Fmoc-L-Hong (Trt)-OH reverse chirality detection method
By using gradient elution program and high performance liquid chromatograph in cellulose inversion system, the problem of difficulty in controlling moisture and solvents of normal phase columns is solved, and the rapid and rapid Fmoc-L-Hgn(Trt)-OH is achieved. Accurate separation is especially suitable for water-soluble amino acid samples, improving separation efficiency and stability.
Patent Information
- Application Number
- CN202411348784.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, it is difficult to achieve effective separation by strict control of moisture and solvent by the normal phase column, resulting in increased column pressure and decreased separation. Especially when the moisture and solvent in the sample are uneven, it is difficult to accommodate and separate. Fmoc-L-Hgn(Trt)-OH sample.
Use pure water to dissolve Fmoc-L-Hgn(Trt)-OH and add a certain proportion of acetonitrile, and detect it through a cellulose reverse phase chiral column, and use gradient elution procedures and high performance liquid chromatograph for separation. The specific steps include Sample solution preparation, filtration and HPLC analysis were performed using an Agilent 1260 chromatograph and DAD detector, combined with a cellulose CHIRALPAKR OJ-RH chiral analysis column.
It realizes simple, fast and accurate separation of Fmoc-L-Hgn(Trt)-OH, has good peak symmetry and high stability, and can effectively separate isomers. It is suitable for amino acid samples that are easily soluble in water.
Smart Images
Figure CN119125368A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting Fmoc-L-Hgn(Trt)-OH, and in particular to a method for detecting the chirality of Fmoc-L-Hgn(Trt)-OH. Background Art
[0002] Normal phase chiral columns have strong specificity, and conventional solvents generally used are n-hexane, ethanol, and isopropanol. The water and solvent content in the sample are strictly controlled, mainly because once water enters the normal phase chiral column or other solvents are used, the column pressure will increase, and the normal phase chiral column has strict control over the column pressure. Once the column pressure is exceeded, the packing in the column is compressed to the point that the column head collapses, the column efficiency is severely reduced, the separation degree is reduced, and the sample may even not be retained and cannot be separated. Summary of the invention
[0003] The present invention provides a reverse phase chiral detection method for Fmoc-L-Hgn(Trt)-OH, which is used to solve the technical problems of difficult tolerance and poor separation of existing samples in normal phase systems. The study found that: dissolving Fmoc-L-Hgn(Trt)-OH in pure water, adding a certain proportion of acetonitrile to make the volume constant, and using a Daicel reverse phase chiral column for detection, the isomers can be separated simply, quickly and accurately. The present invention adopts the following technical scheme: Fmoc-L-Hgn(Trt)-OH reverse chirality detection method, comprising the following steps: Step 1: Prepare the reagents for measurement: acetonitrile-chromatographic grade, ultrapure water, and chemically pure trifluoroacetic acid.
[0004] Step 2: Preparation of sample solution: Weigh the sample and place it in a container, add pure water, dissolve it by ultrasonic at room temperature, and then make up the volume with acetonitrile. Finally, filter the sample with a polypropylene organic filter membrane and perform HPLC analysis.
[0005] Step 3, high performance liquid chromatography determination: the sample chamber temperature is room temperature, the automatic sampler injection volume is 10µL, the chiral chromatographic column is: Daicel CHIRALPAKR OJ-RH chiral analytical column (4.6mm ×150mm, 5.0μm), column temperature: 40°C, flow rate: 0.8 mL / min, mobile phase A is a mass percentage concentration of 0.1% TFA. aqueous solution, mobile phase B is a mass percentage concentration of 100% TFA acetonitrile solution; a gradient elution program is used, 0-30min, the volume ratio of mobile phase B is 40% to 70%, 30.1-45min, the volume ratio of mobile phase B is from 40% to the equilibrium system; that is, the retention time is R type: 20.04in, S type: 23.1min.
[0006] The chromatograph used in the liquid chromatography determination is an Agilent 1260 chromatograph, which uses a DAD detector.
[0007] The present invention has the following beneficial effects: the present invention is simple, rapid, accurate, stable and has good peak symmetry; the present invention adopts accurate quantification of an automatic sample injector of a high performance liquid chromatograph, and utilizes the sensitivity of a DAD detector to have a good response value for Fmoc-L-Hgn(Trt)-OH; the study found that the Daicel CHIRALPAKR OJ-RH chiral analysis column has unique selectivity for various types of molecules, especially for amino acid samples that are easily soluble in water, and can effectively and quickly separate isomers. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 Spectrum of mixed sample detected by Agilent 1260 DAD detector.
[0009] Figure 2 Chiral spectrum of Fmoc-L-Hgn(Trt)-OH detected by Agilent 1260 DAD detector.
[0010] Figure 3 Chiral spectrum of the corresponding isomers of Fmoc-L-Hgn(Trt)-OH detected by Agilent 1260 DAD detector. DETAILED DESCRIPTION
[0011] Main instruments: Agilent 1260 high performance liquid chromatograph, equipped with DAD detector, Daicel CHIRALPAKR OJ-RH chiral analytical column (4.6mm ×150mm, 5.0μm); ultrasonic extractor (KQ5200E, Zhengzhou Ketai Experimental Equipment Co., Ltd.), 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instrument Co., Ltd.), electronic analytical balance (CPA225D, Sartorius Scientific Instrument (Shanghai) Co., Ltd.).
[0012] Main reagents: The reagents used for the measurement include: acetonitrile (chromatographically pure, Merck, Germany), trifluoroacetic acid (chemically pure, Shanghai Sinopharm Company); and ultrapure water (resistivity 18.2 MΩ.cm, Chlamydomonas, USA).
[0013] Sample pretreatment: Weigh about 5 mg of Fmoc-L-Hgn(Trt)-OH and its corresponding isomers, place them in a 5 mL volumetric flask, add 3 mL of pure water, dissolve them by ultrasonication at room temperature for 3 min, and then make up to 5 mL with acetonitrile. Finally, filter the sample with a 0.22 µm polypropylene organic filter membrane for chiral analysis.
[0014] Chromatographic conditions: The sample chamber temperature was room temperature, the injection volume of the autosampler was 10 µL, the chromatographic column was: CHIRALPAKROJ-RH chiral analytical column (4.6mm ×150mm, 5.0μm), the column temperature was 40 ℃, the flow rate was 0.8mL / min, the mobile phase A was a 0.1% TFA aqueous solution by mass percentage, and the mobile phase B was a 100% TFA acetonitrile solution by mass percentage; a gradient elution program was used, 0-30min, the volume ratio of mobile phase B was from 40% to 70%, 30.1-45min, the volume ratio of mobile phase B was from 40% to the equilibrium system. That is, the retention time was R type: 20.04in, S type: 23.1min.
[0015] Selection and optimization of sample pretreatment methods: Currently on the market (Fmoc-L-Hgn(Trt)-OH is a solid powder, and the sample measured in the present invention is Fmoc-L-Hgn(Trt)-OH produced by Jiangsu Jitai Peptide Technology Co., Ltd.
[0016] Optimization of chromatographic conditions: The present invention compares the separation effects of 0.1% TFA aqueous solution-100% TFA acetonitrile solution and 0.025% formic acid aqueous solution-0.025% formic acid acetonitrile solution as mobile phases. When the 0.025% formic acid aqueous solution-0.025% formic acid system is used, it is found that the chromatographic peak shape is asymmetric and easy to tail; when the 0.1% TFA aqueous solution-100% TFA acetonitrile solution system is used, the peak symmetry of Fmoc-L-Hgn(Trt)-OH reaches the standard, and the separation, sensitivity and peak shape of the target compound are better than other elution systems. Therefore, this experiment uses 0.1% TFA aqueous solution-100% TFA acetonitrile solution as eluent, and gradient elution is used for chromatographic separation.
[0017] The temperature of the sample chamber of the present invention is set to room temperature, which is conducive to the preservation of the acetonitrile solvent in the sample chamber. The temperature of the column oven is set to 40°C in the present invention.
[0018] Determination of actual samples: Mixed sample spectrum see Figure 1 , Fmoc-L-Hgn(Trt)-OH single sample chiral spectrum see Figure 2 The chiral spectra of the corresponding isomers of Fmoc-L-Hgn(Trt)-OH are shown in Figure 3 .
[0019] The chiral detection results of Fmoc-L-Hgn(Trt)-OH samples using this method showed that this method was rapid, accurate, highly sensitive, and the sample was easily soluble, requiring a small amount of sample to be tested, and had practical application value.
[0020] The invention establishes a chiral detection method for determining Fmoc-L-Hgn(Trt)-OH. The method is simple, fast, accurate, and stable, and is suitable for providing a new method for chiral separation detection of Fmoc-L-Hgn(Trt)-OH.
Claims
1. A method for detecting the reverse chirality of Fmoc-L-Hgn(Trt)-OH, characterized in that: The following steps are involved: Step 1: Prepare the reagents for measurement: acetonitrile-chromatographic grade, ultrapure water, chemically pure trifluoroacetic acid; Step 2, preparation of sample solution: weigh the sample and place it in a container, add water, dissolve it by ultrasonic at room temperature, then add superacetonitrile to make up the volume, and finally filter the sample with a polypropylene organic filter membrane and perform chiral detection; Step three, perform high performance liquid chromatography determination: the chromatographic conditions are that the sample chamber temperature is room temperature, automatic sample injector, the chromatographic column is: Daicel CHIRALPAKR OJ-RH chiral analytical column, column temperature: 40 ° C, flow rate: 0.8 mL / min, mobile phase A is 0.1% by mass concentration of TFA aqueous solution; mobile phase B is 100% by mass concentration of TFA acetonitrile solution, and a gradient elution program is used.
2. The Fmoc-L-Hgn(Trt)-OH reverse chirality detection method according to claim 1, characterized in that: The chromatograph used in the liquid chromatography determination is an Agilent 1260 liquid chromatograph, which uses DAD as a detector.
3. The reverse chirality detection method of Fmoc-L-Hgn(Trt)-OH according to claim 1, characterized in that: The chromatographic column size of the Daicel CHIRALPAKR OJ-RH chiral analysis column is 4.6 mm × 150 mm, 5.0 μm.
4. The Fmoc-L-Hgn(Trt)-OH reverse chirality detection method according to claim 1, characterized in that: The gradient elution program is: 0-30 min, the volume proportion of mobile phase B is 40% to 70%, 30.1-45 min, the volume proportion of mobile phase B is from 40% to the equilibrium system.