Biochemical Technology Topic: Extraction, Separation and Identification of Radon in Rhubarb

Overview

Rhubarb is recorded in many documents such as Shennong Bencaojing, used for venting, stomach, heat, detoxification, etc.

Since ancient times, rhubarb has occupied an important position in the medicinal diarrhea medicine, and it is a worldwide crude drug that has been collected by the pharmacopoeia of many countries. There are many kinds of rhubarb. The high quality rhubarb is the rhizome and root of rheumatous rhubarb (Rheum palmatclm L), rhubarb (R. officinale Baill) and R. tangutium Maxim. et Regll. Rhubarb contains many kinds of rhubarb. Free hydroxy steroids and their glycosides with sugars. The hydroxy hydrazines already known are mainly the following five:

The aglycone in rhubarb has a different structure, and thus the acidity is also different. Rhein is linked with -COOH, the acidity is the strongest; emodin is linked with β-OH, acidity is second; aloe emodin is linked with benzyl alcohol-OH, acid is the third; emodin methyl ether and chrysophanol have 1,8- Diphenolic hydroxyl group, the former has -OCH3 and -CH3, the latter only has -CH3, so the latter is ranked fourth in acidity.

Experimental purpose and requirements

1. Learn the basic operating techniques of buffer paper chromatography, and design a solution for separating mixtures by liquid-liquid extraction based on the chromatographic results.

2. Master the principle and operation technology of the PH gradient method.

3. The experiment of separating chrysophanol and emodin methyl ether by calcium hydrogen phosphate column chromatography is further familiar with column chromatography operation technology.

4. Learn how to identify terpenoids.

experimental method

1. Extraction of total aglycone from rhubarb

Note: 1 Most of the terpenoids in rhubarb bind to sugar and exist in plant tissues in the form of alfalfa. Therefore, acid hydrolysis is required to form aglycones. The glycosides are soluble in organic solvents such as chloroform, benzene and ether. When using benzene, attention should be paid to the volatilization of benzene vapor to prevent poisoning. 2 If the obtained chloroform solution has an acid water solution, it should be separated by a separatory funnel. Go and rinse back with distilled water to remove acidity so as not to affect the gradient extraction. If the chloroform extract is precipitated and precipitated, it can be filtered out. The precipitate is mostly emodin, and the remaining liquid is subjected to the next separation test.

2. Design of total aglycone separation scheme

1) Paper chromatography of total glucosides of rhubarb

2) Cushioning paper chromatographic test of terpenoids

3) Determination of the best extractant and its dosage

4) Determination of the amount of various extractants of terpenoids

5) Design of the new separation method

3. Separation and purification of terpenoids

1 Separation and purification of rhein

450 ml of chloroform solution containing total free hydrazine was transferred to a 1000 ml large separatory funnel, shaken and extracted with 275 ml of PH8 buffer, allowed to stand until thoroughly layered, and the chloroform solution was discharged, and then the alkali water solution was placed in a 250 ml beaker. Acidified to pH=3 with HCl, after the yellow precipitate is completely precipitated, filtered and dried. The dried sample is heated with 10 ml of glacial acetic acid to dissolve, and the filtrate is allowed to stand. The filtrate is allowed to stand, and the yellow needle crystal is precipitated as rhein. Get pure.

2 Separation and purification of emodin

The chloroform solution containing rhein was continuously transferred to a separatory funnel, and extracted with 275 ml of PH9.9 buffer. After thorough layering, the aqueous layer was separated and acidified to pH = 3 with HCl to precipitate a brownish precipitate. After filtration, the precipitate is dried, heated with 10 ml of glacial acetic acid to dissolve, and filtered while hot to precipitate large orange needles. After filtration, the pure product of emodin is obtained.

3 Separation and purification of aloe-emodin

After the remaining chloroform solution was transferred to a separatory funnel, add 5% NaCO3: 5% NaOH (9:1) 540 ml of alkaline water solution or shake with 0.5% KOH 540 ml, acidify with alkaline water and add HCl, and precipitate the precipitate and filter dry. Recrystallization from 10 ml of ethyl acetate gave a yellow needle crystal of aloe-emodin pure product.

4 Separation of chrysophanol and emodin methyl ether - calcium hydrogen phosphate column chromatography

4. Identification of terpenoids

1) Paper chromatographic identification

2) IR spectrum analysis

The main points and precautions of this experiment

1) Principles and precautions for pH gradient extraction.

2) How to design the procedures and methods for the extraction separation scheme.

3) Characteristics of the infrared spectrum of ruthenium compounds.

4) When separating and extracting, be sure to pay attention to the separation of the emulsion layer, do not mix it, and use fresh CHCl3 for each step to wash back the alkaline water.

5) The preparation of the buffer and the preparation of the lye should be accurate and strictly inspected.

6) At each step of extraction, use paper chromatography to track the extraction effect. If the expected effect is not achieved, it should be corrected in time.