生成可靠的HPLC结果的重要建议

HPLC在分析研究和质量控制实验室中具有明显的存在。关于制造过程以及研究发现的出版的重要决定是基于决策，只有在HPLC系统生成的数据上可以放置高度可靠性时才能达到的决策。

HPLC is a multi component system so the accuracy and reliability of results is dependent on the performance level of each component part. The present article offers some suggestions which will contribute to greater authenticity of results. For simplicity the contribution of each component part is discussed under a separate heading.

Mobile phase

流动相的主要作用是将注入的样品携带到列中，并在列中分离后，以将单个组件转移到检测器中以进行识别和定量。理想情况下，应在开始分析之前准备新的流动阶段，并且HPLC应该使用级溶剂。溶剂应不含任何悬浮颗粒。最初使用0.45μ滤波器在真空下过滤溶剂。建议进一步使用在线过滤器，以去除任何残留的悬浮颗粒。流动相位溶剂应彼此完全混乱，并且注射样品也应该混乱。当使用缓冲液作为流动相分量时，由于干燥时形成的盐会沉积并导致流动相的自由流动，但除了损坏泵组件外，还不会使缓冲液在系统内部干燥。

样本

Like mobile phase solvents it is a good practice to filter the sample before injection. Large volume injections will cause peak shape distortions so columns should not be overloaded by making large volume injections. Whenever a new method is to be developed it is advised to check the compatibility of the sample with the mobile phase. In case the solubility is low try a solvent or combination of solvents matching the polarity of the mobile phase mixture.

柱子

A column is a key component of HPLC system. Its proper care and use will go a long way in providing high reliability of results.

运行流量和压力应逐渐改变，以便不会干扰固定相包装。防护柱的使用还将防止杂质进入主列。由于流动相的流动取决于柱烤箱的温度使用，因此强烈建议保持恒定的柱温度以保持流速的一致性。

泵

The pump is required to provide consistency of flow of mobile phase and maintain the solvent composition throughout the analysis run. In case of gradient analysis a pump is required to maintain the composition of mobile phase as per the software program. Such conditions are achieved by preventing damage to pump components and timely replacement of such components on noticing any visible damage. Gradually flush the pump by allowing rinsing intermediate polarity solvents or pure water as the case may be.

喷油器/自动采样器

注射量的一致性是结果可重复性的主要需求。手动注射器包括固定体积样品循环，如果是自动模式，则注射注射器保持体积的一致性，而无需操作员干预。

Special care needs to be taken to prevent contamination from previous analysis. In case of manual injectors always flush the injection loop with sufficient sample and in case of automated systems make sure that intermediate wash cycles are part of the analysis program. A validation of the cleanliness of sample and standard reusable vials is vital before commencement of analysis.

光源

The majority of HPLC analysis can be carried out using UV light source. Such light sources have a useful life span. Lamp replacement as per recommendations of supplier will contribute to a high degree of reproducibility of absorbance values.

Detectors

A detector is the ultimate part of the HPLC system which confirms the presence of different sample components as well helps in their quantification.

这是recommended that the detector be cleaned periodically from time to time off-line and flushed with water and mobile phase before being re- used. The operating pressure range should never be exceeded to prevent possible damage to detector cell walls.

The suggestions offered in the article will certainly improve the quality of results but it goes without saying that there is no substitute for periodic calibration of the system against reference compounds.