UV/VIS Spectrophotometry 1.

Theory: UV range: 200 nm 400 nm Visible range: 400 nm 800 nm UV/Visible spectroscopy: studying of the electron transitions between molecular orbitals by excitation with UV or visible light. Chromophor: a functional group, which can be assigned to the absorption of a given electron transition The intensity of the incident beam: The intensity of the transmitted beam: Transmittance: Absorption: incident light. Spectrophotometry: quantitative method to obtain the concentration of a compound in a solution by measuring its absorption at a certain wavelength. Lambert-Beer Law: gives the correlation between the measured absorption and the concentration of a given compound: A = c l Where: A : the measured absorption at a given wavelength : the wavelength of the incident light c : the concentration of the compound in the solution (unit: mol/L) l : the path length, i.e. the width of the cuvette in cm, usually it takes 1 cm : the molar absorption coefficient of the absorber at a given . According to the Lambert-Beer law, the absorption is proportional to the concentration of absorbing species in diluted solutions (c<10-3 mol/dm3). I0 I T= I/I0 A= lg (1/T) = lg (I0/I)

Absorption spectrum: the measured absorption plotted against the wavelenght of the

2. The practice of photometry: The most important part of a spectrophotometer is a light source that can cover the required spectral interval. The emitted light is broken up by optical gratings or prisms into its spectral components, one of which is directed onto the sample placed in a cuvette. The intensity of the light beam passing through the sample is measured by an optical detector. Upon conducting optical measurements, one has to comply with the following requirements: a, Intensity measurements are greatly influenced by the scattering light. To avoid that, the solution has to be clear, and the cuvette has to be clean without any fingerprints or scratches on its wall. b, The cuvette should be rinsed thoroughly with the sample solution before each measurement. The sample has to be filled up to of the vial. Before placing the cuvette in the photometer, its walls have to be dried with a piece of tissue paper. c, The absorption of a given solution has to be determined as an average of at least three independent measurements (possibly by re-filling the cuvette with new portions of solution). d, In most cases the exact value of a given material is not known. Therefore, instead of determination of the concentration directly from the Lambert-Beer law, we use the method of calibration in the practice. In this case, a series of `calibration standards and a so-called `blank solution are prepared; the latter contains all of the components of the samples except the substance, the concentration of which has to be determined. The zero value of the photometer has to be set on the blank solution. According to the Lambert-Beer law, the measured absorption values of the calibration series plotted against the concentrations give a linear relationship. A straight line can be fitted (`calibration line) on the measured data either manually, or by applying mathematical methods (method of linear least squares). By measuring the absorption of the unknown sample, its concentration can be interpolated from the calibration curve (by simple reading or by calculating from the equation of the fitted curve).

3. Users guide for the WPA CO7500 Colorimeter: Absorption measurement is possible only at certain wavelengths with this instrument; recording of full absorption spectrum is not possible. Built-in filters provide the following measurement wavelengths: 440, 470, 490, 520, 550, 580, 590 and 680 nm. Steps of the measurement: 1. Switch on the colorimeter with the `ON/OFF button. 2. Set the proper wavelength by turning the wavelength selector on the right side, until the required value is shown up in the control window on the top of the instrument. 3. Choose absorption measurement by pressing the `Abs/%T button (the instrument displays 0.00 in absorption mode). 4. Fill the blank solution into a clean cuvette, and place it into the cuvette hole. The cuvette must be pressed down carefully in order to be held firmly without `jumping out of the hole. 5. Press the `R(Reference) button; the instrument is zero set to the blank solution and ready for the measurement. 6. Fill the standard solutions, as well as the unknown solution into the cuvette, place it into the cuvette holder, and press the ` T (Test) button. Read the measured absorption in each case. Each sample has to be measured three times, possibly by refilling the cuvette with new portions of the sample. PLEASE, CONDUCT THE MEASUREMENTS AS CAREFUL AS POSSIBLE: NEVER PLACE A WET CUVETTE INTO THE COLORIMETER AND AVOID POURING THE SAMPLE SOLUTION INTO THE CUVETTE HOLE!

Picture of the colorimeter used in the practice:

WPA CO7500 colorimeter 4. Evaluation The measured absorptions have to be tabulated as follows: Sample Cal1 Cal2 Cal3 Cal4 Unknown Concentration Abs1 Abs2 Abs3 Abs average

Prepare a plot of the measured Abs values vs. concentrations for the calibration series. You can use a graph paper, or computer programs (e.g. Excel, Origin etc.) can be applied for the graphical projection and linear regression as well. On a graph paper use the whole available area of the sheet in order to achieve the accuracy of the determination. Draw a line on the measurement points with the aid of a ruler. Try to minimize the distance of the line from each point at the same time. Points, which fall far from the trend line can be left out, but they have to be plotted and marked. Always place the names and the units of the plotted data near the axes. Calculate the concentration of the unknown sample by interpolating it from the calibration curve.