Prepared by: ENGR. RENA P.

MORA, LPT 1
Assay of Volatile Oils

MODEL 1: VOLATILE OILS

o Generally, are complex products composed of mixtures of compounds of widely variant chemical
characteristics.
o Also known as ethereal oils or essential oils and, in some cases, as essences
▪ Fixed Oils vs. Volatile Oils
o Fixed Oils
(+) spot test obtained by extraction
Undergo rancidity
Contains glycerylesters of fatty acids
o Volatile Oils
(-) spot test
Obtained by distillation
Tends to be oxidized and resinified
May contain esters but not glycerylesters
▪ Chemical Components of Volatile Oils
o Hydrocarbons
Acyclic series
• heptane • myrcene
Isocyclic series
• pinene • fenchene
• camphene • dipentene
• limonene • sylvestrene
• bornylene • phellandrene
o Alcohols
linalool borneol
geraniol menthol
citronellol santalol
terpineol
o Aldehydes
benzaldehyde citral
cinnamic aldehyde citronellal
salicyl aldehyde
o Ketones
camphor thujone
carvone menthone
fenchone
o Phenols
anethol safrol
eugenol chavicol
carvacrol thymol
o Acids
acetic cinnamic
propionic hydrocyanic
butyric linalyl acetate
valeric bornyl acetate
benzoic menthyl acetate
o Sulfur compounds
allyl isothiocyanate (mustard oil)

MODEL 2: GENERAL METHODS OF ASSAYING VOLATILE OILS

▪ Organoleptic Tests
o Use of senses
o Appearance, odor, color, etc.
▪ Specific Gravity
o Maybe determined by pycnometer or Westphal balance and is expressed as the ratio of the weight
of the volume of oil to that of an equal volume of pure water at 25°C
o The use of pycnometer is the official method and more accurate
 Prepared by: ENGR. RENA P. MORA, LPT 2
Assay of Volatile Oils

o Oils rich in hydrocarbons are usually lighter than water

o Oils rich in aldehydes, phenols and derivatives, and esters usually approach or exceed 1
o Specific gravity for any volatile oil is not constant because it varies from maturity of the plant, age
of the oil, methods of preparation and purification.
▪ Optical Activity and Rotatory Power
o Serves to determine adulteration
o Generally measured with a Laurent half-shadow polarimeter
o Optical activity should be made at 25°C
▪ Refractive Index
o Measured by means of Abbe refractometer
o Serves to detect the presence of extraneous matter
▪ Congealing Temperature or Point
o Most oils have low congealing temperatures, but for oils with large amounts of readily
crystallizable constituents (eucalyptus oil, anise and fennel oil), a higher congealing point is
desired.
o Method is usually applied to the said oils, an abnormally low congealing point for these oils
indicates presence of extraneous matter or partial removal of its constituent for which it is valued.
▪ Distillation Range or Limits
o Volatile oils boil between wide temperature ranges hence, official standards usually designate the
temp or temp range at which a definite percentage of the oil distills over.
o E.g. NLT 10% of pine needle oil should distill below 165°C
▪ Fractional Distillation
o For determining purity by separating the various components of the volatile oil and adulterants.
o Adulterants may be further detected by collecting various fractions of the oil and subjecting them
to rotatory power and index of refraction tests.
▪ Solubility
o Volatile oils are generally miscible in organic solvents such as absolute ether, chloroform, benzene,
and carbon disulfide.
o All are miscible in 70 to 90%
o Dilute can differentiate them according to chemical components and detect purity since
adulterants such as petroleum oil, fatty oils and turpentine oil are not miscible.
o Oils with large amounts of oxygenated substances produce turbid solutions with petroleum ether
and carbon disulfide due to the traces of water they contain.

MODEL 3: ASSAY FOR ESTER CONTENT

o Esters found in volatile oils are mostly acetate esters of with 10 to 15 carbon atoms.
o Detects adulteration and establish qualities of volatile oils valued for their ester content.
o The esters are saponified with 0.5 N KOH.
o Volatile oils with appreciable amounts of aldehydes or phenols cannot be assayed in this way
because these and their decomposition products can react with KOH.
o Ester value or ester number of the oil is the number of milligrams of potassium hydroxide required
to saponify 1 g of the oil.
o Example: Assay of Peppermint Oil for Total Esters
It is more suitable to perform the weighing in dropper bottles.
The total ester of peppermint oil is determined as its methyl acetate content.
KOH is used since the oil is more soluble in it than in aqueous solutions.
An excess of the alkali used and the excess back titrated with standard acid. A blank
determination is performed at the same time.
Sample Calculation: Assay for Ester Content
• Problem:
Calculate the percent menthyl acetate if a 9.120-g sample of peppermint oil,
refluxed with 25.00 mL of about 0.5 N alcoholic KOH, 21.52 mL of 0.4900 N HCl
of the residual titration. The blank was run using the volume of 0.5 N alcoholic
KOH and required 26.00 mL of 0.4900 N HCl to bring about the end point. Each
mL of 0.5 N KOH is equivalent to 99.06 mg of methyl acetate.
 Prepared by: ENGR. RENA P. MORA, LPT 3
Assay of Volatile Oils

• Solution:
1. Determine the number of milliequivalents of KOH consumed by the sample.
0.4900 𝑚𝑒𝑞 𝐻𝐶𝑙
26.00 𝑚𝐿 𝐻𝐶𝑙 𝑥 = 12.740 𝑚𝑒𝑞 𝐻𝐶𝑙 (𝑎𝑙𝑠𝑜 𝐾𝑂𝐻)
1 𝑚𝐿 𝐻𝐶𝑙
0.4900 𝑚𝑒𝑞 𝐻𝐶𝑙
21.52 𝑚𝐿 𝐻𝐶𝑙 𝑥 = 10.545 𝑚𝑒𝑞 𝐻𝐶𝑙 (𝑎𝑙𝑠𝑜 𝐾𝑂𝐻)
1 𝑚𝐿 𝐻𝐶𝑙
12.740 𝑚𝑒𝑞 − 10.545 𝑚𝑒𝑞 = 2.195 𝑚𝑒𝑞 𝑜𝑓 𝐾𝑂𝐻
the number of milliequivalents of KOH required to saponify the total esters
calculated as menthyl acetate.
2. Multiply 2.195 meq of KOH so consumed by 0.1983 g, the weight in grams
of 1 meq of menthyl acetate:
0.1983 𝑔
2.195 𝑚𝑒𝑞 𝐾𝑂𝐻 𝑥 = 0.4353 𝑔 𝑚𝑒𝑡ℎ𝑦𝑙 𝑎𝑐𝑒𝑡𝑎𝑡𝑒
1 𝑚𝑒𝑞
𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 𝑏𝑦 1.532 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
3. Divide the weight of menthyl acetate by the weight of the sample and
multiply by 100 in order to obtain the percentage of total esters:
0.4353 𝑔 𝑚𝑒𝑛𝑡ℎ𝑦𝑙 𝑎𝑐𝑒𝑡𝑎𝑡𝑒 𝑥 100
= 4.77% 𝑤/𝑤
9.120 𝑔 𝑠𝑎𝑚𝑝𝑙𝑒
4. Combining all mathematical operations (steps 1, 2, and 3) and factoring:
(26.00 𝑚𝐿 − 21.52 𝑚𝐿)𝑥 0.4900 𝑁 𝑥0.19831𝑔/𝑚𝑒𝑞𝑥 100
= 𝟒. 𝟕𝟕% 𝒘/𝒘
9.120 𝑔
• Alternative Solution:
1. Determine the number of milliliters of exactly 0.5 N alcoholic KOH consumed
by the sample:
0.4900 𝑚𝑒𝑞
26.00 𝑚𝐿 𝑥
𝑚𝐿 = 25.48 𝑚𝐿 𝐻𝐶𝑙 (𝑎𝑙𝑠𝑜 𝐾𝑂𝐻) 𝑏𝑙𝑎𝑛𝑘 𝑡𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛
0.5000 𝑚𝑒𝑞/𝑚𝐿
0.4900 𝑚𝑒𝑞
21.52 𝑚𝐿 𝑥
𝑚𝐿 = 21.09 𝑚𝐿 𝐻𝐶𝑙 (𝑎𝑙𝑠𝑜 𝐾𝑂𝐻) 𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑡𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛
0.5000 𝑚𝑒𝑞/𝑚𝐿
25.48 𝑚𝐿 − 21.09 𝑚𝐿 = 4.39 𝑚𝐿
2. Multiply 4.39 mL by 0.09915, the number of grams of menthyl acetate
equivalent to 1.00 mL of 0.5 N alcoholic KOH consumed:
0.09915 𝑔
4.39 𝑚𝐿 𝑥 = 0.4353 𝑔 𝑚𝑒𝑛𝑡ℎ𝑦𝑙 𝑎𝑐𝑒𝑡𝑎𝑡𝑒
1 𝑚𝐿
3. Divide the weight of menthyl acetate by the weight of the sample and
multiply by 100 in order to obtain the percentage of total esters:
0.4353 𝑔 𝑚𝑒𝑛𝑡ℎ𝑦𝑙 𝑎𝑐𝑒𝑡𝑎𝑡𝑒
= 𝟒. 𝟕𝟕% 𝐰/𝐰
9.120 𝑔 𝑠𝑎𝑚𝑝𝑙𝑒

Study Guide Question

1. What is the purpose of blank determination in the assay of peppermint oil for total esters?
2. Why is an alcoholic rather than an aqueous solution of KOH used to saponify esters?

MODEL 4: ASSAY FOR ALCOHOL CONTENT

o Usually, the determination of the total alcohol, is sufficient to establish the purity and value of a
volatile oil with respect to its content of alcoholic constituents.
o The total alcohols present are determined by converting them into acetate esters with acetic
anhydride using an acetylization flask with the saponification value of the acetylized product
determined.
o The alcohol of volatile oils usually occurs partly free and partly combined, hence, correction factors
are employed.
o Example: Assay of Peppermint Oil for Total Menthol
After acetylation, the aqueous layer is rejected and the oily layer separated and washed
with Na2CO3, and further dried with anhydrous Na 2SO4.
The acetylated oil is weighed and treated with excess 0.5 N KOH, with the excess back
titrated with standard HCl and a blank determination performed.

Calculate the percentage of total menthol in the oil tested by the formula
 Prepared by: ENGR. RENA P. MORA, LPT 4
Assay of Volatile Oils

𝑨 𝒙 𝟕. 𝟖𝟏𝟑𝟓
𝒙 [𝟏 − (𝑬 𝒙 𝟎. 𝟐𝟏𝟕)]
𝑩 − (𝑨 𝒙 𝟎. 𝟎𝟏𝟎𝟕𝟓)
which A = the result obtained by subtracting the number of milliliters of 0.5 N HCl
required in the above titration from the number of milliliters of 0.5 N HCl
required in the residual titration blank
B = the weight of acetylated oil taken
E = the percentage of esters calculated
o Sample Calculation:
Problem: A sample of peppermint oil found to contain 5% total esters was acetylated and
1.5 g of this was treated with 50 mL of 0.5 KOH. The excess KOH required 21.75 of 0.5 N
HCl and the blank determination required 26.75 mL. Calculate the percentage of total
menthol in the oil.
Solution:
𝐴 𝑥 7.8135
% 𝑡𝑜𝑡𝑎𝑙 𝑚𝑒𝑛𝑡ℎ𝑜𝑙 = 𝑥 [1 − (𝐸 𝑥 0.217)]
𝐵 − (𝐴 𝑥 0.01075)
(26.75 − 21.75)𝑥 7.8135
= 𝑥 [1 − (0.05 𝑥 0.217)]
1.5 − [(26.75 − 21.75)𝑥 0.01075]
39.0675
= 𝑥 0.98915 = 𝟐𝟔. 𝟕𝟐%
1.44625
Study Guide Question
3. If peppermint oil containing 7.2% total esters is found to contain 51.5% total menthol (uncorrected), what
change in the result would the correction 1 - (E x 00.21) cause? Is this correction important?
Answers: 50.72%, yes

MODEL 5: ASSAY FOR ALDEHYDE CONTENT

▪ Bisulfite Method
o Carried out when there are no constituents present capable of reacting with NaHSO3
o The addition product dissolves in water leaving the non-aldehyde constituents undissolved
o The volume of this water insoluble layer is measure in a cassia flask. It rises to the surface up to
the graduated mark when the cassia flask is filled with Na2SO3
o The cassia flask is a glass flask of about 100-mL capacity with a long, narrow neck graduated to 10
mL in 0.1-mL divisions.
o This volume of water insoluble layer is subtracted from the amount of the volatile oil employed.
o Sample Calculation:
Problem: A 20 mL sample of cinnamon oil was assayed for its cinnamaldehyde content.
After treatment with NaHSO3 the sample was transferred to a Cassia flask and filled with
Na2SO3 to raise the oily layer to the graduated mark. The oily layer measured 3.61 mL.
Calculate for the percent of cinnamaldehyde.
Solution:
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑜𝑖𝑙 − 𝑣𝑜𝑙𝑢𝑚𝑒 𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑜𝑖𝑙𝑦 𝑙𝑎𝑦𝑒𝑟
= 20 𝑚𝐿 − 3.61 𝑚𝐿 = 16.39 𝑚𝐿 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 − 𝑠𝑜𝑙𝑢𝑏𝑙𝑒 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 𝑎𝑑𝑑𝑖𝑡𝑖𝑜𝑛 𝑝𝑟𝑜𝑑𝑢𝑐𝑡
𝑣𝑜𝑙𝑢𝑚𝑒 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 16.39 𝑚𝐿
% 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 = 𝑥 100 = 𝑥 100 = 𝟖𝟏. 𝟗𝟓%
𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑜𝑖𝑙 20 𝑚𝐿
▪ Hydroxylamine Method
o For samples with small amounts of aldehyde and those that contain constituents that can form
water-soluble addition products with NaHSO3 aside from its aldehyde.
o The liberated HCl is titrated with NaOH.
o A blank determination is performed.
o Sample Calculation:
Problem: A 1.37 mL sample of benzaldehyde was treated with hydroxylamine
hydrochloride and the liberated HCl required 26.98 mL of 1.5781 NaOH. The blank
determination was ran and required 18.96 mL of the same standard solution. What is the
percent purity?
Solution:
𝑔
(𝑣𝑜𝑙𝑢𝑚𝑒 𝑏𝑎𝑠𝑒 − 𝑣𝑜𝑙𝑢𝑚𝑒 𝑏𝑙𝑎𝑛𝑘 ) 𝑥 𝑁 𝑥 0.10607 𝑥 100
𝑚𝑒𝑞
% 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 =
𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒
 Prepared by: ENGR. RENA P. MORA, LPT 5
Assay of Volatile Oils

𝑔
(26.98 𝑚𝐿 − 18.96 𝑚𝐿 ) 𝑥 1.5781 𝑚𝑒𝑞/𝑚𝐿 𝑥 0.10607
𝑚𝑒𝑞 𝑥 100
% 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 =
1.37 𝑚𝐿
% 𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 = 97.99%

Study Guide Question

4. Compare and contrast between the bisulfite method and hydroxylamine method in the assay of aldehyde
content.

MODEL 6: ASSAY FOR KETONE CONTENT

o Also carried by the same methods and principles (bisulfite and hydroxylamine methods), but only
two volatile oils are assayed for their ketone content. (Caraway and Spearmint Oil)
o Sample Calculation: Bisulfite Method
Problem: A 10 mL sample of caraway oil was assayed for its carvone content. After
treatment with NaHSO3 the sample was transferred to a Cassia flask and filled with Na 2SO3
to raise the oily layer to the graduated mark. The oily layer measured 4.50 mL. Calculate
for the percent of carvone.
Solution:
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑎𝑟𝑣𝑜𝑛𝑒 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑜𝑖𝑙 − 𝑣𝑜𝑙𝑢𝑚𝑒 𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑜𝑖𝑙𝑦 𝑙𝑎𝑦𝑒𝑟
= 10 𝑚𝐿 − 4.50 𝑚𝐿 = 5.50 𝑚𝐿
𝑣𝑜𝑙𝑢𝑚𝑒 𝑐𝑎𝑟𝑣𝑜𝑛𝑒 5.50 𝑚𝐿
% 𝑐𝑎𝑟𝑣𝑜𝑛𝑒 = 𝑥 100 = 𝑥 100 = 𝟓𝟓%
𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑜𝑖𝑙 10 𝑚𝐿
Study Guide Question
5. What is the principal ketone present in the oil?
6. What is a cassia flask?
7. What are the two official volatile oils assayed for their ketone content?

MODEL 7: ASSAY FOR PHENOL CONTENT

o Phenol-containing volatile oils are assayed by shaking with NaOH or KOH.
o The undissolved oily layer represents the non-phenolic portion of the volatile oil.
o On separation, it is transferred to a cassia flask and the oily layer brought to mark by addition of
more alkali.
o In such assays, the mixture of the alkali should be heated in order to saponify the acetate ester of
the phenolic constituent which could add to the oily layer.
o Dilute solution of the alkali is employed to avoid dissolving other oxygenated non-phenolic
constituents.
o The volume of oily layer is subtracted from the amount of volatile oil employed.
o Sample Calculation:
Problem: A 10 mL sample of clove oil was assayed for its eugenol content and gave an oily
residual layer amounting to 1.4 mL. Calculate for the percent of eugenol in the sample.
Solution:
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑢𝑔𝑒𝑛𝑜𝑙 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑜𝑖𝑙 − 𝑣𝑜𝑙𝑢𝑚𝑒 𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑜𝑖𝑙𝑦 𝑙𝑎𝑦𝑒𝑟
= 10 𝑚𝐿 − 1.40 𝑚𝐿 = 8.60 𝑚𝐿
𝑣𝑜𝑙𝑢𝑚𝑒 𝑒𝑢𝑔𝑒𝑛𝑜𝑙 8.60 𝑚𝐿
% 𝑒𝑢𝑔𝑒𝑛𝑜𝑙 = 𝑥 100 = 𝑥 100 = 𝟖𝟔%
𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑜𝑖𝑙 10 𝑚𝐿
Study Guide Question
8. Why phenol-containing volatile oils are assayed by shaking with NaOH or KOH?

MODEL 8: DETERMINATION OF VOLATILE OIL CONTENT OF CRUDE DRUGS AND OLEORESINS

o The method prescribed for these determinations consist of separation of the oil from the other
components of the drug or oleoresin by means of steam distillation and the accurate measurement
of the volume of oil obtained.
o Carried out in a distillation apparatus designed especially for the purpose.
o Do not boil the contents of the flask too rapidly during distillation, as this causes vapors to escape
around the condenser and results in a loss of volatile oil.
o If the oil in the sample is lighter than water, it is measured directly in the receiver.
o If the oil is heavier than water, the oil is transferred to a graduated cylinder after distillation is
complete, and the water with any remaining oil is drawn off into a small separator.
 Prepared by: ENGR. RENA P. MORA, LPT 6
Assay of Volatile Oils

o The volume of oil represents the amount obtained from 100 g of drug.

MODEL 9: ASSAY FOR VOLATILE OIL IN SPIRITS

o The volatile oil is separated by use of a small amount of immiscible solvent such as kerosene.
o The method is carried out by using a Babcock bottle and using only a small amount of the spirit.
o A dehydrating agent such as CaCl2 solution is employed to completely separate the oil from the
spirit.
o HCl is added to neutralize alkali that may render some constituents of the volatile oil water-soluble.
o Both HCl and CaCl2 solution is used to bring the layer of the immiscible solvent with the oil to the
mark.
o The bottle is centrifuged and the volume of the immiscible solvent with the oil to the mark.
o The bottle is centrifuged and the volume of the immiscible solvent employed is deducted from the
volume of the layer that has separated and taken as the amount of the volatile oil.
o Each division of the Babcock bottle is equivalent to 0.20 mL.
o Sample Calculation:
Problem: A 5 mL sample of peppermint spirit was assayed for its peppermint oil content
by extraction with 1 mL kerosene and treatment with acidified CaCl2 solution. After
centrifugal separation, the kerosene layer was read and was found to be equal to 7.4
divisions. What is the percent v/v of peppermint oil in the sample?
Note: The volatile oil-kerosene layer is a little miscible with water hence a correction
factor is employed.
Solution:
➢ If 7.4 divisions represent the length of the column of oil-kerosene in the
graduated neck of the flask, the oil measures 7.4 – 5.0 = 2.4 divisions. Since, 1
mL kerosene is added and 1 mL = 5 divisions (1 mL x 1 division/0.2 mL). 5 divisions
were subtracted from 7.4 divisions.
➢ mL of oil = 2.4 divisions x 0.2 mL/1 division = 0.48 mL of oil
➢ Since a 5-mL sample is employed, 100 mL of the spirit yielded 0.48 mL x 20 = 9.60
mL of oil. 20 = 100 mL/5 mL
➢ The procedure is subject to slight error, which has been experimentally
determined. Consequently, the volume of oil in scale divisions is multiplied by
4.2. Thus, the volume of peppermint oil in 100 mL of the spirit is equal to 2.4 x
4.2 = 10.08 mL of oil in 100 mL of the spirit or simply 10.08%.

Study Guide Question

9. Why the saturated solution of calcium chloride should be acidified with hydrochloric acid?
10. Compare and contrast a cassia flask and Babcock bottle.