S2. INTRODUCTION TO ULTRA-VIOLET / VISIBLE SPECTROSCOPY
PURPOSE
1. To become familiar with the operation of a conventional scanning ultra-violet
spectrophotometer
2. To determine suitable cells and solvents for ultraviolet spectroscopy
3. To examine the relationship between transmission and absorption of light in colours.
4. To determine the relationship between absorption of light and concentration of absorbing
species
PREWORK
1. List the chemicals you will be working with in this practical, (include formulae)
2. Note any safety requirements as indicated by the MSDS
3. Note the disposal procedures for the organic chemicals
INTRODUCTION
The fundamental law of spectrophotometry is known as the Beer-Lambert Law or Beer’s
Law. It may be stated as:
log(Po/P) = A
and
A=axbxc
where A =
a =
b =
c =
absorbance
a constant characteristic of the substance measured
the cell pathlength
the concentration of the solution
Thus for a solution of the same substance in similar cells, Beer’s Law can be simplified to:
Absorbance = constant x concentration
If for a particular system Beer’s Law is obeyed, a graph of absorbance versus concentration
will be linear. If this graph is NOT linear, the relationship does not hold. As a guide for
analytical work absorbance readings should be between 0.1 and 1.0.
I. Cell Materials
Cells for UV-vis spectroscopy are commonly made of plastic, glass or quartz. This section is
designed to enable you to be able to identify the cell material from its scan in a particular
region. A cell is useable in a particular region of the electro-magnetic spectrum if the
absorbance of the cell is below 0.1 absorbance units. Not all cells are appropriate over the
entire UV-visible spectrum.
For Spectra 1-3, the following should be used:
Reference (baseline or blank):
none (air)
Wavelength range:
200-400nm
Samples:
1. Plastic Cell
2. Glass Cell
3. Quartz Cell
II. Solvents
For a solvent to be useful it should have minimum absorbance in the region being scanned.
Colourless solvents can be used in the Visible region. The following is designed to determine
suitable solvents for use in the UV region.
For spectra 4-9, the following should be used:
Reference (baseline or blank):
quartz cell
Sample:
in quartz cell
Wavelength range:
200-400nm
Samples
4. Propanone (acetone)
5. Water
6. Ethanol
7. Methyl benzene (toluene)
8. Trichloromethane (chloroform)
9. Hexane
III. Colourless Organic Compounds
For spectra 10 and 11, the following should be used:
Reference: (baseline or blank)
hexane in quartz cell
Sample:
in quartz cell
Wavelength:
200-400 nm
Samples
10. Napthalene
Dissolve ONE crystal in approx 20 mL hexane
11. Camphor
Dissolve about 100 mg in approx 20 mL hexane
IV. Coloured Compounds
For spectra 12-15, the following should be used:
Reference:
distilled water in plastic cell
Sample:
in plastic cell
Wavelength range:
400-800nm
12. Methyl Red (Acidic)
Add 10 drops of methyl red to approx. 10 mL of 0.1 M HCl
13. Methyl Red (Alkaline)
Add 20 drops of methyl red in approx. 10 mL of 0.1 M NaOH
14. Nickel
Dissolve 1.5 g of nickel nitrate hexahydrate in approx. 20 mL distilled water
15. Copper (II)
Dissolve 1 g of copper (II) sulfate pentahydrate in approx. 20 mL distilled water.
V. Relationship of absorption and concentration
a) From a stock 250 mg/L permanganate solution, prepare 50 mL of 12.5, 25, 50, 125 and
200 mg/L and 100 mL of 2.5 and 5 mg/L solutions
b) Record the visible absorption spectrum of the 50 mg/L solution using plastic cells and
water as the reference.
c) Determine the wavelength of maximum absorption
d) Record the absorbance of each of the solutions at this wavelength and a wavelength at the
base of the absorption peak.
CALCULATIONS
V. Relationship of absorption and concentration
1. Plot absorbance versus concentration for the permanganate solutions at both wavelengths.
Do not attempt to draw a line-of-best-fit. The point of the exercise is to see where linearity
is best obeyed, so a join-the dots graph is better.
2. Calculate the absorption coefficient (a in Beer’s Law) for each of the permanganate
solutions. Calculate the average and relative precision of the coefficients.
DISCUSSION
•
•
•
•
•
discuss the use of the three types of cells in the ultraviolet and visible regions
discuss the use of the various solvents in the ultraviolet region, and explain what structural
features give rise to the observed spectra.
use the results from TWO of the coloured solutions to explain how absorption and
transmission of different colours leads to the actual colour of a solution.
comment of the absorbance graph for the permanganate solutions in terms of Beer’s Law
discuss why absorbance is a better measure to plot than % transmittance for calibration
graphs.
QUESTIONS
1. Where do you think that chlorophyll, the green pigment in plants, would absorb in the
visible region?
2. What features in the spectra of naphthalene and camphor could be used to differentiate
between the two compounds?
3. What are the reasons why two quartz cells filled with the same solution would not give the
same absorbance value?
4. Why are the organic solvents used in ultraviolet spectroscopy special “spectrograde”
standard? How does spectrograde differ from AR grade?
5. Which of the following would be suitable as solvents for UV spectra?
* 1-butanol
* cyclohexane
* ethanenitrile (CH3C-N)
* 1,4-dimethylbenzene
* 1,1,1-trichloroethane
* 3-pentanone
S2 RESULTS SHEET
Identity of instruments
Appearance of Spectrum
I. Cell materials
1. Plastic
2. Glass
3. Quartz
II. Solvents
4. Propanone
5. Water
6. Ethanol
7. Methyl Benzene (toluene)
8. Trichloromethane (chloroform)
9. Hexane
Date of analysis
III. Colourless Organic Compounds
10. Naphthalene
11. Camphor
IV. Coloured Compounds
IV. Coloured Solutions Colour
12. Methyl red (Acidic)
13. Methyl red (Alkaline)
14. Nickel
15. Copper (II)
16. Permanganate
Solution
Absorption wavelength range (nm)
Maximum
Minimum
12. Methyl red (Acidic)
13. Methyl red (Alkaline)
14. Nickel
15. Copper
16. Permanganate
Solution
Colours absorbed (V B G Y O R)
Strongly
Weakly
12. Methyl red (Acidic)
13. Methyl red (Alkaline)
14. Nickel
15. Copper
16. Permanganate
V. Relationship between absorption and concentration
Wavelength of maximum absorption:
Wavelength near base of absorption peak:
Solution
mg/L
2.5
5
12.5
25
50
125
200
Absorbance
At maximum λ
At minimum λ
Have you?
Completed the instrument log
Completed the sample register
Completed the standard register
Teachers signature
Date
Signature