Development of a high resolution UV spectrophotometer for at-line monitoring of bioprocesses

The construction, calibration and application of a direct readout UV spectrophotometer are described. The instrument utilises a concave holographic flat field diffraction grating and a charge coupled device (CCD) as a detector which measures a continuous spectrum, allowing several analytes to be monitored simultaneously. The advantages over conventional spectrophotometers come from miniaturisation and the near-instantaneous capture of full spectra. The spectrophotometer was characterised in terms of spectral range (185.0-380.0 nm), spectral resolution (0.3 nm with 10 μm slit width), absorbance precision (standard deviation (STD):±0.0009), dynamic range (0.00 to 2.00 absorbance unit (AU)), and accuracy (0.5771 AU with 0.0012 STD at 313.0 nm). The efficiency and reliability of the instrument were demonstrated using artificial mixtures of bovine serum albumin (BSA), ribonucleic acid (RNA) and cell debris of defined composition to mimic Saccharomyces cerevisiae homogenate, as well as real Saccharomyces cerevisiae homogenates. The application of the spectrophotometer in conjunction with the partial least squares (PLS) technique in at-line monitoring of selective flocculation processes purification of Saccharomyces cerevisiae homogenates have shown good agreement with the results obtained from off-line chemical assays.