Isolation, Identification and

Analysis
of
MENTHOL
 Menthol
• It an organic compound made synthetically or
obtained from Mint, Peppermint, Mentha.
• B.Source: It is obtained from the fresh flowering
tops and leaves of the plant commonly known as
Mentha piperita or other allied species of
Mentha, belonging to family Labiatae.
• It is a monocyclic monoterpenoid (C5H8) 2
alcohol
 Description

• Colour : Colourless to yellow

• Odour : Characteristic and pleasant
• Taste : Characteristic, pungent followed by
cooling sensation
• Refractive index: 1.4590 to 1.4650
• Shape: Needle like crystals
• Solubility: It is soluble in ethanol, diethyl ether,
acetone, chloroform, acetic acid and slightly
soluble in water.
 Structure and molecular formula of
menthol
• Molecular formula: C10H20O
• Molecular weight: 156.27 g/mol
• Menthol, on oxidation gives menthone.
 Isolation
• Take accurate weighed of coarse powder of Mentha
piperita leaves
• Extract the oil by water/hydro distillation method
using clevenger apparatus
• Collect the oil (present above the water layer) from
the collecting tube or separate the oil from water by
using separating funnel and allow cooling
• Crystals of menthol will separate out
• Collect the crystals by centrifugation.
• Recrystallize menthol from acetone or any other
suitable solvent of low boiling point.
Water/hydro distillation method using Clevenger
apparatus
 Method-2
• Take dried weighed quantity of Mentha piperita
leaves in a stainless steel container of assembly
• Steam is generated with the help of boiler and
allowed to pass through the drug
• The vapour of oil is formed and cools back into
liquid while passing through the condenser
• Essential oil floats on the top of water (since it is
lighter than water) and it is syphoned off.
• Allow cooling to separate the menthol crystals.
• In order to make the isolated oil moisture free, it
is passed through a bed of anhydrous sodium
sulphate followed by filtration.
• The moisture free oil is then frozen to about -60
degree centigrade for about 7 days by packing in
tightly sealed plastic containers and during the
process, menthol in the oil separates out as flaky
crystals.
• The mother liquor still may contain menthol
along with menthone and other terpenes.
• Therefore, for complete recovery of menthol
crystals, the oil is treated with 8 g boric acid in
distillation flask for 3 hours to distill off
menthone.
• Distillation residues containing borates of
menthol are saponified by steam distillation over
70 ml of 15% NaOH solution for about 1 hour
and thereby separating the menthol from liquid
mixture.
• The resultant solution is allowed to cool to
separate remaining menthol
• The crystals are dried in desiccators.
Other methods of isolation of menthol

• Fractional distillation and Chromatographic

adsorption methods can be used to isolate
menthol from extracted essential oil of mint.
 Fractional distillation
• Ordinarily 60% to 85% menthol with 12% to
20% menthone is present in Mentha leaves.
• The following steps are, : (l) By process of
vacuum distillation in a column, menthone and
other fractions are distilled off; (2) isolation of
menthol from non-volatile compounds by steam
distillation; and (3) Cooled and formation of
crystals and drying.
 Chromatographic adsorption
• Quantitative yields of both menthol and menthone can
be acquired in a on a commercial scale using adsorbents
and specific eluents.
• The mint oil is dissolved in a non polar solvent and after
that passed over an adsorbent; the adsorbed material is
then washed with eluents (appropriately chosen solvents)
to isolate the menthone and menthol into obvious
divisions.
• Generally, four fractions are produced: the terpenes,
menthyl esters are cleaned out first, the menthone
fraction is turned out straightaway and the last fraction
contains just menthol.
• The components which are not strongly adsorbed
move quickly and the strongly adsorbed
components move at a low speed. Because of this
differential relocation, the components which are
adsorbed gradually isolated from each other. The
adsorption affinity of main components in mint
oil is in following order menthol, menthone,
methyl esters and terpene hydrocarbons, the last
being least adsorbed
 Identification
• Take few crystals of menthol and mix with 5ml
nitric acid and heat on water bath. Within a few
minutes, the solution develops blue colour and
after sometime, it becomes golden yellow colour
which indicates the presence of menthol.
• 10mg crystals of methanol first dissolved in 4
drops of conc. H2SO4, then add few drops of
Vanilline-H2SO4 reagent. Formation of yellowish
orange colour which on addition to water changes
to violet.
• A few crystals of menthol are dissolved in
acetic acid and a mixture of 3 drops sulphuric
acid and 1 drop nitric acid is added to it. If it
does not produce bluish or greenish colour,
menthol is present.
• Take a few grams of menthol crystals in a
porcelain dish and heat. All the menthol
crystals are evaporated leaving no residue.
 TLC method

• Sample preparation: Dissolve 1mg menthol in

1ml methanol
• Stationary phase: Silica gel-G
• Standard sample: Menthol
• Detecting agent: 1% Vanilline-H2SO4 reagent and
heat the plate at 110o C for 15 minutes.
• Mobile phase: Chloroform
• Rf value: 0.48-0.62
 TLC-2

• Sample preparation: Sample is extracted with

chloroform. The chloroform extract is evaporated
to dryness and dissolved in methanol
• Stationary phase: Plates coated with 0.2 mm layers
of silica gel 60 F254 as stationary phase
• Mobile phase: Hexane : ethyl acetate 8 : 2 (%, v/v)
previously saturated for 30 min at 22°C
• Detecting agent: Anisaldehyde–sulfuric acid
reagent and heat the plate at 110o C for 15 minutes
 
• RF value: 0.34
 Methods of Analysis
• Techniques of chromatography are applicable in the
separation and identification of compounds.
• Gas Chromatography-Mass Spectrometry is used
mostly in the isolation and recognition of compounds
which are present in the essential oils.
• The identification of the components of essential oil is
usually happen with the assistance of gas
chromatography equipped and together with flame
ionization detector (FID) and MS detectors, a capillary
column (30m×0.25mm, film thickness 0.25μm),
• Test conditions may differ according to the
sample of essential oil to be examined and the
column used for its identification or analysis. For
example, GC–MS experiments are often
performed with injector (by which we inject
sample of our own requirements whose
identification are required) and detector while the
temperatures of injector is kept at 250 ºC and
those of detector kept at 270°C, respectively, with
the oven initially at 50 °C for 2–3min and then
risen up of temperature by 3–10°C/min until it
reaches 200–240°C, with a flow of 0.7–1.0μL/min
• Regularly, helium is used as a carrier gas at a flow
rate of 0.7–1.0mL/min, the samples are diluted
(1/10, v/v), and the volume injected is 0.2μL.
• The recognition and quantification can be done by
observing the obtained peaks.
• The retention time of the peaks gives recognition
of the compound while the area of the peak is
responsible for the quantification of the compound.
• The compiled & gathered data can then be
compared against reference standards to find out
the purity.
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