纳升液相色谱仪器的研究进展

Research advances in nano liquid chromatography instrumentation

小型化是液相色谱分离技术发展的重要趋势之一,包括仪器外形尺寸的小型化、分离材料粒径的小型化以及色谱柱内径的小型化。色谱柱内径的减小能够降低样品和流动相的消耗,具有更高的质量灵敏度,特别适合用于复杂样品体系的分离分析。纳升液相色谱一般是指使用内径小于100μm的毛细管色谱柱,流速范围在每分钟几十至几百纳升的色谱技术。由于流速很低,色谱柱体积很小,柱外效应显著,因此对色谱仪器系统各个模块的性能以及系统柱外效应的优化提出了较高的要求。纳升液相色谱的输液装置需要能够准确稳定地输送纳升级流速,具有梯度输液模式,且拥有一定的耐压能力,以适应不同规格的色谱柱类型;进样装置需要能够进行准确重复的进样过程,进样体积及进样方式适合毛细管色谱柱,同时不产生明显的柱外效应;检测装置需要具有较高的灵敏度,且具有较小的柱外扩散;管路与连接系统需要稳定、可靠、易操作,并能够最大限度地减小柱外体积,适配纳升级流速。鉴于目前大多数纳升液相色谱系统与质谱检测器联用,因而本文主要从输液装置、进样装置、管路与连接3个方面对相关技术领域的研究论文、技术专利以及仪器厂商的宣传文件等进行了检索与归纳,综述了这些模块的技术路线与研究进展,同时简要介绍光学吸收型检测装置的优化思路与研究进展,并对部分商品化的纳升液相色谱系统进行了对比。

The miniaturization of liquid chromatography equipment is among the most important focus areas in chromatographic technology.It involves the miniaturization of the physical dimensions of the instrument,size of the separation material,and inner diameter of the column.The advantages of a reduced inner diameter of the column have been investigated for several decades,and can be summarized as follows.First,the sample consumption is lower,which is particularly beneficial when a limited amount of sample is available,as is the case with natural products,and in biochemistry and biomedicine.Second,the consumption of the mobile phase is reduced,making the process environmentally friendly and facilitating green chemistry.This allows the addition of more expensive solvent additives,such as chiral additives or isotopic reagents,while maintaining a low analysis cost.Moreover,the degree of band dilution in the column is lower than that with conventional liquid chromatography under the same sample injection conditions.Thus,enhanced mass sensitivity is achieved.Other benefits of a reduced inner diameter of the column include temperature control due to effective heat transfer through the columns and easier coupling to mass detectors,which is particularly advantageous for analyzing complex samples.Typically,the term“nano liquid chromatography”is associated with liquid chromatography,which employs capillary columns of inner diameters less than 100μm and flow rates in the range of tens to hundreds of nanoliters per minute.Because of the extremely low flow rates and small column volume,the extra-column effect becomes more prominent.Thus,the requirements for every component of liquid chromatographs are augmented toward improving their performance and optimizing the extra-column band broadening of the entire system.The solvent delivery equipment should be able to pump mobile phases accurately and steadily at nanoliter-level flow rates.A gradient mode is required to achieve this,which implies that the lowest flow rate for a single pump unit should reach a few nanoliters per minute.A certain operating pressure is also necessary to employ columns with different inner diameters and particle sizes.A precise and repeatable sample injection procedure is essential for nano liquid chromatography.The injection volume and mode should be suitable for capillary columns,without inducing a significant extra-column effect.A higher-sensitivity detector should be employed,and sample dispersion should be limited.The improved tubing and connection method in nano liquid chromatography should offer stability,reliability,and ease of operation.The extra-column volume should also be restricted to suit nanoliter-level flow rates.Considering that most nano liquid chromatographic instruments have been coupled with a mass detector,this review mainly focused on nanoliter solvent delivery modules,sample injection modules,and tubing and connection modules.By searching and summarizing research articles,technical patents,and brochures of instrument manufacturers,technical routes and research progress on these modules were described in detail.The pump designs can be classified into four types.Pneumatic amplifying pumps have been used in ultra-high-pressure applications.The flow-splitting delivery system,though easy to realize,may lead to a large amount of solvent wastage.Splitless pumps,which are classified based on two main principles,are widely used.Some pumps based on other physical phenomena have been suggested;however,they lacked stability and robustness.Two types of injection modes have been utilized in nano liquid chromatography.The direct nanoliter injection mode typically takes advantage of the groove on the rotor of a switching valve.The trapping injection mode uses trap columns to enable the introduction of large sample volumes.As for the tubing and connection,a few appropriate designs can be acquired from commercial suppliers.The robustness has been improved using some patented technologies.The optimization principles and research progress on optical absorption detection are briefly introduced.Finally,commercial nano liquid chromatographic systems are compared by considering the pumps and injectors.