**5. Performance of CHIRALPAK IG-U®: In terms of enantioselectivity and solvent versatility**

In recent years, column miniaturisation has been investigated and tested in order to achieve highly sensitive chromatography [38]. The miniaturised columns are better for handling minute and/or dilute samples, especially in an area such as forensic science and sport drug trails [38]. The idea of miniaturisation is to provide higher sensitivity and peak capacity than standard columns with minimal dead volume for small sample amounts [39]. Narrow-bore 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 micron CSPs is still not used in the market on conventional HPLC [40–43].

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].

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

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.

**5. Performance of CHIRALPAK IG-U®: In terms of enantioselectivity** 

In recent years, column miniaturisation has been investigated and tested in order to achieve highly sensitive chromatography [38]. The miniaturised columns are better for handling minute and/or dilute samples, especially in an area such as forensic science and sport drug trails [38]. The idea of miniaturisation is to provide higher sensitivity and peak capacity than standard columns with minimal dead volume for small sample amounts [39]. Narrow-bore

**and solvent versatility**

62 New Uses of Micro and Nanomaterials

between increasing peak efficiency and a reduction in particle size [28, 34].

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, sensitivity and peak capacity [45].

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 discussed below [47].

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

achieved under standard and non-standard organic solvents. For example, few separations were achieved for compounds (Naftopidil, Miconazole, Sulconazole, Aminoglutethimide, Tocainide, Propafenone, Flavanone, and 6-Hydroxyflavanone). Compound Naftopidil was

(**Figure 8**) [47]. Similarly, in case of compound Aminoglutethimide; Rs 1.90 and α 1.34 were superior to other separations achieved under standard and non-standard organic solvents. Compound 6-Hydroxyflavanone which was separated under standard and non-standard sol-

According to the following results and VDE Ghanem et al. [46, 47] and VDE, all three parameters (length, i.d., and particle size) reduced in the transition from conventional CHIRALPAK IG® to the sub-2 micron CHIRALPAK IG-U® resulted in an enhanced separation and resolution (**Figure 9**). However, Practical difficulties that one can expect following the sub-2-micron particle approach are twofold: one is inherently related to the decrease of column permeability

**Figure 8.** Naftopidil was baseline separated with a Rs of 2.56 and α 1.62 under reversed phase solvent composition

**Figure 9.** CHIRALPAK IG-U® (left side) shows enhanced separation and resolution in shorter time of 2 minutes

under standard and non-standard solvents, was separated under reversed phase condition (ACN/H<sup>2</sup>

O/TEA 80:20:0.1% v/v/v). Aminoglutethimide with Rs 1.90 and α 1.34 were superior to other separations achieved under standard and non-standard organic solvents. Compound 6-Hydroxyflavanone which was separated

O/TEA 80:20:0.1% v/v/v)

65

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

O/TEA 40:60:0.1% v/v/v) with

O/TEA 40:60:0.1%

baseline separated with a Rs of 2.56 and α 1.62 under (ACN/H<sup>2</sup>

vents, was separated under reversed phase condition (ACN/H<sup>2</sup>

superior Rs 2.39 and α 2.71 (**Figure 8**) [47].

(ACN/H<sup>2</sup>

v/v/v) with superior Rs 2.39 and α 2.71.

compared to 20 minutes using the CHIRALPAK IG® (right side).

**Figure 6.** The effect of EtOH in mobile phase composition on Indoprofen, Naproxen, and Tocainide.

addition of non-standard solvents in mobile phase composition enhanced resolution and separation in several tested racemates for example, in case of Tocainide, Rs 1.44 and α 1.39 in standard solvents namely n-hexane/EtOH 90:10 v/v were enhanced to Rs 1.89 and α 1.66 when using non-standard solvent in mobile phase composition (*n*-hexane/DCM/EtOH 50/50/0.2 *v/v/v*) (**Figure 7**) [47]. Of interest, compound 1-phenyl-2,2,2-trifluorethanol which was not resolved under any standard solvents' combination investigated, was baseline separated under non-standard organic solvent (MtBE/EtOH 40/60% *v/v*) with Rs 1.55 and α 1.78. Similarly, compounds Aminoglutethimide and α-Methyl DOPA were only separated under non-standard organic mobile phase composition (MtBE/EtOH 40:60 *v/v*) and (*n*-hexane/DCM 85%/15% *v/v*) [47]. The chiral recognition of sub 2-micron column CHIRALPAK IG-U® is like that of CHIRALPAK IG® where polarity is playing a role. Another reason might be the stereo environment of the chiral cavities in amylose derivatives which might be favorable in the presence of ethanol. Other researchers have speculated that the configuration of the chiral cavities in the amylose tris (3,5-dimethylphenyl carbamate) is determined by the composition of mobile phase in normal phase mode while the configuration of reversed phase mode remains unchanged. It is of essential to note that the chiral recognition is due to different factors such as hydrogen bonding, π-π interactions and the chiral cavities of CSPs with specific configuration responsible for bonding of varying magnitude between the stationary phase and enantiomers [46, 47]. CHIRALPAK IG-U® was investigated under reversed-phase conditions including ACN and H<sup>2</sup> O ranging from 10 to 90% (v/v) [46]. The resolution and separation factors were enhanced in several compounds compared to other separations

**Figure 7.** Tocainide resolution and separation factor were enhanced from (Rs:1.44 and α:1.39) under standard solvent (n-hexane/EtOH 90:10 v/v) to (Rs:1.89 and α:1.66) under non-standard solvent (*n*-hexane/DCM/EtOH 50:50:0.2 v/v/v).

achieved under standard and non-standard organic solvents. For example, few separations were achieved for compounds (Naftopidil, Miconazole, Sulconazole, Aminoglutethimide, Tocainide, Propafenone, Flavanone, and 6-Hydroxyflavanone). Compound Naftopidil was baseline separated with a Rs of 2.56 and α 1.62 under (ACN/H<sup>2</sup> O/TEA 80:20:0.1% v/v/v) (**Figure 8**) [47]. Similarly, in case of compound Aminoglutethimide; Rs 1.90 and α 1.34 were superior to other separations achieved under standard and non-standard organic solvents. Compound 6-Hydroxyflavanone which was separated under standard and non-standard solvents, was separated under reversed phase condition (ACN/H<sup>2</sup> O/TEA 40:60:0.1% v/v/v) with superior Rs 2.39 and α 2.71 (**Figure 8**) [47].

According to the following results and VDE Ghanem et al. [46, 47] and VDE, all three parameters (length, i.d., and particle size) reduced in the transition from conventional CHIRALPAK IG® to the sub-2 micron CHIRALPAK IG-U® resulted in an enhanced separation and resolution (**Figure 9**). However, Practical difficulties that one can expect following the sub-2-micron particle approach are twofold: one is inherently related to the decrease of column permeability

addition of non-standard solvents in mobile phase composition enhanced resolution and separation in several tested racemates for example, in case of Tocainide, Rs 1.44 and α 1.39 in standard solvents namely n-hexane/EtOH 90:10 v/v were enhanced to Rs 1.89 and α 1.66 when using non-standard solvent in mobile phase composition (*n*-hexane/DCM/EtOH 50/50/0.2 *v/v/v*) (**Figure 7**) [47]. Of interest, compound 1-phenyl-2,2,2-trifluorethanol which was not resolved under any standard solvents' combination investigated, was baseline separated under non-standard organic solvent (MtBE/EtOH 40/60% *v/v*) with Rs 1.55 and α 1.78. Similarly, compounds Aminoglutethimide and α-Methyl DOPA were only separated under non-standard organic mobile phase composition (MtBE/EtOH 40:60 *v/v*) and (*n*-hexane/DCM 85%/15% *v/v*) [47]. The chiral recognition of sub 2-micron column CHIRALPAK IG-U® is like that of CHIRALPAK IG® where polarity is playing a role. Another reason might be the stereo environment of the chiral cavities in amylose derivatives which might be favorable in the presence of ethanol. Other researchers have speculated that the configuration of the chiral cavities in the amylose tris (3,5-dimethylphenyl carbamate) is determined by the composition of mobile phase in normal phase mode while the configuration of reversed phase mode remains unchanged. It is of essential to note that the chiral recognition is due to different factors such as hydrogen bonding, π-π interactions and the chiral cavities of CSPs with specific configuration responsible for bonding of varying magnitude between the stationary phase and enantiomers [46, 47]. CHIRALPAK IG-U® was investigated under reversed-phase

**Figure 6.** The effect of EtOH in mobile phase composition on Indoprofen, Naproxen, and Tocainide.

separation factors were enhanced in several compounds compared to other separations

**Figure 7.** Tocainide resolution and separation factor were enhanced from (Rs:1.44 and α:1.39) under standard solvent (n-hexane/EtOH 90:10 v/v) to (Rs:1.89 and α:1.66) under non-standard solvent (*n*-hexane/DCM/EtOH 50:50:0.2 v/v/v).

O ranging from 10 to 90% (v/v) [46]. The resolution and

conditions including ACN and H<sup>2</sup>

64 New Uses of Micro and Nanomaterials

**Figure 8.** Naftopidil was baseline separated with a Rs of 2.56 and α 1.62 under reversed phase solvent composition (ACN/H<sup>2</sup> O/TEA 80:20:0.1% v/v/v). Aminoglutethimide with Rs 1.90 and α 1.34 were superior to other separations achieved under standard and non-standard organic solvents. Compound 6-Hydroxyflavanone which was separated under standard and non-standard solvents, was separated under reversed phase condition (ACN/H<sup>2</sup> O/TEA 40:60:0.1% v/v/v) with superior Rs 2.39 and α 2.71.

**Figure 9.** CHIRALPAK IG-U® (left side) shows enhanced separation and resolution in shorter time of 2 minutes compared to 20 minutes using the CHIRALPAK IG® (right side).

that accompanies the particle size reduction; the other one is associated to the adaptation of the surface modification chemistry of classical CSPs to smaller particles [21–26].

Ideally, the flow should not be split and used directly from the pump as it is used in standard HPLC [35]. These nano-LC systems are often called splitless systems and are commercially available [53]. The direct flow systems can be divided into two groups: the 'solvent refill' systems and the 'continuous flow' systems [53]. Commonly, these systems also are capable of operating at UHPLC pressures. Whereas UHPLC is mostly used to increase throughput in standard-bore LC, its use in nano LC is mainly aimed at improving separation efficiency through the use of longer nano-LC columns packed with 2-μm (or smaller) particles [53].

Sub-2 μm Silica Particles in Chiral Separation http://dx.doi.org/10.5772/intechopen.79063 67

Further miniaturisation of standard UHPLC instrumentation is possible [50]. Microfluidics has proven a great success as an alternate approach to achieve analytical separations [48]. Such downsizing of the LC experiment would undoubtedly require a major redesign in the column and instrumentation [51, 54]. The use of miniaturised instruments would result in a significant solvent, bench-space, and sample savings, and with mass spectrometry would allow even better interfacing [35, 53–55]. Chip-based LC systems have been extensively investigated, and a limited number of instruments have already been commercialised [55]. However, the adoption rate for commercial instruments has been somewhat slow and, compared to regular analytical columns, in microfluidics column efficiencies are not as high as expected [54]. The packing of microparticles within narrow channels is difficult, and one of the reasons for low column efficiency in microfluidics-based column systems [18, 35, 50–55].

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

**7. Conclusion**

separation efficiency.

**Abbreviations**

CSP chiral stationary phase

HPLC high-performance liquid chromatography

HETP plate height

i.d. internal diameter

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

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 ultrahighpressure instruments [49].

## **6. Future perspectives**

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 higher pressures associated with smaller particles [53].

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. Ideally, the flow should not be split and used directly from the pump as it is used in standard HPLC [35]. These nano-LC systems are often called splitless systems and are commercially available [53]. The direct flow systems can be divided into two groups: the 'solvent refill' systems and the 'continuous flow' systems [53]. Commonly, these systems also are capable of operating at UHPLC pressures. Whereas UHPLC is mostly used to increase throughput in standard-bore LC, its use in nano LC is mainly aimed at improving separation efficiency through the use of longer nano-LC columns packed with 2-μm (or smaller) particles [53].

Further miniaturisation of standard UHPLC instrumentation is possible [50]. Microfluidics has proven a great success as an alternate approach to achieve analytical separations [48]. Such downsizing of the LC experiment would undoubtedly require a major redesign in the column and instrumentation [51, 54]. The use of miniaturised instruments would result in a significant solvent, bench-space, and sample savings, and with mass spectrometry would allow even better interfacing [35, 53–55]. Chip-based LC systems have been extensively investigated, and a limited number of instruments have already been commercialised [55]. However, the adoption rate for commercial instruments has been somewhat slow and, compared to regular analytical columns, in microfluidics column efficiencies are not as high as expected [54]. The packing of microparticles within narrow channels is difficult, and one of the reasons for low column efficiency in microfluidics-based column systems [18, 35, 50–55].
