Amino Acid Project--UV-Visible Spectroscopy

Spectroscopy is the study of the interaction of molecules with light. It has long been known that atoms and molecules absorb light. We discussed this in CH1010 in connection with the energy levels of the hydrogen atom. The light is absorbed because it interacts with the various types of motion that a molecule may undergo. These motions occur in different ranges of frequency, so they interact with light in different wavelength (frequency) ranges. For a molecule to absorb a photon of light, one of its motions must occur with the same frequency that the light wave oscillates with. By measuring the frequencies of light absorbed by the molecules of a substance, we obtain information about the molecular motions and often about the molecular structure.

Table 1 shows the molecular motions that interact with light in the various wavelength regions.

In UV-visible spectroscopy, we prepare a sample, place it in a UV-visible spectrometer (an instrument), and obtain the UV-visible (UV-vis) spectrum. The instrument automatically exposes the sample to all of the wavelengths of light in the infrared region, and measures how much of the light is absorbed by the sample at each wavelength. It then produces a plot of the percentage of the absorbance by the sample at each wavelength. The absorbance, A, is 0 at wavelengths where the sample does not absorb light, and is greater than 0 at wavelengths where the sample absorbs. (The relationship between absorbance, A, and percent transmission, %T, used in IR spectroscopy, is A = 2 - log%T.) A particular substance absorbs UV-vis radiation at characteristic wavelengths; we can use the UV-vis spectrum to help identify the substance. For example, the substance with this UV-vis spectrum absorbs UV-vis radiation in the wavelength regions 200-300, 400, and 500 nm. Normally we report only the wavelengths at which the absorbance maximizes. These wavelengths are called the l_max values for the substance. We also report the strength, e, of absorbance at each l_max. This can be calculated from equation 1.

This equation is called Beer's Law. [substance] is the molarity of the substance that is absorbing the light; l is the length of sample through which the light passes, almost always 1 cm; and e is called the molar absorptivity of the substance at the particular wavelength and has units of M^-1cm^-1. If a 0.001 M solution of a substance has A = 0.90 at l = 375 nm, then e = 900 M^-1cm^-1 for the substance at 375 nm. We report this information as l_max(e), in this case 375(900).

Sample Preparation. UV-visible spectra are most easily obtained from solutions of the substance of interest in a suitable solvent. A solvent is suitable if a) the substance dissolved in it without reaction, and b) the solvent has no absorbance in the wavelength range of interest. Water is a good solvent for UV-visible spectroscopy because it has no absorbance over the entire wavelength range from 1100 to 200 nm.

We start by preparing 5 or 10 mL of a 0.01 M solution of the substance in the chosen solvent, say water. This solution is prepared by accurately weighing a calculated amount of the substance into a 5- or 10-mL volumetric flask; filling the flask about halfway with solvent; swirling to dissolve all of the substance; filling the flask to the mark with solvent; then stoppering the flask and shaking the he__ out of it to obtain a thoroughly mixed solution.Two to three mL of the solution is transferred to a quartz cuvet with a Pasteur pipet. THE CUVETTES ARE PRECISION MADE, HIGHLY POLISHED, AND EXPENSIVE. DO NOT TOUCH THE POLISHED (TRANSPARENT) FACES! IF YOU BREAK A CUVET YOU WILL HAVE TO PAY FOR IT. COST, $100. An identical cuvet may be filled with solvent only. The cuvets are placed in the instrument and the spectrum is scanned. If absorbance bands go "off scale," appropriate dilution of the original solution should enable you to get them "on scale." A series of dilutions (called serial dilution) may be necessary to bring all absorbance bands on scale.

When finished, empty the cell into a waste container. Rinse it several times with distilled water, then a couple of times with small amounts of acetone. CAREFULLY DRY IT BY ASPIRATION and return it to its storage location.

Remember that The UV-vis spectrometers are delicate and expensive. Please exercise utmost care and respect when using them. IF YOU BREAK A UV-VIS SPECTROMETER YOU WILL HAVE TO REPLACE IT. COST: $9,000.

For more information on this subject, see Chapter 4 of Concepts of Chemistry, or take CH1040!