The separation and identification of chiral compounds is a relevant issue in several areas of science like the pharmaceutical, agrochemical or food analysis due to the important rule of chirality for human and the environment. As a result of the dramatic case of the chiral drug thalidomide, it was revealed that enantiomers can exhibit different biological, pharmacokinetic, and pharmacodynamic properties in a chiral environment as the human body, showing different stereospecific recognition to receptors and active sites [1]. Consequently, the biological or pharmacological activity of chiral compounds is generally associated with only one of the enantiomers, while the other can be inactive, less active, toxic or show a completely different activity [2]. For instance, the R-enantiomer of the drug lacosamide shows antiepileptic activity, while the S-configuration is inactive [3]; R-duloxetine presents less antidepressant power than its S-enantiomer [4]; the famous thalidomide, whose R-enantiomer has sedative and antiemetic properties but its S-enantiomer is teratogenic and toxic [5]; or fluoxetine, whose S-enantiomer is effective against migraines while its R-configuration is useful against anxiety and sexual dysfunction [6]. For this reason, it is of the utmost importance to commercialize drugs as enantiomerically pure formulations to avoid exposing the organism to inactive or even harmful compounds. Likewise, the occurrence of inactive or less active stereoisomers in agrochemicals contributes to increased environmental pollution without any benefit on the desired action [7]. On the other hand, pure enantiomers are also required in the preparation of amino acid-based food supplements and additives, as the use of D-enantiomers (R-configuration) is forbidden in the food industry [8]. Consequently, the preference for pure enantiomer formulations is evident. In the case of drugs, during the last decades, the trend to commercialize drugs as enantiomerically pure formulations has risen significantly [1,7]. In fact, this increase has been partly encouraged by the market strategy so-called racemic switch, which enable the companies to achieve a patent on a new-single pure enantiomer formulation of a drug that was first sold as racemate [4]. Nonetheless, many drugs are still marketed as racemic mixtures, even though there is only one active enantiomer. In fact, currently, more than 60% of the drugs commercialized are chiral, and approximately an 88% of them are sold in their racemic form [2]. These racemic formulations should only be administered when enantiomers show complementary biological activity. Moreover, even when both enantiomers are active, it is still more advantageous to use single pure enantiomeric formulations, because lower doses can be used, there is higher safety margin, lower interindividual variability, as well as less drug interactions and side effects [1,2].

Therefore, the growing demand for pure enantiomer formulations requires the development of sensitive analytical methodologies that enable the detection and differentiation of enantiomers and the control of the enantiomeric purity of the marketed products. In this sense, liquid chromatography (HPLC) has been widely applied for the analysis of chiral compounds due to its multiple advantages and suitability to achieve the separation of enantiomers.

In contrast to conventional liquid chromatography with particle packed columns, highly efficient fast separation is usually desired in different fields including but not limited to pharmaceutical, biological, food and environmental analysis. Fast efficient separation in chromatography is conducted by three competing approaches; ultrahigh pressure HPLC, core-shell and monoliths.

Ultrahigh pressure HPLC is based on the use of small sub-2-µm non-porous particle packed columns that can provide large surface area than the classical particle packed stationary phase for more efficient separation thus allowing the use of shorter columns with equivalent resolution to save the analysis time. The accompanied high back pressure of the small particle packed columns is overcoming by running the column in an ultrahigh pressure HPLC instrument that can resist high back pressure of up to 15,000 psi or 10,000 psi for longer column lifetimes. This has been achieved by the introduction of UHPLC system to run the chromatographic process with the sub-2-µm particle packed columns [9].

The second approach to achieve highly efficient fast chromatographic separation depends on changing the type of packed stationary phase particles inside the columns. Instead of using solid sphere particles, a core-shell particle (superficially porous particles) is used. The core shell involves the use of a solid core particles coated with a thin of stationary phase. A short diffusion path length of the shell improves mass transfer and the separation efficiency, as well as it ensures a reduced plate height [10,11]. The smaller eddy dispersion time also contributes to better separation efficiency [12]. However, lower sample load capacity is a common disadvantage and possible weaker retention would be obtained.

The third approach is the use of monolithic HPLC columns, which are columns of stationary phase contain continuous porous material for chromatographic separation. Monolithic silica was introduced first by Nakanishi et al. [13,14] in the early 1990s fabricated by sol-gel phase process transformation. Tanaka et al. [15,16] described the fabrication process to obtain a monolithic column. Based on the filling material, three types of monolithic column are known, i.e., polymeric-based monolithic columns, hybrid-monolithic columns, and monolithic-silica columns. Polymeric-based columns are particularly efficient in separating large molecules such as proteins and are more used in capillary rather than conventional HPLC columns, mostly for the separation or functionalization of large biomolecules, such as enzymes, proteins, and antibodies [17,18]. Hybrid monolithic columns of a mixed modes showed also successful applications [19,20,21]. Nano-LC monolithic capillary columns are also used for enantioseparation. Different types of chiral selectors have been immobilized into monolithic capillary columns either to silica-based [22,23,24] or to polymeric-based [25] or to hybrid monolithic [26] capillary columns for chiral discrimination. Examples of immobilized chiral selectors include cyclodextrins as well as derivatives of cellulose, amylose, and macrocyclic antibiotics, among others [25,27,28]. While chiral selector immobilized into polymeric-based monolithic capillary columns are more reproducible [25,29], silica-based monolithic silica capillary columns show better enantioresolution power [28].

In 2000 monolithic-silica columns were commercialized by two companies Merck KGaA (Darmstadt, Germany) and Phenomenex (Torrance, CA, USA). Merck started first by introducing Chromolith®Performance RP-18e column 4.6 × 100 mm monolithic silica rods enclosed in polyetheretherketone (PEEK) column body. Different modes (Si, RP8, RP18, NH2) then appeared and are these currently commercialized [30]. Phenomenex also produced monolithic silica columns commercially as Onyx^TM monolithic available in C8, C18. The second-generation monolithic (Chromolith^® HighResolution RP-18e columns (100 × 4.6 mm) followed the first by Merck in 2011. It shows improved radial pore distribution and reduced the size of macropores to correspond in performance to sub-2-µm conventional particle packed columns with better peak symmetry than the first generation. Reduced theoretical plate heights are usually obtained with the second-generation monolithic columns. Higher backpressure is usually obtained with second generation compared to first generation monolithic columns but still lower than that of a conventional particle-packed column [31,32]. Macropores of a second-generation column has a diameter of 1.15 µm diameter and is claimed to give a column efficiency exceeding 140,000 plates/meter. The smaller eddy dispersion obtained by smaller macropores size reduces band broadening compared to that possible with the first-generation skeleton. The separation performance of HighResolution is claimed to correspond to sub-3-µm total porous or 2.7-µm core-shell columns [33]. Monolithic silica HPLC columns are currently available in different i.d. (2, 3, 4.6 mm) and different lengths (25, 50, 100, 150 mm). Capillary format monolithic (C8 and C18) of i.d. 50, 100 and 200 µm and 5, 15 and 30 cm lengths are also available.

Given the fact that the molecular modeling provides reasonably good predictions regarding elution orders against experiments, it is also able to offer molecular level insights, including hydrogen bonding behaviors, ring–ring stacking, and sterical effects, which are critical to understand chiral separation recognition between selectors and drug molecules. While there should be other important molecular events and interactions, modern machine learning technology can help to elucidate and reveal better prediction models.