**2. Applications of nanomaterials in HPLC stationary phases**

In conventional HPLC, the stationary phase (SP) plays a pivotal role in the separation technique [5]. The packing particles constituting the SP are of several micrometres in diameter with nanometre-sized pores [6]. Therefore, the industry has pushed researchers to investigate new packing materials as an attempt to achieve high throughput with robust analysis [6]. Packings in HPLC can be divided into three types-polymeric, inorganic, and hybrid materials. At present, inorganic materials, which include silica, hydroxyapatite, graphite, and metal oxides, etc. are widely used in research and applications [7]. Among these materials, silica is almost ideal support given its favourable characteristics, for example, good mechanical strength, high chemical and thermal stability, controllable pore structure and surface area, etc. [7]. Therefore, silica has been developed as the most widely used HPLC packing material [6, 7]. Throughout the years, many silica stationary phases (both porous and non-porous) have been commercialised and widely applied for analysis of pharmaceutical and biological samples [8].

#### **2.1. Porous and non-porous nanomaterials**

Non-porous and porous particles are the two major types of spherical packing materials used in HPLC [8]. The significant difference between both particles is that porous particles

**Figure 1.** The difference in surface area between porous and non-porous microspheres under scanning electron microspray [2].

have resistance to mass transfer contribution from the stagnant mobile phase in the pores [8]. Decreasing the particle size and increasing the diffusion coefficient can improve the mass transfer of solutes in the mobile phase [3, 5]. Non-porous particles can provide lower mass transfer resistance and higher efficiency than porous particles [8]. However, porous particles have higher surface areas (**Figure 1**) and can provide much higher sample loading capacity [8].

On the other hand, in porous particles, solutes transfer from the mobile phase exterior to the particles into the mobile phase within the pores to interact with the chiral stationary phase (CSP) [9]. Following this interaction, the solute molecule must diffuse out of the particle and continue its journey down the column ahead of the solute [5, 9]. This slow rate of mass transfer into and out of the porous particle is a source of HPLC band broadening [9]. **Figure 2** illustrates a reduction in particle size shortens the path length of this diffusion process, improves mass transfer, and provides better efficiency [9].

**Figure 2.** Smaller particle sizes increase efficiency and result in a wider range of flow rates [9].
