Macrocyclic glycopeptide‐based chiral selectors for enantioseparation in sub/supercritical fluid chromatography

Abstract

Increasing number of reported works dealing with macrocyclic glycopeptide‐based columns in sub/supercritical fluid chromatography (SFC) points to the growing interest in this area. With the development and production of sub 2 µm fully porous particles and superficially porous particles with bonded macrocyclic glycopeptides, significant improvements have been made in ultrafast high efficiency chiral SFC. This review article gives an overview of macrocyclic glycopeptide‐based chiral selectors that were used in theoretical studies and/or applications in SFC. The review covers the period from 1997 when macrocyclic glycopeptides were first used in SFC till the end of July 2020 according to Web of Science. This work can also be helpful to analysts searching for an appropriate method for the separation/determination of enantiomers of their interest.

ABBREVIATIONS

2‐PrOH

propan‐2‐ol

ACN

acetonitrile

CSPs

chiral stationary phases

CSs

chiral selectors

DEA

diethylamine

EtOH

ethanol

FPPs

fully porous particles

HAc

acetic acid

IPAM

isopropylamine

LSER

linear solvation energy relationship

MeOH

methanol

MGs

macrocyclic glycopeptides

MP

mobile phase

NPSD

narrow particle size distribution

SFC

sub/supercritical fluid chromatography

SPPs

superficially porous particles

TEA

triethylamine

TFA

trifluoroacetic acid

1. INTRODUCTION

Enantioseparation can be successfully carried out using various separation techniques, that is, capillary electrophoresis, gas chromatography, normal phase, reversed phase, and polar organic modes of liquid chromatography. However, there are some issues such as long equilibration and analysis time or peak broadening that are often associated with those methods. These drawbacks can be overcome by using SFC ¹ , ² that enables faster separation with high sample throughput. ³ Nowadays, SFC becomes more and more popular due to its properties, for example, short analysis time due to the possibility of high flow rate use, the high chromatographic efficiency due to the low mobile phase viscosity, the lower cost and toxicity of mobile phase, ⁴ progress in hyphenation with mass spectrometry, ⁵ , ⁶ , ⁷ and improvement in general understanding of its retention/separation mechanism. ⁸ , ⁹ , ¹⁰ , ¹¹ Commercially available chiral stationary phases (CSPs) are based on a number of different chiral selectors including: polysaccharides, macrocyclic glycopeptides, cyclodextrins, cyclofructans, ion‐exchangers, Pirkle type, ligand exchangers, crown ethers, and chiral polymers. ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶

Macrocyclic glycopeptide (also termed macrocyclic antibiotic ¹⁷ )‐based chiral selectors have been widely used in enantioseparation in liquid chromatography ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² and capillary electrophoresis. ²³ , ²⁴ , ²⁵ They have also been employed in SFC ²⁶ , ²⁷ for various applications – for example, in bioanalysis, ²⁸ , ²⁹ , ³⁰ pharmaceutical analysis, ³¹ , ³² , ³³ and for analysis of amino acids ³⁴ , ³⁵ and psychoactive substances. ³⁶ In the recent years, there has been an increase in use of macrocyclic glycopeptide‐based chiral selectors in SFC. ³⁷ Very encouraging findings illustrate the potential of modern chiral chromatography in enabling enantiopurity analysis of an entire pharmaceutical synthetic route with macrocyclic glycopeptide chiral selectors bonded to core‐shell particles. ³⁸

Macrocyclic glycopeptides (MGs) have been introduced as chiral selectors (CSs) for liquid chromatography by Armstrong and colleagues in 1994. ³⁹ Three years later they have been used in SFC for the first time. ⁴⁰ MGs share a unique structure – they consist of an aglycone “basket” and pendent carbohydrate moieties except for teicoplanin aglycone where carbohydrate moieties are removed. They contain, inter alia, peptide moieties, carbohydrates, ionizable phenolic, carboxylic, and amine moieties, which can interact with analytes through various types of interaction. ⁴¹ , ⁴² For demonstration, the planar structures of teicoplanin, teicoplanin aglycone, vancomycin, and ristocetin A are shown in Figure 1.

FIGURE 1.

Structures of teicoplanin, teicoplanin aglycone, vancomycin and ristocetin A

Because of the presence of multiple active chiral interaction sites within their macromolecular structure, these CSs display a broad enantioselectivity. On the other hand, due to their complex interaction possibilities, it is difficult to recognize/describe the mechanism of enantiodiscrimination on molecular level. ⁴³ MGs possess multiple sites capable of interacting with chiral analyte by hydrogen bonds, π‐π interactions, a variety of dipole, electrostatic, and hydrophobic interactions (hydrophobic inclusion complexes or associates with a hydrophobic “pocket”). On account of complex molecular structure, steric hindrance also plays a role in CS‐solute interactions. ²⁵ , ⁴⁴ , ⁴⁵ , ⁴⁶

Commercially available MG‐based chiral stationary phases (CSPs) are based either on fully porous particles (FPPs) or superficially porous particles (SPPs), also termed core shell particles – see Table 1. Astec Chirobiotic™ columns contain 5 µm FPPs while the MG‐based columns produced by AZYP (TeicoShell, VancoShell, TagShell, and NicoShell) are covalently bonded to 2.7 µm SPPs. ²⁷ , ⁴⁷ , ⁴⁸ An example of fast enantioseparation of α‐pyrrolidinopropiophenone (α‐PPP) on NicoShell (modified MG as CS) column is shown in Figure 2. Sub‐2 µm FPPs‐based macrocyclic glycopeptide CSPs have been also prepared and used in SFC ⁴⁹ but they were not commercialized yet. ⁵⁰ , ⁵¹ Columns packed with SPPs exhibit higher efficiency, lower pressure drops and allow to increase flow rates and/or co‐solvent percentage to higher values than what would be possible with FPPs‐based columns of identical dimensions thanks to lower back pressure. ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ , ⁵⁶ Shorter diffusion path of solutes and consequently higher column efficiency belong to the major advantages of SPPs over FPPs. SPPs‐based materials provide flatter dependence of column performance on the mobile phase flow rate, especially due to decreased resistance to mass transfer compared to FPPs, that is, a smaller C‐term in the van Deemter equation. ⁵⁷ , ⁵⁸ , ⁵⁹ How the morphology of solid core shell particles improves chromatographic performance in terms of individual parameters of the van Deemter equation is reviewed in depth by Hayes and co‐workers. ⁶⁰ Compared to FPPs, SPPs offer two main advantages due to the presence of a solid and inaccessible core: smaller longitudinal diffusion effects (B term in van Deemter equation) and fast solid‐liquid mass transfer (C‐term of van Deemter equation), both contributing to reduce band broadening. ⁶¹ The solid core materials do provide a benefit also in terms of the A term of van Deemter equation that describes the eddy dispersion. This parameter is dependent primarily on the particle size and the packing efficiency. Based on a wealth of experimental data it was concluded that the packing is better with solid core materials than with fully porous materials. Solid core materials have a rougher surface than fully porous materials. As a consequence, there is considerably more shear stress applied to the particles when they are packed. ⁶⁰ Decreased pressure drops and back pressure lead to reduction of frictional heating and enable the use of such stationary phases at higher flow rates without losing efficiency. ⁵⁰ Plate heights of columns produced with SPPs are less dependent on the mobile phase (MP) flow rate compared to FPPs, hence they are better suited for high speed separations. ⁵² , ⁵⁷ , ⁶² , ⁶³

TABLE 1.

Commercially available MG‐based columns and their basic characteristics

Trade name/company	Chiral selector	Column dimension/L × ID (mm)	Particles size (µm)	Particles type	Pore size (Å)	pH range
Astec® CHIROBIOTIC® V (Supelco™)	Vancomycin	50 × 2.1	5	Fully porous	100	3.5‐7.0
		100 × 2.1
		150 × 2.1
		100 × 4.6
		150 × 4.6
		250 × 4.6
		250 × 10
		250 × 21.2
Astec® CHIROBIOTIC® V2 (Supelco™)	Vancomycin	50 × 2.1	5	Fully porous	200	3.5‐7.0
		100 × 2.1
		150 × 2.1
		100 × 4.6
		150 × 4.6
		250 × 4.6
		250 × 10
		250 × 21.2
Astec® CHIROBIOTIC® T (Supelco™)	Teicoplanin	50 × 2.1	5	Fully porous	100	3.8‐6.8
		100 × 2.1
		150 × 2.1
		100 × 3
		50 × 4.6
		100 × 4.6
		150 × 4.6
		250 × 4.6
		250 × 10
Astec® CHIROBIOTIC® T2 (Supelco™)	Teicoplanin	100 × 2.1	5	Fully porous	200	3.8‐6.8
		150 × 2.1
		150 × 4.6
		250 × 4.6
Astec® CHIROBIOTIC® TAG (Supelco™)	Teicoplanin aglycone	50 × 2.1	5	Fully porous	100	3.0‐6.8
		100 × 2.1
		150 × 2.1
		100 × 4.6
		150 × 4.6
		250 × 4.6
		250 × 10
		250 × 21.2
		150 × 10	16
Astec® CHIROBIOTIC® R (Supelco™)	Ristocetin A	150 × 2.1	5	Fully porous	100	3.5‐6.8
		250 × 2.1
		100 × 4.6
		150 × 4.6
		250 × 4.6
VancoShell (AZYP, LLC)	Vancomycin	50 × 2.1	2.7	Superficially porous	–	2.5‐7.0
		100 × 2.1
		150 × 2.1
		50 × 4.6
		100 × 4.6
		150 × 4.6
		50 × 3.0
		100 × 3.0
		150 × 3.0
TeicoShell (AZYP, LLC)	Teicoplanin	50 × 2.1	2.7	Superficially porous	–	2.5‐7.0
		100 × 2.1
		150 × 2.1
		50 × 4.6
		100 × 4.6
		150 × 4.6
		50 × 3.0
		100 × 3.0
		150 × 3.0
TagShell (AZYP, LLC)	Teicoplanin aglycone	50 × 2.1	2.7	Superficially porous	–	2.5‐7.0
		100 × 2.1
		150 × 2.1
		50 × 4.6
		100 × 4.6
		150 × 4.6
		50 × 3.0
		100 × 3.0
		150 × 3.0
NicoShell (AZYP, LLC)	Modified glycopeptide	50 × 2.1	2.7	Superficially porous	–	2.5‐7.0
		100 × 2.1
		150 × 2.1
		50 × 4.6
		100 × 4.6
		150 × 4.6
		50 × 3.0
		100 × 3.0
		150 × 3.0
InfinityLab Poroshell 120 Chiral‐V (Agilent Technologies, Inc.)	Vancomycin	50 × 2.1	2.7	Superficially porous	120	2.5‐7.0
		100 × 2.1
		150 × 2.1
		50 × 4.6
		100 × 4.6
		150 × 4.6
InfinityLab Poroshell 120 Chiral‐T (Agilent Technologies, Inc.)	Teicoplanin	50 × 2.1	2.7	Superficially porous	120	2.5‐7.0
		100 × 2.1
		150 × 2.1
		50 × 4.6
		100 × 4.6
		150 × 4.6

FIGURE 2.

Chromatogram of enantioseparation of α‐PPP on NicoShell column (50 × 2.1 mm ID, 2.7 µm SPP). Mobile phase: CO₂/MeOH/HAc/TEA 90/10/0.5/0.01 (v/v/v/v), flow rate 2 mL/min, back pressure 2000 psi, temperature 40°C, injected volume 1 µL, UV detection at 254 nm. Original work

With the decrease in particle size and use of columns of smaller dimensions arising from it, ultrafast chromatographic sub‐minute enantioseparations are now possible. An example of sub‐minute baseline enantioseparations using different chiral selectors bonded to 2.7 µm SPPs is shown in Figure 3. ⁴⁷

FIGURE 3.

Representative chromatograms on different stationary phases (A) VancoShell (10 cm  ×  0.46 cm) Analyte: Fluoxetine MP‐ 75/25 CO₂/MeOH, 3% water, 0.1% TEA, 0.1% TFA, 4 mL/min, T = 30°C (B) NicoShell (10 cm  ×  0.46 cm) Analyte: Nicotine MP‐ 60/40 CO₂/MeOH, 0.1% TEA, 4  mL/min, T = 30°C (C) NicoShell (10 cm  ×  0.46 cm) Analyte: Tramadol MP‐ 60/40 CO₂/MeOH, 0.2% TEA, 0.3% TFA, 4 mL/min, T = 30°C (D) LarihcShell‐P (10 cm  ×  0.46 cm) Analyte: Norephedrine MP‐ 80/20 CO₂/MeOH, 0.2% TEA, 0.3% TFA, 4  mL/min, T = 25°C (E) LarihcShell (10 cm  ×  0.46 cm) Analyte: 1‐(1‐Naphthyl)ethylamine MP‐ 80/20 CO₂/MeOH, 0.2% TEA, 0.3% TFA, 4 mL/min, T = 25°C (F) TeicoShell (10 cm  ×  0.46 cm) Analyte: Dichloroprop MP‐ 60/40 CO₂/MeOH, 0.1% ammonium formate, 4  mL/min, T = 30°C UV detection at 254 nm. Reproduced from ref. ⁴⁶ with permission from Elsevier

2. THEORETICAL STUDIES OF MACROCYCLIC GLYCOPEPTIDES IN SFC

2.1. Interaction mechanism

Interactions contributing to retention on three FPPs‐MG‐based columns, that is, the Chirobiotic V2 (vancomycin as CS), the Chirobiotic T (teicoplanin as CS) and the Chirobiotic TAG (teicoplanin aglycone as CS) were characterized using linear solvation energy relationship model (LSER). ⁶⁴ Simple mobile phase composed of CO₂/methanol 90/10 (v/v) without additives was used for this purpose. The authors showed that these three CSPs share the same pattern of interactions. Hydrogen bond interactions, interactions with n‐ and π‐electrons, dipole‐dipole and dipole‐induced dipole interactions, and cation‐exchange interactions increase the retention of analytes while dispersion interactions (hydrophobicity) and anion‐exchange interactions contribute to the decrease of retention. Under the same conditions, 67 chiral compounds were measured on all three columns. The complementarity among the columns was examined using Venn diagrams. The results showed that a significant portion of chiral compounds could be separated by two or three CSPs. The teicoplanin aglycone (Chirobiotic TAG) provided a larger number of unique separations compared to Chirobiotic T and V columns. The LSER model was also used to describe interactions which contribute to the retention in the system with the Chirobiotic TAG (teicoplanin aglycone) column with MPs containing high amount of basic (isopropylamine) or acidic (trifluoroacetic acid) additives. ⁶⁵ The authors tested 11 MPs composed of 90 vol% of carbon dioxide and 10 vol% of methanol with or without additives. The concentration of additive in methanol ranged from 0 to 610 mM and 0 to 1220 mM of trifluoroacetic acid (TFA) and isopropylamine (IPAM), respectively. The results showed that highly concentrated additives had negligible effect on the MP polarity. However, the additives affect MP acidity – TFA addition led to strong decrease in the apparent pH value (from 4.5‐5 to 1‐2) while IPAM addition caused moderate increase up to pH 6. On the basis of the results obtained it can be supposed that: (a) in the additive free MP, both the carboxylic acid and amine functions of the Chirobiotic TAG column should be ionized, thus the net charge for the ligand would be zero; if the amine function is unavailable (due to the bonding of TAG molecule to silica gel through this group), ⁶⁶ the net charge is –1; (b) when IPAM is introduced, deprotonation of a free amine moiety would not occur, thus the net charge would still be the same; (c) when TFA is introduced, the carboxylic acid moiety should protonate, thus the net charge would be +1, or zero in the case the amine moiety is not free. Regarding the coefficients of LSER equation, the following trends were observed. Hydrogen bond interactions described by coefficients a and b significantly varied with increasing IPAM concentration in MP with opposite trends, that is, coefficient a (hydrogen bond basicity; related to retention of proton donors) increased while coefficient b (hydrogen bond acidity; related to retention of proton acceptors) decreased. The increase in the coefficient a value is related with adsorption of the IPAM on the CSP surface. Regarding ionic interactions, described by d ⁻ and d ⁺ coefficients related to interactions with anionic and cationic analytes, significant changes were observed with increasing IPAM concentration in MP. The value of d ⁻ coefficient strongly increased when IPAM was first added to the MP. Further increases of IPAM in the MP led to situation that d ⁻ coefficient value reached at maximum before its value again decreased when further increasing the IPAM concentration. The obtained results were explained as follows: (a) at high IPAM concentrations, the surface of CSP is saturated with additive and the excess unadsorbed IPAM remains in the MP, forming ion‐pairs with negatively charged analytes. Thus, the solute‐MP interactions are enhanced that results in d ⁻ coefficient decrease; (b) the methoxycarbonyl acid presented in the MP (formed from CO₂ and MeOH) may be titrated by IPAM that leads to increase of apparent pH of the MP. Introduction of IPAM to the MP caused significant decrease of d ⁺ coefficient, that is, decrease of retention of positively charged analytes. Addition of TFA to the MP affected only d ⁺ coefficient value, that is, interactions with cationic analytes. When using 610 mM TFA in MeOH in MP CO₂/MeOH + additive 90/10 (v/v), this interaction type became the most significant contributor to the retention. This is explained by strong adsorption of TFA on the CSP surface, creating a layer on top of the surface and ligands. Thus, the interactions between the positively charged analytes and the CSP strongly increased. The authors also tested the influence of MP additives on enantioseparation, which will be described in the following section.

2.2. Effect of mobile phase composition on retention and enantioseparation

2.2.1. Effect of co‐solvent

Alcohols such as methanol (MeOH), ethanol (EtOH), and propan‐2‐ol are the most common modifiers (co‐solvents) in sub/supercritical MPs. ⁶⁷ , ⁶⁸ , ⁶⁹ , ⁷⁰ The nature and portion of co‐solvent in MP may affect not only retention, but also enantioselectivity and enantiomer elution order. ⁷¹ , ⁷² Methanol (with or without additives) is the most often used co‐solvent to increase the elution strength of the MP with MG‐based CSPs in SFC. ⁷³ A few works dealing with the effect of co‐solvent type on retention and enantioseparation on MG‐based CSPs can be found in the literature. ⁷⁴ , ⁷⁵ The TeicoShell (teicoplanin as CS), the VancoShell (vancomycin as CS), the TagShell (teicoplanin aglycone as CS), and the NicoShell (modified MG as CS) columns were tested for their enantioselectivity for rod‐like liquid crystals. ⁷⁵ In this work, three co‐solvents were tested, that is, methanol, ethanol, and propan‐2‐ol. The results showed that on the TeicoShell, the VancoShell, and the NicoShell columns, propan‐2‐ol produced the highest enantioselectivity values as well as the highest retention. Surprisingly, one group of liquid crystals could not be resolved on the TagShell column using propan‐2‐ol and MeOH was the optimal co‐solvent instead. No change in elution order was observed under the tested conditions on MG‐based CSPs. In this work, enantioselective potential of polysaccharide‐based CSPs (amylose tris(3,5‐dimethylphenylcarbamate), cellulose tris(3,5‐dimethylphenylcarbamate), and cellulose tris(3‐chloro‐4‐methylphenylcarbamate) for rod‐like liquid crystals was also evaluated. The results showed that polysaccharide‐based CSPs provide generally better enantioseparation and higher enantioselectivity for these liquid crystals compared to MG‐based CSPs. Other work compares enantioselective potential of TeicoShell and VancoShell columns for various groups of chiral analytes, that is, phytoalexins, ketamine, and cathinone derivatives, ⁷⁶ substituted tryptophans, and calcium channel blockers. ⁷⁴ The effect of co‐solvent type, that is, methanol, ethanol, propan‐2‐ol, and propan‐1‐ol on retention and enantioseparation were evaluated. The authors demonstrated that optimal co‐solvent (baseline separation in shortest analysis time) differs for individual columns and the group of chiral compounds with no hint of predictable pattern. For instance, methanol was found to be the optimal co‐solvent for enantioseparation of phytoalexins on the TeicoShell column while on the Vancoshell column the most successful MP contained EtOH as co‐solvent. Enantioseparation of ketamines can serve as another example. MP containing propan‐2‐ol/acetonitrile as a co‐solvent provided the highest resolution of these derivatives on the TeicoShell column, while ethanol was a better choice for ketamines enantioseparation on the VancoShell column. Thus, screening of various alcohols as co‐solvents is an important part of optimization of separation conditions for particular chiral analytes using MG‐based CSPs. Recently, Armstrong et al tested azeotropic ethanol (190 proof ethanol) instead of methanol or absolute ethanol as co‐solvents using SPP‐based chiral stationary phases including macrocyclic glycopeptide‐based TeicoShell, VancoShell, and NicoShell columns. ⁷⁷ The authors showed significant increase in efficiency and for some chiral compounds reduced retention by using a “190 proof” ethanol. This was explained as follows: the small amount of water in the mobile phase is competing for the active sites on the stationary phase and enhancing the rate of mass transfer kinetics. When using methanol and absolute ethanol, the efficiency was directly proportional to their concentration. But, when using the “190 proof” ethanol, the efficiency trend was reversed, that is, the efficiency increased with lower concentration that allows the use of lower amount of organic solvent in the MP. This behavior is valid for 10‐40 vol% of co‐solvent in the mobile phase. Other concentrations of co‐solvents have not been tested. These MPs satisfied three “R” principles in green chemistry: (a) Replacement of methanol with “190 proof” ethanol since it is a less toxic alternative, (b) Reducing solvent consumption as comparable or lower retention times were obtained with the new MP, and (c) Recycling of waste is facilitated due to the fact that “190 proof” ethanol can be obtained by simple distillation and does not require additional steps of purification, hence its recovery is more straightforward compared to absolute ethanol. The advantages of using azeotropic ethanol were described also for preparative SFC. Compared to methanol the detrimental effect of increased analyte loading on the peak shapes was significantly reduced when using the “190 proof” ethanol thereby allowing higher loadability on the same CSP.

The scale up from analytical to preparative SFC is not as easy as in liquid chromatography due to the physical properties of CO₂. ⁷⁸ The main issue is the compressibility of the mobile phase that frequently results in pressure, density, and temperature gradients. ⁷⁹ Scale‐up approaches in SFC are comprehensibly summarized in the review article by Rajendran. ⁸⁰ Tarafder and co‐workers demonstrated that scale‐up and method transfer techniques used in liquid chromatography can be applied to SFC as long as it is ensured that both the original and the target SFC systems operate at the same average density. ⁷⁹ From a practical point of view, however, there is a trouble with the criterion of keeping the same density as the availability of density data is limited, in many cases density data may not be accessible at all. Therefore, a simpler approach, of matching average pressures was developed. ⁸¹ The authors demonstrated that the average‐density rule is not superior to the average‐pressure rule and the latter should be applicable under nearly all the method conditions where the average‐density rule is applicable. ⁸¹

Unfortunately, as only few works dealt with the effect of co‐solvent type in MP on retention and enantioseparation in chiral SFC using MG‐based columns, any general conclusions cannot be drawn. The mentioned conclusions from cited articles are generally only applicable to the series of compounds examined, as enantioseparation mechanisms are very complex.

2.2.2. Effect of basic, acidic, and mixed additives

The effect of MP additives on enantioseparation using MG‐based CSPs is more frequently studied. These chiral selectors contain ionizable groups thus the use of additives can significantly affect retention and enantioseparation even of neutral analytes through influencing the state of the CSPs (eg, ionization of chiral selector) due to the adsorption on the stationary phases. ⁴⁷ , ⁸² , ⁸³ Moreover, the MP additives also affect the peak shape in chiral SFC. ⁸⁴ , ⁸⁵ , ⁸⁶ The effect of MP additives on retention and enantioseparation of phenylalanine on the TeicoShell column is shown in Figure 4. As can be seen from Figure 4, phenylalanine can be baseline separated in the MP without additive. However, the obtained peaks are very broad and analysis time is approximately 6 min. Addition of mixed additive (acid and base) into the MP significantly narrows the peaks and decreases analysis time while maintaining baseline separation. The amount of additives is also significant. A comparison of Figures 4B and 4C shows that higher amount of additive results in narrower peaks and sub‐minute baseline enantioseparation.

FIGURE 4.

The effect of mobile phase additive on enantioseparation and peak shape of d,l‐phenylalanine on Teicoshell column (50 × 2.1 mm ID, 2.7 µm SPP). Mobile phases: A: CO₂/MeOH 60/40 (v/v); B: CO₂/MeOH/TFA/DEA 60/40/0.05/0.05 (v/v/v/v); C: CO₂/MeOH/TFA/DEA 60/40/0.1/0.1 (v/v/v/v). Flow rate 2 mL/min, back pressure 2000 psi, temperature 40°C, injected volume 1 µL, UV detection at 210 nm. Original work

The effects of type of basic additive, that is, isopropylamine or triethylamine (TEA) and its concentration, in methanol used as co‐solvent on retention and enantioseparation of a set of 12 chiral compounds with various functional groups were tested on vancomycin‐based (Chirobiotic V) column. ⁸⁷ The authors demonstrated that most of the analytes did not elute from the column in the absence of basic additive. The addition of basic additive led to elution of all target compounds with higher retention observed with triethylamine than with isopropylamine. Increasing additive concentration tended to have a deleterious effect on enantioresolution. The apparent loss of resolution was considered in the context of retention decrease. The use of amine additives has a significant impact on retention and resolution and the magnitude of these effects varies with the nature of the analytes (structure, presence of functional and ionizable groups) and the CSP. For example, when using MP without basic additives most of the analytes did not elute from the vancomycin‐based column because it contains ionizable groups. In contrast, all analytes eluted from Chiralpak AD column (amylose tris(3,5‐dimethylphenylcarbamate as CS ⁸⁸ )) as this CSP is not ionizable under normal‐phase conditions with additive free MP. For comparison: on the Chiralpak AD column the additives had little effect on retention, but they did foster significant improvements in peak shape and resolution of tested chiral compounds. Some tentative experiments were also performed using ristocetin‐A‐based (Chirobiotic R) column. ⁸⁹ Figure 5 shows an example of fast baseline enantioseparation of efavirenz enantiomers using 250 mm long Chirobiotic R column. Due to the high enantioselectivity for efavirenz enantiomers, the flow rate could be increased to 6 mL/min that resulted in baseline separation within 1.5 min. Based on the analysis of six chiral compounds, it has been demonstrated that enantioselectivity and resolution did not change when using acetic acid as an additive, while triethylamine had enhancing effect on the enantioseparation. The effect of water as an additive to methanol depended on the type of the compound although not on its acidic properties. The authors concluded that water influences the enantioseparation by reduction of interactions between residual silanol groups. A few recent works dealt with the role of water in SFC enantioseparations. ⁴⁷ , ⁹⁰ , ⁹¹ It has been shown to be advantageous in some cases, especially for hydrophilic compounds. The Hildebrandt solubility parameter for water is nearly twice that of methanol, ⁹² making it excellent choice for increasing the solubility of hydrophilic compounds in packed column SFC. Moreover, the addition of water to the MP also enhances the solvating power of the mobile phase thereby facilitating hydrophilic solute separation. ⁹⁰ Makarov and co‐workers recently introduced chaotropic effect mechanism in SFC via ammonium hydroxide in water‐rich modifiers. ⁹³ Armstrong´s group described effective ways of enhancing SFC efficiency, and even predicting the effects of small aqueous additives. ⁹⁴ Recently, Armstrong et al thoroughly tested and reported the effects of water in ternary MPs on different CSPs including TeicoShell (teicoplanin as CS), VancoShell (vancomycin as CS), and NicoShell (modified MG as CS) columns. ⁴⁸ The hydrophilic columns (ie, MG‐based columns) were much more sensitive to small amounts of water in the MP in terms of efficiency increase and reduction of analysis time of enantiomers compared to hydrophobic CSPs (for example, polysaccharide‐based columns). The authors have demonstrated that using water in MP, there can be more than or equal to eightfold improvements in efficiency with hydrophilic CSPs. On the other hand, polysaccharide‐based columns showed a minimal increase in efficiency and decrease in analysis times compared to MG‐based columns. The order for hydrophilicity was experimentally determined using MP ACN/25 mM ammonium acetate, pH 6.8 80/20 (v/v) as follows: TeicoShell ≫ VancoShell > LarihcShell > Bare Silica ≈ NicoShell > (S,S)‐Whelk‐O1 > QShell > Chiralpak IC‐5 > Chiralcel OD‐3 > Chiralpak IA‐3. Water changes the polarity of the MP and sorbs on the stationary phase. Moreover, water acts as a sorption competitor to the analyte on hydrophilic stationary phase that was demonstrated with nonlinear chiral chromatography.

FIGURE 5.

Separation of efavirenz enantiomers on Ristocetin A CSP (Chirobiotic R column 250 × 4.6 mm, 5 µm). Conditions: MP: CO₂/[(MeOH/acetic acid (100:0.5 (v/v)] 75:25 (v/v), flow rate 6 mL/min, 210 bar, 30°C, UV detection at 310 nm. Reproduced from ref. ⁸⁷ with permission from John Wiley and Sons

The effect of high concentration of additives (acidic trifluoroacetic acid and basic isopropylamine) on retention and enantioseparation was tested on Chirobiotic TAG column. ⁶⁵ A loss of enantioseparation was observed when adding high concentrations of basic isopropylamine for most of the compounds that were resolved when no additive was used. The same situation occurred when high concentration of acidic additive was used.

The use of mixed (TEA+TFA) additives led to sharp retention decrease of neutral liquid crystals and reduction or loss of enantioselectivity on SPP‐based macrocyclic glycopeptides. ⁷⁵ This behavior was ascribed to the presence of basic additive in MP as was previously also shown by Khater and West. ⁶⁴ On the other hand the effect of acidic TFA differed for individual CSPs. On VancoShell (vancomycin as CS) and NicoShell (modified MG as CS) columns, adding TFA resulted in significant decrease of retention as opposed to TeicoShell (teicoplanin as CS) and TagShell (teicoplanin aglycone as CS) columns. On VancoShell, NicoShell and TagShell CSPs peak shape was improved and in the case of VancoShell and TagShell the use of acidic additive also resulted in slight increase of enantioresolution. The effect of TFA in MP was negligible in the system with the TeicoShell column. Other work showed positive effect of mixed additives (TEA + TFA, diethylamine (DEA) + TFA, IPAM + TFA) for enantioseparation of biologically active compounds on TeicoShell and VancoShell columns. ⁷⁴ The discussed works show great importance of MP additives in enantioseparation processes on MG‐based CSPs in SFC. The mixed additives seem to be very useful in many cases. Also addition of water to the MP led to significant improvement of enantioseparation in particular cases. However, no generally applicable rules can be defined at this time due to the lack of more systematic experimental and theoretical investigations. The MP additives change differently the state of individual macrocyclic glycopeptides and charged analytes that should be systematically explored and explained in further works.

2.3. Effect of back pressure

Chirobiotic T (teicoplanin as CS) column was one of the set of 10 chiral columns used for thorough study of the effect of back pressure on retention and enantioresolution in SFC. ⁹⁵ Binary mobile phases, composed of carbon dioxide and methanol, were tested. Rate of retention change with pressure, dk/dP, linearly correlated with retention factor, that is, the higher the retention of an analyte, the more sensitive its retention to pressure changes. The change of column back pressure did not affect enantioselectivity. Slight decrease in enantioresolution (on average 6%) was observed when increasing back pressure from 100 to 200 bar especially due to the retention and efficiency decrease. Higher back pressure usually results in decrease of retention and efficiency accompanied by worsening of resolution compared to lower back pressure. ⁷⁰ , ⁹⁶ , ⁹⁷

3. APPLICATIONS

Analytical scale SFC applications, for example, ⁹⁸ , ⁹⁹ , ¹⁰⁰ , ¹⁰¹ , ¹⁰² , ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ including commercial column name, CSP type, separation conditions, and analytes are summarized in Table 2. This presentation offers an easy orientation in the separation systems used for particular enantioselective analysis. Table 2 demonstrates that MG‐based CSPs were applied for enantioseparation of a large scale of structurally different chiral compounds. Roy and Armstrong demonstrated that teicoplanin‐based (Teicoshell) column offers unique enantioseparations for acidic and neutral analytes. ⁴⁷ On the other hand, the Nicoshell and Vancoshell columns were suitable for separating secondary and tertiary amine containing pharmaceutical drugs and controlled substances. The Teicoshell column exhibits higher enantiorecognition ability than the Vancoshell column for a group of structurally different synthetically modified indole phytoalexins and tryptophan derivatives. On the other hand, the Vancoshell column provides higher enantioselective potential for chiral calcium channel blockers than the Teicoshell column under the same conditions. ⁷⁴ In this work, complementary enantioseparation behavior of teicoplanin and vancomycin‐based CSPs was confirmed. Another example of complementary enantioseparation behavior of three MG‐based CSPs can be found in the work by Armstrong´s group. ¹⁰² In this work, 24 chiral dihydrofurocoumarin derivatives and structurally related compounds were enantioseparated on the teicoplanin‐, the teicoplanin aglycone‐ and the ristocetin A‐based CSPs. All the dihydrofurocoumarin derivatives were resolved on at least one of the MG‐based CSPs. The teicoplanin‐based column showed the best enantioselectivity for 21 of the compounds tested. The teicoplanin aglycone‐ and the ristocetin A‐based columns separated similar numbers of chiral compounds tested, but the teicoplanin aglycone‐based column baseline resolved more samples than the ristocetin A‐based column under similar experimental conditions. The teicoplanin‐based column (Chirobiotic T) was also able to baseline resolved enantiomers of imidazoline compound Nutlin 3 while on the vancomycin‐based column (Chirobiotic V) no enantioseparation occurred. ¹⁰⁴ The Nutlin 3 is a promising small molecule antagonist of the MDM2‐p53 interaction and offers a novel strategy for cancer therapy. The Chirobiotic T2 (teicoplanin as CS) column has also found application in the determination of three aromatic amino acids (phenylalanine, tyrosine, tryptophan) in five commercial food supplements. ¹⁰⁰ The quality of the food supplements was confirmed through the absence of enantiomeric impurity, that is, d‐enantiomer in all of them. These works indicate the suitability of MG‐based columns for the analysis of real samples in SFC.

TABLE 2.

Summary of the MG‐based columns, mobile phase composition and enantioseparated compounds in SFC

Column name	CS	MP	Compounds analyzed	Reference
Chirobiotic T	Teicoplanin	gradient 5% to 30%	44 Chiral compounds, eg, acebutolol, fenoterol	40
		MeOH +0.1%DEA or TFA	naproxen, oxprenolol, propranolol, warfarin,
		within 5 min	tropic acid, hexobarbital
Chirobiotic T	Teicoplanin	CO2/MeOH+0.5% IPAM	Indapamide, ketoprofen, N‐CBZ‐phenylalanine,	103
		various volume ratios	propranolol, warfarin
Chirobiotic T	Teicoplanin	CO2/MeOH+TEA or TFA or	111 Chiral compounds ‐ heterocyclic compounds,	34
		H2O or glycerol	propionic acid derivatives, β‐blockers, sulfoxides,
		various volume ratios	N‐protected and native amino acids
Chirobiotic T	Teicoplanin	CO2/MeOH	24 Dihydrofurocoumarin derivatives and related	102
		various volume ratios	compounds
		2 to 25 volume % of MeOH
Chirobitotic T2	Teicoplanin	CO2/MeOH/water (v/v/v)	Aromatic amino acids	100
		MeOH 35‐60 vol %
		water 2‐10 vol %
Chirobiotic T	Teicoplanin	CO2/(MeOH/water 98:2+	24 Chiral compounds, eg, haloxyfop, mandelic acid,	51
Chirobiotic T2	Teicoplanin	20 mM ammonium acetate)	ketorolac, sulfoxide 4, phosphine oxide
In‐house made	Teicoplanin	60/40 (v/v)
UHPC‐Titan120‐
TZWIT‐1.9
In‐house made:		CO2/MeOH/TFA/TEA	Chlorthalidone, 5‐methyl‐5phenylhydantoin,	50
1.9 µm Titan T	Teicoplanin	71/29/0.1/0.1 (v/v/v/v)	5‐5‐diphenyl‐4‐methyl‐2‐oxazolidone
1.7 µm Daisogel T	Teicoplanin	60/40/0.1/0.1 (v/v/v/v)
		CO2/MeOH 60/40 (v/v)
Chirobiotic T	Teicoplanin	CO2/MeOH 60/40 (v/v)	Nutlin 3	104
TeicoShell	Teicoplanin	CO2/MeOH+additive (v/v)	100 Chiral compounds, eg, 4‐methylethcathinone,	47
		various types, amount and	thioridazine, ephedrine, nicotine, fluoxetine, MDMA,
		mixtures of additives ‐ TEA, TFA,	methylphenidate, pseudoephedrine, butylone, ethylone,
		water, ammonium formate,	4‐Methyl‐5‐phenyl‐2‐oxazolidinone, chlorthalidone,
		NH4OH, formic acid	esmolol, sotalol, clenbuterol, keterolac, hydantoin
TeicoShell	Teicoplanin	CO2/MeOH/additive (v/v/v)	29 Biologically active compounds, ie, phytoalexins,	74
		CO2/EtOH/additive (v/v/v)	calcium channel blockers, tryptophans, ketamines,
		CO2/2‐PrOH/additive (v/v/v)	cathinones
		CO2/ACN/2‐PrOH/additive
		(v/v/v/v), various types, amount
		and mixtures of additives ‐
		TFA, DEA, TEA, IPAM
TeicoShell	Teicoplanin	CO2/MeOH/additive (v/v/v)	Rod‐like liquid crystals	75
		CO2/EtOH/additive (v/v/v)
		CO2/2‐PrOH/additive (v/v/v)
		additives: TFA or TFA+TEA
TeicoShell	Teicoplanin	CO2/MeOH+water (v/v)	(±) cis‐4,5‐Diphenyl‐2‐oxazolidinone, 2‐(4‐chlorophenoxy)	48
		CO2/MeOH (v/v)	Propionic acid, metoprolol, tryptophan, fluoxetine,
		CO2/MeOH+additive	tryptophanamide, nicardipine, bupivacaine, proglumide,
		CO2/MeOH+additive+water	trans‐stilbene oxide, ketoprofen, bendroflumethazide,
		additives: ammonium formate,	warfarin, N‐benzoyl‐DL‐valine, dansyl serine, coumachlor,
		TEA+TFA, DEA, ammonium	hydrobenzoin, mianserin, trans‐stilbene oxide
		formate+formic acid
Chirobiotic TAG	Teicoplanin	CO2/MeOH+TEA or TFA or	111 Chiral compounds ‐ heterocyclic compounds,	34
	aglycone	H2O or glycerol	propionic acid derivatives, β‐blockers, sulfoxides,
		various volume ratios	N‐protected and native amino acids
Chirobiotic TAG	Teicoplanin	CO2/MeOH	24 Dihydrofurocoumarin derivatives and related	102
	aglycone	various volume ratios	compounds
		2 to 25 volume % of MeOH
In‐house made:		CO2/MeOH/TFA/TEA	Chlorthalidone, 5‐methyl‐5‐phenylhydantoin,	50
1.9 µm Titan TAG	Teicoplanin	71/29/0.1/0.1 (v/v/v/v)	5‐5‐diphenyl‐4‐methyl‐2‐oxazolidone
	aglycone	60/40/0.1/0.1 (v/v/v/v)
1.7 µm Daisogel TAG	Teicoplanin	CO2/MeOH 60/40 (v/v)
	aglycone
TagShell	Teicoplanin	CO2/MeOH/additive (v/v/v)	Rod‐like liquid crystals	75
	aglycone	CO2/EtOH/additive (v/v/v)
		CO2/2‐PrOH/additive (v/v/v)
		additives: TFA or TFA+TEA
Chirobiotic V	Vancomycin	gradient 5% to 30%	44 Chiral compounds, eg, acebutolol, fenoterol	40
		MeOH +0.1%DEA or TFA	naproxen, oxprenolol, propranolol, warfarin,
		within 5 min	tropic acid, hexobarbital
Chirobiotic V	Vancomycin	CO2/EtOH (91.8/8.2 mole ratio)	1,1´Bi‐2‐naphthol, α‐methyl‐α‐phenyl succinimide,	98
		CO2/MeOH (85/15 mole ratio)	ftorafur, 5‐methyl‐5‐phenyl hydantoin, warfarin,
			1,1´‐binaphthyl‐2,2´‐diyl‐hydrogenphosphate,
			3‐(α‐acetonyl‐4‐chlorobenzyl)‐4‐OH coumarin
In‐house made	Vancomycin	CO2/MeOH, CO2/MeOH+TEA,	28 Chiral compounds, eg, alprenolol, atenolol, benzoin,	99
capillary column		CO2/MeOH+TEA+HAc	binaphthol, bupivacaine, dichlorprop, ethotoin, etidocaine,
		CO2/MeOH+butylamine	fendilin, mepivacaine, metixene, phensuximide
		various volume ratios
Chirobiotic V	Vancomycin	CO2/MeOH+0.5% IPAM	Indapamide, ketoprofen, N‐CBZ‐phenylalanine,	103
		various volume ratios	propranolol, warfarin
Chirobiotic V	Vancomycin	CO2/MeOH+TEA or TFA or	111 Chiral compounds ‐ heterocyclic compounds,	34
		H2O or glycerol	propionic acid derivatives, β‐blockers, sulfoxides,
		various volume ratios	N‐protected and native amino acids
Chirobiotic V	Vancomycin	CO2/MeOH+additive (85/15 (v/v)	Alprenolol, bupivacaine, clenbuterol, fluoxetine,	87
		various amount of IPAM or TEA	indapamide, metoprolol, mianserin, nicardipine,
		as additives	orphenadrine, proglumide, tropicamide, verapamil
Chirobiotic V2	Vancomycin	CO2/(MeOH:aq. ammonia:formic	Clenbuterol	29
		acid 998:1:1 (v/v/v))‐linear gradient
		from 50% to 60% of co‐solvent
		in 4 min
VancoShell	Vancomycin	CO2/MeOH+additive (v/v)	100 Chiral compounds, eg, 4‐methylethcathinone,	47
		various types, amount and	thioridazine, ephedrine, nicotine, fluoxetine, MDMA,
		mixtures of additives ‐ TEA, TFA,	methylphenidate, pseudoephedrine, butylone, ethylone,
		water, ammonium formate,	4‐Methyl‐5‐phenyl‐2‐oxazolidinone, chlorthalidone,
		NH4OH, formic acid	esmolol, sotalol, clenbuterol, keterolac, hydantoin
VancoShell	Vancomycin	CO2/MeOH/additive (v/v/v)	29 Biologically active compounds, ie, phytoalexins,	74
		CO2/EtOH/additive (v/v/v)	calcium channel blockers, tryptophans, ketamines,
		CO2/2‐PrOH/additive (v/v/v)	cathinones
		CO2/ACN/2‐PrOH/additive
		(v/v/v/v), various types, amount
		and mixtures of additives ‐
		TFA, DEA, TEA, IPAM
VancoShell	Vancomycin	CO2/MeOH/additive (v/v/v)	Rod‐like liquid crystals	75
		CO2/EtOH/additive (v/v/v)
		CO2/2‐PrOH/additive (v/v/v)
		additives: TFA or TFA+TEA
VancoShell	Vancomycin	CO2/MeOH+water (v/v)	(±) cis‐4,5‐Diphenyl‐2‐oxazolidinone, 2‐(4‐chlorophenoxy)	48
		CO2/MeOH (v/v)	Propionic acid, metoprolol, tryptophan, fluoxetine,
		CO2/MeOH+additive	tryptophanamide, nicardipine, bupivacaine, proglumide,
		CO2/MeOH+additive+water	trans‐stilbene oxide, ketoprofen, bendroflumethazide,
		additives: ammonium formate,	warfarin, N‐benzoyl‐DL‐valine, dansyl serine, coumachlor,
		TEA+TFA, DEA, ammonium	hydrobenzoin, mianserin, trans‐stilbene oxide
		formate+formic acid
Chirobiotic R	Ristocetin A	CO2/MeOH (75/25 (v/v))	5 Acidic compounds, ie, dichlorprop, ketoprofen,	101
In‐house made	Ristocetin A	CO2/MeOH (80/20 (v/v))	warfarin, coumachlor, thalidomide
capillary column		CO2/MeOH+10 mM TFA
		(75/25 (v/v))
Chirobiotic R	Ristocetin A	CO2/MeOH	24 Dihydrofurocoumarin derivatives and related	102
		various volume ratios	compounds
		2 to 25 volume % of MeOH
Chirobiotic R	Ristocetin A	CO2/MeOH with TEA or HAc	3a,4,5,6‐Tetrahydrosuccinimido[3,4‐b]acenaphten‐10‐one,	89
		or water (v/v); CO2/EtOH with	α‐Methyl‐α‐propyl‐succinimide, warfarin, thalidomide,
		HAc; CO2/2‐PrOH with HAc	5‐methyl‐5‐phenyl‐hydantoin, efavirenz,
		(v/v); CO2/EtOH+2‐PrOH with	1,1´‐binaphtyl‐2´‐dihyl‐hydrogenophosphate
		HAc (85/15 (v/v))‐ various ratios
		of EtOH/2‐PrOH
Nautilus E	Eremomycin	CO2/MeOH/IPAM/TFA	Salbutamol sulfate	119
		various amounts and ratios
		of additives
NicoShell	modified MG	CO2/MeOH+additive (v/v)	100 Chiral compounds, eg, 4‐methylethcathinone,	47
		various types, amount and	thioridazine, ephedrine, nicotine, fluoxetine, MDMA,
		mixtures of additives ‐ TEA, TFA,	methylphenidate, pseudoephedrine, butylone, ethylone,
		water, ammonium formate,	4‐Methyl‐5‐phenyl‐2‐oxazolidinone, chlorthalidone,
		NH4OH, formic acid	esmolol, sotalol, clenbuterol, keterolac, hydantoin
NicoShell	modified MG	CO2/MeOH/additive (v/v/v)	rod‐like liquid crystals	75
		CO2/EtOH/additive (v/v/v)
		CO2/2‐PrOH/additive (v/v/v)
		additives: TFA or TFA+TEA
NicoShell	modified MG	CO2/MeOH+water (v/v)	(±) cis‐4,5‐Diphenyl‐2‐oxazolidinone, 2‐(4‐chlorophenoxy)	48
		CO2/MeOH (v/v)	Propionic acid, metoprolol, tryptophan, fluoxetine,
		CO2/MeOH+additive	tryptophanamide, nicardipine, bupivacaine, proglumide,
		CO2/MeOH+additive+water	trans‐stilbene oxide, ketoprofen, bendroflumethazide,
		additives: ammonium formate,	warfarin, N‐benzoyl‐DL‐valine, dansyl serine, coumachlor,
		TEA+TFA, DEA, ammonium	hydrobenzoin, mianserin, trans‐stilbene oxide
		formate+formic acid

MeOH, methanol; DEA, diethylamine; TFA, trifluoroacetic acid; EtOH, ethanol; TEA, triethylamine; HAc, acetic acid; IPAM, isopropylamine; 2‐PrOH, propan‐2‐ol; ACN, acetonitrile.

4. OTHER STUDIES

Teicoplanin‐based (Chirobiotic T) column was included in a set of other columns tested in simulated moving bed SFC for separation of binaphthol enantiomers and phytol isomers. ¹⁰⁶ Chirobiotic V (vancomycin as CS) column was used as one of a set of ten chiral columns for new SFC tandem column screening tool. ¹⁰⁷ The authors modified commercially available SFC instrument to be able to screen ten different columns and twenty‐five different tandem column arrangements. This instrument was used for separation of stereoisomers prepared by coupling of racemic ibuprofen and racemic 1‐phenylethylamine using peptide coupling reagent. The automated multicolumn and multieluent screenings are essential methodology in the pharmaceutical industry. ¹⁰⁸ , ¹⁰⁹ SFC has quickly evolved to become the preferred method for the enantiomeric analysis in pharmaceutical research and development. Improved peak shape, resolution, and analysis time were a significant driver underlying this expansion of SFC., ¹¹⁰ As the SFC is subject to continuous development, both by upgrades of instrumentation as well as by expanding selection of commercially available columns, the automated enantioselective SFC method development evolved from run times of more than half hour to current speed of just few minutes. ⁴⁹ , ¹¹⁰ , ¹¹¹ , ¹¹²

An original approach for achieving enantioseparation in SFC was presented by Frantz and Thurbide. ¹¹³ The authors filled empty tubing with water that set on the surface of capillary and by this “coating” process created water stationary phase. Analyzed compounds could partition between the water stationary phase and carbon dioxide MP without co‐solvent. ¹¹⁴ The water enriched with vancomycin as a stationary phase enabled enantioseparation of 2‐phenoxypropionic acid and 2‐(3‐chlorophenoxy)propionic acid with NaCl or triethylamine as additives. ¹¹³ The enantioseparation at low temperature, that is, 0°C brought the best results. Nowadays, the preferred method for enatioseparation in SFC is the use of chiral stationary phase. ²⁷ , ¹¹⁵ , ¹¹⁶

Armstrong et al demonstrated unusual effects of connection tubing with tubing between 50 and 500 µm ID and described the complex relation of dead time, retention time, efficiency, and optimum velocity with the tubing diameter (via column outlet pressure) in ultrafast chiral and achiral SFC. ¹¹⁷ 20, 30, and 50 mm long, 4.6 mm ID columns packed with 1.9 µm narrow particle size distribution fully porous silica particles (NPSD FPP) with a teicoplanin or teicoplanin aglycone as chiral selectors were used. Reduced plate heights were poor and no better than 5.8. Surprisingly, the highest efficiency was obtained with 250 µm tubing. The 50 µm tubing was by far the worst efficient, while 75 µm tubing was slightly worse than the 250 µm tubes. This seems counter‐intuitive. ¹¹⁷ , ¹¹⁸ Further studies in this direction are still needed.

Eremomycin‐based CSP was used for enantioseparation of salbutamol enantiomers in SFC. ¹¹⁹ The use of this selector is unique in SFC. No further use of this selector in SFC can be found in the literature. The authors showed that the manipulation of ionic interactions through choice and amount of additives in MP was significant for enantioresolution of salbutamol sulfate enantiomers. Enantioseparation occurred only when mixed additives were used, that is, isopropylamine and trifluoroacetic acid or isopropylamine and ammonium acetate. Unfortunately, the baseline separation was not achieved in any of the MP tested.

5. SUMMARY AND OUTLOOK

SFC is becoming a chiral separation technique of future because it offers efficient, fast and ultrafast analyses. Macrocyclic glycopeptide‐based CSPs provide versatile enantioseparation abilities toward a wide range of structurally various chiral compounds in SFC. Use of azeotropic ethanol as a MP co‐solvent for enantioseparation on MG‐based columns seems to be very promising and it can be considered as a green solvent. Improvement in chiral column technology, especially a decrease in particle size and use of columns of smaller dimensions arising from it, enables fast or ultrafast enantioseparations with MG‐based columns in SFC. Recent research in this field is promising.

The detailed description and understanding of interaction/enantiorecognition mechanism of these CSPs in SFC is a still challenging area and only a few works have been published so far. Moreover, the retention/interaction mechanism in these separation systems is studied rather than enantiorecognition mechanism. Therefore, further studies are required for a more detailed understanding of the chiral recognition mechanisms in these separation systems in SFC.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Folprechtová D, Kalíková K. Macrocyclic glycopeptide‐based chiral selectors for enantioseparation in sub/supercritical fluid chromatography. Anal Sci Adv. 2021;2:15–32. 10.1002/ansa.202000099