Initial design

Work carried out by Rachel Marrington (a previous PhD student within the Rodger group) acted as the initial basis for this investigation. Her investigation proved the concept of using capillaries for polarised spectroscopic applications, which ultimately

The initial caCD cell (caCDRM) (figure 2.1) was made by a company called Dioptica Scientific Limited for researcher Rachel Marrington. Aluminium was used for the construction of the cell, with the lens holders, capillary holder and the base-plate itself all fabricated from that material. The slide-way was also constructed from aluminium, with a series of holes for securing the optical component holders in place. For the optical elements themselves (i.e. the lens holders and the capillary holder) a

further step of anodising the aluminium was taken, in order to minimise any scattering effects that may take place should any stray light hit the metal of the optical component holders.

The original design for the caCD cell consisted of two double convex (DCX) lenses, produced by optical suppliers Edmund Optics in series, with a holder for the capillary placed between them. The light path enters the first lens (seen here furthest from the camera) which is then focused on to the capillary. This first lens is named the

focusing lens (Figure 2.1). This takes the slightly diverging light beam of the

spectrophotometer and focuses it onto a central point in the capillary. Once the light beam has passed through the capillary the now significantly divergent light passes through the second lens, named the collecting lens. This lens takes the light and

limits the degree of divergence experienced by the light beam, so that it enters the detector.

This cell design worked on the principle that the focusing lens would ensure the light beam went through the sample held in the capillary. After leaving the capillary, the now divergent light beam would be harvested by the collecting lens. The light beam would remain within the confines of the optical system due to the parallelism of the optical components (Figure 2.2). This method of caCD had many advantages. First

and foremost, light passed through the capillary in a manner which would generate CD data. In addition to this, the use of the collecting lens meant that the photon flux was reasonably high for the system (photon flux is the number of photons over a time period, with SI units of s-1, see http://goldbook.iupac.org/P04636.html for more information). For a polarised spectrophotometer, this term is shown by the HT (or more formally known as the high tension voltage), which is proportional to the absorbance of the sample.

Figure 2.1: The focusing lens of the caCDRM instrument. Note that the lens is positioned precisely in the centre of the capillary.

Figure 2.2: caCDRM image showing the slide-way of the instrument. The groove permits the addition of different optical components into the light path.

As the photon flux of a CD instrument decreases, the voltage passed through the detector increases to compensate. An HT reading higher than 600 V does not produce reliable CD data on a bench-top instrument, as the photon flux reaching the photomultiplier tube is too low to be accurately ‘multiplied’. This creates a non- linear relationship between CD signal recorded and the actual sample absorbance. Keeping the HT as low as possible is important to ensure the widest possible wavelength range for data collection: as wavelength decreases, the amount of information accessible for the structure of the biological macromolecules contained within the sample increases. For this reason, the design of the caCD cell should maximise photon flux whilst retaining spectral shape and quality.

In order to test the instrument, Na [Co (Edds).H2O] 12 spectra were gathered to ensure

the suitability of the caCDRM cell. This standard, developed by the Scott group in collaboration with the Rodger group at Warwick University, allows CD spectroscopists to test the effectiveness of a CD instrument across a wide wavelength range (160 – 750 nm). The standard consists of solutions of independent R,R

enantiomers and a S,S enantiomers, which give an equal but opposite spectrum to one another. Used in conjunction, the two enantiomers can show any errors with the performance of either the CD instrument or, critically for this application, any optical systems accessories placed within the light path of the spectrophotometer. In this case, we wished to compare the data quality between a standard CD cuvette and the prototype caCD cell. If the cell design worked then there should have been no difference between the caCD and the cuvette spectra, after differences in path-length had been accounted for.

Figure 2.3: Na [Co (Edds)].H2O at 0.55 mM spectra acquired through both cuvette CD and caCD. The caCD spectra were acquired using the Marrington design of caCD base plate.

The path-length of the capillary was 1.12 mm, determined through analysis with potassium

dichromate. Capillary spectra scaled using a 0.892 correction factor to allow for differences in path- length.

The path-length of the cuvette was 1 mm, verified via analysis with potassium dichromate.

As can be seen from figure 2.3, the caCDRM cell did not produce sufficiently high quality CD spectra in terms of data correlation between the capillary cell and the cuvette. The low-energy data values are in fairly good agreement, but the wildly varying magnitude and shape of the CD signal at lower wavelength shows a poor correlation between the cuvette spectrum and the capillary spectrum of the S,S CoEdds stereoisomer (the R,R data by contrast was in reasonable agreement). Whilst the concentrations of the two enantiomers were not the same (hence the difference in spectral magnitude between the R, R and S, S forms), the caCD data does not overlay

-20 -10 0 10 20 30 240 320 400 480 560 640 720 Capillary CoEdds R,R Capillary CoEdds S,S Cuvette CoEdds R,R Cuvette CoEdds S,S Cuvette water baseline

Capillary water baseline

C D / m d e g Wavelength / nm

the cuvette data with any single scaling factor, showing that there was an issue with the design of the caCDRM cell.

After investigating the optical setup of the caCDRM, it became apparent that the amount of light hitting the detector after it had passed through the caCDRM was not sufficient to generate a high-quality CD spectrum. The relatively short focal length of the focusing lens resulted in a highly divergent beam once it had passed beyond the capillary. The collecting lens was therefore not sufficient to ensure all the light reached the detector. The problem with the system was that the capillary itself was exerting a lensing effect upon the light beam. The approach taken in the design of the caCDRM would be effective in a system where the only optical changes experienced by the system were from that of the lenses – this was clearly not the case here. As a result, it was decided that this cell design would be abandoned in favour of a first principles approach to caCD, based upon an investigation into the optical behaviour of a capillary cross-section.