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

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 frits [20, 21].

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].

As shown in **Figure 4**, sub-2 micron particles are noted to be highly efficient and hence, the column length can be shortened while maintaining resolution in a shorter analysis time [23]. To achieve fast separations, short columns and high flow rates are necessary. In such columns, it can be practically difficult to achieve axially and radially homogenous beds [24]. It seems more logical to use smaller diameter columns, such as 3 mm i.d. when using sub-2 micron particles [23, 24]. This significantly reduces the flow rate required to achieve optimum efficiency, which in turn minimises the extra-column dispersion caused by the tubing [25]. Higher linear velocities can be achieved at lower flow rates, with much lower pressure drops in the tubing [25].

with the square of the column radius and with the square root of column length [29]. Thus, a reduction in column diameter results in a significantly lower dilution factor, thereby increasing the concentration in the eluted peak [29]. Downscaling the column used in an analytical method should result in an almost 4000-fold gain in sensitivity [28]. With the reduction in particle size, column efficiency improved further allowing a reduction in column length. Shorter columns are now in vogue [28]. Not only does a shorter column provide faster separations but also solvent use is reduced [28]. Columns of 50 mm in length now provide plate counts formerly obtained on 15- and 25-cm columns packed with larger particles [25, 28, 29]. The sub-2-μm columns have struck the fancy of those who wish to decrease their analysis times by shortening the column length and to those who want to have greater plate count by using

Sub-2 μm Silica Particles in Chiral Separation http://dx.doi.org/10.5772/intechopen.79063 61

The equation demonstrated the obvious advantage of using small particles to decrease plate height [30]. The chromatographic separating power of HPLC is dependent upon the selectivity of the mobile/stationary phase and the efficiency of the column [12, 13]. The column efficiency is dependent upon multiple factors most importantly: the column length and the packing particle size together with the mobile phase velocity [22]. At a fixed velocity, the column efficiency increases in direct proportion to the column length [24]. At the minimum of the plate height versus velocity curve, the column efficiency increases in inverse proportion

As seen in **Figure 5**, van Deemter equation describes that efficiency varies with the linear velocity, and the nature of the second and third terms of the equation indicates a minimum value for plate height (HETP) [31]. In the third term of van Deemter equation, the particle size is squared, and so the curve is steeper for larger particles at high linear velocities [31]. The A term depends

**Figure 5.** Van Deemter equation describes that efficiency varies with the linear velocity, and the second and third terms

longer columns, albeit at the higher pressure [25, 28, 29].

with the particle size [22, 24].

indicates a minimum value for HETP [31].

If the main goal is reducing analysis time, an increase in the flow rate above the optimum rate allows for robust separations, while maintaining resolution due to the small particles lower mass transfer resistance [24]. On the other side, if the primary goal is higher resolution, maintaining column length can increase resolution with a subsequent increase in analysis time [24]. Particle size reduction has more effect than column length, gradient time, or flow-rate to improve peak capacity in gradient mode [26]. However, these small particles can generate a high bp incompatible with conventional instrumentation [27]. It is evident that a reduction in particle size reduces backpressure because of the inverse square relationship between them [27]. As a result, new HPLC instrumentation such as ultra-high performance liquid chromatography (UHPLC) has been developed to handle elevated pressures above 400 bar [27]. However, employing shorter columns with smaller particles (i.e. sub-2 micron) have the ability to combine high resolution without exceeding the pressure limit of 400 bar associated with conventional HPLC [23–27].

#### **4.1. Van Deemter analysis of sub-2 micron CSPs**

According to VDE, the A- and C-terms are directly proportional to the particle size [28]. Therefore, the use of smaller particles provides a decrease of the plate height together with a flatter profile of the right branch of the van Deemter curve [28]. A reduction in column i.d. results in less chromatographic dilution and, consequently, increased concentration of the injected sample on LC system [25]. The chromatographic dilution increases proportionally

**Figure 4.** A reduction in particle size results in an increase in column efficiency, a wider range of flow rates is applicable for small particles [23].

with the square of the column radius and with the square root of column length [29]. Thus, a reduction in column diameter results in a significantly lower dilution factor, thereby increasing the concentration in the eluted peak [29]. Downscaling the column used in an analytical method should result in an almost 4000-fold gain in sensitivity [28]. With the reduction in particle size, column efficiency improved further allowing a reduction in column length. Shorter columns are now in vogue [28]. Not only does a shorter column provide faster separations but also solvent use is reduced [28]. Columns of 50 mm in length now provide plate counts formerly obtained on 15- and 25-cm columns packed with larger particles [25, 28, 29]. The sub-2-μm columns have struck the fancy of those who wish to decrease their analysis times by shortening the column length and to those who want to have greater plate count by using longer columns, albeit at the higher pressure [25, 28, 29].

As shown in **Figure 4**, sub-2 micron particles are noted to be highly efficient and hence, the column length can be shortened while maintaining resolution in a shorter analysis time [23]. To achieve fast separations, short columns and high flow rates are necessary. In such columns, it can be practically difficult to achieve axially and radially homogenous beds [24]. It seems more logical to use smaller diameter columns, such as 3 mm i.d. when using sub-2 micron particles [23, 24]. This significantly reduces the flow rate required to achieve optimum efficiency, which in turn minimises the extra-column dispersion caused by the tubing [25]. Higher linear velocities can be achieved at lower flow rates, with much lower pressure drops in the tubing [25].

If the main goal is reducing analysis time, an increase in the flow rate above the optimum rate allows for robust separations, while maintaining resolution due to the small particles lower mass transfer resistance [24]. On the other side, if the primary goal is higher resolution, maintaining column length can increase resolution with a subsequent increase in analysis time [24]. Particle size reduction has more effect than column length, gradient time, or flow-rate to improve peak capacity in gradient mode [26]. However, these small particles can generate a high bp incompatible with conventional instrumentation [27]. It is evident that a reduction in particle size reduces backpressure because of the inverse square relationship between them [27]. As a result, new HPLC instrumentation such as ultra-high performance liquid chromatography (UHPLC) has been developed to handle elevated pressures above 400 bar [27]. However, employing shorter columns with smaller particles (i.e. sub-2 micron) have the ability to combine high resolution without exceeding the pressure limit of 400 bar associated with conventional HPLC [23–27].

According to VDE, the A- and C-terms are directly proportional to the particle size [28]. Therefore, the use of smaller particles provides a decrease of the plate height together with a flatter profile of the right branch of the van Deemter curve [28]. A reduction in column i.d. results in less chromatographic dilution and, consequently, increased concentration of the injected sample on LC system [25]. The chromatographic dilution increases proportionally

**Figure 4.** A reduction in particle size results in an increase in column efficiency, a wider range of flow rates is applicable

**4.1. Van Deemter analysis of sub-2 micron CSPs**

60 New Uses of Micro and Nanomaterials

for small particles [23].

The equation demonstrated the obvious advantage of using small particles to decrease plate height [30]. The chromatographic separating power of HPLC is dependent upon the selectivity of the mobile/stationary phase and the efficiency of the column [12, 13]. The column efficiency is dependent upon multiple factors most importantly: the column length and the packing particle size together with the mobile phase velocity [22]. At a fixed velocity, the column efficiency increases in direct proportion to the column length [24]. At the minimum of the plate height versus velocity curve, the column efficiency increases in inverse proportion with the particle size [22, 24].

As seen in **Figure 5**, van Deemter equation describes that efficiency varies with the linear velocity, and the nature of the second and third terms of the equation indicates a minimum value for plate height (HETP) [31]. In the third term of van Deemter equation, the particle size is squared, and so the curve is steeper for larger particles at high linear velocities [31]. The A term depends

**Figure 5.** Van Deemter equation describes that efficiency varies with the linear velocity, and the second and third terms indicates a minimum value for HETP [31].

on both the quality of the column packing and the contribution values of the coefficients A, B and C for the different columns, can be accounted for by the minor contribution of several effects (packing characteristics and the combined effects of frictional heating and high pressure) on the velocity-dependence of the plate height. B- and C-terms of the equation depend on analyte retention [32]. The B-term is expected to increase with analyte retention as more time is available for diffusion to take place in the mobile phase [32]. According to the theory, smaller particles should perform lower plate heights and higher optimum linear velocity [31, 32].

columns are used on a conventional HPLC system [24]. The sub-2 micron silica-based stationary phases have established themselves as an effective analytical tool in achiral applications, but in the field of chiral separations, the technology related to the development of sub-2

Sub-2 μm Silica Particles in Chiral Separation http://dx.doi.org/10.5772/intechopen.79063 63

Daicel group recently commercialised a sub-2 micron 5 cm column applicable for use on conventional HPLC [44]. The design is based on the simple way of reducing solvent usage by using a shorter column. A more dynamic saving in solvent usage is made by reducing the i.d. of the column, together with an appropriate scaling down in the flow rate [44]. Separation efficiencies are also recovered by reducing particle size down to sub-2 micron; because columns packed with sub-2 micron particles offer advantages over the more traditional systems containing 3 and 5 micron particles by allowing operation at higher flow rates without compromising efficiency [45]. Consequently, this results in shorter analysis times and a reduction in solvent consumption, together with associated improvements in resolving power, sensitiv-

Ghanem et al. [46] investigated the impact of reducing the three VDE parameters (Length, i.d. and particle size) on separation and efficiency via the transition from conventional CHIRALPAK IG® to the sub-2 micron CHIRALPAK IG-U®. The effects of miniaturising the three column parameters (i.d., length and particle size) five times from CHIRALPAK IG® (250 mm length, 4.6 mm i.d., and 5 mm particle size) to CHIRALPAK IG-U® (50 mm length, 3 mm i.d., and 1.6 μm or sub-2 micron particle size) using similar CSP amylose *tris* (3-chloro-5-methylphenylcarbamate) for the enantioselective separation of racemates under normal standard, non-standard organic phase, and reversed-phase chromatographic conditions are

Pore size influences several factors such as: the retention factor, the separation factor, and the resolution of racemates was also examined. CHIRALPAK IG-U® has been tested in normal-phase mode chromatographic separation consisting of *n*-hexane/ethanol screened from 90:10 to 10:90 v/v at 1 mL/min flow rate on CHIRALPAK IG-U® at fixed UV detection 245 nm. Out of the twenty-eight compounds tested, eleven compounds (Naftopidil, Naproxen, Indoprofen, Cizolitrine, Carprofen, Miconazole, Nomifensine, Tocainide, Propafenone, Flavanone, and 6-Hydroxyflavanone) were partially, or baseline separated under either 90:10 or 80:20 v/v *n*-hexane/ethanol mobile phase. Substituting ethanol (EtOH) with 2-propanol (2-PrOH) resulted in the separation of only seven compounds (Naftopidil, Carprofen, Sulconazole, Propafenone, Flavanone, 6-Hydroxyflavanone, and 1-Acenaphthenol) under either 90:10, 80:20, 70:30 or 60:40 v/v n-hexane/2-PrOH. Regarding resolution (Rs) and separation factor (α), EtOH in mobile phase system showed better results than 2-PrOH where (Naproxen, Indoprofen, Miconazole, Nomifensine, and Tocainide) were all separated when 2-PrOH was replaced by n-hexane (See **Figure 6** for examples). This is mainly because the mobile phase travels easily in large spaces between particles and hence particle size affect permeability; smaller particles can be packed closer together, thus, using ethanol resulted in

the best separation with the highest resolution and separation factor [46, 47].

CHIRALPAK IG-U® showed great performance in different solvents such as non-standard solvents namely dichloromethane, tetrahydrofuran and Methyl *tert* Butyl Ether [46]. The

micron CSPs is still not used in the market on conventional HPLC [40–43].

ity and peak capacity [45].

discussed below [47].

The dependence of the third term (C-term), is considered to represent mainly the resistance to mass transfer in the mobile phase, on the square of the particle size translates into a substantial decrease in the plate height with smaller particles, especially at high linear velocities [33]. Small particle diameters induce an increase in efficiency, optimal velocity and mass transfer. Sub-2 micron particles packed into shorter column permit shorter analysis time along with using less solvent without compromising the resolution between closely eluting peaks [34]. Because the H-u curves are flatter (lower C term) for the smaller particle diameters, they also allow conducting separations at linear velocities higher than the optimum without significant loss of efficiency [34]. Using small diameter packing material reduces eddy diffusion and mass transfer resistance in the mobile phase [28, 33]. Van Deemter realised a correlation between increasing peak efficiency and a reduction in particle size [28, 34].

Optimization of efficiency and analysis time can be useful but generally leads to a large increase in the analysis time [35]. Instead, tuning the column length together with the stationary phase morphology (e.g. particle size) can result in a better compromise between the plate count and analysis time [36, 37]. Column length and plate count are related through the height equivalent to one theoretical plate with the relationship between the theoretical plate and mobile phase velocity described by VDE as the sum of different band-broadening contributions [34–36].

Based on VDE, the solution for enhancing chromatographic performance is to use shorter columns with small particle diameters (i.e. sub-2 micron particles) to induce a simultaneous improvement in efficiency, optimal velocity and mass transfer. The use of these sub-2 micron materials in LC is examined, including their applications in normal-phase LC, reversed-phase LC. In this study, the possibilities and restrictions of chromatographic separations obtained with 5 cm long bore columns packed with sub-2 micron particles is presented. Performance of columns immobilised on silica gel with different internal diameters and different lengths will be briefly mentioned to provide an overview of the miniaturisation of HPLC column technology.
