Conclusions

Having utilised the Protasis DFGF supporting equipment and software, standard procedures have been developed for the use of the high voltage supply.

Implementing the Rheos Flux 2200 pump8 _{has enabled dynamic control over the}

flow rate of buffer to ±0.1µLmin-1 _{and therefore the ability to adjust the position of}

82 This is particularly important in this DFGF configuration as in later DFGF devices developed by Ivory et al.6,31,32_{, there are up to 32 individually driven electrodes to} provide an electric field with a high resolution across the entire channel compared to only using four electrodes as in this case. However even though only four high voltage supplies were used, effective separations and focusing of samples has been observed with relatively smaller dyes and larger proteins. Controlling these

electrodes with the use of the Protasis9 _{Labview Ucontroller software has offered a}

high level of control over the four electrodes with facile adjustments using the automatically calculated voltages for a specified electric field gradient, and the manual mode enabling independent adjustments to fine tune the dynamic electric field to improve and manipulate individual bands of sample in situ.

Observing the separation and focusing of samples was previously monitored by, and the data interpreted from, manually processing a sequence of more than 200 images. Development detailed above has seen significant improvements in speed of data interpretation from the analysis of images by utilising a series of software tools to automate each step. For the first time an entire DFGF experiment has been plotted on a graph to enable the viewing of the changes in focal point and relative intensity of the bands over the entire separation. In addition, the processed data can also be interpreted further, and individual profile plots from each image are available after analysis without the need for further processing. With the new graphical presentation of the data, the method of which the images were captured was also improved to ensure stability in detection over time. Nevertheless, the reflectance method of data capture has limitations when detecting samples which are less visible against the packing material. Also this detection is only qualitative.

83 Innovations in previous work saw the addition of a quartz window to the separation channel of a DFGF device which facilitated imaging in the UV end of the

spectrum by fluorescence quenching25_{. This gave advantages in detection by}

increasing the resolution and enabling the detection of unstained species, however, this technique was limited by image capture. In the devices discussed in this chapter the simple reflectance method was the only method available because of the material used in their construction being opaque to UV light.

The packing method described in this chapter was an improvement on the previous method of solely driving the slurry with compressed air. The addition of the vibrating motor and the sonic probe significantly lowered the number of failed packing attempts.

Further improvements in packing were observed when packing the monodispersed 80µm particles. The synthesis of this material explored a range of different drop generating techniques.

The most accurate and high turnover droplet generator was the Domino30 _industrial

printer giving highly accurate well defined droplets at a high velocity and volume. On the other hand, there were issues with the Domino printer as the narrow tubing and complex components often became blocked by the silica sol. A simple method for drop generation was explored utilising a pneumatic spray bottle, which delivered high volumes of particles, but at the cost of lower monodispersity. Experimentation with the monodispersed particles packed in the first generation device yielded faster and more reliable packing and a more uniform flow profile through the separation channel. This was observed from the improved intensity of focused bands, and the reduction in the time taken for analytes to focus into bands.

84 Finally, with limitations and issues with both the first generation and Protasis devices being inherently caused by the size and materials they are constructed from, a new approach was needed to solve these limitations. As shown in the theory discussed at the beginning of this chapter, the technique is independent of total column length, therefore, a smaller channel with a length long enough to house the minimum of five electrodes with space between them to form a gradient will be sufficient. This is shown experimentally by the fact that separations have been manipulated to take place in an area ~3-4cm long and as the electric field is dynamic it can be manipulated to a high extent over that small range. Moreover these larger channels require more power, meaning higher voltages, more joule heating, and more supporting equipment. Considering these points, and following

on from work done previously by Bartle and Myers21_{, a new approach lends itself to}

a microfluidic device which will be described in the next chapter. Earlier DFGF devices depended on a packing material or monolith to narrow the diffusion pathway for the flow thought the separation channel.

85 2.8 References

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87