CH 4212 Instrumental Analysis UV-VIS Absorption Spectrophotometry Background:

Fall, 2012

This exploration will introduce you to the process of taking UV-Vis spectra using the PerkinElmer Lambda 35 Spectrophotometer in Room 408. Fundamentally, this instrument is no different from the Ocean Optics Spectrophotometers that you used in Quantitative Analysis. The same principles hold: Beers Law determines the absorbance at any wavelength. You get the same information (absorption spectra, quantitative measurements, etc.) What is different is that this Lambda 35 has powerful (and complicated) software attached to it that requires you to learn specific operations to perform what should be a simple task collecting spectra. Because the learning curve for the software is so steep, your actual laboratory work is reduced to the preparation of a simple dilution series of caffeine in water, from which you will generate the associated UV spectra. Using the same solutions, you will use the Lambda 35 to generate a calibration curve using absorbance measurements obtained at a single analytical wavelength. Exploration: Familiarization with the Lambda 35 You will each have a copy of the UV-Vis SOP written for the Instrumental Analysis lab. You will learn to Perform pre-analysis cleanup and system checks Turn on the instrument Handle quartz cuvettes Create a personal folder on the instrument computer Generate spectrum; preparation (caffeine in water); store Set up a data table in the software Generate a calibration plot; store; print Shut down procedure Cuvette Care The quartz cuvettes you will be using are expensive (~$350 for a matched set) so be careful when handling the cuvettes. Keep them in the poly cuvette rack between measurements and return them cleaned to the storage box when you are through for the day. Clean the outer optical faces with lens paper (not Kimwipes or paper towels). Rinse the inside with water and dry with a Q-tip. Preliminary spectrum and solution prep Your instructor will provide you with a stock solution of caffeine in water. Make a rough dilution using a graduated cylinder and a beaker. Obtain the absorbance spectrum from 310 to 240 nm. (You may have to do this more than once.) From the absorbance at the wavelength of maximum absorbance (max), calculate the solution concentrations needed to make up a series of 1

CH 4212 Instrumental Analysis

Fall, 2010

five solutions that will give you absorbances at max in the range of 0.2 to 1.0 AU. Prepare these solutions with volumetric glassware using proper lab technique learned in Quantitative Analysis. All volumes should individually be less than 100 mL. Solution spectra Obtain the absorbance spectra (310 240 nm) of the solutions you prepared, using the Scan method on the Lambda 35. If the maximum absorbances fall outside the AU range specified, you must prepare new solutions. Store each of the spectra according to the SOP and then produce a plot of all the spectra on one graph. Calibration plot Using the solutions prepared above, switch to Wavelength method and generate a calibration plot from the absorbance at max. Moving files Create a personal folder in the CH 4212 folder. Move your Method and Task files out of the common directory to your personal folder. Shut down Pay special attention to the shut down procedure in the UV-Vis SOP. There is a specific order in which operations must be performed to protect the source. Ending the exploration Have your notebook signed by your instructor. Sign the log book. Lab Practicum: At some point, you will schedule your lab practicum to prove your competence on UV-Vis. From this exploration, you should know how to do the following: 1. Instrument turn-on 2. Care, handling of quartz cuvettes 3. Create personal folder; move Method and Task files to this folder 4. Calculation of needed solution concentration from initial spectrum 5. Generate spectra; store; overlay in printout 6. Generation of calibration curve 7. Shut down procedure 8. Post-analysis cleanup; storing of materials; proper disposal of waste