Laboratory 4: Worksheet Learning Check Questions Learning Check Exercise 4.1 HomeworkUnanswered Explain the use of blank and standards in a spectrophotometric analysis.
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What is the purpose of the blank? To determine the protein concentration using a spectrophotometer we insert the blank as label control. 2. If you're trying to find the maximum absorbance for compound A which is dissolved in the following solution: 10% acetone and 2% ether, 88% alcohol, what would be used for the blank? Explain. 3. Which of the test tubes (listed in Table 1) is used as the blank in this procedure? What is its content? Test tube B 4. Formulate an idea as to why is it important to wipe your fingerprints from the cuvettes before you place it in the spectrophotometer and record the absorbance/transmittance. It is important to wipe fingerprints off of the cuvette before placing it in the spectrophotometer. Wipe the cuvette with a Kimwipe to remove any fingerprints on the outside of the cuvette. This will interfere with light transmission and will cause erroneous readings. This technique prevents scratching of the cuvette in those areas through which the light will pass. 5. Why did an indirect method have to be adopted to measure the protein concentration? 6. What is the reagent used to quantify the protein? 7. What is the color of light that is being used in this experiment? Explain why this wavelength of light(color of light)was used in this experiment?(Hint think about previous lab with plant pigments.)
Adi S.
Tube number Standard albumin (mg/ml) Absorbance @ 540 nm 1 0.00 0.00 2 0.20 0.033 3 0.50 0.095 4 0.70 0.180 5 1.0 0.183 U1 0.125 U2 0.172 2.1. When using the spectrophotometer, what is the purpose of the reaction in Test tube 1? 2.2. Explain in detail the principle the spectrophotometer. 2.3. Provide a clearly annotated standard curve of absorbance @540 nm versus standard albumin (mg/ml) concentrations. 2.4. Determine the concentration (mg/ml) of albumin in the unknown samples (U1 and U2). Clearly show how you have come to your answer.
After setting the spectrophotometer to 0 absorbance (100% transmittance) at 580 nm with a cuvette containing water (blank), measure the absorption of the samples in sample tubes #1-#5 at 580 nm. 1. Set the Spec-20 (spectrophotometer) to a wavelength of 580 nanometers. 2. Zero (Blank) the Spec-20 using water as the blank. 3. Read the absorbance of your solutions from the lowest concentration to the highest concentration. Pour the solution from tube #5 into a clean, dry cuvette. Wipe any fingerprints from the outside of the cuvette with a Kimwipe. Place the cuvette into the Spec-20, making sure the clear side of the cuvette faces you. 4. Dispose of the solution. 5. Rinse the cuvette with the next standard and dispose of the rinse. 6. Repeat steps 3-5 for the remaining four diluted standard solutions. Determine the absorbance of test tube #4, then #3, then #2, and lastly #1. 7. Obtain two solutions of BPB of unknown concentration from your instructor. Procedure Part B: Graphing the Calibration/Standard Curve Plot the absorbance (y-axis) versus concentration of the BPB (x-axis). Draw a best-fit line using your data points. Do this by hand and with a computer. Determine the slope of the lines for each graph. You now have a working relationship between the absorbance and the concentration of BPB. Data Table 2: Absorbance and Concentration of Unknown Solutions Unknown # Absorbance Concentration (ppm) 1 0.220 2 0.632 1. Calculate the ppm of each sample using: a) The slope of your hand-drawn graph b) The slope of your computer-drawn graph c) The Beer-Lambert Law using the molar absorptivity coefficient determined in part A 2. Does the calibration curve obey the Beer-Lambert Law? Why? 3. What is the percent error for the hand-drawn slope compared to the computer-generated slope (actual)? 4. If you rinsed each solution with water between each reading and neglected to dry it completely, how would that affect your results.
Dominador T.
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