I. II. III. IV.

Tittle of experiment Date of experiment Objectives Basic Theory

: Determination glucose persentage in the blood : 24th October 2013

: To determine glucose persentage in the blood

Blood is a liquid that is red and slightly thickened, of all living things (except plants) tinggi.Darah level flow throughout our bodies, and direct contact with the cells in the body is the main kita.Fungsi carry oxygen needed by the cell -cells throughout the body. Human blood is red, the bright red when oxygenated to dark red when starved of oxygen. The red color of the blood caused by hemoglobin, the protein breathing (respiratory protein) containing iron in the form of heme, which is bonded with oxygen molecules. Components of blood chemistry is very complex due to a large amount of nutrients the blood carries metabolic waste products from ionanorganik. The liquid part of blood is called blood plasma yangterdiri of 90% and 10% H2O dissolved components. One of the major organic component of non-protein in blood plasma are glucose, which has a normal range of 70-90 mg/100 mL.Glukosa, a monosaccharide sugar, is one of the most important carbohydrate used as a source of energy for animals and plants. Glucose is the result of photosynthesis in plants and some prokaryotes and early bagirespirasi.Glukosa also formed in the liver danotot order of glycogen breakdown (glucose polymers), and synthesized in the liver and kidney of intermediates through a process called gluconeogenesis natural form (D-glucose) is also called dextrose, especially in the food industry (Wikipedia, 2013). Glucose is a aldohexoses and is often called dextrose karenamempunyai properties can rotate polarized light to the right. In nature, the glucose found in fruits and honey bees (Poedjiadi 1994). When blood sugar levels more than or less than the normal limit then the system will be disturbed metabolism in the body. Normal human blood glucose remains in the amount or concentration of between 70-100 mg per 100 ml of blood. This can increase blood glucose after you eat carbohydrate foods, but about 2 hours after that, the amount of blood glucose will go back to the original condition. One of the main examples of diseases caused by disorders such as diabetes mellitus glucose levels....

absorbance Measure the absorbance of the solution If you read the part about how the absorption spectrometer works, you will know that the range of wavelengths of light will pass through a solution (sample cell) and another identical container that contains only the solvent (cell comparator / references). For each wavelength of light passing through the spectrometer, the intensity of the light passing through the cell count comparison. Commonly referred to as Io - the I is the intensity. The intensity of the light passing through the sample cell is also calculated for the same wavelength - symbolized I. If I is less than Io, meaning the sample absorbs some of the light. Further simple calculations performed by the computer to turn it into what is called the absorbance - the I is the intensity. The intensity of the light passing through the sample cell is also calculated for the same wavelength - symbolized by A. Generally obtained absorbance range from 0 to 1, but may also be higher than that. Absorbance at a wavelength of 0 means there is no light at certain wavelengths are absorbed. Comparison sample beam intensity and the same, so the ratio Io / I is 1. Log10 of one is zero. Absorbance of 1 occurs when 90% of the light at a wavelength absorbed - 10% are either not absorbed. In this case, Io / I is 100/I0 (= 10) and log10 of 10 is 1. Absorbance is not suitable for making comparisons The importance of concentration Part of the light is absorbed will depend on how many molecules are interacting with light. Imagine you have a strong organic dye / sharp. If the dye in the form of a concentrated solution, it will obtain the absorbance is very high because there are many molecules that interact dengam rays. However, in very dilute solutions, it is very difficult to see the color. Absorbance is very low. Suppose you want to compare the dye with other compounds, but you do not know the concentration, then you will not be able to make a good comparison with the compound which absorbs more light. thats why the wavelength that use is 660 nm hukum Beer-Lambert. This solution is measured absorbance maximum wavelength is 660 nm. By using the Lambert-Beer law in the blood glucose concentration dap; at specified. Lambert-Beer law states: A=kCl

where: A = absorbance (light absorption) k = molar extinction coefficient of the solution l = thick cuvette C = concentration of sample Based on the Beer-Lambert law, light absorption is proportional to the concentration. Concentration of glucose in the blood is a very important factor for the smooth working of the body. Normal levels of glucose in the blood is 70-90 mg/100 mL. Circumstances in which glucose levels are under 70-90 mg/100 mL called hipeglisemia while above 90mg / 100 mL is called hyperglycemia. V. TOOLS AND MATERIALS a TOOLS 1. Spektronik-20 2. Hot water 3. Sentrifus 4. Pippete 5. glass 6. Test tube MATERIALS : 1. Ba(OH)2 0,3 N 2. standard solution 0,005 mg/ml 3. ZnSO4 5% solution 4. Cu Alkalis Reactant 5. arsenomolibdat reactant :

VI.

PROCEDURE 1. Deproteinase blood filtrate

0,1 mL oxalated blood Entered in centrifuge tubes containing distilled water 1.90 - + 1.5 mL Ba (OH) 2 0.3 N stirring until blended - + 1.5 mL ZnSO4.7H2O 5%, mixed and allowed to stand 5 minutes - Centrifuged for 10 minutes Decanted Filtrate - Taken last few tested Biuret

Result

2. Determination of Blood Glucose Level

1 mL blood filtrated

- Put in a test tube - Cu + 1.0 mL reagent alkalis - Entered into boiling water for 20 minutes - Put in cold water - + 1.0 mL molybdate reagent arseno - Stir until evenly -

Absorbantion

3. Standard curves making

1,0 mL larutan glukosa dengan konsentrasi 0,01 mg/L; 0,02 mg/L; 0,03 mg/L; 0,04 mg/L; 0,05 mg/L

- Inserted in each test tube - 1.0 mL alkaline reagent Cu - Entered immersion in boiling water 20 minutes - Put in cold water - 1.0 arseno molybdate reagent - Stir until evenly - Tested with 20 spektronik tool with alpha = 660 nm

Absorbantion

4. Determination of the solution absorbance Blanko

1 mL aquades

- Put in a test tube - Entered immersion in boiling water 20 minutes - Put in cold water - 1.0 mL molybdate reagent arseno - Stir until evenly - Tested with 20 spektronik tool at alpha = 660 nm

Absorbantion

V. OBSERVATION RESULT

PROCEDURE 1. Deproteinase Filtrat Darah

0,1 mL oxalated blood
Entered in centrifuge tubes containing distilled water 1.90 - + 1.5 mL Ba (OH) 2 0.3 N stirring until blended Filtrate - + 1.5 mL ZnSO4.7H2O 5%, mixed and allowed to stand 5 minutes - Taken last few tested - Centrifuged for 10 minutes Biuret

OBSERVATION - Blood oxalated: clear red - Distilled: colorless - Ba (OH) 2: colorless - ZnSO4: colorless mixing results - Ba (OH) 2: dark green solution and a green precipitate - ZnSO4: dark green solution and a white blob

REACTION Normal glucose levels in the blood: 70-90 mg / L

CONCLUSION

Based

on

the

ResultDecanted

regression equation - The filtrate: clear colorless - Residue: dark green - Biuret: clear dark blue - after added biuret: colorless on the graph is y = 1.004 x 0.1054.

value obtained in blood glucose

levels at = - 0.0139 mg / L, which

indicates that the glucose content in

2. Determination of Blood Glucose Levels

1 mL blood filtrated

the blood is very small levels The function of Cu

- Put in a test tube - Cu + 1.0 mL reagent alkalis

alkalis is as oxidator the - Blood filtrate: colorless - Cu alkalis: clear blue - Cu + alkalis: blue - Heated: there are deposits of red brick - + Arsenomolibdat: clear blue function is of as

- Entered into boiling water for 20 minutes - Put in cold water - + 1.0 mL molybdate reagent arseno - Stir until evenly

arsenomolibdat

khromatogen that make the solution color

become blue

- Absorbance: 0.104
- Tested with appliance Absorbantion nm

3. Standard curve making

1,0 mL larutan glukosa dengan konsentrasi 0,01 mg/L; 0,02 mg/L; 0,03 mg/L; 0,04 mg/L; 0,05 mg/L

- Glucose solution: colorless - Cu alkalis: light blue - Cu alkalis: blue

- Inserted in each test tube - 1.0 mL alkaline reagent Cu - Entered immersion in boiling water 20 minutes - Put in cold water - 1.0 arseno molybdate reagent - Stir until evenly

- Heated: there are deposits of red brick - Arsenomolibdat: clear blue - absorbance 0,1 mg / L: 0,193 0,2 mg / L: 0.341 0,3 mg / L: 0,387 0,4 mg / L: 0,493 0,5 mg / L: 0,619 With correlation coeficient R2 = 0,97970

Absorbantion - Tested with 20

spektronik tool with alpha = 660 nm

4.

Determination

of

the

solution - Aquades : colorless - Cu alkalis : colorless blue - + Cu alkalis : blue - + arsenomolibdat : colorless blue

absorbance Blanko
1 mL aquades

- Put in a test tube - Entered immersion in boiling water 20 minutes - Put in cold water - 1.0 mL molybdate reagent arseno - Stir until evenly

- Absorbantion: 0,032

Absorbantion - Tested with 20

spektronik tool at alpha = 660 nm

VI . Discussion

Determination of blood glucose levels is done by several stages as follows: 1. Deprotenation blood filtrate In this experiment aims to obtain samples of the protein-free blood filtrate. At trial, oxalated blood were included in the reaction tube was centrifuged and added to distilled water. Function is the addition of distilled water thins the blood so that the albumin in the blood will be dissolved by distilled water. Albumin is a protein that is soluble in water and can be coagulated by heat. Then added 1.5 mL Ba(OH)2 0.3 N in the form of a colorless solution of the dark green solution and a green precipitate. Function of the addition of Ba(OH)2 0.3 N is to precipitate albumin dissolved in water. Then the addition of 1.5 mL of 5% ZnSO4 solution in the form of the colorless solution became dark green solution and a white blob. The addition of ZnSO4 serves as a catalyst to accelerate the reaction by precipitation of albumin Ba(OH)2. The next solution was made centrifuged for 30 minutes in order to completely precipitates albumin, so that when the sediment is separated by decantation to separate completely. Results of decantation the filtrate which is a colorless solution that has shown that protein-free blood filtrate. While the residue obtained was a dark green precipitate. Furthermore filtrate tested with the addition of biuret in the form of a clear blue solution then the filtrate remained colorless. It is indicative that the filtrate is free of protein.

2. Determination of Blood Glucose Levels In this experiment aims to determine blood glucose levels in the sample use the proteinfree filtrate 1 mL of protein-free blood filtrate from the previous experiment and put into a test tube. Then added 1 mL of a solution of Cu alkalis and blue. This function is the addition of Cu alkalis as oxidator that will reducing glucose in the sample. Then the solution is put in boiling water for 20 minutes to increase the reaction rate by Cu alkalis, and there are deposits of red brick. This is because the blood sample was heated with a solution of Cu2+ under alkaline conditions. At alkaline conditions, on glucose enediol form became dominant and reduce cupric ions (Cu2+) and then the solution was cooled. Then added 1 mL reagent arsenomolibdat yielding a clear blue solution because a solution containing lactic acid and Cu2+ ions. This is in accordance with the principles Tauber test that will give positive results in a solution that is blue solution containing monosaccharides (glucose). Cupric ions (Cu+) will be reduced by the sugar into kupro

(Cu2+) and precipitate as Cu2O (kuprooksida). Arsenomolibdat reagent additions are aimed at making Cu2O can redissolve and The function of arsenomolibdat is as khromatogen that make the solution color become blue. The next solution absorbance was measured with the 20 spektronik wavelength used 660 nm, because at this wavelength a solution absorbs blue light is the maximum. Blood glucose testing at the lab using spectophotometric. Absorption spectrometer is an instrument for measuring the absorption / absorption of light energy (wavelength) given by an atom / molecule. Observations with spektronik-20 using the Beer Lambert law principles. Factors affecting the concentration of the solution and is the shape of the container. Part of the light is absorbed will depend on how many molecules are interacting with light. Blood glucose concentration can be determined using a standard curve. Numbers listed on spektronik-20 shows the absorbance level solution. Absorbance level was noted that the solution will then be calculated to determine the glucose levels in the blood. Absorbance obtained is equal to 0.104. Sensitivity is defined as the concentration of the element in aqueous solution expressed in ppm which gives absorbance of 0.104 is comparable to the absorption of 1% of radiation transmitted so as to prove the calculation to find the amount of concentration to the absorbance value. From the calculations, that the magnitude of the concentration is equal to the absorbance -0.001 0.104 ppm. Minus sign (-) indicates that for the absorbance of 0.104 was not detected by the tool

3. Determination of Standard Curve In this experiment aims to create a standard curve that would be obtained curve has the regression equation. From the regression equation it can be used as a determination of blood glucose levels. In this experiment the first step is to create a standard solution. Standard solution is made menghsilkan dilution standard solution of glucose 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL 0.4 mg / mL and 0.5 mg / mL dilution formula used is as the following:M1 x V1 = M2 x V2 Furthermore, each standard solution that has been made pipetted 1 mL and placed in each test tube. Then added 1 mL Cu alkalis in each tube and the resulting clear blue solution. The addition of cupric ions Cu resulted Alkalis (Cu +) will be reduced by glucose to kupro (Cu
2+

) and precipitate as Cu2O (kuprooksida). Then all the test tube

was heated in boiling water for 20 minutes. Warming aims to increase the rate of reaction by alkaline Cu and then cooled. Then added 1 mL reagent arsenomolibdat

yielding a clear bluish solution because a solution containing lactic acid and Cu 2 + ions. This is in accordance with the principles Tauber test that will give positive results in a solution that is blue solution containing monosaccharides (glucose). Cupric ions (Cu +) will be reduced by the sugar into kupro (Cu2+) and precipitate as Cu2O (kuprooksida). Arsenomolibdat reagent additions are aimed at making Cu2O can redissolve and solution color change to blue is due to the oxidation of molybdate. the function of arsenomolibdat is as khromatogen that make the solution color become greenish blue The next solution absorbance was measured with the 20 spektronik wavelenght 660 nm long. The absorbantion obtained are as follows: Concentration (mg/mL) Absorbantion 0,1 mg / L 0,2 mg / L 0,3 mg / L 0,4 mg / L 0,5 mg / L 0,193 0.341 0,387 0,493 0,619

Based on the absorbance obtained for each standard solution of the calibration curve as follows:

curve of absorbantion
0.7 0.6 Absorbantion 0.5 0.4 0.3 0.2 0.1 0 0 0.2 0.4 0.6 Concentration absorbantion Linear (absorbantion) y = 1.004x + 0.1054 R = 0.9797

Based on the absorbance obtained for each standard solution of the calibration curve as follows: y = 1,004x + 0,1054

R2 = 0.97970

from the regression equation we can find the glucose percentage: y = 1,004x + 0,1054 and absorbantion sample = 0,104 as y So :

X = 0,0139 mg/mL (glucose persentage) Minus sign (-) indicates that for the absorbance of 0.104 was not detected by the tool. Normal levels of glucose in the blood is 70-90 mg/100 mL, whereas glucose levels in the blood that we use is 0,0139 mg/mL (In 100 mL of blood). This indicates that the owner of the blood of suffering hypoglycemia, or glucose levels below 90 mg / 100ml. This is because the owner did not eat the blood of more than two hours before practice. Drop in blood glucose levels because the body does not occur in the glucose metabolism process 4. Blanko solution In this experiment aims to determine the actual value of the solution absorbance. Blank solution blinded manner dengtan 1 mL of distilled water was then added Cu alkalis in each tube and the resulting clear blue solution. The addition of cupric ions Cu resulted Alkalis (Cu+) will be reduced by glucose to kupro (Cu2+) and precipitate as Cu2O (kuprooksida). Then all the test tube was heated in boiling water for 20 minutes. Warming aims to increase the rate of reaction by alkaline Cu and then cooled. Then added 1 mL reagent arsenomolibdat yielding a clear bluish solution because a solution containing lactic acid and Cu
2+

ions. This is in accordance with the principles Tauber test that will
2 +

give positive results in a solution that is blue solution containing monosaccharides (glucose). Cupric ions (Cu +) will be reduced by the sugar into kupro (Cu ) and

precipitate as Cu2O (kuprooksida). Arsenomolibdat reagent additions are aimed at making Cu2O can redissolve and solution color change to blue is due to the oxidation of molybdate. Subsequently the solution was measured by absorbance at length gelombaang spektronik 20 660 nm and absorbance values obtained at 0.032.

VII. CONCLUSION From experiment that done, it can conclude value obtained in blood glucose levels at 0.0139 mg / L, which indicates that the glucose content in the blood is very small levels

ANSWER QUESTIONS 1. Determine the levels of glucose in the blood mg mL glukosa/100! 2. What is the function of the trial process is boiling over? 3. Explain the role of the hormone insulin in the regulation of glucose levels! ANSWER 1. from the regression equation : y = 1,004x + 0,1054 and absorbantion sample = 0,104 as y So :

X = -0,0139 mg/mL 2. Function of the boiling process to accelerate the rate of reaction in the presence of Cucatalyst.

3. Functioning of the hormone insulin to stimulate the conversion of glucose to glycogen for storage in the liver and stimulates glucose oxidation in the cells for the purpose of respiration. If so glucose levels too low, less than the normal amount, Langerhans alpha cells of the glands will secrete more of the hormone glucagon, glucose levels in the blood will rise, this process will continue so that blood glucose levels are at normal levels. DAFTAR PUSTAKA Dhiena. 2012. Analisis protein. http://id.scribd.com/doc/95629531/Analisis-KadarGlukosa (October 26th 2013) Hanum, Farida. 2012. Analisis protein. http://faridahanumgm47.blogspot. com/2012/03/analisis-glukosa.html (October 26th 2013) Lehninger. 1982. dasar dasar Biokimia. Surabaya : Erlangga Tim. 2013. PETUNJUK PRAKTIKUM BIOKIMIA I. Surabaya : Unesa Press

ATTACHMENT

precipitation of blood which free protein

sample (blood which free protein

standard solution

sentrifuse