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Mass Spectrometry & Spectroscopy
From Containment to Immersion A history of sample handling in optical analysis
Author: Ray Wood, Hellma UK Ltd
Whether your application is for simple photometry or UV/Vis spectroscopy; for fl uorescence measurement or light scattering; for polarimetry, circular dichroism, cytometry, imaging, or any of the many other forms of optical analysis, your sample must always be presented to the instrument in a container that is as transparent to the light source as possible and one that has a minimal impact on the measurement process.
In the earliest photometers, like the EEL (Evans Electroselenium Ltd) model shown in Figure 1, ordinary test-tubes were used, because that was the only transparent sample holder available at the time; and the resolution of the instrument was too low to detect the problems and errors they introduced anyway.
Figure 1: An early photometer from Evans Electroselenium Ltd
As instrument performance improved specially selected and matched test-tubes, with index marked orientation had to be used. But eventually the benefi ts of fl at optical-grade windows in square-section cuvettes were the
only way to keep up with the ever improving specifi cations. To make the most of the more sensitive detectors being developed cuvettes manufactured with special optical glass and high-quality quartz were introduced to expand the measurable range further into the UV and infra-red regions.
The earliest cuvettes were nearly all manufactured from windows glued to a frame, but many soon came un-stuck, especially when used for applications with acids, organic solvents and other aggressive samples! With the development of novel manufacturing techniques, introduced by Hellma GmbH, modern cuvettes no longer needed to be manufactured with the use of glue or adhesive. The special wringing and fusion processes that were developed resulted in cuvettes being produced using just one material and in what is effectively a one-piece construction.
From large blocks of glass or quartz, a section is fi rst cut down to a workable size using various techniques (Figure 2). Special cutting and lapping procedures are then used to get the parts to the exact size. After this the optical surfaces, and the edges to be joined, are polished to a very fi ne fi nish ensuring they are completely free from defects and have a level of fl atness that is better than 1µm. To ensure these demanding requirements can be met specially designed machinery, operated by
experienced and highly qualifi ed operators has to be used.
Finally, before assembly all the parts are cleaned in an automated, multi-stage, precision-cleaning process that is computer controlled and under clean-room conditions. Because of the flatness and cleanness of the surface finish the edges can then be joined by wringing; a process that uses the forces of molecular attraction to hold together and seal the joints.
Figure 3: Cuvettes being stacked in an oven for the fusion process
To ensure these wrung joints can withstand the physical, mechanical and chemical demands, that they may be subject to in use, the joints are then thermally bonded. This is a fusion process that is carried out by heating the parts to a level well below the material’s melting point to ensure no changes occur to the quality of the prepared optical surfaces (Figure 3). Complex items often go through this cycle many times, while special temperature controlled annealing processes are required for stress- relief in those items destined for use in applications such as polarimetry and circular dichroism. Even for a standard cuvette around 100 work steps may be needed from raw material to the fi nished item.
Throughout the production process Quality Control must ensure that the dimensions and tolerances of the original design are met. An obvious candidate is the optical path-length that will have a direct bearing on the accuracy of your readings, for short path-length quartz cuvettes tolerances as low as +/-0.003mm can be achieved and even up to path-lengths of 100mm they can be as small as +/- 0.02mm. But a spot measurement of the path-length is pointless if the windows are not parallel and their surfaces are not fl at. The interferometry scans of window fl atness in Figure 4 compare a top quality quartz cuvette (a) with a similar one of lower quality (b).
The transmission of quartz cuvettes is around 87%, even down into the UV region, this ensures a good level of light passes through your sample and on to the instrument’s detector (most of these losses are due to refl ection off the external and internal surfaces). Compared to this, modern UV plastic cuvettes have transmission levels as low as 50% in the UV, signifi cantly reducing the dynamic range of your instrument at these wavelengths. A limited choice of path-lengths is available in plastic versions, while the path-length tolerances, inherent in the moulding process for them, are also much greater. Some manufacturers of plastic cuvettes ensure every cuvette in each pack comes from the same mould cavity to help reduce this variation, while regular re- blanking is advised, especially when changing from one batch to another.
Figure 2: Cutting a block of Quartz to a workable size
Reducing the width of the central chamber of a cuvette is one way to reduce the overall sample volume required to make a measurement. However where the light beam is wider than this aperture light can pass through the walls of the cuvette without going through the sample, leading to signifi cant measurement error. Although many plastic cuvettes have frosted areas around the central sample chamber light can still pass through this to reach the detector. For this type of low-volume cuvette a separate masking aperture should always be fi tted to the cuvette holder; however many low volume glass and quartz cuvettes are supplied with black material
LABMATE UK & IRELAND - SEPTEMBER 2014
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