Lab 2 Spectrophotometric Measurement of Glucose - Employees ...

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CEBDCABA. On/Off and Zero KnobB. Wavelength DialC. Sample compartmentD. Light control knobE. Mode ButtonDAFig. 2-2. Diagram of Spec 20 and Spec 20D SpectrophotometersGlucose AssayNow we will use the spectrophotometer to measure the concentration of glucose (sugar) in Kool-Aid. When you measured the absorbance of Kool-Aid, you made a direct measurement. TheKool-Aid was a colored product that the spectrophotometer could easily measure. In the glucoseassay we will use, you will not measure the concentration of glucose directly. Instead, you willmeasure the absorbance of colored product created when glucose reacts with the Glucose AssayReagent. The amount of colored substance is directly proportional to the amount of glucosepresent. You will create a standard curve that translates the absorbance of the colored substanceinto concentration of glucose and will use it to determine the amount of glucose in Kool-Aid.1. First you will make several dilutions of a glucose standard solution that has an initialconcentration of 500 mg/dl of glucose. See Fig. 2-3.a. Label three large mixing tubes A, B, C.b. Into each of the large mixing tubes, pipette 5 ml of deionized water.c. To mixing tube A, add 5 ml of glucose standard solution. Cover with Parafilmand mix well.d. Into mixing tube B, pipette 5 ml of standard dilution A. Cover with Parafilm andmix well.M:\Macvol\Courses\Biol 114.F03\Lab\Lab2.spec\lab.2.writeup.03.doc - 4 -
e. Into mixing tube C, pipette 5 ml of standard dilution B. Cover with Parafilm andmix well.GlucoseStandardStandardDilution A StandardDilution BStandardDilution CFig. 2-3. Illustration of the dilutions made.2. Calculate the concentration of glucose in each tube using the following equation andrecord it in your assignment:V 1 C 1 = V 2 C 2Where V 1 = volume of the stock solutionC 1 = the concentration of the stock solutionV 2 = the final diluted volume (volume of stock solution and diluent)C 2 = the final diluted concentration.For example, we want to find the final diluted concentration (C 2 ) of Standard Dilution A.V 1 is the volume of the stock solution we are adding to Standard Dilution A. ThereforeV 1 is 5 ml or 0.5 dl. The concentration of the stock solution is 500 mg/dl; this is C 1 . V 2is the final diluted volume of Standard Dilution A. Since we are adding 5 ml of stocksolution to 5 ml of water, the final diluted volume of Standard Dilution A is 10 ml or 1 dl.Therefore, V 2 is 10 ml or 1 dl. Inserting these values into the formula, we see that:(0.5 dl)(500 mg/dl) = (1 dl) C 2C 2 = 250 mg/dlWhat is your stock solution for Standard Solution B? What about Standard Solution C?3. For the glucose assay, we will need seven more mixing tubes. One tube will be labeled“blank.” Three additional tubes will be used to test the different concentrations ofglucose standard. These tubes can be labeled Standard A, Standard B, and Standard C.Finally, three more tubes will be used to test a sample of Kool-Aid that your instructor orTA will give you. Your instructor or TA will give each lab group a different Kool-Aidsample to assay. Make sure to record in your assignment, which Kool-Aid sample youwere assigned. You will test your Kool-Aid sample three times in three different tubes.When the same sample is tested more than one time, the individual tests of that sampleare called replicates. By testing the glucose concentration more than one time andaveraging the results, we will get a more accurate estimate of its true glucoseconcentration. For these replicates of your Kool-Aid sample, you can label the remainingmixing tubes Kool-Aid 1, Kool-Aid 2, and Kool-Aid 3.M:\Macvol\Courses\Biol 114.F03\Lab\Lab2.spec\lab.2.writeup.03.doc - 5 -
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Lab 2 Spectrophotometric Measurement of Glucose - Employees ...

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