Bergström E T, Goodall D M, Pokrić B, Allinson N M
Department of Chemistry, University of York, York YO10 5DD, UK.
Anal Chem. 1999 Oct 1;71(19):4376-84. doi: 10.1021/ac990035l.
A fundamental limitation to the use of single-point absorbance detection for capillary electrophoresis is irradiance, since it is not possible to create an image at the detection point on capillary that is brighter than the light source. This limitation may be overcome by illuminating a length of the capillary using a fiber-optic bundle and using a charge coupled device (CCD) camera that can image the full length of the illuminated zone. The present paper describes design and development of a CCD detector for UV absorbance that can be used in both multiwavelength and single-wavelength modes. The CCD camera images analyte peaks in the capillary dimension, together with wavelength-resolved absorbance in the dimension perpendicular to the capillary. Successive snapshots of the peaks are added together, after appropriate correction for time-dependent peak displacement, without sacrificing spatial resolution. Measured baseline rms noise values at 200 nm are 34 μAU using a holographic grating in multiwavelength mode and 8 μAU with the addition of a band-pass filter. Both values are in excellent agreement with calculations of limiting shot noise. Performance in multiwavelength mode is constrained by the 470-ms readout time of the CCD used, which sets a maximum duty cycle of 2.3%. Noise contributions from source intensity fluctuations are reduced by using a portion of the CCD image to provide a baseline reference signal. With 4-hydroxybenzoate as test analyte, the linear dynamic range in multiwavelength mode is shown to be between 3 and 4 orders of magnitude. High-quality spectra of 2-, 3-, and 4-methylbenzoates are obtained on capillary and used in deconvolution of closely migrating peaks of the 2- and 3-isomers.
毛细管电泳单点吸光度检测的一个基本限制是辐照度，因为不可能在毛细管的检测点创建比光源更亮的图像。通过使用光纤束照亮一段毛细管并使用能够对被照亮区域的全长进行成像的电荷耦合器件（CCD）相机，可以克服这一限制。本文描述了一种可用于多波长和单波长模式的紫外吸光度CCD探测器的设计与开发。CCD相机对毛细管维度上的分析物峰以及垂直于毛细管维度上的波长分辨吸光度进行成像。在对与时间相关的峰位移进行适当校正后，将峰的连续快照相加，而不牺牲空间分辨率。在多波长模式下，使用全息光栅时，在200 nm处测得的基线均方根噪声值为34 μAU，添加带通滤波器后为8 μAU。这两个值与极限散粒噪声的计算结果非常吻合。多波长模式下的性能受到所用CCD 470毫秒读出时间的限制，这设定了最大占空比为2.3%。通过使用一部分CCD图像提供基线参考信号，可降低源强度波动产生的噪声贡献。以4-羟基苯甲酸作为测试分析物，多波长模式下的线性动态范围显示在3到4个数量级之间。在毛细管上获得了2-、3-和4-甲基苯甲酸的高质量光谱，并用于对2-和3-异构体紧密迁移峰的去卷积。
