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Ultraviolet-Visible (UV-Vis) Spectroscopy
Standard Operating Procedure
INSTRUMENT:
Hewlett Packard Agilent 8453 UV-Vis Spectrometer
INSTRUMENT START-UP:
A. Turn on the instrument
1. The switch located left of the stage on the bottom left corner
2. Let the instrument warm up for ½ an hour before running any samples
B. Log on to the computer
1. User Name: user
2. No Password
C. Open the software Start→ Programs → HP UV-Vis ChemStation →Instrument 1
Online
D. A password screen will appear
1. Click OK – there is no password
E. An Instrument Time dialogue box may appear
1. Click Yes
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--------------------------------------------------------------------------------------------------------------------PREPARING A SAMPLE:
A. Use a quartz cuvette that is frosted on two sides – DO NOT USE PLASTIC
B. Rinse the cuvette 3-5 times with the solvent you will be using
C. Fill the cuvette ¾ full with sample or blank
1. With volatile samples that will evaporate quickly, make sure the cuvette is
capped
D. Wipe the outside of the cuvette with a Kim-Wipe to remove any fingerprints
--------------------------------------------------------------------------------------------------------------------CREATING A METHOD:
A. Make sure the instrument is in standard mode
1. Indicated in the main tool bar on the lower left side of the screen
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B. Select desired analysis mode from the method menu
1. Fixed Wavelengths – analyzes a sample at a maximum of 6 different
wavelengths
2. Spectrum/Peaks – analyzes absorbance minima and maxima
a.
Enter desired number of peaks and valleys
b.
Select data type:
 Transmittance
 Absorbance
 1st Derivative
 2nd Derivative
 3rd Derivative
 4th Derivative
c.
Enter range for the spectrum to be displayed
 Maximum range 190-1100 nm
3. Ratio/Equation – analyzes sample using a self defined equation based on
previous sample data and information
a.
Analyzes up to 6 different wavelengths
4. Quantification – single component analysis based on 4 different calibration
curves and a set of standards
a. Used for Instrumental Chemistry
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C. Solvent Ranges
Limit (nm)
180-195
200-210
210-220
245-260
265-275
280-290
Above 300
Solvent
Sulfuric Acid 96%
Water
Acetonitrile
Cyclopentane
n-Hexane
Glycerol
2,2,4-Trimenthylpentane
Methanol
n-Butyl Alcohol
Isopropyl Alcohol
Cyclohexane
n-Ethyl Ether
Chloroform
Ethyl Acetate
Methyl Formate
Carbon Tetrachloride
Dimethyl Sulfoxide
Dimethyl Formamide
Acetic Acid
Benzene
Toluene
m-Xylene
Pyridine
Acetone
Carbon Disulfide
--------------------------------------------------------------------------------------------------------------------MEASURING A BLANK:
A. Fill the cuvette with the solvent used in the experiment
B. Pull the black handle forward
C. Place the sample in the cell holder
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D. Push the black handle down
E. Select Measure → Blank
--------------------------------------------------------------------------------------------------------------------RUNNING A QUANTIFICATION EXPERIMENT:
A. Measuring a Standard
1. Fill the cuvette with the standard solution
2. Wipe the outside of the cuvette with a Kim-Wipe
3. Place the cuvette in the cell holder
4. Select the Measure → Standard
B. Measuring a Sample
1. Flush the cell three times with the sample solution
2. Wipe the outside of the cuvette with a Kim-Wipe
3. Place the cuvette in the cell holder
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4. Select Measure → Sample
--------------------------------------------------------------------------------------------------------------------DATA ANALYSIS
A. Fixed Wavelength – The absorbance will be displayed at the specified
wavelengths
B. Spectrum/Peaks – The absorbance for the peaks and valleys will be displayed
C. Ratio/Equation – The results will be displayed in the results table
D. Quantification – Results will be displayed in a sample/result table
--------------------------------------------------------------------------------------------------------------------INSTRUMENT SHUT-DOWN:
A. Remove cuvettes from cell holder
B. Clean cuvettes with acetone
C. Turn off the instrument
D. Close the Instrument 1 online software program
E. Log off the computer
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WASTE CONSIDERATIONS FOR INSTRUMENTAL CHEMISTRY COURSE:
A. Standards and samples may be disposed in waste container A in the waste hood
B. Container is denoted by Blue Label tape
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