SPECIAL METHODS

USED IN
PHARMACEUTICAL
ANALYSES
 FINAL COVERAGE
1.Analysis of Crude Drugs
2.Ash /Water content determination
3.Extractive Content
4.Constant, Fats and fixed oils tests
5.Assay of Volatile oils
6.Assay of Alkaloids
Course Objective:
• Demonstrate competence in analysis of
natural products and crude drugs using
gravimetric and miscellaneous methods.
 Volatile Oils
- The odorous principles found in various
plant parts.
- They evaporate when exposed to air at
ordinary temperature, hence, are called
volatile oils, ethereal oils, or essential
oils (represent “essences” or the
odoriferous constituents of the plants.)
- Colorless when fresh, but on long
standing they may oxidize and may
resinify, thus darkening in color.
Characteristics of volatile oils:
- They posses characteristic odors
- Characterized by high refractive indices
- Optically active
- Their specific rotation is a valuable
diagnostic property
- Volatile oils are immiscible with water,
but they are sufficiently soluble to impart
their odor to water, aromatic water are
dependent on this slight solubility
- Volatile oils are soluble in ether, alcohol
and most organic solvents
Divisions in w/c volatile oils and volatile-
containing drugs are placed:
1.Hydrocarbons – turpentine oil, rectified
turpentine oil, terpene hydrate or terpinol
2. Alcohols – peppermint oil, menthol
3. Aldehydes – cinnamon, lemon peel/oil
4. Ketones – camphor, spearmint
5. Phenols – thymol, clove
6. Phenolic ethers – nutmeg oil or myristica oil
7. Oxides – eucalyptus oil
8. Esters – gaultheria oil
Analysis of Volatile Oils
Physical Characteristics of VO:
•Odor
•Appearance
•Color
•Consistency
•pH
•solubility
 Important Properties of Volatile
Oils are analyzed by the ff:

• Specific Gravity
• Rotatory power
• Refractive index
• Solidifying point (Congealing point)
• Behavior on distillation (distillation
range or limit)
• Assay for ester content
 Specific gravity
• SG = weight of the volume of the oil
weight of equal volume of water

**specific gravities of official oils is

between 0.84 to 1.2.
*may be influenced by the maturity of the
plant from which the oils is obtained and
the age of the oil, as well as the methods
of preparation and purification.
• Oils lighter than water:
orange, caraway, coriander, lemon,
turpentine, and rosemary oils (usually rich in
hydrocarbons, alcohols, ester, and ketones.

• Oils heavier than water:

anise, cinnamon, clove, sassafras oil
(usually contain aldehydes, phenols,
phenolic derivatives, and certain esters.
 Rotatory Power:
• Measured using Laurent half-shadow
polarimeter.
• Serves as a valuable means of
detecting adulteration with inactive
substances such as alcohol, or with
substances of rotatory power different
from the oil being examined.
• Optically active
 Refractive Index:
• Measured by means of
Abbe Refractometer.
• Refractive index
• Serve in the detection of
extraneous matter.
 Congealing Temperature or
Point:
• congealing temperature is that point at
which there exists a mixture of the liquid
(fused) phase of a substance and a
small but increasing proportion of the
solid phase. It is distinct from the
freezing point which is the temperature
at which the liquid and solid phases of a
substance are in equilibrium.
CONGEALING POINT
•The congealing point offers a distinct advantage
over the melting point and the titer, in the case of
mixtures, such as essential oils. In determining
the congealing point, the oil is supercooled so
that, upon congelation, immediate crystallization
with liberation of heat occurs. This results in a
rapid rise of temperature, which soon approaches
a constant value and remains at this temperature
for a period of time.
CONGEALING POINT
•This point is known as the "congealing
point." With increasing percentage of
crystalline material in an oil, the congealing
point will approach a maximum.40 Hence,
this physical property is a good criterion of
the percentage of such material. The
congealing point is important in the
evaluation of anise, sassafras and fennel
oils.
CONGEALING POINT
•Procedure:

1.Place about 10 cc. of the oil in a dry test tube of 18 to

20 nun. diameter.
2.Cool in water or in a suitable freezing mixture, the
temperature of which should be about 5 lower than the
supposed congealing point of the liquid.
3.To initiate congelation, rub the inner walls of the tube
with a thermometer, or add a small amount of the
substance previously solidified by excessive freezing.
4.The thermometer should be rubbed quickly up and
down in the mixture in order to cause a rapid
congelation throughout, with its subsequent liberation of
heat.
CONGEALING POINT

•Procedure:

5. The temperature should be read frequently; at first

the rise of temperature is rapid, but soon approaches a
constant value for a brief interval of time. This value is
taken as the congealing point of the oil.

•The process described above should be repeated

several times to assure obtaining the true congealing
point.
Distillation Range or
Limits
• Methods of obtaining volatile oils:

1.Obtained by distillation of the

plant parts containing the oil.
Types of distillation:
a.Water distillation – applied to plant
material that is dried and not subject
to injury by boiling.
e.g. turpentine oil
b. Water and steam distillation
– employed for either dried or
fresh substances that may be
injured by boiling.
e.g. cinnamon oil, clove oil

c. Direct steam distillation –

applicable to fresh plant drugs.
e.g. peppermint, spearmint
*Destructive distillation – means of
obtaining “empyreumatic oils”
e.g. wood or resin members of Pinaceae
or Cupressaceous is heated w/o access
to air, decomposition takes place and
volatile compounds are driven off. The
resultant mass is charcoal.
2 layers:
1.Aqueous layer containing wood naphtha
(methyl alcohol)
2.Pyroligneous acid (crude acetic)
• Medicinal and commercial
uses of volatile oils
- Used for flavoring purposes
- Possesses carminative action
- Analgesic (wintergreen oil)
- Used in perfumery
- For masking or providing odor
to household cleaners,
polishes and insecticides
 Solubility
• Volatile oils are generally soluble in
organic solvents such as absolute
alcohol, ether, CHCl3, benzene, CS2,
etc.
• VO are soluble in 90, 80, or 70% alcohol.
• the test of solubility of VO in dilute
alcohol gives valuable data relative to its
purity since the commonly used
adulterants such as turpentine oil,
petroleum oils and fatty oils are slightly
soluble in 80 or 70% alcohol.
Assay of VO for ester content
• Ester content of VO are refluxed w/
alcoholic KOH, they are saponified
and yield free alcohol and the
potassium salt corresponding to the
acid component of the ester.
• Determination of total ester serves to
detect adulteration and to establish
the quality of oils valued for their ester
content.
 Sample problem:
• Calculate the percent menthyl
salicylate if a 9.120g sample of
peppermint oil, refluxed w/ 25mL of
about 0.5N alcoholic KOH, required
21.52mL of 0.4900N HCl for bthe
residual titration. The blank was run
using the same volume of 0.5N
alcoholic KOH and required 26mL of
0.4900N HCl to bring about the end
point.
• Solution:
1.Determine the number of meq of KOH
required to saponify the esters in the
sample:
26mL x 0.4900N = 12.740meq HCL
21.52mL x 0.4900 = 10.545 meq HCl
12.740meq - 10.545meq = 2.195meq of
KOH
*the no of meq of KOH required to saponify
the total esters calculated as menthyl
acetate.
2) Multiple 2.195 meq of KOH consumed
by 0.1983, the weight in grams of 1meq of
menthyl acetate.
2.195 meq x 0.1983 g/.eq = 0.4353g MA

3) Divide the weight of menthyl acetate by

the weight of the sample and multiply by
100 to obtain the percentage of total
esters:
0.4353g menthyl acetate x 100
9.120g sample
= 4.77% w/w
4) Combine all mathematical operations
(steps 1,2, and 3) and factoring:

(26mL - 21.52mL) x 0.4900N x 0.19831g/meq x

9.120g
100

= 4.77%w/w
 Assay of Alkaloids
• Are nitrogenous crystalline or oily
compounds, usually basic in character:
atropine, morphine, quinine, cocaine,
reserpine
• Xanthine alkaloid = found in coffee
• Free alkaloids = insol in water
soluble in organic solvents
*Alkaloidal salts = soluble in water
insol in org. solvents
 Assay of Alkaloids

• Alkaloids are a group of nitrogen-

containing bases. But few of them
are derived from purines or
pyrimidines, while the majority of
them are produced from amino acids.
 Tests for alkaloids

• Precipitation tests with reagents

1. Mayer’s- mercuric potassium iodide (MAMEKI)
most sensitive test for alkaloids
2. Hager’s –saturated solution of picric acid /
trinitrophenol test solution
3. Wagner’s- iodine in potassium iodide (WIKI)
4. Dragendorff ’s- solution of bismuth potassium iodide
(BIKI)
5. Valser’s – HgI2 (VMI)
6. Marme’s cadmium potassium iodide (MarCadKI)
7. Sonnenchein’s – phosphomolybdic acid (PMA)
8. Scheibler’s – phosphotungstic acid (PTA)
9. Gold compound
10.Tannic acid
Structure and Properties of Caffeine
Procedure:
1. Accurately weigh 15 g of coffee powder in a tared
beaker
2. Dissolve it in 100 mL of hot distilled water
3. Add 10 mL of diluted sodium hydroxide solution
4. Cool and transfer the content in a separatory funnel
5.Add three (3) successive portions, one at a time of 15
mL each of chloroform
6. For every extraction, shake the separator for about 5
minutes
7. Filter the combined chloroform extracts through a
small filter paper into an Erlenmeyer flask
8. Wash the stem of the separator, the filter paper and
the funnel with 30 mL of chloroform, adding the washing
to the flask
Procedure:

9. Evaporate the combined chloroform extracts

on a water bath
10. Add 20 mL of standard sulfuric acid and mix
with #9
11. Add three (3) drops of methyl red TS
12. Titrate the excess acid with standard sodium
hydroxide solution to a yellow color endpoint
13. Calculate the % caffeine content
 Formula to compute for % Caffeine content:

% Caffeine = (NxmL)H2SO4 – (NxmL)NaOH x titer Caffeine x 100

wt sample

*Each mL of 0.02 N H2SO4 is equivalent to 3.8858 mg of

caffeine (C9H10O2).

*Please refer to experiment no. 18 for the assay

procedure
 Reference:
• Guevarra, Beatrice Q. ed., A Guidebook to
Plant Screening: Phytochemical and
Biological. Research Center for the Natural
and Applied Sciences, UST, Manila, 2004.
• Knevel, A.M. and Digangi, F.E., Jenkin’s
Quantitative Pharmaceutical Chemistry.
USA: Mc Graw Hill. 1977.
• Handbook of Modern Pharmaceutical
Analysis (2011) Amsterdam; Boston:
Academic Press/Elsevier.
• Tyler, V.E., L.R. Brady and J.E. Roberts,
Pharmacognosy 9th ed. Lea and
Febiger, Philadephia, 1988.