Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
J Chromatogr A. 2012 Aug 17;1251:54-62. doi: 10.1016/j.chroma.2012.06.002. Epub 2012 Jun 9.
The speed of a separation defines the best time resolution possible in online measurements using chromatography. The desired time resolution multiplied by the flow rate of the stream of analyte being sampled defines the maximum volume of sample per injection. The best concentration sensitivity in chromatography is obtained by injecting the largest volume of sample that is consistent with achieving a satisfactory separation, and thus measurement accuracy. Taking these facts together, it is easy to understand that separation speed and concentration sensitivity are linked in this type of measurement. To address the problem of how to achieve the best sensitivity and shortest measurement time simultaneously, we have combined recent approaches to the optimization of the separation itself with an analysis of method sensitivity. This analysis leads to the column diameter becoming an important parameter in the optimization process. We use these ideas in one particular problem presented by online microdialysis sampling/liquid chromatography/electrochemical detection for measuring concentrations of serotonin in the dialysate. In this case the problem becomes the optimization of conditions to yield maximum signal for a given sample volume under the highest speed conditions with a certain required number of theoretical plates. It turns out that the observed concentration sensitivity at an electrochemical detector can be regulated by temperature, particle size, injection volume/column diameter, and void time. The theory was successfully used for optimization of neurotransmitter serotonin measurement by capillary HPLC when sampling from a microdialysis flow stream. The final conditions are: 150 μm i.d., 3.1cm long columns with 1.7 μm particle diameter working at a flow rate of 12 μL/min, an injection volume of 500 nL, and a temperature of 343 K. The retention time for serotonin is 22.7s, the analysis time is about 36 s (which allows for determination of 3-methoxytyramine), and the sampling time is about 0.8 min with a perfusion flow rate of 0.6 μL/min.
分离速度决定了使用色谱法进行在线测量时的最佳时间分辨率。所需的时间分辨率乘以被采样分析物流的流速，即可定义每次注射的最大样品体积。色谱法中最佳的浓度灵敏度是通过注入与实现令人满意的分离和测量精度一致的最大样品量来获得的。考虑到这些事实，很容易理解分离速度和浓度灵敏度在这种测量类型中是相互关联的。为了解决如何同时实现最佳灵敏度和最短测量时间的问题，我们将分离本身优化的最新方法与方法灵敏度分析结合起来。这种分析导致柱径成为优化过程中的一个重要参数。我们在一个特定的问题中使用了这些想法，即在线微透析采样/液相色谱/电化学检测用于测量透析液中 5-羟色胺的浓度。在这种情况下，问题变成了在最高速度条件下，用一定数量的理论塔板，优化条件以获得给定样品体积的最大信号。事实证明，在电化学检测器中观察到的浓度灵敏度可以通过温度、粒径、进样体积/柱径和空体积时间来调节。该理论成功地用于优化毛细管 HPLC 从微透析流中采样时神经递质 5-羟色胺的测量。最终条件为：内径 150μm、长 3.1cm 的柱，粒径 1.7μm，流速 12μL/min，进样体积 500nL，温度 343K。5-羟色胺的保留时间为 22.7s，分析时间约为 36s（允许测定 3-甲氧基酪胺），采样时间约为 0.8min，灌注流速为 0.6μL/min。
