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:

1. Weigh 500mg dye on electric balance
2. Dissolving all the solid dye in beaker
3. Transfer all the solution into a 500ml volumetric flask
4. Fill up the volumetric flask to the tick mark
5. 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

1. Vortex vial to complete mix
2. Transfer Standard solutions to cuvette

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1. Measure the transmissivity at a peak measure three times and make a note of your results in your lab notebook
2. 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

1. Pour about 11 ml unknown concentration of methylene blue solution from the bottle to your vial
2. Dilute the dye solution 10 fold by using pipettes : Pipette 1 ml above sample solution to your vial
3. Fill the solution to 10 ml
4. Transfer Standard solutions to cuvette
5. 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

1. Power on instrument (switch is on left side).
2. Turn on monitor and double-click ‘Cary win UV’
3. Instrument will run through a start-up check for about 1 minutes.
4. In the toolbar frame, select the ‘Concentration’ icon.
5. In dialog box, enter wavelength 369 nm.

Serial Dilution	Absorbance			Wavelength
	1	1/10	1/100
Sample 1
Sample 2
Sample 3

a.

b.

1. Clicka. b.
2. Click
3. Click
4. 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.

1. Fill cuvette 3⁄4 with standard samples and cleaning cuvette surface with chem wipes.
2. Place cuvette in the slot.
3. Click ‘Start’
4. Record Abs(absorbance)
5. Drawing standard graph
6. Fill cuvette 3⁄4 with unknown samples and cleaning cuvette surface with chem wipes.
7. Place cuvette in the slot.
8. Click ‘Start’
9. 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