Determinación de los indicadores dinámicos de rentabilidad.

In their work on fluorescence detected circular dichroism (F DCD), Muto et al.

used the Stoke’s shift of fluorescence emissions to separate them from transmitted incident light by placing 100 mm of saturated NaNO₃ solution between the sample and the detector [49]. We use similar reasoning in our F DLD experiments, where we use long-pass edge filters with a high optical density through their attenuated wavelengths and as high a cut-off gradient as possible, to maintain the PMT facing the propagation direction of the incident light. The initial motivation for this was that when using a mechanical stretcher to orient a sample (Figure 2.3), it is not possible to detect a signal at 90 as it is blocked by the apparatus, however, we

have found that it also gives better results when using solution samples oriented by Couette flow.

When selecting a long-pass filter, the first thing that needs to be considered is the sample’s excitation and emission wavelength maxima. These can be measured using standard absorbance and fluorescence spectrophotometers. The cut-off wavelength of the long-pass filter is selected to lie between these maxima. It is preferable that the Stoke’s shift of the fluorophore is large, so that that there is no overlap between the excitation and emission peaks. This is generally the case when the fluorophore is excited into the S2 state (Figure 2.5), although it is less common when exciting into S1. When there is overlap between the excitation and emission spectra, a trade-off has to be made between the wavelength range of the measured spectrum and its signal intensity. Selecting a filter with a steep cut-off gradient reduces the sacrifice of both these parameters. The long-pass filters used in this work are given in Table 2.1.

Table 2.1: Long-pass filters used for fluorescence detected linear dichroism

Filter Cut-off Wavelength (nm)

SCHOTT OG570 570 ThorLabs FEL0400 400 ThorLabs FELH0450 450 ThorLabs FEL0500 500 ThorLabs FEL0550 550 Semrock FF01-300/LP-25 300 Semrock FF01-341/LP-25 341 Semrock FF02-409/LP-25 409 Semrock LP02-568RU-25 568

In Table 2.1, the SCHOTT OG570 filter is included as an example of the coloured glass filters we used. This type of filter performs very poorly in our setup, as they transmit too much light through their attenuated wavelengths, and so spectra

measured using these are a mixture of LD and F DLD. All of the ThorLabs filters listed performed very well, with their maximum transmission intensity through their attenuated wavelengths being 0.01% (optical density (OD) 4). These filters blocked light over a large wavelength range, though their cut-off gradient was found to be quite shallow, with ⇠15 nm between their maximum transmission and rejection regions.

The steepest cut-off gradient filter in Table 2.1 is the Semrock Razor Edge LP02-568RU-25, with only 3 nm between its maximum transmission and rejection regions. This filter, however, is designed for Raman spectroscopy and has a poor range of blocked wavelengths. Much better performers in this respect are the other Semrock filters, which are all from the BrightLine series. They have the range of blocked light achieved by the ThorLabs filters (with an increased blocking efficiency of OD 5-7), with only a 6 nm cut-off gradient. On occasions when an individual filter’s blocking range did not cover the region we wished to measure, it was found to be very effective to ‘piggyback’ one filter on top of another, such that the incident light hit the filter with the longest cut-off wavelength first. This technique was used frequently throughout this work.

Once an appropriate long-pass filter has been selected, it needs to be fitted directly in front of the detector. As the filters given in Table 2.1 require that they are set at exactly 90 to the incident light to transmit all polarisations of light equally, we custom made a holder that fit them into a Jasco J-815 spectrometer. A diagram of the filter holder is given in Figure 2.7.

40

29 24

25

4.5 4.5

Figure 2.7: Drawing of the custom made long-pass filter holder used to fit filters into a Jasco J-815 spectrometer. Once the 25 mm filter was placed in the mouth of the fitting, a rubber o-ring was used to hold it in place. All dimensions shown are in millimetres.

Once the filter and sample are in place, the other adjustments are made using the instrument’s software. So that transmitted light is not recorded, the wavelength range must start before the cut-off wavelength of the long-pass filter that is in place in the instrument — the minimum number of wavelengths before depends on the steepness of its cut-off gradient — and the stop wavelength is set at the shortest wavelength of the range. Channel two of the instrument is set to record either the high tension voltage (HT) or the DC output from the PMT, depending on whether the DC is set to be constant (using Equation 2.15) or the HT is set at a constant value (normally 600 V), respectively. In either case, it is obvious from the trace of this channel whether the filters being used block transmitted light over the selected wavelength range, as regions where they transmit light will be seen as a sharp decrease in the HT value or a sharp increase in the DC output.

Whether to set the instrument to measure in fixed HT or fixed DC mode depends on the sample one is using. The reason for this, and an introduction to the theory of F DLD is given below.