For the separation of chiral compounds, the choice of chiral chromatographic column plays an absolute role. However, in order to achieve enantiomeric separation, the selection of appropriate mobile phase and elution mode is also an indispensable step. Commonly used mobile phase dependent elution modes can be divided into normal phase liquid chromatography (NPLC), polar and polar ion mode mobile phase and reversed phase liquid chromatography (RPLC). The types and characteristics of compounds suitable for each mobile phase are different and also limited by the combination of chromatographic column and stationary phase matrix. In addition, no matter which elution mode or mobile phase is used, it is necessary to ensure that the column pressure does not exceed the maximum tolerance range, and the column should be fully equilibrated when performing method transfer or changing mobile phases.
Normal phase mobile phase can be subdivided into neutral mobile phase, basic mobile phase and acidic mobile phase. The neutral mobile phase is generally based on hexane or heptane, with appropriate proportions of EtOH, IPA, MTBE, EtOAc, DCM, etc. Which polar solvent to add will vary with the compound being analyzed and also with the type of column used. For example, the coated polysaccharide derivative column can only use standard mobile phase. The acidic or basic mobile phase is achieved by adding a certain amount of organic acid-base additives to the neutral mobile phase, and the addition ratio is generally not more than 0.5%.
In the normal phase elution mode, the retention time of the chiral compound to be separated and the selectivity factor α (or resolution) between the enantiomers generally increase with the decrease of polar solvent ratio in the mobile phase. In addition, it is also necessary to pay attention to whether the elution order of enantiomers is reversed with the change of alcohol ratio. When using the normal phase elution mode, blindly increasing the selectivity factor alpha by lowering the alcohol ratio should be avoided.
In general, hydrogen bonding interactions, π-π interactions, and inclusion interactions (polysaccharide derivatives and cyclodextrin derivatives chiral columns) are the main driving forces for chiral resolution in normal phase elution mode. The isopropanol added to the normal phase mobile phase is a hydrogen bond donor and acceptor, methanol and ethanol act as hydrogen bond donors, and acetonitrile acts as a hydrogen bond acceptor. In addition, adding less than 5% acetonitrile to the normal phase mobile phase can also reduce the tailing factor.
The compounds used for normal phase mobile phase and normal phase mode separations are moderately polar. Ethanol and isopropanol can be used as basic polar additives. Which organic alcohol will give the best separation depends on the type of compound and chiral column. When developing the first resolution method using normal phase mode, polar additives such as ethanol or isopropanol can be first immobilized to obtain a suitable capacity factor k (k ≥ 6 more appropriate ) for the enantiomer by adjusting the ratio of the polar additive. Meanwhile, the chromatographic retention behavior of the chiral compound is collected as much as possible. If the enantiomer is still completely co-eluted in this case, it indicates that the chiral column has no chiral selectivity or weak selectivity for the chiral compound.
The polar mode can be tried when the capacity factor k is still much larger than 6 when using absolutely large amounts of polar additives. When using an absolute small amount of polar additive, the reversed phase mode can be tried when the capacity factor is still small. In addition, the bonded chiral chromatographic column of the same filler type can be replaced when there is a certain degree of separation, and solvents such as MTBE or EtOAc with weaker elution ability can be selected.
Polar mode means using a single or mixed polar solvent as the mobile phase, and polar ion mode means adding a certain proportion of organic acid-base additives to the polar mobile phase. Polar or polar ion mode has higher column efficiency, shorter analysis time and better method tolerance than normal phase mode. Chiral compounds resolved using polar or polar ion modes are generally more polar. In addition, there are fewer impurities, especially related impurities, in the sample to be separated.
Polysaccharide derivative chiral columns, including first-generation coated chiral columns, can only be used with standard mobile phases. Therefore, in polar or polar ion mode, the range of mobile phases that can be selected for polysaccharide derivative chiral chromatography columns is limited, mainly including methanol, ethanol, isopropanol, acetonitrile and their mixtures. The second-generation bonded polysaccharide chiral column is not limited by the type of mobile phase, and the polar mobile phases that can be used include organic alcohol, dichloromethane, chloroform, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate and 1, 4-dioxane and other solvents.
Compounds used for separation in reversed-phase mode are generally chiral compounds with hydrophobic ability, especially compounds with larger hydrophobic groups at the α or β position of chiral carbon atom. The polysaccharide derivative chiral columns that can use reversed-phase mode mainly include coated-RH chiral columns and bonded I chiral columns, and other types include Chiralbiotic chiral columns, Regis pirkle chiral column and Cyclobond chiral column, etc.
Commonly used reversed-phase mobile phases can be divided into water-organic phase types, wherein the organic phases are generally acetonitrile, methanol, ethanol and isopropanol. Buffer salts-organic phase types, wherein the buffer salts can be divided into MS compatible and incompatible. Among them, MS-compatible buffer salts include ammonium formate and amine acetate, while incompatible buffer salts are mainly salts with high chaotropic order, such as NaBF4, KPF6, NaClO4, Borate, Na2HPO3, and NaH2PO3. Generally, the concentration and pH of buffer salts also affect the separation of chiral compounds.
For the chiral stationary phase and mobile phase, it is necessary to ensure that the column pressure of the chromatographic column is within the allowable range at any time. Generally, the column pressure is related to the type of stationary phase, the flow rate of the mobile phase, the composition of the mobile phase and the temperature of the chromatographic column. In addition, after changing the mobile phase or switching the method, the chromatographic column needs to be fully equilibrated, and the length of the equilibration time is also related to the type of stationary phase and mobile phase.
Chiral chromatography can easily, accurately and quickly measure the optical purity and absolute configuration of samples. As a leading service provider for the synthesis and resolution of chiral compounds, BOC Sciences not only provides screening services for chiral columns, but also provides services such as mobile phase screening, elution mode, column pressure screening and temperature screening for specific compounds. If you are interested in our chiral resolution services, please contact us for more information.
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