Appendix B: UV-vis Guidelines

 

Instrument Operation - Hewlett Packard Model 8452A

A. Enter software by typing "HPUV" at the C: prompt.

B. The steps are fairly self explanatory. Enter "General Scanning" after changing "Operator Name".

C. Run a background (the neat solvent): "Meas. Blank F2"

D. Run a sample: "Meas. Sample F1"

E. By entering "Register", you can enter sample data such as molarity and solvent.  You can move the arrow down so that you designate an active register. Each session can then consist of several registers.

F. Enter "File" F6 to save data "Store Data File".  You must provide your own disk so that everything is saved to the a: directory.

G. "Cursor" (F2 of the second window) feature will either peak pick or you can move it to obtain absorbance and wavelength.

H. "Return" and "Exit" when done.

 

 

Sample Preparation

 

Method A

In a research laboratory setting where new compounds are frequently synthesized, small quantities of solid are dissolved in various solvents to determine solubility.  The UV cut-off of the best solvent is verified (CRC Handbook or various solvent catalogs).  There is often a trade-off at this point: the solvent that dissolves the compound best does not always have the optimum UV cut-off.

To prepare UV-vis samples, all glassware is volumetric (flasks and pipettes) and four-place analytical balances are used to weigh solid samples.  Once the solvent has been selected, the concentrations are a matter of trial-and-error. A small amount of solid is placed in a small volumetric flask (25-50 mL).  This sample is diluted with the chosen solvent (or a larger volumetric flask).  If the solid does not dissolve, the process begins again with a smaller amount of solid.  When the compound has dissolved, a UV-vis spectrum is obtained.  If the peaks do not fall below 1.0 absorbance where Beer's Law (see Method B) is linear, the sample is volumetrically diluted and another UV-vis spectrum is obtained. This process continues until the peaks are below 1.0 absorbance.

In the teaching laboratory situation, known compounds are synthesized and their UV-vis spectra are available in the literature.  This is the case with ferrocene, acetylferrocene and the ferrocenium cation.  Method B may be used for these samples.  Method A is used for the UV-vis spectra of W(CO)₆ and W(CO)₄en, where literature values are more difficult to obtain.  The product, W(CO)₄en, is compared with the starting material W(CO)₆.  Your teaching assistant or laboratory instructor will advise you as to which method is optimum for each experiment.  Regardless of the method employed, the experimental absorbance, wavelength and extinction coefficient are reported in the laboratory write-up.  If possible, the experimental values are compared with literature values.

 

Method B

For previously synthesized compounds, library work is essential before lab work in order to obtain and interpret a UV-vis spectrum.  This means that you can look in the Dictionary of Organometallic Compounds or other sources (such as the references in your lab notebook) to find the peaks, the solvent, and the expected extinction coefficients before you make your solutions.

For instance: Jolly, W. L., The Synthesis and Characterization of Inorganic Compounds, Prentice-Hall, New Jersey, 1970, provides a variety of information on the ferrocene complex including the following UV-vis information (in ethanol or hexanes):

Wavelength (mm) Extinction Coefficient (e) (L/mol-cm)

        225                            5250

        325                                50

        440                                87

 

Using Beer's Law, A = ebc, you can back-calculate the concentration, c, in moles/L, by setting absorbance, A, arbitrarily between 0.8 and 1.0 and the pathlength, b, of the cuvette at 1.0 cm. When the spectra are obtained, the experimental concentration, absorbance and path length are used to calculate the experimental extinction coefficient. The experimental extinction coefficient and wavelength are then compared to the literature values.

 

For Method A or B:

ALL SOLUTIONS MUST BE MADE VOLUMETRICALLY USING VOLUMETRIC FLASKS AND PIPETTES. YOU WILL LIKELY START WITH MORE CONCENTRATED SOLUTIONS AND BY SERIAL DILUTION ARRIVE AT A UV-VIS SPECTRUM WHERE THE ABSORBANCE IS LESS THAN ONE. THIS IS DESIRABLE, SINCE IT IS IN THIS REGION THAT BEER'S LAW IS LINEAR. YOU CAN THEN CALCULATE THE EXPERIMENTAL EXTINCTION COEFFICIENT FROM THE OBTAINED ABSORBANCE, SINCE YOU KNOW THE CONCENTRATION.