The objectives of this experiment are: 1. to master the basic principles and operation methods of high performance liquid chromatography (HPLC). 2. to master the application of HPLC analytical methods for the identification and determination of active ingredients of natural drugs. 3. to familiarize with the operation methods for the determination of NMR samples.

Operation method

High Performance Liquid Chromatography

Materials and Instruments

Rhododendron
Ethanol Methanol Deuterated CHCl3 Salvia divinorum Extract
Volumetric flasks Chromatographic columns Nuclear magnetic tubes Brown volumetric flasks

Move

1. Purity analysis of rhodopsin (Experiment V) obtained from the isolation of C. tigrinus

(1) Solution preparation

Preparation of control solution: weigh 10 mg of rhodopsin control product, put it in a 50 ml volumetric flask, dissolve and dilute it with ethanol to the scale, shake well. Measure 2 ml in 25 ml volumetric flask, dissolve with methanol and dilute to scale, shake well, it is obtained.

Preparation of test solution: weigh 10 mg of test material, put in a 10 ml volumetric flask, dissolve with ethanol and dilute to scale, shake well, that is to obtain.

(2) Chromatographic conditions (refer to Pharmacopoeia method)

Column: ODS, 4.6×250, 5 μM.

Mobile phase: methanol: water (0.1% phosphoric acid) = 80:20

Detection wavelength: 254 nm, flow rate: 1 ml/min

Column temperature: room temperature

Injection volume: 10 μL

2. 1H-NMR determination of rhodopsin isolated from C. tigris (experiment 5)

(1) Determination of sample

10 mg of dry sample was dissolved in 0.5 ml of deuterated _CHCl3 and transferred to a NMR tube for 1H-NMR determination.

(2) Spectral analysis

3. Analysis of tanshinone IIA in Salvia miltiorrhiza extract (Experiment 6)

(1) Solution preparation

Preparation of control solution: weigh 10 mg of rhodopsin control product, put it in a 50 ml brown volumetric flask, dissolve with methanol and dilute to the scale, shake well. Measure 2 ml in 25 ml brown volumetric flask, dissolve with methanol and dilute to scale, shake well, it is obtained.

Preparation of test solution: weigh 10 mg of test material, put in 50 ml brown volumetric flask, dissolve with ethanol and dilute to scale, shake well, that is to obtain.

(2) Chromatographic conditions (refer to Pharmacopoeia method)

Column: ODS, 4.6×250, 5 μM.

Mobile phase: methanol: water = 75:25

Detection wavelength: 270 nm, flow rate: 1 ml/min

Column temperature: room temperature

Injection volume: 5 μL

(3) Data processing: Based on the results of HPLC analysis, determine the optimal process conditions for the orthogonal design experiment in Experiment VI.

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