UV-Vis Spectrophotometers¶

Like most spectroscopy, UV-Vis respects the beer lambert law and its limitations, Including:

Linearity breaks down when a solution is too concentrated
- As intermolecular distances decrease, the charge distribution of the molecule changes and so does \(\varepsilon\)
Linearity can break down when a reaction is taking place
- Something as simple as protonation/deprotonation of the analyte with the solvent can drastically change the linearity of the sample.
As discussed in Monochromator, it is practically impossible to select an individual wavelength of light from a broad source, so any
reading of absorbance/transmittance will be the average over the provided range, drastically reducing the resolution
While increasing the path length can negate a lower concentration solution, it can also increase scattering, causing a loss of linearity to occur

The UV-Vis Spectrophotometer¶

There are four types of UV-Vis spectrophotometer:

These are the most basic, 0% T is read with the shutter closed 100% with the shutter open and no sample
The blank spectrum must be recorded first and is subtracted from the measure spectrum
Can be slow to operate, requires more intervention

The monochromatic beam is split with a beam splitter and one beam passes through the sample while the other passes through the blank
Two photodetectors are utilised and the difference between the two is compared

The double-beam in space has the disadvantage of using two different photodetectors, each with their own response curves and tolerances. It also assumes that the beam splitter will allow exactly 50% of the light in either direction
The double-beam in time physically changes the beam to pass through the blank, then the sample with a certain periodicity using a rotating shutter
The photodetector knows this periodicity and will take intermittent readings for the blank and sample individually

Multi-channel instruments are Photodiode Array based and will measure the entire spectrum at once with whatever accuracy is provided by the array utilised

The lamps usually consists of two types of bulbs

Provides full coverage of the visible band (350 - 800 nm)

Used for the UV range (160 - 350 nm)

An alternative for high intensity spectroscopy is to use a xenon lamp (200 - 1000 nm), however these are expensive and short-lived

Must be completely transparent to the wavelengths being measured, as such quartz is used rather than glass
They can come in a variety of path lengths to accommodate various concentrations of solutions

Photometers can only measure light at a single wavelength using filters, however are cheap and have a high energy throughput
Spectrophotometers use a monochromator to scan through a range of wavelengths making them more versatile, and more expensive. Complex optics give them a lower S/N

Use a mercury lamp and filter specifically for the 254 mm emission reading
Useful for identification of organic molecules, since they absorb around this region