S2. INTRODUCTION TO ULTRA-VIOLET / VISIBLE SPECTROSCOPY AIM 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 SDS 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 = a x b x c where A = absorbance a = a constant characteristic of the substance measured b = the cell pathlength c = 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. 1
PROCEDURE: 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 2
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. 3
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. 4
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
Identity of instruments I. Cell materials 1. Plastic S2 RESULTS SHEET Appearance of Spectrum Date of analysis 2. Glass 3. Quartz II. Solvents 4. Propanone 5. Water 6. Ethanol 6
7. Methyl Benzene (toluene) 8. Trichloromethane (chloroform) 9. Hexane III. Colourless Organic Compounds 10. Naphthalene 11. Camphor 7
IV. Coloured Compounds IV. Coloured Solutions 12. Methyl red (Acidic) 13. Methyl red (Alkaline) 14. Nickel 15. Copper (II) 16. Permanganate Colour Solution 12. Methyl red (Acidic) 13. Methyl red (Alkaline) 14. Nickel 15. Copper 16. Permanganate Absorption wavelength range (nm) Maximum Minimum 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 8
V. Relationship between absorption and concentration Wavelength of maximum absorption: Wavelength near base of absorption peak: Solution Absorbance mg/l At maximum λ At minimum λ 2.5 5 12.5 25 50 125 200 Have you? Completed the instrument log Completed the sample register Completed the standard register Date Signature Teachers signature 9