Analytical Techniques

5.7.1. Spectrophotometry

Spectrophotometry was used to determine the OD of an algal culture by light scattering. OD, measured in absorbance units (AU), has a linear correlation with the Chl content and the algal cell count of that culture (Figure 5.01). A Lambda 40 UV/Vis Spectrometer (Perkin-Elmer Instruments) running the UV WinLab software was used to measure OD at the algal PSII absorption peak of 663 nm. The OD reading at this photosynthetic wavelength maximum is the most responsive measurement to changes in the optical thickness of the culture (Tamburic et al., 2011b). At 663 nm, 1 AU is equal to 17.9 mg·l-1 _{of Chl, or approximately 26 million algal cells. Algal samples were} analysed in a 1.5 ml cuvette and the instrument was auto-zeroed with the light scattering measurement from a cuvette filled with deionised water. Spectrophotometric OD measurements were also used to calibrate the flat-plate reactor photodiode for experiments at specific light intensities.

5.7.2. Ion Chromatography

Ion chromatography (IC) was carried out using the 882 Compact IC Plus instrument with an 863 Compact Autosampler (Metrohm) running the MagIC Net software. The technique used a Metrosep A Supp 10 separation column, which is capable of analysing compounds whose concentrations differ widely. The concentrations of compounds coming off the column were measured by means of a conductivity detector in units of microSiemens per centimetre (µS·cm-1_{). The sodium-based anion} solution (liquid phase) consisted of 3.2 mM sodium carbonate, 1.0 mM sodium bicarbonate and 5.0 µM sodium perchlorate. Deionised water was used as the rinse and 0.1 M sulphuric acid as the regenerator. The liquid phase was pumped through the instrument using a peristaltic pump at a flow rate of 1.0 ml·min-1_{. Algal samples were spun down in a mini-centrifuge at 13,000 rpm for 10 min:} the purified medium was used for IC and high-performance liquid chromatography and the algal pellet was discarded. Samples of 25 µl were injected into the IC instrument. The anion concentrations corresponded to the area occupied by their respective peaks. These areas were calibrated against concentration using CertiPUR Anion Multi-Element Standards (Merck) at

concentrations of 1 ppm, 10 ppm, 50 ppm, 100 ppm and 200 ppm (1 ppm = 1 mg·l-1_{). The anions} coming off the column included PO43- and SO42- with a retention time of 12.2 min and 22.0 min respectively. It was subsequently determined that the phosphate concentrations present in the algal growth medium were not sufficiently high to be tracked by this technique. IC was therefore primarily used to measure the sulphate uptake during algal growth. The IC signal also contained peaks at 2.1 min and 3.9 min, which were believed to correspond to chloride (Cl-_{) and CH3COO}- respectively. It was not possible to calculate the concentrations of these ions from the conductivity signals generated using the A Supp 10 column. A representative IC result, showing the Cl-_, CH3COO-, PO43- and SO42- conduction peaks, is presented in Figure 5.06.

5.7.3. High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) was performed using the Hewlett Packard Series 1050 instrument running HPCORE ChemStation software. The technique was based on a well- established procedure designed to investigate the concentration of fermentative products such as organic acids and alcohols (Ding et al., 1995). The Aminex HPX-87H Ion Exchange Column from BioRad (HPLC Organic Acid Analysis Column) was used at a temperature of 58.0ºC together with a BioRad guard column. Compounds were detected by means of an ultra-violet (UV) detector operating at 210 nm and measuring light absorption units of milliwatts per centimetre squared (mW·cm-2_{). The liquid phase consisted of 4 mM sulphuric acid, which was peristaltically pumped} through the column at a flow rate of 0.6 ml·min-1_{. Having been spun down at 13,000 rpm for 10} min, 50 µl medium samples were injected into the instrument by means of an autosampler. HPLC was principally used to measure the acetate (CH3COO-) uptake during algal growth. A strong acetate peak was observed with a residence time of 15.1 min and the peak area was calibrated against acetate concentration using 20,000 ppm, 5,000 ppm 1,000 ppm and 200 ppm standards prepared from 99.99% pure acetic acid. This HPLC technique was also used to measure the fermentation products during anaerobic H2 production, but those experiments fall outside the scope of this thesis. A representative HPLC result is depicted in Figure 5.07; the CH3COO- UV peak dominates all other signals.

Figure 5.06: Representative ion chromatography (IC) results

5.7.4. Mass Spectrometry

The mass spectrometer (MS) is an instrument that identifies the chemical composition of a compound or sample by measuring the ionic mass-to-charge ratio of its constituent elements. The MS contains an ioniser to chemically fragment the compound into its charged particles, a detector to capture those particles and a suction pump that creates a vacuum system between the ioniser and the detector. Ions are separated on a mass basis; they impact the detector, inducing a current or a charge (Beckmann K. et al., 2009). A compact quadrupole Pfeiffer Vacuum Prisma MS running Windows Quadstar 422 software was used in this thesis.

Injection MS was used to identify the components of the gaseous product emitted by a sulphur- deprived C. reinhardtii culture. An argon (Ar) carrier gas flow of 25 ml·min-1 _{was fed into the MS,} which was set to continuously scan across all mass-to-charge ratios and take measurements of the relevant atomic masses (H2, water, N2, O2, Ar and CO2; atomic mass = 2, 18, 28, 32, 40 and 44, respectively). Figure 5.08 shows a representative result of this scan; it represents the injection MS background measurement. A gas-tight syringe was used to inject 0.5 ml of gas phase from the sulphur-deprived culture into the Ar carrier gas stream via a septum. This produced a number of peaks in the MS readings corresponding to the presence of particular atomic masses in the injection. Injections were repeated three times to verify the consistency of the measurements. The syringe was dipped into acetone to sterilise it before injections were taken. It was important to use a heated (110°C) MS capillary to prevent any moisture in the injection from blocking the carrier gas path to the MS ioniser. Injection MS results will be described in Chapter III. Membrane-inlet mass spectrometry (MIMS) is an analytical technique used to measure in situ concentrations of dissolved gasses or volatile organic compound in aqueous solutions (Scott et al., 1983). The development of a MIMS system for the ICL Chemical Engineering PBR will be described in Chapter I.

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