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.
| Molecular Motion | Wavelength range of Electromagnetic Radiation | Type of Spectroscopy |
|---|---|---|
| spinning of an atomic nucleus | radio | Nuclear Magnetic Resonance |
| rotation of the molecule in space | microwave | Rotational Spectroscopy |
| vibrations of the molecule (bond stretches and bends) | infrared | Vibrational (Infrared) spectroscopy |
| motions of the electrons from one energy level to another | ultraviolet, visible | UV-Visible Spectroscopy |
In infrared spectroscopy, we prepare a sample, place it in an infrared spectrometer (an instrument), and obtain the infrared (IR) 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 transmitted by the sample at each wavelength. It then produces a plot of the percentage of the light transmitted versus wavelength. Of course the percent transmission, or %T, is 100 at wavelengths where the sample does not absorb light, and is less than 100 at wavelengths where the sample absorbs. The smaller the amount of light transmitted, the stronger the absorption by the molecules of the sample. A particular substance absorbs infrared radiation at very characteristic wavelengths; we can then use the infrared spectrum to identify the substance! For example, the substance with this IR spectrum absorbs light of frequency in the range 3200-3400 cm-1 and at quite a few frequencies below 1400 cm-1. The strong absorptions in the neighborhood of 3000 and 400-600 cm-1 are due to materials used to contain the sample, and so must be ignored when examining the IR spectrum. Here is the neat thing about IR spectroscopy: no two substances in the world have exactly the same IR spectrum. This spectrum is thus an excellent way to "fingerprint" a substance. In The Amino Acid Project, we are interested in your obtaining the IR spectrum for your acid, then comparing it with the IR spectra of amino acids that we will provide. You look for a match, and thus identify your acid. It is possible to get a lot more information from the IR spectrum, but we save that for later.
Sample Preparation. Place a small amount of your amino acid in an agate mortar. By "small amount" we mean enough to cover the tip of a small spatula. A pile about 1 mm deep and 2 mm in diameter would be good. Grind the sample with an agate pestle until it no longer looks crystalline. Add 1 drop of Nujol (light mineral oil) to the mortar, and use the pestle to mix the solid amino acid with the mineral oil to obtain a paste. The paste is called a "mull". It is somewhat of an art to get this paste; it must be neither to thick nor too thin! Something with about the same viscosity as cake batter is pretty good. You may have to add a little more substance or a little more Nujol to achieve this. Using a flat spatula, scrape some of the paste out of the mortar onto the end of the spatula and CAREFULLY transfer the paste from the spatula to the surface of a sodium chloride plate. A sodium chloride plate is a disc of sodium chloride that is about 2 cm in diameter and 5 mm thick. It looks like glass, but it is actually table salt! PLEASE HANDLE THE SODIUM CHLORIDE PLATES BY THEIR EDGES ONLY--DO NOT PUT YOUR FINGERS ON THE FLAT CIRCULAR SURFACES. Place a second NaCl plate over the sample and gently rotate the top plate to spread the sample around. Place the plates in a plate holder and carefully screw in the plastic male piece of the plate holder. IT IS VERY EASY TO GET THIS PIECE CROSS THREADED--IF YOU FEEL ANY RESISTANCE AT ALL WHEN YOU TRY TO TURN IT, STOP!!! IF YOU FORCE THE TURN YOU WILL RUIN THE PLASTIC PIECE. IF YOU RUIN THE PLASTIC PIECE YOU WILL HAVE TO PAY FOR IT. COST: $80. Then take the loaded sample to the instrument and run the spectrum as instructed.
The IR spectrometers are delicate and expensive. Please exercise utmost care and respect when using them. IF YOU BREAK AN IR SPECTROMETER YOU WILL HAVE TO REPLACE IT. COST: $16,000.
After obtaining the spectrum, disassemble the plate holder and carefully remove the plates. Separate them and gently wipe the sample side of each place on a Kimwipe that is placed flat on the bench. Often this is sufficient to clean the plates. IF NECESSARY, squirt a little acetone on the Kimwipe and wipe the plate surface across the acetone. Let the plates dry for a couple of minutes, then return them to the plate holder and return the plate holder to the storage desiccator. DO NOT UNDER ANY CIRCUMSTANCES TRY TO CLEAN THE PLATES WITH WATER--THEY WILL DISSOLVE AND DISAPPEAR BEFORE YOUR EYES! IF YOU RUIN A PLATE YOU WILL HAVE TO PAY FOR IT. COST: $50.
For more information on this subject, see Chapter 4 of Concepts of Chemistry, or take CH1040!
