Question

A) One of the natural products in plant material shows antibacterial activity. Researchers are planning to utilize it in pharmaceutical applications. You are asked to contribute to this research in isolating the active ingredients in the plant material. Explain how do you plan to isolate and characterize the active ingredients in the plant material. (highlight the extraction procedure and characterization methods).

B) If the isolated active ingredient is a solid residue, explain how you purify the isolated crude product to use in pharmaceutical applications.

Answer 1

Ans: Isolation of AI (active ingredient) from natural products is a tedious process as the extract contains multiple components, active ingredients as well as impurities.

Step1: Preparation of Crude Extract by Extraction

Extraction is the process where we extract or separate the natural product from the raw material. There are different types of extraction methods depends on the nature of substance you want to extract and the most commonly used methods in extraction of natural products are Liquid-Liquid extraction and Solid-Liquid extraction.

Liquid-Liquid Extraction: Is the process where two immiscible liquids are mixed together in a separating funnel to separate the solute. After vigorous mixing separate the two layers (Organic & Water) and concentrate under reduced pressure to get the crude extract.

Solid-Liquid Extraction: Is a separation process based on the preferential dissolution of one or more components of a solid mixture in a liquid solvent. It can also be a multistage extraction where we extract the solute using limited quantity of solvent to get a concentrated extract. Once the extraction is complete the solvent can be removed and the desired product be collected.

Step2: Isolation of AI from Crude Extract

Once the crude extract is prepared the major task is to identify the major compounds. In order to do that 1st we need to remove any traces of water if present, by adding sodium sulfate and then filter off sodium sulfate from the crude extract using a funnel.

B) If the isolated AI is solid, which need to dissolve in Polar solvents like methanol/ethanol etc.

It can either purify by Column Chromatography or Thin Layer Chromatography.

Purification Procedure: Add proper amount of silica gel to the crude extract and gently heat the mixture using a hair dryer until the silica gel becomes free flowing. Transfer the adsorbed crude material into the solvent layer above the silica gel in the packed column. Continue filling the column with suitable solvent and elute it until the major spots runs down the column and collect this in a conical flask. After collecting all the spots, fill the column with acetone and elute completely. The major spots collected from the column are then concentrated by removing the solvents using a rotary evaporator and check for the active ingredient.

Determination of Molecular Weight using LC-MS can be done.

The crude material obtained here is then screened for the possible class of bioactive compounds using various chemical tests. For Example: Molisch test for sugars, Ferric Chloride for Flavonoids etc.