Chapter Eleven Ultraviolet-Visible Spectrophotometry Spectrophotometry is one of the most widely used methods of analysis in clinical chemistry, hygiene analysis, medicine analysis. It is used to measure the absorption capabilities of certain compounds with reference to wavelengths of light on the ultraviolet and visible light ranges. 2008-11-19

High sensitive, can used microanalysis 10-3～10-6mol/L e.g. There is 10-8g Fe in 10ml solution, if titrate it using KMnO4 standard solution (c=2.0×10-4 mol/L), only consumed 0.02ml KMnO4 standard solution. 2008-11-19

11-3 The Spectrophotometer 11-4 Typical Analysis Procedures 11-1 Properties of Light 11-2 Absorption of Light * 11-3 The Spectrophotometer 11-4 Typical Analysis Procedures 2008-11-19

11-1 Properties of Light *Wavenumber Light is electromagnetic wave The dual nature of particles and waves *Wavelength λ unit: nm *Frequencyν (Hz) unit of frequency is s-1 Hertz: One oscillations per second is called 1Hz 106 s-1 is therefore said to be 106 Hz, or one megahertz (MHz). *Wavenumber unit: nm-1, m-1, cm-1 2008-11-19

c is the speed of 1ight in vacuum. c=2.99792458 ×108 m s-1 The relationship between wavelength and frequency c is the speed of 1ight in vacuum. c=2.99792458 ×108 m s-1 2008-11-19

The light with certain wavelength or frequency have definite energy Photons E=hv = Planck's constant h=6.626176×10-34J·s One mole of photons is called one einstein. 2008-11-19

Complement Color: The observed color is said to be the complement of the absorbed color. Any substance that absorbs visible light will appear colored red purple orange white light yellow blue greenish blue green Bluish green 2008-11-19

Wavelength Color Color Table 11-1 Colors of visible light and complementary hue Wavelength Color Color Absorbed/nm absorbed observed 380-420 Violet Green-yellow 420-440 Violet-blue Yellow 440-470 Blue Orange 470-500 Blue-green Red 500-520 Green Purple 520-550 Yellow-green Violet 550-580 Yellow Violet-blue 580-620 Orange Blue 620-680 Red Blue-green 680-780 Purple Green             2008-11-19

11-2 Absorption of Light = abc c b Lambert – Beer,s law Ia Absorbance The absorbance (A) of a sample is the logarithm of the ratio of Io / I . 2008-11-19

= abc Lambert–Beer,s law A- absorbance a - absorptivity (L·g-1·cm-1 ) b - path length (cm) c - mass concentration (g/L) c：mass concentration(g/L)→ concentration (mol·L-1) a：absorptivity (L/g·cm ) → ε (L·mol-1·cm-1 ) ε：the molar absorptivity 2008-11-19

Transmittance (T) is the ratio of I/Io . 2008-11-19

l00·T : (the percent transmittance) varies between 0 and 100%. T: 　range 0 ~ 1 l00·T : (the percent transmittance) varies between 0 and 100%. When no light is absorbed T=1=100% , P=P0 and A=0 I= I0 and A=0 2008-11-19

Monochromatic light Lambert – Beer,s law, commonly called Beer's law: Absorbance is directly proportional to the concentration of light-absorbing species in the sample and the pathlength of the solution. Lambert–Beer,s law is strictly valid for purely monochromatic radiation; that is, for radiation consisting of only one wavelength. Monochromatic light 2008-11-19

Absorption spectrum depicts what wavelengths of light are absorbed by a sample. One can readily see what wavelengths of light are absorbed (peaks). Absorption spectrum of iron(Ⅱ) -o-phenanthroline complex ……………… The maximum absorption at 508nm was characteristic of the complex solution. When the concentration of complex increased, the absorption spectrum increased. λmax 2008-11-19

11-3 The Spectrophotometer Major Components Wavelength Selector monochromator Sample cell Light source cuvette tungsten lamp a grating Light detector indicator scale phototube 2008-11-19

11-4 Typical Analytical Procedures Methods and Applications of Spectrophotometry Ⅰ. Direct comparison method Direct comparison method A1=εbc1 A2=εbc2 2008-11-19

Ⅱ. Standard Curve Method · Ax ········· ··········· cx c0 c1 c2 c3 c4 c5 A0 A1 A2 A3 A4 A5 The graph should be a straight line. 2008-11-19

Analytical Procedures 1) Selection of monochromatic light (Choice of Wavelength) 2) Prepare a reagent blank containing all reagents, but with analyte replaced by distilled water. 3) Prepare a series of standards solution and measure their absorbance 4) Establish a calibration curve 5) the unknowns be prepared using the same procedure as for standards solution 6) Calculate the content of unknown 2008-11-19

Analytical Procedures Choice of λmax Prepare solutions Measure their absorbance Establish a calibration curve Calculate the content of unknown 2008-11-19

Example: A sample in a 1.0 cm cell is determined with a spectrometer to transmit 80% light at a certain wavelength. If the absorptivity of this substance at this wavelength is 2.0, 1) what is the concentration of the substance(g/L). 2) what is the absorbance of the substance 2008-11-19

Solution: A=abc = - logT = -log0.80 = 0.0969 The percent transmittance is 80%, and so T=0.80，a=2.0，b=1.0 A=abc = - logT = -log0.80 = 0.0969 2008-11-19

Problems: The transmittance of a solution is found to be 35.0%. What is the transmittance if the solution is diluted in half? 2.The transmittance of a solution is found to be 85.0% when measured in a cell whose path length is 1.00cm. What is the transmittance if the path length is increased to 4.00 cm ? 2008-11-19

Calculate the molar absorptivity of M at 520 nm. 3. A colored substance M has an absorption maximum at 520 nm. A solution containing 2.00 mg M per liter has an absorbance of 0.840 using a 2.00 cm cell. The formula weight of M is 150. Calculate the molar absorptivity of M at 520 nm. How many milligrams of M are contained in 25.00 mL of a solution giving an absorbance of 0.250 at 520 nm when measured with a 1.00 cm cell ? 4. The molar absorptivity of benzoic acid (M=122.1g·mol-1) in methanol at 275 nm is about 1950 L·mol-1·cm-1. If it is desired to use an absorbance not exceeding 1.25, what is the maximum allowable concentration in g·L-1 that can be used in a 2.00 cm cell. 2008-11-19