pigments of the beetroot to leak out.

Variables Manipulated variable Responding variable Fixed variable

: : the temperature of water bath used : the reading of the colorimeter (the absorbency of solution) : the type of beetroot used, the size of each section of beetroot, the time 30-minute time used. :

Apparatus

Cork borer, white tile, knife, ruler, beaker, boiling tubes, forceps, thermometer, colorimeter, cuvette, measuring cylinder, pipette, stopwatch. Material :

Beetroot, distilled water, water bath at 350C, 450C, 550C and 650C.

Procedure

1. The raw beetroot is first cut into half using a knife and lined with a white tile. Then, four sections of beetroot are cut using a cork borer. The sections then are measured to 1cm length using a ruler and put aside. Be alert when handling the beetroot as the beetroot juice can stain the clothes and skin badly. 2. The sections then are put in a beaker and rinsed with distilled water. 3. Four boiling tubes labeled 350C, 450C, 550C, and 650C are prepared and 5cm3 of distilled water is added to each of the boiling tube. 4. The boiling tubes then are placed in water bath with temperature ; 350C, 450C, 550C, and 650C respectively for 5 minutes so thet the distilled water would reach the required temperature. 5. One of the sections of beetroot then placed in each boiling tube and left for 30 minutes in the water bath. The time is set using a stopwatch. 6. After 30 minutes, the beetroot sections from each of the boiling tube is taken out and the solution in the boiling tubes are shaken to disperse the dye. 7. The colorimeter is adjusted to read % absorbance. 8. A 3cm3 of distilled water is added into a cuvette and placed into the colorimeter with the smooth side facing the light ray. 9. The colorimeter is adjusted to read zero absorbance for the clear distilled water. The setting is remained untouch during the experiment. 10. 3cm3 of each dye solution is measured and added into the cuvettes separately. The cuvette then is put into the colorimeter and the reading is taken. 11. The smooth sides of the cuvette is wiped clean and dry to make sure theres no dust, water and finger prints on it. The reading is repeated for three times to obtain a more reliable result. 12. This step is done on each of the cuvette for dye solutions. 13. All of the readings are recorded in a table and interpreted into a graph. 14. The apparatus are cleaned with soap and water and rinsed thoroughly with distilled water.

Result

Temperature, C

1 reading 0.205 0.377 0.449 0.525

st

The absorbency of solution, A.U 2nd reading 3rd reading 0.205 0.376 0.450 0.525 0.203 0.378 0.450 0.525

Average reading 0.204 0.377 0.450 0.525

35 45 55 65

Table of the results for colorimeter reading of the absorbency of dye solution at different temperatures.

Discussion

The graph tabulated shows that when the temperature is higher, the absorbency of the solutions is higher. This mean that, there are more beetroot dye leaks into the solution when the temperature is higher. From 350C to 450C, the line of the graph is increasing rapidly. This is because, the high temperature started to denature the proteins in the cell membrane causing the red dye to leak. The higher the temperature exposed to the cell as in to the beetroot, more kinetic energy will be gained by the red pigments inside of the beetroot cells. Thus, this will lead to diffusion out of red pigments from the internal cell of beetroot into the environment or surrounding through cell membrane. Besides, proteins are formed of amino acids coiled and attached to each other like strings. The bond between the amino acid is held by hydrogen bonds and disulphide bridge. The hydrogen bonds however weaken when high temperature is applied and the disulphide bridge or known as SS-bond is a type of covalent bond which has a low boiling point thus, easily disrupted when exposed to high temperature. This will result the damaging of the vacuoles and leaking in the cell membrane causing the dye or red pigments inside the vacuole to leaks out or diffuse out of the cell into the surrounding. This diffusion is assist by the concentration gradient law that a substance will move from a place with high concentration to lower concentration. The enhancement is because at this temperature, more protein will be denatured and also the gaps in the cell membrane getting bigger as the red pigments of beetroot rush out of the cell into the environment or surrounding. Besides that, as we know, the cell membrane is made up of phospolipid bilayer. These lipid bilayers are consist of water-loving head and water-hating tail or simply known as hydrophilic and hydrophobic. The head (water-loving or hydrophilic is the only side that is exposed to water.

When this lipid bilayer is heated or supplied heat, the bond will break down as lipid become more fluid. The water also expands and the molecules vibrate faster. This will resulted disrupting the membrane. From 450C to 650C, the graph is increasing steadily showing that the leaking of red pigments of the beetroot cell is still happening. But, the gradient is not as steep as the gradient from 350C to 450C. This is because, some of the denatured proteins are blocking the holes made at first resulting low flow out of red pigments from the internal cell into the surrounding. In conclusion, this is all about kinetic energy. Energy is supplied to the molecules especially the red pigments of beetroot by heat. Thus, increase the rate of diffusion of the molecules into surrounding. Other than the pigments gaining energy, the cell membrane (proteins and lipid bilayers) will be damaged and denatured due to the high temperature. This, will cause the red pigments molecules that gained energy to rush out of the cell. Although the red pigments might gained a lot of energy because of the high temperature, it is the gaps between the membrane) lipid bilayer) which causes it to pass through and as the pigments move faster, it will increase the rate of diffusion resulting in faster rate of diffusion as the temperature increase.

Limitation Limitations are factors that you cannot control in experiments. The limitation of this experiment is the beetroot used. There are many type of beetroot and certain species of beetroot might only gives out little dye as its red pigments is not as many as the other beetroot. Besides, the beetroot used is also not fresh. This might affect the result as wilting beetroot doesnt have so many red pigments stored in the vacuole. Other than that, there might be unseen scratch on the smooth sides of the cuvettes. As we know, the colorimeter is a very light-sensitive device. The scratches that cannot be avoided will make the result differ from what it should be as the light passing through it will be diffracted causing a wrong absorbency. Sources of Error and Ways to Overcome Errors do happen in every experiment. The errors that always happen are when cutting the sections of beetroot. The size might be slightly varied when you measure the 1cm length. This will affect the surface area exposed to the surrounding and the result might be different than it should be. Besides, the part of beetroot took also must be from the same part. When the sections are taken for example, from the part that is near to the skin of the beetroot, the flesh is not juicy. So, to make it standardize and fixed, the sections should be taken from the middle part which is the thickest and juiciest as the volume of betalain (red pigments) might differ at different parts of the beetroot. Other than that, when the beetroot is cut, there are dyes that leak out. The dye should have been washed away by soaking the section of beetroot overnight. By just washing it and simply rinse it with distilled water, the dye might still be on the surface of the beetroot. This will affect

the result because the red pigments that enter the solution may come from the cuts not entirely because of the broken down cell membrane. Last but not least, the water bath heated is not consistently at the temperature required. Means that the water baths are fluctuated. This will result in the pigments getting more or less kinetic energy that what they are supposed to get. Hence, the result is affected.

Further Study There are other ways to investigate the properties of cell membrane or what will affect the permeability of cell membranes. For example, the pH of the solute. The level of pH affects the pigment of the beetroot, the dye in the beetroot (betalain) diffuses out of the cell when the membrane proteins are damaged due to high acidic level of pH. The pH level are responsible for regulating the function of the protein in the cell membrane. The protein will be denatured at high alkalinity and acidity, and breaks down the lipid bilayer. For example, if we use alcohol to investigate the effect of pH on the cell membrane, more alcohol used, will result in more leakage of red pigments of beetroot into the surrounding. This is because, alcohol is a fat-solvent. Thus, it will dissolve the cell membrane is it is made up of lipids.

Safety Precautions A lab coat must be wear during the experiment as the beetroot juice is a good dye which can stain your skin and clothes badly. Besides, you must use all the apparatus carefully especially the cork borer and the knife to avoid any harm or injuries. While handling the test tubes with water bath, make sure you are in a suitable and safe distance with the water bath to avoid scalding or burns. When you are taking measurements such as the volume of water or the length of the beetroot, make sure you read the reading carefully. Your eyes must be at parallel with the instrument to avoid any parallax error and affect the experiment. The cuvettes also must be handled with care. Make sure you wipe the smooth sides of the cuvette and no fingerprints are left behind as it may disturb the reading of colorimeter. The reading of the colorimeter must be repeated three times to get a reliable result. Last but not least, you must wash the sections of beetroot thoroughly to make sure there are no red pigments left behind so that the result will be much more reliable.

Conclusion When the cell membrane is exposed to high temperature, the membrane of the cell membrane will be broken down as the proteins and lipid bilayer is denatured and resulting in becoming permeable to substances. The hypothesis is accepted.

References http://en.wikipedia.org/wiki/Cell_membrane http://www.wisegeek.com Edexcel AS Biology, Students book, Pearson company, 2008 Edexcel Biology for AS, CJ Clegg, Hodder Education, 2008 www.biology-online.org www.wiki-answer.com http://www.scienceteacherprogram.org http://www.cliffsnotes.com