Department of Chemistry and Biochemistry, University of Texas at Arlington, 700 Planetarium Place, Arlington, TX 76019-0065, United States.
Department of Chemistry and Biochemistry, University of Texas at Arlington, 700 Planetarium Place, Arlington, TX 76019-0065, United States.
Anal Chim Acta. 2016 Feb 11;907:31-44. doi: 10.1016/j.aca.2015.11.043. Epub 2015 Dec 17.
With increasingly efficient columns, eluite peaks are increasingly narrower. To take full advantage of this, choice of the detector response time and the data acquisition rate a.k.a. detector sampling frequency, have become increasingly important. In this work, we revisit the concept of data sampling from the theorem variously attributed to Whittaker, Nyquist, Kotelnikov, and Shannon. Focusing on time scales relevant to the current practice of high performance liquid chromatography (HPLC) and optical absorbance detection (the most commonly used method), even for very narrow simulated peaks Fourier transformation shows that theoretical minimum sampling frequency is still relatively low (<10 Hz). However, this consideration alone may not be adequate for real chromatograms when an appreciable amount of noise is present. Further, depending on the instrument, the manufacturer's choice of a particular data bunching/integration/response time condition may be integrally coupled to the sampling frequency. In any case, the exact nature of signal filtration often occurs in a manner neither transparent to nor controllable by the user. Using fast chromatography on a state-of-the-art column (38,000 plates), we evaluate the responses produced by different present generation instruments, each with their unique black box digital filters. We show that the common wisdom of sampling 20 points per peak can be inadequate for high efficiency columns and that the sampling frequency and response choices do affect the peak shape. If the sampling frequency is too low or response time is too large, the observed peak shapes will not remain as narrow as they really are - this is especially true for high efficiency and high speed separations. It is shown that both sampling frequency and digital filtering affect the retention time, noise amplitude, peak shape and width in a complex fashion. We show how a square-wave driven light emitting diode source can reveal the nature of the embedded filter. We discuss time uncertainties related to the choice of sampling frequency. Finally, we suggest steps to obtain optimum results from a given system.
随着柱子效率的不断提高，洗脱峰变得越来越窄。为了充分利用这一点，选择检测器的响应时间和数据采集率（即检测器采样频率）变得越来越重要。在这项工作中，我们重新审视了从 Whittaker、Nyquist、Kotelnikov 和 Shannon 等不同人提出的定理中得出的数据采样概念。我们关注与高效液相色谱（HPLC）和光吸收检测的当前实践相关的时间尺度（这是最常用的方法），即使是非常窄的模拟峰，傅里叶变换也表明理论上的最小采样频率仍然相对较低（<10 Hz）。然而，当存在相当数量的噪声时，仅考虑这一点可能不足以用于真实的色谱图。此外，根据仪器的不同，制造商选择特定的数据聚集/积分/响应时间条件可能与采样频率紧密结合。在任何情况下，信号滤波的精确性质通常以用户既不可见也无法控制的方式发生。使用最先进的柱子进行快速色谱分析（38000 块板），我们评估了不同现有仪器产生的响应，每个仪器都有其独特的黑盒数字滤波器。我们表明，对于高效柱子，每峰采样 20 点的常识可能不足，并且采样频率和响应选择确实会影响峰形。如果采样频率太低或响应时间太长，则观察到的峰形将不会保持与实际一样窄 - 对于高效和高速分离尤其如此。结果表明，采样频率和数字滤波都会以复杂的方式影响保留时间、噪声幅度、峰形和宽度。我们展示了如何使用方波驱动的发光二极管源揭示嵌入式滤波器的性质。我们讨论了与采样频率选择相关的时间不确定性。最后，我们建议从给定系统中获得最佳结果的步骤。
