¶ 26 December 2020 Frank Lee
Goal: To learn to use and sanity-check the operation of Boslab’s Milton-Roy SPECTRONIC 21 DUV spectrophotometer.
Method: Serially dilute vitamin B12 solution and read the absorbance at 530 nm of each dilution, comparing to expectations.
Data: This table logs the steps I took and the measurements made. Each row represents a step. Time proceeds downward in the following table.
| Tube 1 | Tube 2 | Tube 3 | Tube 4 | Control | |
|---|---|---|---|---|---|
| Initial conditions | 6000uL B12 hydroxycobalamin solution | 3000uL H2O | 3000uL H2O | 3000uL H2O | 3000uL H2O |
| Read absorbance | .052 | Not read | .000 | ||
| Serial dilution step | Pipetted 2000uL from tube 1 to tube 2 | No action | |||
| Read absorbance | .048 | .018 | Not read | .000 | |
| Serial dilution step | No action | Pipetted 2000uL from tube 2 to tube 3 | No action | ||
| Read absorbance | .045 | .016 | .006 | Not read | -.000 |
| Serial dilution step | No action | Pipetted 2000uL from tube 3 to tube 4 | No action | ||
| Read absorbance | .045 | .016 | .007 | .001 | -.000 |
| Wait 5 minutes | No action | ||||
| Read absorbance | .047 | .017 | .007 | .001 | .000 |
¶ 28 December 2020 Frank Lee
Repeated experiment with changes:
- Used 330nm wavelength to test deuterium lamp. The previous measurements, 12/26/2020, were made at 530nm using the tungsten lamp.
- Used more concentrated vitamin B12 solution to check performance at higher absorbance values.
- I chose to use vitamin B12 solutions for this experiment because the solutions are both strongly colored and edible, thus making possible a strong absorbance signal while obviating the chemical/biohazard disposal protocols of most colorful transition-metal salt solutions.
- Solution was made by crushing seven Amazon Elements brand 5000 mcg methylcobalamin B12 supplement tablets in a mortar and pestle and dissolving in 150 mL of filtered and boiled tap water, then reboiled until the tablets appeared fully dissolved.
- When making the serial dilutions, I pipetted each dilution up and down five times to help make the solutions evenly well-mixed.
- Read both transmission and absorbance data.
Data: This table logs the steps I took and the measurements made. Each row represents a step. Time proceeds downward in the following table.
| Tube 1 | Tube 2 | Tube 3 | Tube 4 | Tube5 | Tube6 | Tube7 | Control (blank) | |
|---|---|---|---|---|---|---|---|---|
| Initial conditions | 5000uL B12 methylcobalamin solution | 3000uL H2O | 3000uL H2O | 3000uL H2O | 3000uL H2O | 3000uL H2O | 3000uL H2O | 3000uL H2O |
| Read Transmittance | 0.0% | 99.3% | 99.4% | 99.5% | 99.9% | 99.7% | 100.2% | 100.0% |
| Read absorbance | 1999 = over range | .003 | .002 | .002 | .000 | .001 | -.000 | -.000 |
| Serial dilution step | Pipetted 2000uL from tube 1 to tube 2 | No action | ||||||
| Read Transmittance | 0.0% | 0.7% | 99.6% | 99.6% | 99.8% | 99.7% | 100.0% | 99.9% |
| Read absorbance | 1999 = over range | 1999 = over range | .002 | .002 | .001 | .002 | .000 | .000 |
| Serial dilution step | No action | Pipetted 2000uL from tube 2 to tube 3 | No action | |||||
| Read Transmittance | 0.0% | 0.8% | 16.2% | 99.7% | 100.0% | 99.7% | 100.2% | 100.0% |
| Read absorbance | 1999 = over range | 1999 = over range | .788 | .001 | -.000 | .001 | -.001 | -.000 |
| Serial dilution step | No action | Pipetted 2000uL from tube 3 to tube 4 | No action | |||||
| Read Transmittance | 0.0% | 0.9% | 16.9% | 49.0% | 100.0% | 99.6% | 100.2% | 100.0% |
| Read absorbance | 1999 = over range | 1999 = over range | .772 | .310 | .000 | .001 | -.001 | -.000 |
| Serial dilution step | No action | Pipetted 2000uL from tube 4 to tube 5 | No action | |||||
| Read Transmittance | 0.0 | 1.0% | 17.7% | 50.0% | 75.9% | 99.8% | 100.2% | 100.0% |
| Read absorbance | 1999 = over range | 1999 = over range | .752 | .301 | .120 | .001 | -.001 | .000 |
| Serial dilution step | No action | Pipetted 2000uL from tube 5 to tube 6 | No action | |||||
| Read Transmittance | 0.0% | 1.1% | 18.0% | 50.3% | 75.9% | 89.6% | 100.3% | 100.0% |
| Read absorbance | 1999 = over range | 1.980 | .742 | .298 | .120 | .048 | -.001 | -.000 |
| Serial dilution step | No action | Pipetted 2000uL from tube 6 to tube 7 | No action | |||||
| Read Transmittance | 0.0% | 1.2% | 18.4% | 50.7% | 76.2% | 89.6% | 95.9% | 100.0% |
| Read absorbance | 1999 = over range | 1.960 | .736 | .296 | .118 | .048 | .018 | -.000 |
| Wait 5 minutes | No action | |||||||
| Read Transmittance | 0.0% | 1.2% | 18.5% | 50.7% | 76.1% | 89.5% | 95.9% | 100.0% |
| Read absorbance | 1999 = over range | 1.920 | .732 | .295 | .119 | .048 | .018 | .000 |
Notes:
- I used the 10mm Milton-Roy glass tubes because that's what we have in the lab.
- The operator's manual says the glass tubes are useable above 325 nm, so my choice of 330 nm should work.
- I had to change the Sensitivity to MED from LO to be able to get 100% transmission on the blank tube at 330 nm.
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