As discussed in Chapter 1, the majority of modern LC techniques are based on the use of silica based stationary phases, with polymer sorbents being the second most abundant. Since these silica and polymeric sorbents share some certain disadvantages in terms of their applicability under conditions of high temperature, pressure and aggressive mobile phases, carbon materials, especially diamond, are considered as a promising alternative.
Advantages of diamond based adsorbents for application in HPLC, such as chemical and mechanical stability, thermal conductivity and the possibility of surface modification have been discussed previously in Chapter 1. However, there are also several problems related to the use of HPHT diamond in HPLC, which need to be solved in order to prepare a suitable chromatographic column. First of all, HPHT diamond is a hard but brittle material, and no spherical particles are commercially available. Secondly, due to its non-porous structure, the surface area of HPHT diamond is quite small (5.1 m2·g-1 for 1.55 µm particles), so small particles have to be used in order to achieve sufficient column loading capacity. This also means that the application of ultra high pressure (UHPLC conditions) should be considered due to high backpressure associated with small particles. Furthermore, the properties of the diamond surface are hard to control, since they depend largely on manufacturing process and conditions of synthesis. Until now no standard approach has been accepted for the preparation of a uniform diamond surface [1]. Despite the fact that the development of a standardisation technique for diamond materials is outside of scope of this thesis, it is important to acquire all possible information about HPHT diamond properties. This information is especially important since chromatographic selectivity and adsorption properties, which are investigated here, cannot be explained without a complete understanding of the surface chemistry. Additionally, such information is required for the comparison of results obtained here with those obtained by other research groups using different sources of diamond. Overall, in the first part of this Chapter, the preparation and surface characterisation of HPHT diamond as a stationary phase will be presented. Special attention will be paid to the purification of diamond material and size fractionation of particles.
66 Another important aspect in the development of a new stationary phase is optimisation of the column packing procedure, which will be considered in the second part of this Chapter. The ultimate goal will be to elucidate a repeatable and reliable procedure for the preparation of columns with maximum possible efficiency. There is a plethora of information in the literature on column packing with conventional silica and polymeric stationary phases [2]. However, diamond is a very different material, and column packing conditions have still not been optimised. The development of a packing procedure for diamond particles is a non- trivial challenge which requires resolution of several complicating issues.
Firstly, diamond is a brittle material, and the pressure wave packing procedure, which is commonly used for packing silica, is not applicable. There are already some indications in the literature that a pressure wave may result in diamond particle fractures and formation of the ultrafine fragments which would negatively affect separation efficiency [3]. Another important issue is the high density of diamond (3.5 g·cm-3), which makes preparation of the stable diamond slurries complicated. Therefore, it is necessary to choose an appropriate slurry solvent with the required density and viscosity in order to avoid fast sedimentation during column packing. Otherwise, sedimentation can influence the quality of the bed and resultant column efficiency. Finally, other parameters, such as temperature, pressure and column conditioning need to be optimised, since they can also significantly influence both column efficiency and chromatographic peak shape.
Overall, there were a number of research problems that required addressing, and they are, therefore, investigated in this Chapter, including:
Development of a purification procedure for HPHT diamond
Characterisation of the purified diamond with various physical and chemical methods in order to establish its surface properties
Selection of an appropriate slurry solvent for HPHT diamond, to reduce sedimentation rate and provide stable suspensions and dense firm sediments
Preparation of HPHT diamond particles with improved shape and size distribution
Elucidation of column packing procedure for the HPHT diamond stationary phase. Special attention will be paid to the investigation of the influence of packing pressure, temperature and after-packing column conditioning on the column performance.
Comparison of the prepared HPHT diamond columns with other commercial and test column in terms of their performance and efficiency.
67 3.2. Experimental
General information on materials, chemicals and instrumentation is summarised in Chapter 2. Information on the preparation of HPHT diamond material (including purification and fractionation), methods of investigation (titrations and sedimentation analysis), calculations (particle size distribution, void volume and phase ratio) and column packing procedure are also given in Chapter 2.
3.2.1. Calcination of HPHT diamond
Calcination of HPHT diamond was done using Woodrow AF-3 furnace (Woodrow Kilns, Bankstown, NSW, Australia). Air and UHP N2 were used. 1 g of diamond was used for each run. The heating rate was 5 °C per minute until a target temperature was reached (400 °C, 600 °C, 700 °C, 800 °C or 900 °C). After holding the target temperature for 1 hour, the sample was cooled down to room temperature at a rate of 10 °C per minute.
3.2.2. Determination of adsorption capacity of diamond
Adsorption of aurintricarboxylic acid (ATA) was used in order to determine the adsorption capacity of HPHT diamond and how it is affected by calcination. ATA was purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). 10 mL of 0.1 mM solution of ATA (0.422 mg of ATA) in water was added to 1 g of the HPHT diamond sample. After sonication for 30 s the mixture was set to equilibrate for ~10 min and finally centrifuged. The supernatant was separated and used for the determination of the remaining ATA concentration.
In a 25 mL volumetric flask, 5 mL of pH 5.0 sodium phosphate buffer (0.05 M) was placed, to which 15 mL of 0.25 mM Cu(NO3)2 was added and mixed well. 2.5 mL of the supernatant with the unknown ATA concentration was added, and the volumetric flasks was filled with DIW up to 25 mL. The flask was shaken vigorously and the formed Cu-ATA complex was detected at 520 nm using Metertech SP-8001 spectrophotometer (Nankang, Taipei, Taiwan).