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Estelle Osida

on 10 October 2012

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Transcript of BLOOD

Blood is the familiar red fluid in the body that contains white and red blood cells, platelets, proteins, and other elements. Inorganic and organic constituents of blood are determined through tests where blood and serum are added with several reagents to test for the presence of certain substances. Some procedures require heating and filtration. A green color is evident for the presence of cholesterol and cholesterol ethers following the Liebermman Burchard reaction. Blood is a body fluid made up of formed elements-- the erythrocytes (red blood cells), leukocytes (white blood cells), plasma proteins and blood platelets. These elements are suspended in the blood plasma. Besides the organic constituents of blood, inorganic substituents are given emphasis in this experiment. [1] [2]

The biological significances of organic substituents (carbohydrate, proteins and cholesterol) are noted in this experiment. Cholesterol, a product of animal metabolism associated with lipoproteins, is determined by extraction of alcohol ether mixture which would exhibit a green color which indicates its presence. Same test is done to determine presence cholesterol esters. A deviation from the normal value of glucose may result to hyperglycemia and hypoglycemia. These conditions denote that the glucose amount is high and that there is a decrease in the blood value of glucose respectively. Under inorganic constituents, chlorides act as buffers in the blood when oxygen and carbon dioxide substitute and iron aids in the synthesis of hemoglobin, myoglobin and cytochrome. [1]

Through the vertebrate vascular system, blood is circulated by the heart by carrying oxygen and nutrients to and waste materials away from all body tissues. The transfer of oxygen from inhaled air into the blood and the transfer of carbon dioxide from the blood into the exhaled air defines gas exchange which is an important process concerned with blood. [1] [3] EXPERIMENTAL

With an expert in blood extraction, the extracted blood from one experimenter was added on a small test tube and had it placed on the centrifuge machine. Then, the separated serum from the blood was divided into four portions on 4 test tubes. A. Separation of serum from whole blood B. Test for the presence of Carbohydrates On a test tube with the 1st portion of the serum, 5 drops of Benedict’s reagent were added and had it boiled in a water bath. When positive result was obtained, the outcome was noted by the experimenter. C. Test for the presence of Protein. 1. Test for serum albumin and globulin

An equal volume of saturated ammonium sulfate solution was added on the test tube with the 2nd portion of the serum. Then, drop by drop of 1% of sodium chloride solution were added on the solution and shook. The result was described by the experimenters. Lastly, crystals of ammonium sulfate were added to the solution until saturated. D. Chloride determination On the test tube with the 3rd portion of the serum, 2 drops of 10% HNO3 were added for acidification. Afterwards, 5 drops of 5% silver nitrate were added to the solution. E. Phosphate determination 2 drops of 10% nitric acid were added on the test tube with the 4th portion of the serum for acidification. Then, 2 mL of 5% amoonium molybdate solution was added. Finally, had it heated in a water bath and the formation of yellow precipitate was noted by the experimenter. F. Test for fibrin in the clotted blood The enclosed serum was pressed out in the filter paper by the means of glass rod. The fibrin was dried on the filter paper by pressing it thoroughly. Then, the remaining serum was removed after the fibrin was squeezed in a mortar and a little water was added and had it washed with fresh portion of water. After, the experimenter had it dried between the filter paper and the color was noted. After which, the fibrin was divided into 2 portions. On the test tube with the first portion, 2 mL of Millon’s reagent was added and placed it in a water bath for 10 minutes. Lastly, on the test tube with the 2nd portion, a mixture of 3 mL of Hopkin’s Cole reagent and drop by drop of 3 mL concentrated sulfuric acid was added. G. Test for the presence of Cholesterol On a dry test tube, 12 mL of ethyl alcohol-ether mixture and 0.2 mL of whole blood were added. Then, a cork was placed and shook vigorously for 1 minute and placed it in a horizontal position for 30 minutes with the sediments evenly distributed. After, decantation and evaporation was made in a steam bath. The residue was extracted twice with 2.5 mL of chloroform and placed on a test tube. Next, on the test tube with the extract, 2 mL of acetic anhydride, 0.1 mL of concentrated sulfuric acid were added and had it mixed by inverting the test tube several times. The test tube was placed in the dark for 15 minutes. On another test tube, 5 mL of cholesterol standard solution was measured and 2 mL of acetic anhydride, 0.1 mL of concentrated sulfuric acid were added. Lastly, had it mixed by inverting the test tube several times and placed in the dark for 15 minutes. The color intensity of the chloroform extract and standard cholesterol solution were compared by the experimenter. RESULTS AND DISCUSSION A.Separation of serum from a whole blood

Centrifugation of the whole blood separates the serum B. Test for the presence of Carbohydrates To the first portion of the serum, 5 drops of Benedict’s reagent was added. After boiling in a water bath, the solution turned yellow first, then it turned orange after a while then finally there was a formation of brick red precipitate. Benedict's reagent has blue copper (II) ions (Cu2+) which are reduced to copper (I) ions (Cu+) that gave the red precipitate. This red precipitate is a positive result which indicates the presence of reducing sugars in blood. C. Test for the presence of Protein An equal volume of saturated ammonium sulfate solution was added to the second portion of the serum. The solution becomes thicker. Then 1% of sodium chloride solution was also added drop by drop to the thick solution. After shaking it, there was a formation of clear layer on top of the solution. Then ammonium sulfate crystals were added also to the solution. There was no precipitate formed however, there should be a precipitate. This may be due to human errors. Ammonium sulfate purifies proteins and it can “salt out” proteins. And NaCl help precipitates proteins D. Chloride determination The third portion of the serum was acidify with 2 drops of 10% HNO3. And 5 drops of 5% silver nitrate was added to the solution. The solution turned creamy white. The chemical equation involved in the reaction is
Ag +(aq) + Cl-(aq) AgCl(s) white E. Phosphate Determination In the fourth portion of the serum, 2 drops of 10% nitric acid and 2ml of 5% ammonium molybdate solution were added then the solution was heated in water bath then a yellow precipitate was formed. There was a formation of a web-like yellow precipitate in the solution. The chemical composition of the yellow precipitate is names as ammonium phosphomolybdate (NH4)3PMo12O40, which is known to be an inorganic salt of phosphomolybdic acid and contains the remarkable phosphomolybdate ion complex. F.Test for Fibrin in the clotted blood The color and texture of fibrin after it was pressed, squeezed and dried was dark red and has a smooth texture. The test using the Millon’s reagent was not performed due to the inavailability of the reagent. The portion of the fibrin with the added 3ml of Hopkin’s Cole Reagent and a drop by drop of 3ml concentrated sulphuric acid yielded a positive result which was the formation of a small irregular black solid at the top of the solution. G.Test for the presence of Cholesterol Result:

a.Chloroform Extract

Formation of 3 layers:

•Light green on top
•Clear green at the middle
•Red at the bottom b.Standard Cholesterol

Formation of 6 layers:
(from top to bottom)
•Dark green
•Light orange
•Dark violet
•Dark red H.Iron Determination After 2 ml of blood was heated until it turned to ashes, cooled, diluted in HCl, filtered and added with ammonium tiocyanate, a division of layers were seen, the orange yellow color of the solution turned slightly white on top, rusty in the middle and orange yellow at the bottom. The purpose of adding the ammonium thiocyanate is to indicate the presence of iron which is found in blood. I.Blood Gases After the tube of the oxyhemoglobin solution with the added few drops of the Stoke’s reagent was shaken vigorously it yielded a solution that is brown-black with web-like preceipitate. The reaction that took place is caused of the failure of gases to go out while shaking that causes for it to have a change in color. CONCLUSION REFERENCES [1] Biochemistry Laboratory Workbook
[2] http://www.answers.com/topic/blood
[3]http://www.medterms.com/script/main/art.asp?articlekey=10673 G R O U P 6 Blood The study was therefore concluded that blood contains organic and inorganic constituents. Presence of Lipids- specifically cholesterol, carbohydrates and proteins which is used for ATP production, growth and maintenance of cells was detected. Ion is also found in the blood which contributes to the osmotic pressure of body fluids. The phosphate ions affect the level of Calcium in the blood and Chloride ions are known as the principal anion and interstitial fluid. Blood gases oxygen and carbon dioxide are found in the blood. Blood carries oxygen from the lungs to the tissues and carries carbon dioxide from the tissues to the lungs. Conversely, a rise in the partial pressure of CO2 or a lower pH will cause offloading of oxygen from hemoglobin. For future studies, the researchers would recommend reduce in the sources of errors; said errors encountered during the experiment would be insufficient blood extracted from the individual. Blood extraction should be done by a specialist like a Medical technologist to meet the required aseptic preparation and process. The time allotted for the experiment should be fulfilled immediately to avoid blood clot. It is also recommended to use another subject to be able to point-out if there is a difference in the composition of blood of the other individual. Proper disposal of the used syringe and proper handling of the blood should be observed while doing this experiment. Also, during this experiment, Millon’s reagent was not provided thus, a part of the experiment was not done. ABSTRACT INTRODUCTION
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