Practical 1 Spectrophotometry

Name: STEPHENIE KOH KIM HOI FONG AI HSING LOH YING XIN TAN CHAN MING WONG MANN RU TEH CI ZHUANG MOHD NAZRIN BIN HANAFI FATIN AMANINA BT MOHAMAD NUR IZZATI BT MOHAMED ZULKIFLY

BMD 200112 BMD 200912 BMD 204112 BMD 204512 BMD 207912 BMD 208212 BMD 204312 BMD 204612 BMD 201312

DATE: 21 /9/ 2012 LECTURER: DR MYO WINT ZAW

Please answer the following questions. 1. Write down the results of this experiment in the table below Test Tube Blank 1 2 3 4 5 Sample X Questions: 1. What is blank solution? Why the spectrophotometer is set to read zero absorbance 9 100 % transmission) with the blank solution in place? The blank solution is a solution identical to the sample except that the blank solution does not contain solute that absorbs light. It is set to zero absorbance to correct any absorption of light by the solvents used to develop the color. It is used to estimate the concentration of the sample. OD (600 nm) 0.000 0.042 0.071 0.117 0.145 0.173 0.053 Concentration of CuSO4.5H2O (g / 100ml) Distilled water 1 2 3 4 5 1.467

2. What is the normal range for plasma protein (total) ? The normal range of plasma protein level is between 6.0 to 8.3 gm / dL

3. Name three (3) different substances in blood that can be measured using this technique. Substances such as blood glucose level, hemoglobin concentration and glycerol level in the blood can be measured using a spectrophotometer. Glucose is the primary source of energy for body cells. It is being absorbed in the small intestine and transported to body cells via bloodstream. Hemoglobin is found exclusively in the erythrocytes where it plays a vital role in the transport of oxygen, carbon dioxide, nitrogen oxide and hydrogen ions in the body. Besides, glycerol is one of the component that made up triglyceride in the body which function as heat insulator, electrical insulator, shock absorber and energy source of the body during insufficient carbohydrates in the body.

4. State the principle of spectrophotometry. Spectrophotometer consist of 2 instruments which is a spectrometer for producing light of any selected color (wavelength) , and a photometer for measuring the intensity of light. The principle of spectrophotometer is that absorbance is proportional to the length of light passing through the sample (l) and to the molar concentration of substance that absorbs light. There is a direct relationship between absorbance and concentration as shown:

5. Describe the Beer-Lamberts Law Many compounds absorb either ultraviolet light or visible light. In spectrophotometry, a beam of monochromatic light of incident light, I , directed at a sample solution. Absorption takes place and the beam of light leaving the sample as light intensity , I. The amount of light absorb may be measured in a number of ways: Transmittance, T = I / I % Transmittance, %T = 100 T If A=0, there will be no absorption, the light pass through the solution without any absorption and percent transmittance is 100 %. If all light is absorbed, then the percent transmittance is zero. The Beer-Lambert law is the linear relationship between absorbance together with concentration of the sample and the length of light path. The Beer-Lamberts law is usually written as:

Absorbance length of light path x concentration

6. What is standard solution ? A standard solution is a solution containing precisely known concentration of an element or a substance, a known weight of solute is dissolved to make a specific volume. It is prepared using a standard substance. The absorbance of the standard solution are measured and used to prepare a calibration curve, which is a graph showing how the experimental observable varies with the concentration.