Performance of a fast fiber based UV/Vis multiwavelength detector for the analytical ultracentrifuge

Abstract

The optical setup and the performance of a prototype UV/Vis multiwavelength analytical ultracentrifuge (MWL-AUC) is described and compared to the commercially available Optima XL-A from Beckman Coulter. Slight modifications have been made to the optical path of the MWL-AUC. With respect to wavelength accuracy and radial resolution, the new MWL-AUC is found to be comparable to the existing XL-A. Absorbance accuracy is dependent on the light intensity available at the detection wavelength as well as the intrinsic noise of the data. Measurements from single flashes of light are more noisy for the MWL-AUC, potentially due to the absence of flash-to-flash normalization in the current design. However, the possibility of both wavelength and scan averaging can compensate for this and still give much faster scan rates than the XL-A. Some further improvements of the existing design are suggested based on these findings.

Fig. 1

The detector arm. 1 A 600-μm patch fiber UV/Vis (Ocean Optics); 2 The collimating lens system (self-built), f = 20.6-mm biconvex; 3 90° Quartz prism; 4 Iris diaphragm for reducing light intensity; 5 Focusing biconvex lens (40 mm); 6 Spectrometer. The light path is also shown schematically

Fig. 2

a Photograph of the detector arm: 1 Spectrometer; 2 Table with the possibility of x–y movement; 3 Step motor; 4 Lens (40-mm biconvex); 5 Iris and 6 90° Quartz prism. b The arm fitted in the centrifuge. c Photograph of the vacuum feedthrough: 1 Electronic feedthrough for step motor; 2 Electronic feedthrough for spectrometer; 3 Electronic connection for TTL pulse for rpm measurements; 4 Optical feedthrough for fibers

Fig. 3

Intensity distributions for USB2000 spectrometers with different in-built diffraction gratings as compared to the XL-A. Vis Diffraction grating optimized for the Visible spectral range, high/low intensity, the iris opened maximally/minimally. With the iris maximally opened, the maximum intensity of the spectrometer (4,000 counts) is reached, and the spectrometer is maxed out at these wavelengths. UV Diffraction grating optimized for the UV range. With the iris maximally opened, about 70% of the available channels are saturated; these data are therefore not shown. Note that the flash lamp used in the MWL-AUC is different from the one of the XL-A. Due to the design of the spectral dispersion/detection system in the USB2000 spectrometers, the raw intensity spectra for the different spectrometers are a convoluted function of both the emission spectrum of the flash lamp and the preinstalled diffraction grating of the spectrometer itself

Fig. 4

A slit of 200 μm was imaged using the MWL-AUC and the XL-A at different wavelengths. The thick solid bar represents a distance of 200 μm. The wavelength resolution is dependent on wavelength. It is higher in the UV wavelength range for the MWL-AUC, but higher in the Visible range for the XL-A

Fig. 5

Reproducibility of the step size for the MWL and the XL-A AUCs for 10- and 50-μm step size, respectively, for a scan of a cell. Not all data points of the MWL detector (50 μm) are shown for clarity reasons

Fig. 6

Wavelength accuracy of the XL-A and the MWL-AUC. An HO₂O₃ centerpiece was used to record absorbance spectra as shown. The dotted vertical lines indicate the positions for three characteristic peaks at 361, 446, and 537 nm, respectively

Fig. 7

Absorbance accuracy of the MWL-AUC at different wavelengths. The reference are data measured by a benchtop double-beam spectrometer (model lambda 2 from Perkin Elmer)

Fig. 8

Reference intensities I₀ of the absorbance measurements shown in Fig. 6. The dotted vertical lines indicate the two wavelengths at which data were recorded

Fig. 9

Noise comparison between the XL-A and the MWL-AUC. Spectra were taken with 1/10/100 point averaging, shown as the bottom/middle/top spectrum, respectively. Dotted horizontal lines indicate the offset true baselines. Due to the higher point density for the USB2000 spectrometers, the spectra appear broader as they are in reality. Note the differences in scale of the abscissae. The respective statistical data are: UV(1), −0.016 ± 0.029; UV(10), 0.007 ± 0.009; UV(100), 0.001 ± 0.005; Vis(1), −0.011 ± 0.059; Vis(10), 0.004 ± 0.010; Vis(100), −0.002 ± 0.003; XL-A(1), −0.003 ± 0.017; XL-A(10), −0.001 ± 0.006; XL-A(100), −0.002 ± 0.004. UV scanned 200–500 nm; Vis scanned 250–800 nm, and XL-I scanned 200–800 nm