1.
Prepare Mg standards: Dilute 100-ppm Mg standard stock solution (you made previously) to the following concentrations in the previously cleaned test tubes. Use volumetric pipets to transfer the liquids. Again this is your standard solutions whose concentration accuracy is critical to your results. Treat the pipet used to transfer the standard solution with all the proper cleaning/coating steps. Read the markings on the pipet carefully during transfers.
[Mg] made Volume of 100-ppm Mg Volume of d.i. water
(use 5-mL grd. Pipet) (use 10-mL grd. Pipet)
50 ppm 5.00 mL 5.00 mL
25 ppm 2.50 mL 7.50 mL
12.5 ppm 1.25 mL 8.75 mL
Mix the solution in each test tube well by capping and inverting the text tubes.
At this point, place another seven (7) clean test tubes in a test tube rack. Label them 1 to 7.
2. In test tube 1 pipet 0.5-mL d.i. water. In Test Tubes 2-4 pipet 0.50-mL of three Mg standards you just made, one solution to one test tube. In Test Tubes 5-7 pipet 0.5-mL of the bovine serum sample.
3. Add 2.5-mL of TCA to each test tube prepared in Step 2, cap and mix well, and let stand for 5 minutes.
4. Centrifuge all 7 test tubes for 5 minutes. Make sure that you place the test tubes in the centrifuge in a symmetrical pattern so that the spinning will not be off balance. The test tubes will break if off-balance spinning occurs.
5. Use a pipet to transfer 2.00-mL of each solution or the supernatant solution from Step 4 into another set of 7 clean test tubes. To each transferred solution, add 2.00-mL of the TY working solution you made today and 1.00-mL (with a pipet) of 2.5M NaOH solution. Cap, mix well, and let stand for 5 minutes.
6. Bring these seven solutions to a Spectronics 20D spectrophotometer. Set the monochromator to 540nm. Zero the Transmittance with nothing in the cuvet chamber and then zero the Absorbance with water in the cuvet chamber. Measure the absorbance of each of the seven solutions, standards first from the least concentrated to the most concentrated, and then the serum samples.
Based on the procedures delineated in Steps (1)-(6), draw a "flow chart" to follow the path of your analyte Mg. Establish proportional relationships between (1) ppm Mg in serum calculated from the calibration curve with that in the original serum sample; (2) ppm Mg from the calibration curve with that in the curvet. (E.g., if you calculated 10-ppm Mg off the graph, what is it in the serum sample? In the cuvet?)
1.
Prepare Mg standards: Dilute 100-ppm Mg standard stock solution (you made previously) to the following concentrations in the previously cleaned test tubes. Use volumetric pipets to transfer the liquids. Again this is your standard solutions whose concentration accuracy is critical to your results. Treat the pipet used to transfer the standard solution with all the proper cleaning/coating steps. Read the markings on the pipet carefully during transfers.
[Mg] made Volume of 100-ppm Mg Volume of d.i. water
(use 5-mL grd. Pipet) (use 10-mL grd. Pipet)
50 ppm 5.00 mL 5.00 mL
25 ppm 2.50 mL 7.50 mL
12.5 ppm 1.25 mL 8.75 mL
Mix the solution in each test tube well by capping and inverting the text tubes.
At this point, place another seven (7) clean test tubes in a test tube rack. Label them 1 to 7.
2. In test tube 1 pipet 0.5-mL d.i. water. In Test Tubes 2-4 pipet 0.50-mL of three Mg standards you just made, one solution to one test tube. In Test Tubes 5-7 pipet 0.5-mL of the bovine serum sample.
3. Add 2.5-mL of TCA to each test tube prepared in Step 2, cap and mix well, and let stand for 5 minutes.
4. Centrifuge all 7 test tubes for 5 minutes. Make sure that you place the test tubes in the centrifuge in a symmetrical pattern so that the spinning will not be off balance. The test tubes will break if off-balance spinning occurs.
5. Use a pipet to transfer 2.00-mL of each solution or the supernatant solution from Step 4 into another set of 7 clean test tubes. To each transferred solution, add 2.00-mL of the TY working solution you made today and 1.00-mL (with a pipet) of 2.5M NaOH solution. Cap, mix well, and let stand for 5 minutes.
6. Bring these seven solutions to a Spectronics 20D spectrophotometer. Set the monochromator to 540nm. Zero the Transmittance with nothing in the cuvet chamber and then zero the Absorbance with water in the cuvet chamber. Measure the absorbance of each of the seven solutions, standards first from the least concentrated to the most concentrated, and then the serum samples.
Based on the procedures delineated in Steps (1)-(6), draw a "flow chart" to follow the path of your analyte Mg. Establish proportional relationships between (1) ppm Mg in serum calculated from the calibration curve with that in the original serum sample; (2) ppm Mg from the calibration curve with that in the curvet. (E.g., if you calculated 10-ppm Mg off the graph, what is it in the serum sample? In the cuvet?)
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