ANALYSIS OF VOLATILE OILS
Cabrega/Caseres/Celiz/Divino/Leano/Lomibao
Volatile Oils
◦ Complex products composed of mixtures of compouds
◦ Almost any type of organic compound may be found
◦ Specific gravity, optical rotation, refractive index
◦ Fractional distillation, low-temperature crystallization, and fractional crystallization,
chromatography, removal by chemical reaction
◦ Volatile, do not form soaps with alkalis, water-soluble, do not become rancid
Volatile oils
◦ Important chemical components
◦ Hydrocarbons
◦ Alcohols
◦ Aldehydes
◦ Ketones
◦ Phenols
◦ Acids
◦ Sulfur compounds
Classes of Volatile Oils
◦ Terpenoid
◦ Derived from Mevalonate pathway and methylerythritol phosphate pathway
◦ Aromatic
◦ Derived from the Shikimate pathway
Secretory structures of plant families
◦ Glandular hairs ◦ Secretory cavities
◦ Glandular trichomes ◦ Rutaceae, Myrtaceae
◦ Fam. Lamiaceae ◦ Oil tubes / vitae
◦ Nectaries ◦ Apiaceae
◦ Resin ducts ◦ Schizolysigenousducts
◦ Laticifers ◦ Rutaceae, Pinaceae
◦ Secretory ducts ◦ Glandular trichomes
◦ Apiaceae, Asteraceae, Clusiaceae, ◦ Lamiaceae(mint family)
Pinaceae ◦ Modified parenchymal cells
◦ Epidermal cells ◦ Piperaceae
◦ Rosaceae (Evert, 2006)
Peppermint Oil
◦ Mentha x piperita L. (Lamiaceae)
◦ Fresh leaf
◦ flavouring, carminative, aromatherapy
◦ 1-3% oil content
◦ menthol (30–50%)
◦ menthone (15–32%)
◦ menthyl acetate (2–10%)
◦ menthofuran (1–9%)
(Dewick, 2009)
Lemon Oil
◦ Citrus limon (Rutaaceae)
◦ Dried fruit peel
◦ flavouring, perfumery, aromatherapy; terpeneless lemon oil is obtained by removing much of
the terpenes under reduced pressure; this oil is more stable and contains 40–50% citral
◦ 0.1-3% oil content
◦ (+)-limonene (60–80%)
◦ β-pinene (8–12%)
◦ γ-terpinene (8–10%)
◦ citral (= geranial + neral)(2–3%)
(Dewick, 2009)
Spearmint Oil
◦ Mentha spicata L. (Lamiaaceae)
◦ Fresh leaves
◦ flavouring, carminative, aromatherapy
◦ 1-2% oil content
◦ (−)-carvone (50–70%)
◦ (−)-limonene (2–25%)
(Dewick, 2009)
Clove Oil
◦ Syzygium aromaticum L. (Myrtaceae)
◦ Dried flower buds
◦ Flavour, aromatherapy, antiseptic
◦ 15-20% oil content
◦ eugenol (75–90%)
◦ eugenyl acetate (10–15%)
◦ β-caryophyllene (3%)
(Dewick, 2009)
Limonene
◦ Component of many sources of volatile oils
◦ Bergamot, Caraway, Juniper, Lemon, Orange (bitter and sweet), Orange flower, Sage,
Spearmint
◦ Major component of Orange Oil together with Myrcene
(Dewick, 2009)
METHODOLOGY
TLC OF LIMONENE
Sample and Standard Solution
1mL 1:9 1mL 1:9
ethyl ethyl
Experiment acetate - Limonene acetate -
10A hexane standard hexane
extracted
Limonene
Mobile Phase 90mL
10mLethyl hexane
acetate
Development
11.5cm x
3cm silica
GF254
Standard
(20uL)
Sample
0.5cm
1cm
Evaluation
1.5g
K2MNO4
10g
Na2CO3
1.25mL
aqueous 150mL
NAOH distilled
water
DETERMINATION OF TOTAL
ESTERS IN PEPPERMINT OIL,
NF
10g Peppermint Oil –
250mL Erlenmeyer
flask
+10gtt neutralized
Reflux and heat on
alcohol boiling water (1hr)
+2gtt phenolphthalein
cool
+dropwise 0.1N NaOH –
until faint pink color
+20mL water
+ 25mL alcoholic KOH
+ phenolphthalein
Titrate with 0.5N HCl VS
*perform blank (disregard 0.1N
NaOH
DETERMINATION OF TOTAL
MENTHOL IN PEPPERMINT
OIL
10mL Peppermint Oil –
250mL Acetylation
flask
+10mL acetic
Until last washing is
anhydride alakaline to
+1g anhydrous phenolphthalein TS
◦ Cool
NACHCOO
Boil (1hr), cool, Dry oil with sodium sulfate,
disconnect from ◦ +10gtt phenolphthalein TS
filter
condenser
Transfer to separator Transfer 5mL dry
* Rinse acetylation acetylated oil to tared
flask with 3 5mL warm Erlenmeyer
Discard water layer
water +50mL alcocolic KOH VS,
reflux, boil in steam bath
Wash oil with NaOH TS (1hr)
Cool
portions
+10gtt phenolphthalein
TS
Titrate with 0.5N HCL
Perform blank
DETERMINATION OF
ALDEHYDE CONTENT
IN LEMON OIL
Reagent solution
Adjust pH to 3.4
with 0.5N KOH
13mL
water 85mL t-butyl
alcohol
Reagent
solution
50mL
Allow to stand
for 30 minutes
with occasional
shaking
5mL Lemon Oil
0.5N
alcoholic
KOH VS
Titrate
to pH
3.4
Liberated
HCl with
indicator
DETERMINATION OF KETONE
CONTENT IN SPEARMINT OIL
50mL saturated solution Phenolphthalein
of sodium sulfite
(neutral to 2gtt
phenolphthalein TS
Heat in boiling
water (5mins),
shake
*neutralize solution (+gtt
of sodium bisulfite
solution)
Without coloration? Cool to room
temp.
Allow liquids to separate
+ sufficient sodium sulfite
solution to raise oily layer
within graduated portion of
the flask
DETERMINATION OF
PHENOL CONTENT IN
CLOVE OIL
10mL Clove oil – cassia
flask
+75mL 1N KOH, shake
(5mins)
Heat in boiling water
(10mins), shake
Cool to room temp, allow
liquids to separate
+ sufficient 1N KOH to raise oily
layer within graduated portion of
the flask
Measure volume
after standing for
18hours
RESULTS AND
DISCUSSION
THIN LAYER
CHROMATOGRAPHY OF
LIMONENE
TLC of Limonene
Limonene (1-methyl-4-prop-1-en-2-yl-cyclohexene)
◦ Unsaturated hydrocarbon
◦ Monoterpene
◦ Chiral molecule with 2 enantiomers:
a) R = (+)-Limonene – oranges
b) S = (-)-Limonene – lemons
◦ Derived from the mevalonate pathway
Permanganate dip/stain
• For functional groups which are sensitive to oxidation
(oxidative stain)
• Requires strong heating
Functional group present Color of spot
alkene, alkyne, alcohol, brown-yellow spots (MnO2)
ether, ester,, alkyl aromatic,
ketone, carboxylic acid,
amine, amide
thiols, phosphines, dienes white spots before heating
(Mn+2)
alkanes and pyridine no spot
not compatible with entire plate will be yellow
elutants containing before spots develop
triethylamine
Examples of Chromatograms
Figure 1. Before Figure 2. After Drying
Drying
Actual Results Chromatograms
F K
E J
D I
C H
B
G
A
Figure 3. Before Drying Figure 4. Under UV
Characterization of Spots
DETERMINATION OF TOTAL
ESTERS IN PEPPERMINT OIL
Total Esters in Peppermint Oil, NF
◦Type of Titration: Residual with blank
- 0.5 N alcoholic KOH VS (excess)
- 0.5 N HCl VS (back titrant)
◦Type of Reaction: Saponification
◦Acceptance Criteria: NLT 5.0% of esters,
calculated as menthyl acetate (C12H22O2)
◦Indicator used: Phenolphthalein TS
◦Endpoint: colorless solution (USPCI, 2012)
◦ Chemical reaction of menthyl acetate with potassium
hydroxide
menthyl potassium menthol potassium
acetate hydroxide acetate
(Knevel and DiGangi, 1977)
◦ Computations
𝑀𝑊 𝑚𝑒𝑛𝑡ℎ𝑦𝑙 𝑎𝑐𝑒𝑡𝑎𝑡𝑒
(𝑉𝐾𝑂𝐻 − 𝑉𝐻2𝑆04) × × 100
𝑓 × 1000
%𝑒𝑠𝑡𝑒𝑟𝑠 =
𝑊𝑎𝑐𝑒𝑡𝑦𝑙𝑎𝑡𝑒𝑑 𝑜𝑖𝑙
Where:
MW of menthyl acetate = 198.24g/mol (Knevel and DiGangi, 1977)
F=2
DETERMINATION OF TOTAL
MENTHOL IN PEPPERMINT
OIL
Principle Involved
◦ Residual Alkalimetric Titration with Blank
◦ Endpoint:
◦ Phenolphthalein TS – pink to colorless solution
◦ Official Requirement: It yields NLT 5.0% of esters, calculated as menthyl acetate
(C12H22O2), and NLT 50.0% of total menthol (C10H20O), free and as esters.
(USPCI, 2012)
Chemical Equations
◦ Acetylization of Menthol
Acetic Acetic
Menthol Menthyl
Anhydride Acid
Acetate
(Knevel and DiGangi, 1977)
Chemical Equations
◦ Saponification of Menthyl Acetate
Menthyl Potassium Menthol Potassium
Acetate Hydroxide Acetate
◦ Neutralization
HCl + KOH → KCl + H2O
(Knevel and DiGangi, 1977)
Computations
◦ % Total Menthol = 7.831 A (1-0.0021E) / (B-0.021A)
Where:
7.831 MWmenthol = 156.27
B Weight of acetylated
oil taken
Differences in volume
A of 0.5 N HCl VS used in
actual and blank. (mL)
(Knevel and DiGangi, 1977; USPCI, 2012)
Computations
◦ % Total Menthol = 7.831 A (1-0.0021E) / (B-0.021A)
Where:
MWmenthyl acetate = 198.31
0.021 g/mol
MWmenthol = 156.27g/mol
E Percentage of esters
calculated as menthyl
acetate
MWacetyl radical / Mwacetyl radical = 42.04
0.002 (MWmenthyl acetate x 100 %) g/mol
1 MWmenthyl acetate = 198.31
g/mol
(Knevel and DiGangi, 1977; USPCI, 2012)
DETERMINATION OF
ALDEHYDE CONTENT IN
LEMON OIL
Principle Involved
◦ Indirect Alkalimetric Titration
◦ Neutralization Reaction
◦ Endpoint
◦ pH = 3.4
◦ For bromophenol blue: yellow to purple color transition
◦ Official Definition: The aldehyde content, calculates as citral (C10H16O), is not less than
2.2 % and not more than 3.8 % for California-type Lemon Oil, and not less than 3.0 %
and not more than 5.5 % for Italian-type Lemon Oil.
(USPCI, 2012)
Chemical Equations
◦ Liberation of HCl
Citral Citral
Oxime
◦ Neutralization
HCl + KOH → KCl + H2O
(Knevel and DiGangi, 1977)
Titration Data and Computations
Weight of Potassium 35 g
Hydroxide
Nf of 0.5 N Hydrochloric 0.9848
Acid VS
Final Volume of 0.5 N 23.31 mL
Alcoholic Potassium
Hydroxide VS
Nf of 0.5 N Alcoholic 1.0562
Potassium Hydroxide VS
• MWCITRAL= 152.24 g/mol
• f=1
Titration Data and Computations
◦ % Aldehyde Content =
{[NT ● VT ● NFTITRANT ● (MWANALYTE/( f x 1000))] / vol (mL) of sample}
X 100%
Trial Volume of Volume of % Aldehyde
Lemon Oil 0.5 N Content
(Sample) Alcoholic
Potassium
Hydroxide
VS (Titrant)
1 5 mL 1.22 mL 1.9615 %
2 5 mL 0.91 mL 1.4632 %
Titration Data and Computations
◦ Both trials did not conform to the USP standards.
◦ Since the % deviation between two trials is 14.55 % which is greater than the preferred 3
%, the conclusion based on the obtained results is deemed inconclusive.
◦ % deviation between trials:
= (|1.9615%- 1.4632%|/ 3.4247%) ● 100%
= 14.55 %
(Knevel and DiGangi, 1977)
DETERMINATION OF KETONE
CONTENT IN SPEARMINT OIL
Discussion
◦ Carvone, the principal ketone present in the oil, reacts
with sodium bisulfite, forming a water-soluble addition
product as follows:
Carvone
(Knevel, 1977, p.265)
Discussion
Na2SO3 + H2O ↔ NaHSO3 + NaOH Pink
solution
HSO3- + H2O ↔ H3O+ + SO32-
(Knevel, 1977, p.266)
Computations
𝑉𝑠𝑎𝑚𝑝𝑙𝑒 −𝑉𝑝𝑟𝑜𝑑𝑢𝑐𝑡
𝑘𝑒𝑡𝑜𝑛𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = x 100
𝑉𝑠𝑎𝑚𝑝𝑙𝑒
Official requirement: not less than 55.0% by volume of carvone
(Knevel, 1977, p.265)
DETERMINATION OF
PHENOL CONTENT IN
CLOVE OIL
Discussion
◦ KOH reacts with the phenol, eugenol, as follows:
Eugenol
Potassium
eugenolate
(Knevel, 1977, p.267)
Computations
𝑉𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑉𝑢𝑛𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑𝑙𝑖𝑞
𝑝ℎ𝑒𝑛𝑜𝑙 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑥100
𝑉𝑠𝑎𝑚𝑝𝑙𝑒
Official requirement by the USP: not less than 85.0% by
volume
Conclusion and Recommendation
◦ Sources of volatile oils were identified
◦ TLC analysis
◦ Volatile oils were classified based on their chemical structures
◦ Aldehyde Content in Lemon Oil was determined – Aldehyde Content: (USP: 2.2% - 3.8%)
◦ T1= 1.9615 %
◦ T2=1.4632 %
◦ The sample did not conform to the USP range
◦ TLC Analysis of limonene produced golden yellows spots; Most of the spots of the sample
did not have the same Rf values as the spots of the standard but is close to the Rf values of
the standard – Indicates that sample may not be purely limonene, or TLC was done
incorrectly
References
◦ Dewick, P.M. (2009). Medicinal Natural Products: A Biosynthetic Approach (3rd ed.). UK:
John Wiley & Sons Ltd.
◦ DiGangi, F. & Knevel, A. (1977). Jenkins’ Qualitative Pharmaceutical Chemistry. NY: Mc
Graw-Hill Book Company.
◦ United States Pharmacopoeial Convention (2012). United States Pharmacopoeia 35th
Revision ond the National Formluary30th edition. Maryland: USPCI.
