**3. Effect of particle size on the separation efficiency, speed and resolution of chiral separations**

#### **3.1. Relationship between particle size, and column efficiency**

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

coefficient between two distinct phases namely the stationary and the mobile phases [1–3]. The components separate as they migrate with different rates depending on their unique distribution coefficients, they separate [4]. Different chromatographic techniques are available depending on the type of the phases [4]. The chromatography is known to be liquid chromatography (LC) when it employs a liquid mobile phase [5]. The most sophisticated form of LC is High-performance liquid chromatography (HPLC) where the mobile phase passes through

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].

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

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

the stationary via a pump at high pressures [4, 5].

56 New Uses of Micro and Nanomaterials

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

microspray [2].

Particle size is known to be the mean diameter of the spherical support employed in column packing [6]. This physical dimension significantly impacts HPLC column performance [9]. A decrease in particle size increases peak efficiencies (**Figure 2**). This is based on the resolution equation (Eq. (1)) which comprise of three terms: selectivity, retention capacity, and efficiency [10]. The components of an analytical method alter each of these terms. In particular, the column's particle size affects the efficiency factor from the equation [10]. Efficiency is a qualitative term used to measure the number of theoretical plates in a column. Put simply, as particle size is lowered, efficiency increases, and more resolution is achieved [10].

$$\mathbf{R}\_{\rm s} = \mathcal{Z}(\mathbf{t}\_{\rm k,2} - \mathbf{t}\_{\rm k,1}) / \langle \mathbf{w}\_{\rm b,1} + \mathbf{w}\_{\rm b,2} \rangle \tag{1}$$

Burns et al. observed a nearly linear correlation between the width of the particle size distribution of commercially available HPLC particles and the minimum reduced plate height, the van Deemter equation (VDE) A-term and the minimum reduced separation impedance [10, 11]. Column efficiency in HPLC is influenced by a number of factors such as particle size, flow rate and degree of cross-linkage of gels [12]. Particle size distribution (PSD) of packing materials is also considered one of the important factors [12]. It has been empirically found that column efficiency is improved by narrowing the PSD [11, 12]. Because the greatest the achievable plate height is, the more effective the PSD has on the separation efficiency [12]. On one side, column efficiency or plate number is dependent on particle size, and the pore size controls the surface area where retention is controlled primarily by the surface area [9–12].

### **3.2. The impact of the pore size of silica gel on the CSPs**

Retention is directly related to surface area; therefore, the use of large-pore columns is not desirable when small-pore columns can be used [13]. Selection of the pore size is based on providing easy access for the molecules to the pores in the column [10–12]. Consequently, the higher surface area associated with small pore-columns is preferred mainly because the analytes are small enough to pass through the pores [12, 13].

The performance of the CSP increases with pore size when the pore diameter is large enough for the penetration of macromolecules [13]. Specific surface area and, therefore, the number of silanol groups on the surface of the silica gel, decreases with increasing of pore size; consequently, the bound amount of the chiral selector depends on the pore size of silica matrix [13]. The particle size of silica gel has a major effect on the performance of the column, increasing the particle size from 3 to 10 μ decreased the theoretical plate number [13]. A reduction in particle size can lead to more compact and stable packing and thus better column efficiency.

**4. Small-particle (sub-2-μm) columns for high efficiency and speed**

**Figure 3.** The reduction of particle packing throughout the years in hope for the shortest fastest chromatogram [16].

Sub-2 μm Silica Particles in Chiral Separation http://dx.doi.org/10.5772/intechopen.79063 59

frits [20, 21].

Separation efficiency is inversely proportional to the stationary phase diameter [17]. Stationary phase manufacturers have reduced particles for packing down to micron-sized [17]. If packing materials could be further reduced in the future down to the nanometre scale, the band dispersion would consequently be reduced further by 6 magnitude orders [17, 18]. As a consequence, in 2004, the first available porous silica with small particle size was commercialised (1.7 μ), which allowed better resolution compared to the current 5 or 3.5 μ [18]. Several column suppliers now offer columns packed with particles in the range of 1.5–2 μ [18]. The term sub-2 micron, including particles of 2 μ, is used in this work for the sake of clarity [19]. Different works, dealing with drug and metabolites analysis, bioanalytical as well as environmental separations, compared columns packed with 5 μ and sub-2 micron supports and demonstrated that the latter clearly reduced the analysis time with comparable efficiency [20]. However, the quest towards the use of nanomaterials in chromatography has encountered serious challenges such as extremely high back pressures and problems associated with

The high back pressure is induced by the friction of the mobile phase percolating through the sub-2 μm particles stationary phase, generating heat that can be detrimental to the separations [22]. Studies suggest that reducing column internal diameter (i.d.) minimised frictional heating effect from the radial temperature gradient [22, 23]. This is due to fast heat dissipation within such a narrow-bore column [23]. As column length is proportional to the particle size,

shortening the columns lead to fast separation with sub-2 μm particles [23].

### **3.3. Chromatographic effects of varying particle size and size distributions**

In the late 1960s, Horvath and coworkers introduced columns packed with rigid pellicular particles (40–50 μ) applicable under high pressures [14]. The thin porous coat allowed for robust solute mass transfer through the packing, hence, improving column efficiency. However, pellicular particles had a drawback of low sample capacity [16]. In the 1970s, large porous/pellicular particles were reduced down to smaller porous particles of 10 μ to eliminate the drawbacks of pellicular materials [16]. However, particles of silica smaller than 40 μ have demonstrated some difficulties with packing reproducibility [17]. Irregular shapes of microporous particles were used throughout the 1970s until the spherical material was obtained and improved. In the 1980s, 5 μ became the standard particle diameter, and in the early 1990s, 3–3.5 μ particle diameters were also commercially available [16, 17]. The latter demonstrated 30–50% faster analysis times and higher efficiencies compared to 5 μ. Methods can be easily transferred from 5 μ to similar 3 stationary phases [14–17] (**Figure 3**) [15].


Burns et al. observed a nearly linear correlation between the width of the particle size distribution of commercially available HPLC particles and the minimum reduced plate height, the van Deemter equation (VDE) A-term and the minimum reduced separation impedance [10, 11]. Column efficiency in HPLC is influenced by a number of factors such as particle size, flow rate and degree of cross-linkage of gels [12]. Particle size distribution (PSD) of packing materials is also considered one of the important factors [12]. It has been empirically found that column efficiency is improved by narrowing the PSD [11, 12]. Because the greatest the achievable plate height is, the more effective the PSD has on the separation efficiency [12]. On one side, column efficiency or plate number is dependent on particle size, and the pore size controls the surface area where retention is controlled primarily by

Retention is directly related to surface area; therefore, the use of large-pore columns is not desirable when small-pore columns can be used [13]. Selection of the pore size is based on providing easy access for the molecules to the pores in the column [10–12]. Consequently, the higher surface area associated with small pore-columns is preferred mainly because the

The performance of the CSP increases with pore size when the pore diameter is large enough for the penetration of macromolecules [13]. Specific surface area and, therefore, the number of silanol groups on the surface of the silica gel, decreases with increasing of pore size; consequently, the bound amount of the chiral selector depends on the pore size of silica matrix [13]. The particle size of silica gel has a major effect on the performance of the column, increasing the particle size from 3 to 10 μ decreased the theoretical plate number [13]. A reduction in particle size can lead to more compact and stable packing and thus better

In the late 1960s, Horvath and coworkers introduced columns packed with rigid pellicular particles (40–50 μ) applicable under high pressures [14]. The thin porous coat allowed for robust solute mass transfer through the packing, hence, improving column efficiency. However, pellicular particles had a drawback of low sample capacity [16]. In the 1970s, large porous/pellicular particles were reduced down to smaller porous particles of 10 μ to eliminate the drawbacks of pellicular materials [16]. However, particles of silica smaller than 40 μ have demonstrated some difficulties with packing reproducibility [17]. Irregular shapes of microporous particles were used throughout the 1970s until the spherical material was obtained and improved. In the 1980s, 5 μ became the standard particle diameter, and in the early 1990s, 3–3.5 μ particle diameters were also commercially available [16, 17]. The latter demonstrated 30–50% faster analysis times and higher efficiencies compared to 5 μ. Methods can be easily transferred from 5 μ to similar 3 stationary phases [14–17]

**3.3. Chromatographic effects of varying particle size and size distributions**

the surface area [9–12].

58 New Uses of Micro and Nanomaterials

column efficiency.

(**Figure 3**) [15].

**3.2. The impact of the pore size of silica gel on the CSPs**

analytes are small enough to pass through the pores [12, 13].

**Figure 3.** The reduction of particle packing throughout the years in hope for the shortest fastest chromatogram [16].
