Aim : To Distillate volatile oils and identify phytoconstituents using TLC 


Material/ Requirement

Round bottom flask, special Still head known as Clevenger’s apparatus containing condenser, 

receiver and return tube. It also has side tube to introduce water in graduated receiver and return tube.   

 

Theory:

Generally volatile oils are extracted from the plant material by four methods i.e. distillation, steam distillation, expression, and enfleurage which are already discussed in chapter 1. Most oils are distilled in a single process within 2-5 hours, while a few of them require second step to purify through fractional distillation. Maceration, where the plant material is macerated in a warm water to release enzyme-bound essential oils, is a very specific method applicable to limited number of essential oils like onion, garlic, Wintergreen, bitter almond etc. 

 

 Solvent extraction ' one of the most promising methods of essential oil extraction but recently newer

 techniques like microwave assisted or supercritical fluid extraction (SCF) became more popular. Microwave extraction is a mild and controllable processing tool which allows oil extraction with or

 without solvent or even dry extraction within a very short time. The use of SCF in essential oil

 extraction has gained much more attention recently due to the high selectivity in separation, no toxic

 residue and thermal degradation which produces highest quality essential oil than conventional

 distillation methods.

 

There are many factors which are responsible for quality of distilled essential oils like time, temperature, pressure, and distillation equipment. Essential oils are composed of fairly delicate components which can be altered or destroyed or there activity loss by improper methods of extractions. 

 

General Method :

Select the appropriate Clevenger apparatus for volatile oils having density either higher than or lower

 than water. Take the accurately weighed quantity of fresh or dried plant material consisting of the

 flowers, leaves, wood, bark, roots, seeds, or peel (e. g. eucalyptus leaves, lemongrass leaves, clove

 buds etc). Reduce the material to coarse sized particles. Place the material in RBF of Clevenger

 apparatus. Add sufficient quantity of water. Attach the assembly as in the figure. Heat the mixture for 1

 hr. vapours of both water and volatile oil will pass towards condenser and then the collector tube.

 Allow the mixture to cool and separate. Read the volume of oil directly from the graduated tube.


                                            Lighter than water                          Heavier than water

Fig. Clevenger apparatus for volatile oil determination

 

Note 1: If the volume is heavier than water, a known volume of xylene is added to the graduated tube by a pipette through the side tube. The increase in volume of xylene gives the volume of oil distilled.

 

Note 2: Instead of pure water, are 150ml glycerin + 150ml of water mixture for dill, coriander, clove, fennel, dry orange peels etc.


Observation

·         Weight of plant taken (gm) (a):

·         Weight of volatile oil obtained (ml) (b):

·          % yield = (b*100)/a


(TLC) Rf Value = Distance travelled by solute / distance travelled by solvent .


Note:

For the extraction of volatile oil from coriander, dill, cloves and fennel, the mixture of glycerine

and water gives better yield instead of water.

The volatile oil which are heavier than water (clove oil), a known volume of xylene is added in

graduated receiver tube through side tube. The volatile oils are soluble in xylene and are

dissolved in xylene at upper part of graduated tube. The subtraction of xylene from final

volume of volatile oil collected gives the actual amount of volatile oil.  


Detection of Phytoconstituents by TLC

1.     Prepare a suspension of the silica gel G in  distilled water, preapare the plate either of spreading,

      pouring, dipping method. Allow the coated plates to dry in air, heat at 100º to 105º for at least 1

          hour and allow to cool

2.      Allow the saturate the developing chamber with the vapours of solvents of mobile phase by

          keeping this chamber aside for 30 min

3.     Apply the spot on the activated silica gel plate using capillaries and mark it with pencil

4.     Place this plate into the developing chamber and put lid on it

5.      Allow to run the mobile phase on the silica gel plate (stationary phase) to its 80% of height

6.     Mark the level of solvent front immediately after removing of plate from developing chamber

7.      Dry the plate to room temperature and observe the plate either in iodine chamber of UV chamber

        at 254 nm.

 

Rf. Value

Measure and record the distance of each spot from the point of its application and calculate

the Rf. value by dividing the distance travelled by the spots by the distance travelled by

the front of the mobile phase.


Experimental condition for TLC 

Stationary phase     :   silica gel GF254

Mobile phase             : Toluene (100)

visualizing agent      :  Vanillin sulphuric acid      

Phytoconstituents identification - Eugenol – Rf. 0.4; orange brown Aceteugenol- pinkish

yellow below eugenol.


 Rf Value = Distance travelled by solute / distance travelled by solvent