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Spectrophotometers

Spectrophotometers


Spectrophotometers

A spectrophotometer is an instrument which is able to transmit light of a given wavelength into a sample and analyse the output. The difference between the two readings gives the light absorbed by the sample. The most common spectrophotometers are used to measure absorbance, but there are machines which also measure transmission and reflectance from surfaces.

In simple terms, a spectrophotometer is a light source which shines light onto a device which is able to split it up into its component wavelengths. Light of the required wavelength can then be focussed onto the sample. The light absorbed by the sample can be measured and the concentration of the sample can be determined. Some reactions can absorb light in the visible range of the spectrum while others absorb in the ultra violet range.

The absorption of light by the sample is directly related to the concentration of the analyte in solution. Every analyte in solution has a maximum absorption at one particular wavelength and at this wavelength it should have minimum transmission. This means that when light is incident upon it of that wavelength, then most of the photons are absorbed by the solution with very few getting through. If there is a variation in the expected absorption profile, then this means that the solution is either a different concentration, or it is contaminated.

Many different types of spectrophotometer are available to suit all budgets. The simplest types of machines are colorimeters. These are dedicated to simple determination of the concentration of a solution. A colorimeter consists of a light source, which may be a single, low-voltage lamp. A set of colour filters provide the wavelengths of light required. These may be in the form of an optic which is inserted into the machine as required through an external port, or they might be already located within the colorimeter and selected by means of a dial. 

Monochromatic filters allow a narrow wavelength of light to pass through to the solution. These work in the visible range, that is 400 to 700nm. If wavelengths are required outside this range, then a different light source is needed in addition to different filters.

A more advanced spectrophotometer may consist of two or more light paths and detectors, to allow for the measurement of more than one sample and a blank solution to be measured alongside them. Samples are held in containers known as cuvettes. 

A cuvette is a small circular or more usually, square tube which is made from glass, plastic or quartz. Glass and plastic cuvettes are used when light in the visible range is used. For ultra violet light, below 380nm, quartz cuvettes are used because the glass will absorb much of the light below the visible range. The dimensions of the cuvette are important. In order to determine the concentration of the solution accurately, a known path length is needed so most cuvettes provide a 1cm path length through the solution. Each cuvette has clear sides and  serated or opaque sides. They must always be held using the opaque or serated sides to leave the clear ones clean.

Needless to say that the cuvettes used must be completely clean. Any amount of dust or marks or fingerprints on the sides will refract the light away from the solution and cause a false reading. It is for this reason that plastic cuvettes are usually single-use, disposable ones and glass and quartz cuvettes are meticulously cleaned before and after use.

The concentration of a solution is calculated using a relationship known as the Beer-Lambert law. This law states that at a given wavelength the absorbance of light through a sample increases linearly as the concentration of the sample increase. Transmission through the sample decreases exponentially as concentration increases.

The Beer-Lambert law provides a way to determine the concentration of a solution. It depends on knowing the molar absorption coefficient for any compound. This is the amount of light which would be absorbed if it is travelling through a path length of 1cm of a molar solution. From this, we have the following equation:

A = cl

Where A is the absorbance of the sample. This is a logarithmic relationship between the light entering and emerging from the sample.
 (the greek letter epsilon) is the molar absorption coefficient
c is the concentration and l is the path length. This is usually 1cm.

From this law, it can be seen that the concentration of a solution can be easily worked out by measuring the absorbance of a solution.

Visible and UV spectrophotometers are available as single beam and double beam instruments. They have a working wavelength range of 190 to 1100nm. Detectors can be a silicon photodiode or a photomultiplier. Both of these types of detectors convert incident photons into an electrical output which can be used to determine how much light has been absorbed by the sample.

Atomic Absorption and scanning spectrophotometers are able to use oxygen air-acetylene flame analyses to provide high precision analysis of the sample. This includes the ability to determine the optimum wavelength to analyse any given compound. 

Atomic emission spectrophotometers work by using light to excite atoms within a solution. The light emitted as these excited atoms revert back to their lower levels can then be analysed. This can be used to examination contamination within substances such as food items or natural minerals. Spectral analysis of this type requires a high level of sensitivity because of the narrow range of the emission lines in the spectra.

Fluorescence spectrophotomers work under similar conditions. Atoms within the solution are excited to release electrons by incident light. As they decay back to their original states the emitted light is captured by the detectors in the spectrophotometers. They are able to make use of laser or xenon arc lamps to emit light within the correct wavelengths.

Spectrophotometers can be simple devices which are used for single functions, or extremely complex, flexible instruments which can provide sophisticated, precise and high-level information about the substances under examination.