Why don't children like broccoli and broccoli? Chromatographic exploration

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Chiral chromatography became a routine technique in pharmaceutical, food and environmental applications in the 1980s, and now it has largely replaced the earlier techniques that used chiral reagents to derivatize enantiomers and separate them on standard achiral HPLC columns. . For amino acids, the ability to separate into free amino acid enantiomers eliminates the uncertainty in determining the configuration of the enantiomers compared to the use of chiral derivatization reagents. There are several chiral stationary phases (CSP) that can be used for this separation (Table 1), but although HPLC stationary phases have revolutionized with the introduction of sub-2μm and superficially porous particles (SPP), CSP usually remains at 3 and Supported on 5μm silica gel. This article reviews current methods, techniques, and new research that should bring the advantages of UHPLC to chiral HPLC.

Although all protein amino acids exist in the form of L-enantiomers in nature (so their biological interactions are stereochemically predictable), enzymatic post-translational modifications can lead to the incorporation of D-amino acids into certain proteins In, especially in small molluscs [1]. They are also a rich component of bacterial peptidoglycan cell walls [2]. In addition, it has been known for some time that D-serine acts as a neurotransmitter, activating N-methyl-D-aspartate (NMDA) receptors: it originates from the synthesis of L-enantiomers in the brain [ 3,4]. Therefore, the ability to easily separate amino acids from enantiomers is important for our understanding of natural and human biology. Enantiomerically pure amino acids are often used as chiral building blocks in asymmetric synthesis, so monitoring purity and reaction progress is an important application of chiral separation [5,6]. In addition, due to the clinical monitoring of biomarkers [5] and therapeutically important peptides [7], interest in the effective isolation of chiral amino acids has continued to grow in recent years. For biologically active peptides, the analysis of their amino acid sequence is the key to their biological functions. Although amino acid analysis and tandem MS sequencing are possible, determining the stereochemistry of amino acids in peptides is essential for understanding the homochirality of possible post-translational modifications and accidental digressions (natural L-isomer replaced by D-isomer) . For example, the action of peptidyl-aminoacyl-L/D-isomerase can convert the L-enantiomer into its D-correspondence during the synthesis of biological peptides [8]. D residues in peptide bonds may also be generated by age-dependent racemization [9].

Several different CSPs make it possible to separate free amino acids, including crown ethers and ligand exchange, while N-protected amino acids can be separated using brush and cyclodextrin C​​​SP. Macrocyclic glycopeptide and cinchona alkaloid CSP usually have the ability to separate the two. Table 1 lists the most commonly used CSPs for the direct separation of free amino acids and N-protected amino acids. The mechanism of crown ether CSP, which can be coated on 5μm silica and (recently added) 3μm immobilization (commercially, CROWNPAK CR-I), depends on multiple hydrogen bonds between the primary amine of the amino acid and the crown ether part It interacts with ether, so it is necessary to make the amine cationic by using an acid (pH 2-3) in the mobile phase. In fact, due to insufficient interaction, amino acids with secondary amine functions (such as proline) are usually not separated [10]. The CSP is compatible with LC-MS, as shown in a recent study [10], in which LC-TOFMS using isocratic ACN/water/TFA mobile phase can separate 18 protein amino acids without derivatization. Ligand exchange separation uses the more unusual CuSO4 mobile phase, which is very useful if the amino acid does not have a UV chromophore. These CSPs use bonded D- or L-amino acids (phenylalanine, such as Supelco CLC columns), so they can be used to reverse the elution order in trace analysis by simply replacing the column [11,12]. Brushing and protein-based CSP have also been used to isolate some derivative amino acids [13]. However, phases based on ionizable teicoplanin and zwitterionic quinine/quinidine selectors are now the broadest and most useful phases (see the following sections on these). The main reason for this situation is that in addition to small peptides (and other amphiphilic molecules), free natural and synthetic α, β, and γ-amino acids can also be separated, whether they are primary or secondary, aliphatic or aromatic, Cyclic or acyclic compound). The popularity of LC-MS is another reason; the two are fully compatible. Interestingly, teicoplanin-bonded CSP has also been used in clinical applications to isolate isobaric biomarker amino acids (eg, glutamine and lysine [14].

Most mobile phases used for CSP based on macrocyclic glycopeptides and cinchona alkaloids are polar organic or aqueous organic components. The polar organic mobile phase is mainly a mixture of non-aqueous methanol/acetonitrile, and acid and alkali are added when it is necessary to control the glycopeptide CSP and the ionizable part of the solute. Polar organics were first developed by Armstrong in 1993 for use with cyclodextrin C​​SP (commercially CYCLOBOND, [15]), and then expanded to the macrocyclic glycopeptide phase when it was introduced in 1994. In recent years, several other CSPs have been used for polar organics, including brush CSP for general chiral separation [16]. For the amino acid separation of CSP based on cinchona alkaloids [17], usually a small amount of formic acid, diethylamine and water are added to control the relative strength of anions and cations. A simple alcohol-water mixture is also used in the macrocyclic glycopeptide phase. Add a buffer when the amino acid has an ionizable functional group other than the α zwitterionic moiety.

The various functional groups of the macrocyclic glycopeptide phase (commercially known as CHIROBIOTIC) provide high selectivity for ionizable and zwitterionic compounds (including amino acids). Since its introduction in 1994, the use of the macrocyclic glycopeptide teicoplanin has been successfully used to separate a variety of amino acid types and small peptides [18]. The enantioselective range of teicoplanin is based on its ability to provide multiple mechanisms, including hydrogen bonding, dipole-dipole, π-π, van der Waals, hydrophobic, ionic, and steric interactions. NMR and HPLC studies by Gasparrini et al. [19] showed that free carboxyl groups can interact strongly with the hydrogen bonds of the amide portion of CSP peptides to provide high selectivity for various free and N-protected amino acids and peptides. The amine groups of amino acids can be free or blocked by N-bonded functional groups, such as 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC, trademark Waters), benzoyl, N- Tert-butoxycarbonyl (tBOC), carboxybenzyl (CBZ), 5-(dimethylamino)naphthalene-1-sulphonyl (dansyl) and 9-fluorenylmethoxycarbonyl (FMOC) make this method suitable for peptide synthesis Monitoring. In each case of all amino acids, the elution order is L before D and mobile phase alcohol/water, acidic amino acids are added to formic acid, and basic amino acids are added to ammonium acetate buffer. During a study to determine the importance of the glycosyl moiety in the mechanism of separating the teicoplanin bonded phase [20], it is expected that the enantioselectivity will disappear as it is removed. In fact, the selectivity remains the same, but has changed, so the aglycon version can provide alternative separation and mobile phases for different amino acids. It was marketed as Chirobiotic TAG in 2000. In contrast to teicoplanin, this CSP contains a single primary amine and remains charged under conventional HPLC conditions. The interest in the therapeutic effect of changing the conformation of one or more amino acids in a peptide has led to the use of teicoplanin to separate peptide isoforms using a simple mobile phase of acetonitrile/formic acid (Figure 1). This method can achieve selectivity between peptides with only one amino acid difference, even if they are not in the terminal position [7].

By combining quinine with (S,S)-trans-2-aminocyclohexanesulfonic acid (ACHSA) or quinidine with (R,R) form, and by carbamate bond in the C-9 position Combined, two new 3 and 5μm brush type cinchona alkaloids CSP (commercially CHIRALPAK ZWIX() and ZWIX(-)) were put into commercial use in 2012 [21,22,23]. These phases combine weak anion and strong cation interaction sites. By selecting specific mobile phase conditions, both amphoteric amino acid analytes and zwitterionic CSP can be charged; the combination of polar organic solvents with added acids and bases ensures ionization, and the protonated nitrogen atom of the bicyclic quinuclide is positively charged , Has a negative charge at the sulfonic acid functional group. These CSPs separate amino acids through electrostatic interactions between charged substances, supported by hydrogen bonding, van der Waals forces, pp stacking, and hydrophobic interactions (structure dependent). A recent study [17] showed the influence of spatial effects. When linear, the enantioselectivity increases as the length of the side chain increases, and when branched, it increases with the volume and rigidity of the side chain. Spatial effects may also lead to longer retention times of b-amino acids on these CSPs. There are indications that amino groups may not always be necessary for enantioselectivity, as evidenced by the separation achieved by N-protected amino acids, so CSP in this case behaves as a chiral anion exchange rather than a zwitterionic CSP. The main advantage of these stationary phases is the ability to reverse the elution order by switching the column [Figure 2], although the resolution may be slightly different, mainly because the two CSPs are not exact enantiomeric versions of the same selector.

In recent years, higher-performance, smaller-sized CSPs using 3μm and sub-2μm particles have been available; in contrast, CSPs used for amino acid separations were mostly in 5 and 3 μm formats until recently. Since the introduction of reversed phase separation in 2006 [24], surface porous particles (SPP) have continued to be successful and widely used. The porous layer on the SPP solid core improves the mass transfer kinetics because the analyte cannot diffuse into the particles. It is recognized that by reducing the band broadening, higher efficiency and faster separation can be easily achieved [25]. The narrower particle size distribution provided by this technology leads to a more uniform packed bed and consequently higher back pressure (although lower than sub-2μm columns, [26]), so these stationary phases can be used in HPLC and then New UHPLC instrument. Later studies [27] also showed that the efficiency of these particles is mainly due to the improvement of the A and B terms of the van Deemter equation (vortex and longitudinal diffusion, respectively) due to the homogeneous packed column. Recently, several studies have been conducted to incorporate this technique into chiral applications. A study by Sciascera et al. overcomes the practical difficulties of combining larger-volume chiral selectors on sub-2μm particles and the tendency of particle aggregation to create a sub-2μm version of the brush CSP, Whelk-O1 [28]. When the mass transfer effect is more prominent, the axial and radial temperature gradients caused by friction heating in SPP [29] will have a significant impact on the CSP made of these phases. Further research [30] showed that in high water content mobile phases, axial temperature gradients improve mass transfer and offset any efficiency loss due to radial temperature gradients and eddy diffusion. This led to a significant increase in the efficiency of teicoplanin bonding to SPP: for a series of amino acids in MeOH/H2O, a high resolution of 1.6 to 3.0 was achieved in less than one minute. Recently, a new fully porous particle (FPP) has been developed, which has extremely high column efficiency, and the height of the plate in the narrow-pore chromatographic column has been reduced by 1.7 [31]. This has been studied as a potential particle of CSP, leading to very fast separations in seconds instead of minutes [32,33]. The traditional 5μm and new 1.9μm TPP teicoplanin bonded phases were compared [33], and the results showed that the efficiency (N/m) was increased by 3-4 times, and the board height was reduced from 3.5 to 2.5. For example, on a 5 x 4.6mm 1.9μm FPP teicoplanin column, the resolution of methionine in MeOH/H2O is 3.0 in 40 seconds. It is worth noting that the increased permeability of this CSP can achieve rapid separation at high flow rates without excessive frictional heat. It also demonstrated high-speed peptide separation; the dipeptide DL-Leu-DL-Ala can be separated in less than one minute (Figure 3) [30]. In contrast, Gasparrini et al. [32] used a new bonding chemistry on the same 1.9μm TPP, resulting in a protonated amino group on the teicoplanin structure, allowing the CSP to maintain zwitterionic properties, such as a Neutral and permanently charged solutes as evidenced by a series of hydrophobic separations. This developed CSP has a high selectivity of 2.25 to 10.7 for a series of N-protected amino acids in RP, and the column length is 10 cm, which can minimize the influence of extra-column effects. Reducing it to 2 cm maintains relative efficiency and achieves extremely fast separation. For example, the separation time of BOC-D, L-Met in RP is less than 1 minute, the resolution is 2.20, and the average efficiency of 2 mL/min is (93,575 N/m). The ultra-fast separation using a 1cm column was also explored and provided the same separation in 11 seconds in HILIC mode (Rs 1.04). Interestingly, the unusual repulsion effects occasionally seen in teicoplanin CSP are caused by the repulsion of negatively charged carboxylic acid groups on the negatively charged analyte phase, but are not present in this phase . Conclusion The further development of the application of SPP and FPP CSP in the separation of free amino acids will arouse great interest. Instrument optimization is likely to be as important as selective optimization. Compared with particle efficiency, the injection cycle time may have a greater impact on the method because they may be longer than the actual separation time. It is also critical to use low-dispersion syringes, small-volume detector flow cells, and small-in-diameter connecting pipes to take full advantage of these developments. Fast separations such as these can also be used for preparative chromatography, which has the potential to reduce solvent consumption. Perhaps most importantly, the separation speed is so fast that online monitoring of asymmetric synthesis becomes feasible.

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Modern and practical applications in this edition-Analysis of cannabidiol in CBD products by SFC-CD-MS-Use SFC to analyze choline and acetylcholine in rat brain faster...

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