1. Reflection and scattering losses with a solution contained in a typical glass cell. Losses by reflection can occur at all the boundaries that separate the different materials. In this example, the light passes through the fo1Jowing boundaries, ca1Jed interfaces : air-glass, glass-solution, solution- glass, and glass-air.

2. Colorimetry When white light passes through a colored substance, a characteristic portion of the mixed wavelengths is absorbed. Complementary colors are diametrically opposite each other. Thus, absorption of 420-430 nm light renders a substance yellow, and absorption of 500-520 nm light makes it red. The remaining light will then assume the complementary color to the wavelength(s) absorbed.

3. Colored substances appear colored because they selectively absorbed some of wavelengths of visible light and transmitted other wavelengths or colors (apparent color), Red substances absorb the blue- green wavelengths from the visible region, so the transmitted light appears red Red substances absorb the blue- green wavelengths from the visible region, so the transmitted light appears red Blue substances absorb the yellow wavelengths, so the transmitted light appears blue. Blue substances absorb the yellow wavelengths, so the transmitted light appears blue. wavelength region, nmcolorcomplementary color 400-435VioletYellow-green 435-480BlueYellow 480-490Blue-greenOrange 490-500Green-blueRed 500-560GreenPurple 560-580Yellow-greenViolet 580-595YellowBlue 595-650OrangeBlue-green 650-750RedGreen-blue

4. The Absorption Process

6. Transmittance T = P/P o P o : incident light power P : transmitted light power %T = P/P o x 100 =……… % Absorbance A = - log T

7. Beer’s-Lambert law A =absorbance b = pathlength (cm) c =concentration

8. A = abC where a is the analyte’s absorptivity with units of cm –1 conc –1. If we express the concentration using molarity, then we replace a with the molar absorptivity, ε, which has units of (L mol -1 cm -1 ). A = εbC The absorptivity and molar absorptivity are proportional to the probability that the analyte absorbs a photon of a given energy. As a result, values for both a and ε depend on the wavelength of the absorbed photon A = abC where a is the analyte’s absorptivity with units of cm –1 conc –1. If we express the concentration using molarity, then we replace a with the molar absorptivity, ε, which has units of (L mol -1 cm -1 ). A = εbC The absorptivity and molar absorptivity are proportional to the probability that the analyte absorbs a photon of a given energy. As a result, values for both a and ε depend on the wavelength of the absorbed photon

9. Increase  increase sensitivity for the absorption method so the method can be used to determine very low concentration of this substance. Increase the concentration will increase the absorbance and decrease transmittance. Absorbance and transmittance depends on the wave length of incident light Increase  increase sensitivity for the absorption method so the method can be used to determine very low concentration of this substance. Increase the concentration will increase the absorbance and decrease transmittance. Absorbance and transmittance depends on the wave length of incident light

10. Beer's law also applies to solutions containing more than one kind of absorbing substance. Provided that there is no interaction among the various species, the total absorbance for a multicomponent system at a single wavelength is the sum of the individual absorbances. In other words, where the subscripts refer to absorbing components 1, 2,..., n. Applying Beer's Law to Mixtures

11. 5.00 × 10 –4 M solution of an analyte is placed in a sample cell with a pathlength of 1.00 cm. When measured at a wavelength of 490 nm, the solution’s absorbance is 0.338. What is the analyte’s molar absorptivity at this wavelength? problems

13. Beer’s law suggests that a calibration curve is a straight line with a y-intercept of zero and a slope of ab or εb. In many cases a calibration curve deviates from this ideal behavior Deviations from linearity are divided into three categories: fundamental, chemical, and instrumental. Beer’s law suggests that a calibration curve is a straight line with a y-intercept of zero and a slope of ab or εb. In many cases a calibration curve deviates from this ideal behavior Deviations from linearity are divided into three categories: fundamental, chemical, and instrumental. Limitations to Beer’s Law

14. Calibration curves showing positive and negative deviations from the ideal Beer’s law calibration curve, which is a straight line.

15. 1- FUNDAMENTAL LIMITATIONS TO BEER’S LAW a-Beer’s law is good for dilute analyte solutions only. High concentrations (>0.01M) will cause a negative error since as the distance between molecules become smaller the charge distribution will be affected which alter the molecules ability to absorb a specific wavelength. The same phenomenon is also observed for solutions with high electrolyte concentration, even at low analyte concentration. The molar absorptivity is altered due to electrostatic interactions. b. In the derivation of Beer’s law we have introduced a constant (ε). However, ε is dependent on the refractive index and the refractive index is a function of concentration. Therefore, ε will be concentration dependent. However, the refractive index changes very slightly for dilute solutions and thus we can practically assume that ε is constant.

16. c. In rare cases, the molar absorptivity changes widely with concentration, even at dilute solutions. Therefore, Beer’s law is never a linear relation for such compounds, like methylene blue. We have seen earlier that validation of Beer’s law is dependent on the nature of the molar absorptivity. It was found that the molar absorptivity is influenced by: a. The wavelength of radiation b. The refractive index and is thus indirectly related to concentration c. Electrostatic interactions taking place in solution; and thus electronic distribution d. In rare cases, like methylene blue, the molar absorptivity is directly dependent on concentration We have seen earlier that validation of Beer’s law is dependent on the nature of the molar absorptivity. It was found that the molar absorptivity is influenced by: a. The wavelength of radiation b. The refractive index and is thus indirectly related to concentration c. Electrostatic interactions taking place in solution; and thus electronic distribution d. In rare cases, like methylene blue, the molar absorptivity is directly dependent on concentration

17. Deviations from Beer’s law appear when the absorbing species undergoes association, dissociation, or reaction with the solvent to give products that absorb differently from the analyte. 2- Chemical Deviations

19. 3. Instrumental Deviations a.Beer’s law is good for monochromatic light only since ε is wavelength dependent to avoid deviations, it is advisable to select a wavelength band near the wavelength of maximum absorption, where the analyte absorptivity changes little with wavelength. b. Stray Radiation Stray radiation resulting from scattering or various reflections in the instrument will reach the detector without passing through the sample

20. The determination of an analyte’s concentration based on its absorption of ultraviolet or visible radiation is one of the most frequently encountered quantitative analytical methods. One reason for its popularity is that many organic and inorganic compounds have strong absorption bands in the UV/Vis region of the electromagnetic spectrum. In addition, if an analyte does not absorb UV/Vis radiation—or if its absorbance is too weak—we often can react it with another species that is strongly absorbing. For example, a dilute solution of Fe 2+ does not absorb visible light. Reacting Fe 2+ with ophenanthroline, however, forms an orange–red complex of Fe(phen)3 2+ that has a strong, broad absorbance band near 500 nm. An additional advantage to UV/Vis absorption is that in most cases it is relatively easy to adjust experimental and instrumental conditions so that Beer’s law is obeyed Quantitative Applications

21. ENVIRONMENTAL APPLICATIONS The analysis of waters and wastewaters often relies on the absorption of ultraviolet and visible radiation. use to determine trace metals inorganic non metals(Cl - ) and organics Many of these methods are outlined in the following table table: Examples of the Molecular UV/Vis Analysis of Waters and Wastewaters

22. UV/Vis molecular absorption is used for the analysis of a diverse array of industrial samples including pharmaceuticals, food, paint, glass, and metals. INDUSTRIAL ANALYSIS For example, the amount of iron in food can be determined by bringing the iron into solution and analyzing using the o-phenanthroline method listed in Table Many pharmaceutical compounds contain chromophores that make them suitable for analysis by UV/Vis absorption. Products that have been analyzed in this fashion include antibiotics, hormones, vitamins, and analgesics. One example of the use of UV absorption is in determining the purity of aspirin tablets

23. FORENSIC APPLICATIONS One interesting forensic application is the determination of blood alcohol using the Breathalyzer test CLINICAL APPLICATIONS

24. Chromogen : is a compound containing chromophoric group Requirements for ideal chromogen 1-Should be colorless or easily separated from the colored product 2-It Should be selective 3-Its reaction to produce colored product, should be of known mechanism and proceed stoichiometrically 4-The full development of color must be rapid. 5-It must produce only one color of specified max. The part of a molecule responsible for light absorption is called a chromophore.

25. Requirements for coloured product 1-Should be of intense color, to increase the sensitivity 2-Should be unaffected by pH or the pH must be specified and maintained by suitable buffer or the measurement is carried out at of isosbestic 3-Should be stable with time 4.The reaction of its formation, must be rapid and quantitative. 5-The colored product, should obey Beer-lambert’s law, i.e on plotting A versus C at fixed b, we obtain straight line passing through the origin.