UV/Vis Spectroscopy. Varka Evi-Maria Ph.D. Chemist AUTH Thessaloniki 2012

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1 UV/Vis Spectroscopy Varka Evi-Maria Ph.D. Chemist AUTH Thessaloniki 2012

2 Introduction of Spectroscopy The structure of new synthesised molecules or isolated compounds from natural sources in the lab must be determined (and/or verified). Chemical analysis (Classical methods) Spectroscopy (Modern techniques) Main advantages: Spectroscopic techniques are non-destructive and generally require small amounts of sample

3 Electronic Excitation by UV/Vis Spectroscopy : X-ray: core electron excitation UV: valance electronic excitation IR: molecular vibrations Radio waves: Nuclear spin states (in a magnetic field) The visible spectrum constitutes but a small part of the total radiation spectrum. Most of the radiation that surrounds us cannot be seen, but can be detected by dedicated sensing instruments. This electromagnetic spectrum ranges from very short wavelengths (including gamma and x-rays) to very long wavelengths (including microwaves and broadcast radio waves).

4 A diagram showing the various kinds of electronic excitation that may occur in organic molecules is shown on the left. Of the six transitions outlined, only the two lowest energy ones (left-most, colored blue) are achieved by the energies available in the 200 to 800 nm spectrum. As a rule, energetically favored electron promotion will be from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), and the resulting species is called an excited state. When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. An optical spectrometer records the wavelengths at which absorption occurs, together with the degree of absorption at each wavelength.

5 UV-Vis Spectroscopy When electromagnetic radiation in the UV/Visible interacts with a sample, four effects are possible to occur: Electromagnetic Radiation Diffuse Reflectance Fluorescence The radiation can be absorbed transmitted reflected scattered Specular Reflectance Absorbance Interior reflectance Transmitted light

6 UV/Vis spectrometers are equipped to measure the: transmittance or absorbance of transparent solid or homogeneous solution, and the reflected and scattered energy from a sample

7 Quantitative Spectroscopy Beer s Law A = ebc e is molar absorptivity (unique for a given compound ) b is path length c concentration

8 Beer s Law source slit cuvette detector A = -logt = log(p0/p) = ebc T = Psolution/Psolvent = P/P0 Works for monochromatic light Compound x has a unique e at different wavelengths

9 One wavelength Characteristics of Beer s Law Plots Good plots have a range of absorbances from to Absorbances over are not that valid and should be avoided 2 orders of magnitude

10 Standard Practice Prepare standards of known concentration Measure absorbance at λmax Plot A vs. concentration Obtain slope Use slope (and intercept) to determine the concentration of the analyte in the unknown

11 Typical Beer s Law Plot A y = 0.02x concentration (um)

12 Our laboratory is found with: the Lambda 35 Perkin Elmer UV/Vis spectrometer The Lambda 35 is controlled from a Windows based computer using UV Winlab TM software

13 Integrating sphere Integrating sphere is a tool for : collecting and measuring specular and/or diffuse reflectance. capturing the scattered light from a sample

14 Integrating sphere The integrating sphere is coated with a film of a material which can diffuse «ideally» the light that interacts, so as to ensure the maximum is possible and the most isotropic diffuse of the income light for all the wavelength range of the visible light.

15 Integrating sphere The reflected ability of a solid sample is affected from the texture of its surface. However, the color is independent from the texture of the surface. Therefore, the color calculation should be based on the measurements of the diffused reflectance and exclude while calculating the specular reflectance. The integrating sphere is equipped with diaphragm that is capturing the light that would do specular reflectance.

16 Integrating sphere The integrating sphere is placed in the sample beam of the spectrometer and is used in place of the sample beam detector. The instrument detector, external to the sphere, detects the reference beam.

17 Optical schematics of the 50mm integrating sphere for Lambda 35

18 Measurement of diffuse Reflectance By placing a sample at the exit of the sphere, reflected light is collected and measured at the detector in diffuse reflectance mode

19 Reflectance spectra Hansa yellow (acrylic color) %R nm

20 Measurement of Transmittance By placing the sample at the entrance to the sphere, transmitted light enters the sphere and is collected and measured at the detector in diffuse transmittance mode

21 Transmittance spectra 10.0 Paper used as background to acrylic paints 8 6 %T nm

22 Creating a method When we open the program, the Method Editor window appear. This window has four tabs at the bottom which relate to the four types of method available: Scan methods for scanning spectra Td methods for time dependent measurements Wp methods for running wavelength programs Conc methods for concentration measurements Others other methods

23 Creating a method Beneath the Method Editor window are minimized the windows: Data Region where data is stored as long as the method is running. Result Window where the numerical results of the method are displayed. Graph 1 where the graphical results are displayed.

24 Creating a method For scanning the reflectance or the transmittance spectra of a solid sample: 1. We chose the Scan tab of the method Editor, we select: Start/End wavelength Data interval Number of cycles

25 Creating a method 2. We select the Inst tab and we choose : the parameter that we want to calculate, (for example R%) the UV and Vis lamps and the scanning speed

26 Creating a method 3. We select the Sample tab and we put the number of the samples that we want to save and their names. 4. We save the method

27 Using the method 5. Click start the blank sample is request Blank sample : For R% : is the background of a sample or the standard blank sample for reflectance measurements of Lambda 35 For T%: is the background of a sample or the air

28 Using the method 6. Then the first sample is requested and the analysis is performed. During the analysis the current readings are shown on the Graph window. 7. We save the spectra

29 Advanced Spectroscopy Software Advanced Spectroscopy Software has two important applications for processing the reflectance and transmittance spectra : Arithmetic Color

30 Arithmetic Arithmetic application is used for : spectral manipulation data conversion and comparison data reduction One Graph window shows the original spectrum while a second displays the spectral or the results of the Calculations.

31 Arithmetic applications Calculate the area beneath the spectral curve Perform mathematical operations on spectra (e.g. add two spectra) Perform mathematical operations on spectral data Calculate the average and standard deviation from spectra Calculate the average, standard deviation and rms at specified wavelength range Build a spectrum via input values Calculate the total height and the height to a specified base

32 Arithmetic applications Convert the abscissa unit (e.g. convert the abscissa unit from nm to cm -1 ) Convert the ordinate unit (e.g. converts the spectral data from T% to A) Calculate the 1 st, 2 nd,3 rd,4 th derivative of a spectral curve Normalize spectral curve to a given ordinate value Correct a reflectance spectrum for dark and white values Display ordinate values at defined abscissa values Smooth spectral data Calculate the film thickness of single layers

33 Color The color Application allows the reflectance spectra of opaque materials to be evaluated with industry standard CIE, ASTM, DIN and ISO color methodologies.

34 Creating a Method We create a Method in order to use it every time we want to make the same calculations. While we create the method we can select many parameters, e.g. : The wavelength range The observer The calculations (e.g. CIE Yxy, CIE L*a*b*, Yindex, Windex) The graphic (e.g. CIE Yxy, CIE L*a*b* ) The illuminate

35 Color When the calculations have been performed the results ( text and graphic) are displayed according to the templates defined in the Method Editor.

36 CIE L*a*b*

37 CIE Yxy

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