Abstract: This article intends to introduce the application of polysaccharide chiral stationary phases in the separation of enantiomers by high performance liquid chromatography through some specific examples. The types of chiral stationary phases of polysaccharides, the mechanism of chiral recognition, the factors affecting chiral resolution, and the solubility of samples during preparation and separation were elaborated and discussed in detail.
Realistic needs: Different enantiomers of chiral drugs often show different pharmacological, toxicological and pharmacokinetic properties. For drug safety considerations, drug regulatory authorities require that each enantiomer of a potential chiral drug must be Perform separation and activity (toxicity) tests. Therefore, the acquisition of a single configuration chiral compound is extremely important for the development of pharmacological and toxicological experiments. In general, single isomers can be obtained through chiral synthesis and chiral resolution. Among the various chiral resolution techniques, chromatography is widely used because of its advantages such as fast, efficient, and economical. (For chiral synthesis, please refer to related monographs.)
Case study: mg-50g single configuration chiral compounds are pure products (that is, both configurations are required). One of them is probably a safe and effective drug candidate. In order to obtain the various isomers as soon as possible to carry out pharmacological and toxicological tests as early as possible, many pharmaceutical companies have delayed their investment in asymmetric synthesis during the drug discovery stage, but instead quickly and keenly turned to chiral chromatographic separation, quickly becoming less expensive The high-purity enantiomer was separated from the racemic mixture. At the early stage of development of chiral drugs, it is not bad! At this time, the most important thing is time and enantiomeric purity. Time is money, and it took the lead in the early days, and then the money is rolling!
Solution: In the drug discovery stage, due to the low demand for chiral compounds (mg-50g level, compared with the full-scale development of the kilogram level and the tonnage level production in the later period, the little witch sees the big witch ...), in order to obtain a single optically pure substance as soon as possible , Using chiral chromatography to prepare separation strategy.
Specific method: To adopt polysaccharide chiral stationary phase high performance liquid chromatography (PreparativeChiralHPLC)
Method steps: Chiral HPLC to prepare the enantiomers. For a specific sample, how to start the chiralHPLC method? Can the enantiomers be separated directly on the commercial column at hand, can it be amplified, etc.?
For the chiral preparation of the resolution enantiomers by liquid chromatography, the steps are roughly as follows: 1), understand the sample structure information of the compound to be separated; 2), select the appropriate chiral stationary phase (chiral analytical column); 3), Screen the selected analytical column and optimize the chromatographic conditions; 4) Transfer the analytical conditions to the preparative column and make final adjustments to the scale-up separation conditions (analyticalchiralHPLC → preparativechiralHPLC); 5) Start the preparative resolution if conditions permit If necessary, the sample can be injected automatically; 6) Remove the solvent and recover the product. The specific operation can be considered as follows: 1), understand the sample structure information of the compound to be separated: the first step of the establishment of the chiral method is to check the chemical structure of the analyte, and obtain the following information of the sample as much as possible-in different solvents Solubility (because of preparative separation, we expect as much of the sample as possible to dissolve in a relatively small solvent. In addition to separation selectivity, sample solubility is usually the main obstacle to establishing a PreparativeChiralHPLC method. In many cases, the sample is not sufficient in the mobile phase. Large solubility, which affects the amount of single needle loading, and even deposits on the preparative column and cannot be eluted.); The ability to form hydrogen bond H-, π bond or dipole interaction; whether there are polar functional groups (whether Contains amino groups NH2-, carboxyl-COOH or both acidic groups and basic groups); whether there are large substituents near the chiral center; whether the UV spectrum can be detected. Sample structure information such as this is of great help in predicting the chiral separation ability and the selection of chiral stationary phases.
2) How to choose a suitable chiral column: Since the chiral method is used for preparation and separation, the column capacity (column sample load) should be considered when selecting the chiral column. Considering the versatility of chiral stationary phases, the general order of the commercial chiral columns is: polysaccharide column ï¹¥ protein column ï¹¥ glycopeptide column ï¹¥ ligand exchange column.
Polysaccharide chiral stationary phases show excellent performance in chiral separation based on their unique molecular structure characteristics, and have been widely used in the analysis and preparation of various enantiomers of chiral molecules. Therefore, the polysaccharide column was selected as the chiral column for preparation and separation.
3). Optimization of chiral chromatography conditions: Chiral liquid chromatography is similar to ordinary liquid chromatography. The establishment of the method mainly includes the screening of chiral columns and the optimization of mobile phase conditions.
In terms of chiral column screening, even when a polysaccharide chiral column is selected, it is difficult to determine which column is best to start the experiment because of the variety of column types to choose from. According to most of the reported application studies, Daicel's "Four King Kong" starch ChiralpakAD-H, ChiralpakAS-H and cellulose Chiralcel-OD-H, ChiralpakOJ-H can successfully separate 80% of unknown samples. However, based on personal experience, the most wise decision is to reuse the "two protective methods" -ChiralpakAD-H and Chiralcel-OD-H (or new covalently bonded chiral columns ChiralpakIA and ChiralpakIC), both of which are in polysaccharides. The most widely used in the column, can be used for the separation of a variety of structural samples. The helical structure of amylose and the linear structure of cellulose have the characteristics of chiral separation and complementarity in enantiomeric resolution. ChiralpakAD-H and Chiralcel-OD-H (or ChiralpakIA and ChiralpakIC) are used together to complement each other. Greatly good. . .
For optimization of mobile phase conditions, polysaccharide chiral columns are mostly separated in normal phase mode. The initial stage of the method was established. First, try to use absolute ethanol / n-hexane as the mobile phase on the ChiralpakAD-H column (or ChiralpakIA column) to enhance the separation of enantiomers by adjusting the alcohol content in the mobile phase or changing the type of alcohol. Sex. If there is only partial separation or there is still no separation, modifiers such as organic acids and bases can be added to the mobile phase to optimize the separation. At the same time, the effect of column temperature on the separation can be investigated. If the separation is found to be unsatisfactory, another killer is used, and the Chiralcel-OD-H (or ChiralpakIC) column is used, and the previous influencing factors are repeated. If you are back and drink cold water to stop your teeth-using a polysaccharide chiral column fails to achieve satisfactory resolution, then make another plan to try other types of chiral columns.
The chiral chromatographic conditions optimization process is summarized as follows: 4). Transfer of analytical methods: Once the appropriate analytical conditions are determined, the first step of chiral preparative separation is triumphant, and the subsequent work progress is quite smooth. In general, the particle size (20um) of the packing used to prepare the separation is larger than the particle size (5um) of the chiral stationary phase for analysis purposes, and the size of the column is also different. The analysis conditions are switched to semi-preparative and preparative scale, mainly for the adjustment of flow rate and injection volume. Before preparation, it is best to make a "theoretical analysis" (or pre-judgment based on past actual combat experience), followed by small batch experiments to gradually verify and enlarge the separation.
5) Preparative separation: Preparative Chiral Liquid Chromatography (PreparativeChiralHPLC), in addition to exploring the separation conditions on analytical AnalyticalHPLC, generally speaking, the solubility of the sample is very important. If the sample does not have sufficient solubility in the mobile phase, this makes the sample concentration not high and severely restricts the amount of single needle loading. Polysaccharide chiral columns are mostly separated in normal phase mode, using alcohol / n-hexane as the mobile phase system and isocratic elution. In practical applications, the more you worry about what will happen, you will often be entangled in the problem that the sample solubility is not large enough in the normal phase separation mode. Aiming at the problem of sample solubility, specific analysis of specific problems, special circumstances and special care, try to improve the solubility of samples.
Strategies for improving the solubility of samples: The general principle is to do everything possible to find ways to increase the solubility of the samples, but without changing the elution strength of the solvent or affecting the separation selectivity.
Strategy 1: Start with the sample compound structure itself, through structural modification, introduce a protective group to make a derivative to enhance its solubility in organic solvents. The principle is to make the solubility better by derivatives without affecting the separation selectivity of the enantiomers or improving the selectivity, and at the same time the protecting groups are easily introduced and removed. For example, certain types of chiral amines (primary and secondary amines), plus a Boc or Cbz protecting group, the solubility will be dramatically improved, and it is convenient to separate well.
Strategy 2: If the mobile phase cannot dissolve a sufficient amount of sample, you can explore a sample solvent different from the mobile phase. For example, first use a single solvent that can dissolve more samples (such as isopropanol or absolute ethanol) to dissolve the material to be separated, and then dilute it with the mobile phase to prevent precipitation. The change of the sample solvent ionic strength increases the sample solubility. For example, when dissolving the sample, a certain volume of organic acid (glacial acetic acid, trifluoroacetic acid), organic base (diethylamine, triethylamine), ion pair reagent ( Methyl sulfonic acid, ethyl sulfonic acid), etc. to help solubility, but the premise is not to affect the stability of the compound and the separation selectivity of enantiomeric chromatography. The effect of sample solvent on chromatographic separation can be investigated on analytical chiralHPLC and then transferred to preparation.
Strategy 3: Use stack injection. We know that under RP-HPLC gradient elution conditions, after injection, the analyte must completely flow out of the column before the next injection, that is, the injection is intermittent. However, the chiral separation in normal phase mode, isocratic elution, according to the peak time of the sample compound, you can selectively choose intermittent single needle injection or overlapping injection (that is, the last needle has not eluted , Pinch to calculate the peak time, seize the opportunity to enter the next stitch). Stackinjection saves time and increases efficiency, saves energy and reduces emissions, is green and environmentally friendly, and meets the requirements of the era of building a resource-saving and environment-friendly society. For example, for a chiral preparative resolution, if a single needle injection is needed, it takes 32 minutes to run. The two isomers peak in the period of 16.5-30 minutes. The separation on the first 16 minutes of the column is similar to walking the baseline, while paying attention to the two chromatography The peak can completely flow out of the column within 16min. Similar to this case, we can consider the use of overlapping injection strategies to shorten the separation time, save solvent consumption, and reduce waste liquid emissions.
6). Recovered products: Removal of solvent is similar to reversed-phase preparative chromatography, but chiral preparative separation is carried out under isocratic conditions in normal phase mode. After recovery of the solvent, it can be recycled and reused for continued use Separate items. Practice the concept of circular economy development in practical work and build an ecological and civilized society. How to determine the stereo configuration of the two isomers obtained, and what is the elution order of R- and S-types? This can be used to determine the direction of polarized light after the solvent is removed. By comparing the reported values ​​in the literature, the R- and S-configurations can be determined. If the conditions are good, you can use an online optical rotation detector or a circular dichroic detector to track the elution order of the enantiomers.
Summary: Chiral chromatography is one of the most important chiral separation techniques. It can not only quickly determine the enantiomeric purity (enantiomeric excess value), but also can be used to prepare and resolve optical isomers. Among many commercial chiral stationary phases, polysaccharide chiral stationary phases are widely used because of their good separation selectivity and large column capacity. This article discusses the application of polysaccharide chiral stationary phases in the preparation and separation. Rapid screening of chiral stationary phases (chiral columns) and optimization of mobile phase conditions (alcohol and other modifiers and modifiers) are the keys to success. At the same time, the solubility of the sample is an important factor affecting the resolution speed. It can be combined with other cheaper purification methods (recrystallization, chemical resolution) to reduce the overall cost of operation.
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