SPECTROPHOTOMETER THEORY

  SPECTROPHOTOMETER THEORY

 
A spectrophotometer is an instrument that measures the quantity of electromagnetic radiation transmitted through a sample as a function of wavelength. We will use the term intensity for quantity. Depending upon which model (particle or wave) of light is used, intensity is related to the density of photons in the beam or to the amplitude of the light wave.

The spectrophotometer allows one to select a narrow band of wavelengths from a spectral region, and to measure the intensity of the radiation that has passed through a sample. In this way, one can obtain a picture of how absorption varies throughout a given spectral region. A plot of absorption versus wavelength in the visible region is shown in Figure 8-6 for a sample of red dye solution. Plots such as this are called absorption spectra. This particular spectrum shows that red (longer) wavelengths are absorbed very little (transmitted easily), but that other visible wavelengths are strongly absorbed.

There are many different varieties of spectrophotometers. Some are especially designed for measuring absorption in specific regions of the electromagnetic spectrum, but all contain certain common components:
a.A source of electromagnetic radiation.
b. Amonochromator (one color) which produces beams of radiation consisting of a very narrow range of wavelengths within a large spectral region.
c.A detector, which measures the intensity of the radiation it receives.

Many other components, such as sample holders, optical devices (prisms or lenses), and display devices (recorders or meters) vary from instrument to instrument.

Spectrophotometers can be used for both qualitative and quantitative analysis. For example, the interaction of certain substances with certain frequencies of radiation produces characteristic absorption spectra. Using a spectrophotometer, one can easily identify these substances. Quantitative analysis relating the absorption process to the number of absorbing particles present in a given sample can also be developed.


Several companies produce spectrophotometers that operate in the visible range. Rather than examine a specific model, we will consider instrument components in a general way. While reading the text, refer to Figure 8-7.

a. Source of electromagnetic radiation-a tungsten filament lamp provides light of the proper wavelengths and intensity. Usually the lamp is powered by a constant voltage transformer to minimize fluctuations in the output intensity of the lamp.

b.Monochromator-a simple device consisting of an opening, called the entrance slit, through which some of the white light from the source enters. The light is focused by one or more lenses or mirrors onto the surface of a dispersing element. This element can be a prism (Figure 8-7), a reflectance grating, or transmission grating (Figures 8-8a, b). In any case, the white light is dispersed into a spectrum, a portion of which falls on a second opening called the exit slit. With a wave length control knob one can select which portion of the spectrum leaves the monochromator through the exit slit. This can be accomplished by rotating the dispersing element or by leaving the dispersing element stationary and moving the position of the exit slit.



c. Sample container-Glass or quartz containers called cuvettes are suitable for liquid samples. The walls of cuvettes should be of uniform thickness. This is required because the glass does absorb some visible radiation and any irregularities will change the intensity of light ultimately reaching the detector.

d. Detector-a device that responds to the amount of radiation striking it. A common detector for visible light is a phototube. It responds to the intensity of light falling on it by generating an electrical signal (current). The signal is then amplified to a sufficient magnitude to be registered on a meter or other display device.

The quality of an instrument depends to a large extent on the quality of the dispersing element. Most instruments utilize gratings rather than prisms although both produce basically the same result. A transmission grating is a flat piece of glass with a series of closely spaced lines (100 to 1400 lines per millimeter) etched into its surface. The white light passes through the transmission grating, and by a phenomenon called diffraction, is dispersed into a spectrum. A reflectance grating operates in essentially the same way except that its surface is mirrored as well as etched, and the diffracted light is reflected by, rather than transmitted through, the grating itself.

Carefully follow the light path shown in Figure 8-7 beginning at the source, through the monochromator and sample, and onto the detector. With the sample placed between the second slit and the detector, some fraction of the light may be absorbed by the sample. The light not absorbed by the sample is transmitted and reaches the detector. The ratio of I (the light intensity with the sample in position) to I₀ (the light intensity without the sample in position) is called transmittance.

You can plot A versus wavelength or % T versus wavelength (both of these plots are referred to as absorption spectra); but they are quite different in appearance, as you can see from Figure 8-10.

  In this experiment, you will learn how to use a spectrophotometer that operates in the visible region of the spectrum. Several solutions will be prepared and their absorption spectra determined.

Since there are several types of spectrophotometers commonly in use, your instructor will demonstrate the use of your particular instrument. The following points apply to most spectrophotometers:
1. Warm-up time for the instrument: This is the time required for the electrical components and the light source in the instrument to become stabilized. Modern solid state components reduce this time to a few seconds.

2. Wavelength selection: The desired wavelength is set by turning a knob that rotates the dispersing element in the monochromator.

3. Zero transmittance:I The instrument must be set so that the meter reads zero transmittance (or infinite absorbance) when no light is striking the detector. Before setting zero transmittance, a shutter is placed in the light path.

4. 100% transmittance setting: The meter must be set to read 100% transmittance (zero absorbance) when the blank cuvette is in place. If a solution is investigated, the blank cuvette is a cuvette filled with solvent only (no solute). The 100% transmittance must be set each time the wavelength is changed because both the solvent absorption and detector response differ from wavelength to wavelength.

5. Handling of cuvettes: Although some cuvettes look like ordinary test tubes, they are specially constructed for optical work. You should always place them into the sample holder in the same orientation. Remember that fingerprints and dust absorb light, so always be sure that the surfaces of the cuvettes are kept clean.