**7. Conclusion**

that accompanies the particle size reduction; the other one is associated to the adaptation of

The column permeability reduction is linked to the increase in pressure that is proportional to the inverse of the particle diameter squared: thus, reducing the particle diameter by a factor of 3 will result in a ninefold increase in the column back pressure [48]. As a consequence, depending on the column length and eluents viscosity, the full potential of high-speed separations can only be exploited on chromatographic hardware that can withstand elevated pressures (UHPLC) [49]. An additional complication may arise from the pronounced propensity of the smaller particles to aggregate during synthetic steps leading to a final stationary phase with non-optimal performances primarily regarding permeability and/or efficiency [18]. Mechanical resistance and long-term stability of the packed bed are also of significant concern

This area needs considerable attention as solvent efficient narrow-bore columns have already become mainstream for 'greener' chromatography [50]. For ultrafast separations, the sub-2- μm totally porous particles provide better solutions [18, 48]. Thus it can be a viable option to achieve ultrafast separations with slightly lower efficiency, but without a large investment in ultrahigh-

Instruments have been trying to follow the footpath of column developments [51]. The life cycle for instrument development is much longer than what is required for new packings and columns [52]. An area that has been delaying further improvements in column efficiency is the instrument contribution to band dispersion associated with HPLC and UHPLC instruments and their column-instrument interface designs [53]. Integration of column hardware and instrument connections are essential to eliminate dead volumes, much like what has been achieved in some nano and chip instruments [53]. The area of frit and end fitting design needs attention since the column packing where the separation takes place should be located at or near the injector device and the detector measurement device [52, 53]. This may necessitate a new column design that not only cuts down on this extra-column volume but can handle

Reducing the column i.d. is the first of several critical steps in miniaturising a LC system. Extra column peak broadening must be reduced accordingly to preserve optimal performance [54]. Excessive extra column band-broadening causes considerable loss of separation efficiency and, thereby, sensitivity. Connection tubing should be kept as short and especially as narrow as possible to minimise extra column band broadening and result in an acceptable increase in back pressure [35, 54]. Making connections with silica capillaries can be a challenge to lessexperienced users and often has been considered the most difficult part of setting up a nanoliquid chromatography (nano-LC) system [55].LC system implies that all system components should be downscaled, including column, connecting tubing, connections, injector, and the interface to the detector [55]. Nano-LC columns typically require flow rates of 500 nL/min or less [35]. Achieving reproducible flow and gradient formation requires dedicated approaches.

the surface modification chemistry of classical CSPs to smaller particles [21–26].

when high flow (and hence high pressure) applications are planned [18].

pressure instruments [49].

66 New Uses of Micro and Nanomaterials

**6. Future perspectives**

higher pressures associated with smaller particles [53].

Chromatographic technique development has always strived towards higher efficiency and more rapid resolution in diverse areas such as clinical, pharmaceutical and toxicology analysis, as well as enantioselective separation, to reduce costs and enhance throughput. Conventional HPLC, could not fully satisfy these requirements due to the relatively low efficiency and lengthy analysis time. Several approaches have been undertaken to achieve these goals, such as increasing flow rates and shortening the column length by using monolithic columns. However, these approaches may result in low phase ratio and low capacity factor. One promising approach is to use smaller size silica particle (less than 2 μm, as compared to conventional 3 and 5 μm size column packing materials. This is motivated by VDE that shows an inversely proportional relationship between the separation efficiency and particle size. Therefore, nano-or sub-micron size supporting materials may be promising to improve separation efficiency.
