Isolation: Method-1

• Aerial parts of the plant are extracted with petroleum ether (Boiling point 40-60°C) and concentrate the petroleum ether under vacuum.

• The concentrated extract is redissolved in chloroform and acetonitrile is added to separate out inert plant components like waxes and sugars.

• Column chromatography of this concentrated extract was performed using silica gel as stationary phase.

• High artemisnin content fraction will crystallize rapidly.

• Then recrystallised this fraction using cyclohexane or 50 percent ethanol.

• Artemisinin is less water soluble, colourless or white, crystalline compound having molecular weight 282.3 with odourless crystalline powder.

• Melting point: 156-157°C.

Thin layer chromatography of Artemisin:

• Dissolve the standard and sample both in chloroform and perform the TLC using mobile phase petroleum ether: Ethyl acetate (1:2).

• Dry the plate and sprayed with either ρ-di methylamino benzaldehyde (densitometric scan at 600 nm) or 2 % solution of vanilline sulphuric acid (densitometric scan 560 nm).

• Compare the spot of standard and sample. Sesquiterpene can also be estimated by HPLC with UV detection (220 nm).

• Sesquiterpene can or cannot be converted into their derivative which can absorb UV radiation.

• In an alkaline medium open the oxygen-containing heterocycles are then converted into lactol in an acidic medium.

• It can be also estimated using GC-MS technique by estimating the degradation product (artemisinin is thermal degradable).

Analysis Artemisin can be analysed by the following techniques:

• IR Spectrophotometer:

  • • 2mg of isolates are crushed and mixed with 98mg of KBr (dried for 24 hours at 105 ºC temperature).

    • The isolates are analysed at wave number 4000 -400cm-1 using KBr as the baseline.

    • The spectrum of isolates obtained is compared with the spectrum of standard artemisin.

• UV Spectrophotometer:

  • • 1mg of isolates is dissolved in 10ml of methanol and analysed at wavelength of 200-400nm.

    • The spectrum of isolates obtained is compared with the spectrum of standard artemisin.

• Thin Layer Chromatography (TLC):

  • • 1mg of isolates is dissolved in 5ml of ethyl acetate.

    • Isolates are spotted with a solution using the capillary tube on a silica gel 60 F254 (the stationary phase).

    • Ethyl acetate: Hexane (3:97) and Ethyl acetate: Hexane (7:93) are used as the mobile phase.

• Liquid Chromatography-Mass Spectrometry (LC-MS):

  • • 1mg of sample is dissolved in 1ml of methanol and then 20μl is injected and eluted using methanol:water (9:1).

    • With a flow rate of 1ml/min, separation is done through C18 column (RP 18).

    • The samples are analysed separately as per their retention times.

• HNMR Spectroscopy:

  • • Isolates are analysed using CDCl3 as the solvent and tetramethylsilane as the reference compound.

    • The spectrum of isolates obtained is compared with the spectrum of standard artemisin.

Identification

1 gm finely powdered drug is boiled with 10 ml alcohol and filtered. Then add sodium hydroxide to the filtrate and heat again. The red colour develops in liquid.