Light, Spectroscopy and the Spectrophotometer

Transcription

1 Wavelengths of Light Light, Spectroscopy and the Spectrophotometer Spectroscopy is the study of the interaction between matter and radiated energy. In the past, spectroscopy was basically the study of how visible light dispersed according to its wavelength by a prism. Later, the concept was expanded to comprise any interaction with radiative energy as a function of its wavelength or frequency. Spectroscopic data is often represented by - you guessed it - a spectrum, which is a plot of the response of interest as a function of wavelength or frequency. Penguins love rainbows! The quantitative measurement of the absorption or transmission properties of a material as a function of wavelength is called spectrophotometry, and the instrument we use is called a spectrophotometer. So we re going to be looking at how light and matter interact. The nature of this interaction depends on two things: the wavelength of the light (which we will control) and the structure of the matter (which is what we are trying to study). Here s you new best friend, the electromagnetic spectrum. Isn t it pretty? 1

2 Ready, Set Choose Your Wavelength! We want to use wavelengths which can excite electrons and move them to a higher energy state. These wavelengths are in the visible / UV range, but even within that range, we need to be more selective. It is important to note that molecules absorb only those wavelengths of light that contain just the right amount of energy to move the electrons to a higher energy state, resulting in peaks and valleys when absorbance is plotted against wavelength. Students often ask me what happens if you use longer or shorter wavelengths (i.e., outside the UV / visible range). Well, you can - but you ll need different instrumentation and you would measure different properties of matter. Absorption of longer, low energy wavelengths (infrared) stretches and bends bonds in molecules; consequently IR spectroscopy is used to identify chemical structures. Shorter wavelengths (X-rays and gamma rays) are sufficiently energetic to strip electrons from molecules completely. Wow. Light vs. Matter - Quantified You probably already know that when you pass light through a substance, some of the incident energy is absorbed and some is transmitted. The spectrophotometer measures transmitted light through a sample with respect to the incident light very precisely. Check this out: OK. As you can see, the spectrophotometer measures how much light is transmitted through our sample (compared to how much light entered the sample). Transmittance is simply the percentage of incident light reaching the photocell: %T = 100 (I / Io) where I is the light reaching the photocell and Io is the light striking the cuvette. 2

3 Wait a second here s a graph of transmittance as a function of concentration: As you can see, the relationship is exponential, which is awkward to work with. The good news is that since transmittance decreases exponentially with concentration, log 1/T increases linearly with increasing concentration! That s great because straight lines are a lot more convenient than curved ones! Here s a graph of absorbance as a function of concentration: Absorbance Penguins like straight lines! The best news of all is that YOU don t have to do the math - the spectrophotometer does it for you! All YOU have to do is tell the spectrophotometer that you want to know absorbance (ABS) not %T. But just so you know A = - log %T The relationship between absorbance and concentration is known as the Beer-Lambert law: A = elc, where e is the extinction coefficient (a property of the light-absorbing substance), l is the light path in cm and C is the concentration of the light-absorbing substance. Most cuvettes have a light path of 1 cm. Just FYI. 3

4 Using the Spectronic 20 Genesys Spectrophotometer Say Hello! 1. Check that the cell holder is empty. 2. Turn on the power switch at the back of the unit and allow the spectrophotometer to warm up for 30 minutes. 3. Press the A/T/C key to select ABS (absorbance) mode. If you take your readings in %T, YOU LL have to do the math yourself later!!! 4. Set the wavelength to the desired value. Holding the nm arrow keys will cause the wavelength to change more quickly 4

5 5. Wipe the cuvette with a kimwipe to remove fingerprints. Holding the cuvette at the top, insert the blank into the cell holder with the clear walls of the cuvette to the front and back of the instrument (if not using glass test tubes). Remember - setting the blank is like hitting the TARE button on a balance! The blank is used to zero readings due to imperfections in the cuvette and for the absorbance of light by the medium or carrier solution. Close the sample compartment. 6. Press 0ABS/100%T to blank the instrument. You will see A in the window (as well as the wavelength you selected). This is like the TARE button on a balance! 7. Remove the blank from the cell holder. Mix contents of your sample if necessary. Wipe fingerprints from your sample cuvette, insert your sample and close the sample compartment. 8. Record the instrument reading in your lab notebook. 9. Remove the sample from the cell holder. 10. Say TA-DAH! Or do a hula. Your choice. 5