enviromontal engineering lab
5. UV-Vis Spectroscopy5.1 Introduction
Objective
Review basic analytical chemistry skills, including solution making, transferring and linear diluting. Understand the error involved and error propagation.
Materials:
Methylene Blue C16H18ClN3S, MW 319.85.Also called Swiss blue. One gram dissolves in about 1000 ml of water. Peak absorption at 369 nm
5.2 Create Calibration Curve from standard solutions
Procedures:
- Weigh 500mg dye on electric balance
- Dissolving all the solid dye in beaker
- Transfer all the solution into a 500ml volumetric flask
- Fill up the volumetric flask to the tick mark
- Prepare standard solutions of 500, 200, 100, 50 and 20 ppm from 1000 ppm stock
solution
Table 5-1 Preparation of standard solutions
PPM |
STD |
||
Stock |
DI water |
Total Volume |
|
20 |
0.2 |
9.8 |
10ml |
50 |
0.5 |
9.5 |
10ml |
100 |
1 |
9 |
10ml |
200 |
2 |
8 |
10ml |
500 |
5 |
5 |
10ml |
- Vortex vial to complete mix
- Transfer Standard solutions to cuvette
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- Measure the transmissivity at a peak measure three times and make a note of your results in your lab notebook
- Create calibration curves from data.
Table 5-2 Absorbance of standard solutions
PPM |
Wavelength |
Absorbance |
||
1 |
2 |
Average |
||
20 |
||||
50 |
||||
100 |
||||
200 |
||||
500 |
1 0.8 0.6 0.4 0.2 0
UV/VIS
0 50 100 150 200 250 300 350 400 450 500
Concentration (ppm)
Figure 5-1 Calibration curve from standard solutions
5.3 Two steps of a serial dilution and measure the concentrations of unknown samples
- Pour about 11 ml unknown concentration of methylene blue solution from the bottle to your vial
- Dilute the dye solution 10 fold by using pipettes : Pipette 1 ml above sample solution to your vial
- Fill the solution to 10 ml
- Transfer Standard solutions to cuvette
- Measure absorbance of each
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Absorbance
Figure 5-2 Serial Dilution of unknown samples Table 5-3 Absorbance of unknown samples
Instrument Use
- Power on instrument (switch is on left side).
- Turn on monitor and double-click ‘Cary win UV’
- Instrument will run through a start-up check for about 1 minutes.
- In the toolbar frame, select the ‘Concentration’ icon.
- In dialog box, enter wavelength 369 nm.
Serial Dilution |
Absorbance |
Wavelength |
||
1 |
1/10 |
1/100 |
||
Sample 1 |
||||
Sample 2 |
||||
Sample 3 |
a.
b.
- Clicka. b.
- Click
- Click
- Click
Choose Abs (absorbance).
Replicate is 2on ‘Standard’.
unit : mg/L
number of standard and concentration of standard solutionon ‘Sample’ and enter sample number
‘OK’
on Zero icon after insert Blank cuvette into UV slot.
- Fill cuvette 3⁄4 with standard samples and cleaning cuvette surface with chem wipes.
- Place cuvette in the slot.
- Click ‘Start’
- Record Abs(absorbance)
- Drawing standard graph
- Fill cuvette 3⁄4 with unknown samples and cleaning cuvette surface with chem wipes.
- Place cuvette in the slot.
- Click ‘Start’
- Record concentrations
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Lab report requirement
o Calculate solution concentration by ppm for all solutions
o Discuss source of data errors
o How do you improve your skill to get better accuracy and precision of solution makingo Describe the key steps of the experiment
o Attach all the original data and spectrum
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