Abstract:

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 100 μm 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.

Key words: miniaturization, nano liquid chromatography, extra-column effect, solvent delivery equipment, injection equipment, tubing and connection