In a double beam system monochromatic light from either a single or two identical monochromators pass through both a reference and sample compartment. The intensity of these two light beams is then measured by one or two photo detectors. The sample beam intensity is compared with the reference one as a ratio. Double-beam designs include double beam in space and double beam in time.

In a double beam in space spectrophotometer, the light is split with a half-silvered mirror, called a dichroic mirror. The dichroic mirror splits the beam so half the light passes through the specimen aliquot and the other half to the reference cell. Both beams pass simultaneously through different components separated in space. This arrangement compensates for changes in intensity of the light source. However, photo detectors may age differently, resulting in different responses. This design also does not compensate for fluctuation in the photo detector output. In a double beam in time spectrophotometer, the light beam is split with a rotating chopper that alternately presents a mirror and an opening. One beam passes through the sample and the other through a reference solution or blank. The open part of the chopper passes the beam directly to one path, while the mirrored part reflects the beam to the second path. Each beam, consisting of a pulse of electromagnetic radiation separated in time by a dark interval, is directed onto a photo detector. The output of the detector is an alternating current that has amplitude proportional to the difference in intensity of the two beams.

To produce a spectral absorbance curve, a drive motor is geared to the diffraction grating. It is turned slowly in the High path so that light of various wavelengths pass through the exit slit of the monochromator. The light is passed through the cuvette compartment in succession. Spectral scans require adequate resolution for interpretation. To resolve absorbance peaks, the bandpass must be short. Often, a very sharp, narrow peak may be characteristic of a compound. This peak may be completely missed if a wide bandpass were selected. It is possible to use, as a calibcaSmg standard, almost any substance that has a spectral absoebance curve.

It is sound practice to calibrate at more than one wavelength on any spectrophotometers. Didymium is a commercially available calibration material. Its principal absorption peak is around 500 nm, and there are four wavelength maxima that may be chosen as calibration points. Another widely used wavelength calibration material is holmium oxide glass. It has about 10 sharp major absorption peaks. Two frequently used wavelengths are 360 and 536 nm. Therefore, an advantage of using this filter is that your spectrophotometer can be calibrated near the UV region.

Spectrophotometry Emission Flame Photometry

When a metallic salt is burned in a flame, the heat energy that the atom absorbs derives one or more of the electrons out of their usual orbits. As the excited electrons return to a lower or ground electronic state, they eriut-electromagnetic radiation. The heat energy absorbed and the lighTenntted-aFe characteristic of the atom under consideration. Each metal has its own emission spectrum showing emission at characteristic wavelengths.