Theory of UV spectrum of Tetraphenylcyclopentadienone.

Practical Aspects of UV-Vis spectroscopy or "How to get a good UV-VIS spectrum"

The UV-Vis instrument (HP 8453) used in Chem 30BL is located in YH 1102.

In order to acquire a UV-Vis spectrum, the solid has to be dissolved in a suitable solvent. Note that not every solvent is suitable for UV spectroscopy (for optical windows see SKR). Generally, spectrograde solvents are used that have undergone special purification steps to remove trace impurities of aromatic and other compounds. A list of commonly used solvents and their lower limits are given in the theoretical part (see link above). The same applies to the cuvette material. While quartz cuvettes allow measurements as far down as l~200 nm, Pyrex cuvettes already show a significant absorption around l=260 nm. Various 'plastic cuvettes' absorb already at higher wavelengths. In addition, many of them are not compatible with organic solvents (the windows are not translucent anymore or they are plainly dissolved). The cuvette used in Chem 30 BL are polyethylene cuvettes which have a cutoff at l~240 nm .

Since UV-Vis spectroscopy is an absorption technique, Beer's Law can be applied. It is only valid at low concentrations (<10^-4 mol/L). At higher concentrations, a negative deviation is observed due to association of the molecules and other effects. Another critical factor is the detector of the UV-Vis instrument that only works linear within a certain range as well. A meaningful absorbance reading should be between A=0.1 and A=1.0. In essence, the concentration has to be low enough, so that the maximum absorbance does not exceed A=1. And yes, the exact concentration of the sample has to be known in order to determine the molar extinction coefficient (e). Just preparing a solution that produces a reading in the desired range is not enough to obtain all the relevant information, which are the l_max and the e_max!!

General Procedure:

In the lab, prepare a solution of the compound (based on the given extinction coefficients above) in a graduated cylinder. Fill the cuvette about 3/4 with the solution. Then place the cuvette in the holder shown in figure 2. At this piont, the TA usually acquired the background spectrum already.

1. Start-up procedure (if you are the first one to run a sample):

Make sure that the instrument is turned on. It has to warm up for 10-15 minutes. Start the program by double clicking the Chemstation icon icon on the desktop. Skip the request for a admininstrator password. Go to the FILE LOAD METHOD option and load the method "130AL". The software tested if the instrument is working properly. Make sure that both lamps are on (see lower left corner!).

2. Blanking the spectrum

First make sure that you clear the spectrum window if the previous student did not delete his spectrum by checking the CLEAR button on the upper left corner. Choose the Samples option. This should remove any spectra in the spectral window.

Then pour the solvent in the cuvette (3/4 full). Place the cuvette with the transparent window pointing towards you, in the holder above. Then click on the BLANK ICON to acquire the spectrum of the solvent. The solvent spectrum should be relatively flat, except in the lower range due to the absorption of water in the air.

3. Measurement of sample

Place the sample in the cuvette and then place the cuvette in the holder. Then click on the "SAMPLE ICON". The instrument will acquire the spectrum of the solution. The method will automatically assign some peaks and valleys. You can print the spectrum by using the "FILE PRINT CURRENT SCREEN". Make sure that the maximum absorbance of your sample is around A~1 and that you print the range from 280-600 nm.

4. Data Processing

Make sure that the the relevant peaks are labeled. If this is the case, go to the PRINT OPTION,. Chose PRINT CURRENT WINDOW. This will print the spectrum with a table of peaks. If the printout looks weird (="wingdings"), go to the printer preferences menu and check the HP-5 printer as active printer (the printer attached is the HP-N 4100 model, but the printer driver does not work properly with the UV-Vis software!)