Faculty of pharmacy

Delta University for Science and Technology

Practical
Phytochemistry I (PG 503, Pharm D)

Laboratory Manual
1
 Contents
Item # Page#

Carbohydrates ................................................... 3
1. Qualitative Tests of Monosaccharides ................................. 8
2. Qualitative Tests of Disaccharides ........................................ 13
3. Qualitative Tests of Polysaccarides ..................................... 18
4. Estimation of reducing: sugars by the copper reduction
method .................................................................................33
5. Iodometric estimation of Aldoses ......................................... 34
6. Estimation of Non-reducing sugars ....................................... 35
7. Estimation of a mixture of glucose, fructose and sucrose .... 37
8. Estimation of a mixture of glucose and maltose ..................... 37
9. Estimation of Starch ..............................................................39
10. Colorimetric Estimation of sugars by reaction with ortho
toluidine ............................................................................ 40

Volatile oils ........................................................... 42

11. Physical methods of examination of volatile oils… ................ 42
12. Assay of bitter almond oil by hydroxylamine method………46
13. Assay of Caraway oil by the sulfite method ........................ 50
14.Separation of a mixture of menthol and thymol
And test for their identification ................................................ 53
15. Assay of clove oil ........................................................................ 55
16. Assay of chenopodium oil ......................................................... 58
17. Assay of eucalyptus oil .............................................................. 62

2
 Carbohydrates
The name carbohydrates which means hydrated carbon, implies that carbohydrates consist of

carbon, hydrogen and oxygen, with the latter two elements being in the same proportion as in water.

So, almost all carbohydrates comply with the formula [Cn (H2O)y]. Carbohydrates are among the first

products to arise as a result of the process of photosynthesis.

I) General physical characters:
Sugars: are crystalline, sweet and soluble in water. Polysaccharides: are amorphous, tasteless,
and insoluble in water.

II) General chemical properties:

- All monosaccharides exhibit reducing properties (being either aldehydes and or α-OH-ketones);
and all other classes of carbohydrates would yield reducing monosaccharides upon hydrolysis.
- Being generally represented by the formula Cn(H2O)y; they can be easily dehydrated.

. Partial dehydration would yield furfural derivatives (responsible for many qualitative and

quantitative color tests).

. Complete dehydration would yield free carbon (charring reaction with H2SO4).

III) General qualitative chemical tests:

1- Molisch's test:

This is a specific general test for all carbohydrates (whether sugars or polysaccharides) as

well as glycosides, in a test tube take 2 ml of the aqueous solution of carbohydrates, add 2 drops of

10% alcoholic α-naphthol, mix, then run carefully (on the walls of the test tube) about 1 ml of conc.

H2SO4, so as to form a layer below the aqueous sugar solution; a violet ring appears at the junction

of the two layers. Upon dilution with water, a bluish violet precipitate is given.

Notes :
. If the material is H2O insoluble, few mg of the material are boiled with few ml of dil. H2SO4 (to
affect hydrolysis); then few ml of the resulting solution is used.
. Conc. H2SO4 in Molisch's test acts, both as a hydrolyzing (for oligo-and polysaccharide) and as
a dehydrating agent. The latter effect would transform the monosaccharide to furfural derivatives),
which would in turn give a violet condensation product with α-naphthol.

3
4
 2- Reduction tests (depending on the reducing action of sugars)

i) Fehling's Test:
Boil few m1 of an aqueous solution of the sample with equal volumes of Fehling's A & B, a red
ppt. of cuprous oxide is formed with reducing sugars.

Notes :

. Any change in color from blue to green, yellow or red indicates a positive reduction.

. The test is given with mono. or oligo-saccharides containing a free reducing group (aldehydic

or ketonic), It is also given by the hydrolytic products of all other carbohydrates (winch yield

on hydrolysis reducing sugars)

. Fehling's solution consists of two separate solutions.

Fehling A: consists of 69.28 g of pure crystalline CuSO4 in 1 L of H2O.

Fehling B: consists of 350 g of sodium potassium tartarate (Roschelle's salt), 100 g of NaOH in 1 L
of H2O.

ii) Reduction of ammoniacal silver nitrate:

5
To few ml of silver nitrate add few ml of dilute ammonia and few ml of the carbohydrate solution,
and heat for few minutes in a water bath. The formation of a black precipitate of metallic silver, or
silver mirror on the walls of the test tube, indicates a reducing sugar.

iii) Reduction of Barfoed's solution:

To about 2 ml of sugar solution add an equal volume of Barfoed's reagent, and heat in a
boiling water bath for only 5 minutes. The appearance of a red ppt. of cuprous oxide indicates

reduction and the presence of reducing monosaccharides.

Notes:

. This test distinguishes monosaccharides from reducing disaccharides the test is given by

monosaccharides only, but not by disaccharides, within the indicated time (5 minutes).

. Barfoed's solution is a solution of 5% cupric acetate in acetic acid.

3- Special Identity Test:

- Osazone test (phenylhydrazine test)

In a test tube mix 0.2 g of the sugar with 0.5 g of phenyl

hydrazine hydrochloride and 1 g of crystalline sodium acetate and 5 rnI of water. Dissolve by

shaking and filter the solution if not clear. Heat in a boiling water bath for 45-60 minutes and allow

to cool at room temperature (observe the time after which the yellow crystalline osazone is formed).

Then examine the yellow crystals of the given osazone under the microscope.

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Notes:
 The osazone test is given only by sugars having, alpha hydroxyl aldehyde or alpha hydroxy
ketone functions.
 Monosaccharides give crystalline osazones on hot after about 15 minutes of heating.
However, osazones of reducing disaccharides are formed after heating for about 45 minutes and
crystals appear after cooling.

 Glucose, fructose and mannose give the same osazone (The same chemical structure &
form (needles or raphides).
 Maltosazone ................................................................. plates (rosettes).
 Lactosazone ................................................................. clusters of needles (tufts).
 Arabinosazone ............................................................. crystals of indefinite shape.

7
 Laboratory # 1

Qualitative tests of monosaccharides

Test for pentoses: (e.g. xylose and arabinose).

Physical characters:
White crystalline sweet powder soluble in water and insoluble in alcohol.

Tests for identity:

- Molish's test: positive reaction.

- Reduction of Fehling's solution: positive reaction.

- Reduction of Barfoed's solution: positive reaction.

- Osazone test: The crystals are formed on hot within 15 minutes (crystals of indefinite shape).

Special Tests :

- Bial's test: Heat a small amount of the pentose sugar or a pentosan (e.g. gum arabic) with 2

ml of Bial's reagent, a green color is produced.

(Bial's reagent is a solution of orcinol in conc. HCI to which traces of ferric chloride is added).

- Furfural reaction:
In a test tube boil a small quantity of the pentose or pentosan with few ml of 12% solution of
HCI. Cover the mouth of the tube with a piece of filter paper soaked with aniline acetate solution.
The liberated furfural will produce a brilliant red stain on the filter paper.

Notes:

Methyl pentoses (e.g. rhamnose), produce a yellow stain due to methyl furfural.

- Phloroglucin Test:
Heat few mg of the pentose or pentosan in test tube with 0.1 g phloroglucin and 1 ml HCI,
a red color that changes gradually to brown-violet is given.

8
 Tests for Hexoses:

1- Glucose
Physical characters:

White crystalline sweet powder, soluble in water, and insoluble in alcohol.

Tests for identity:

- Molisch' s test: positive reaction.

- Reduction of Fehling's solution: positive reaction.

- Reduction of Barfoed's solution: positive reaction.

- Osazone test:
The crystals are formed on hot within 15 minutes in the form of raphides (needles).
Special test:
To about 2 mI glucose solution in distilled water add few drops of lead subacetate and one
drop of conc. ammonia, a faint ppt. is formed that changes into pink color by heating.

2- Fructose

Physical characters:

White, crystaIline, sweet, soluble in water and insoluble in alcohol.

Test for identity:

- Molisch' s test: positive reaction.

- Reduction of Fehling's solution: positive reaction.

- Reduction of Barfoed's solution: positive reaction.

- Osazone test: The same as glucose.

Special test:

- Resorcinol test:
Boil 0.2 g sugar with few crystals of resorcin and 2 ml 12% HCI a deep red color is formed
within few seconds that changes into dark brown ppt. on cooling.

9
-Cobalt Nitrate Test:
. To about 2 ml of sugar solution add 3 drops of cobalt nitrate and excess of NaOH, a violet color is
formed that gradually changes into blue.

10
 Laboratory # 1
Work sheet # 1
Identify the glucose sample you are provided with and write down full identification scheme

11
 Laboratory # 1
Work sheet # 2
Identify the fructose sample you are provided with and write down full identification scheme

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 Laboratory # 2
Qualitative tests of disaccharides

(A) Non reducing disaccharides:

Sucrose

Physical characters:
White, crystalline, sweet taste, soluble in water and insoluble in alcohol.
Tests for identity:
- Molisch's test: positive reaction.
- Reduction of Fehling's solution: negative reaction (gives positive test after acid hydrolysis).

Special tests:

- Cobalt nitrate test:

Add 3 drops of cobalt nitrate to about 2 mI of sugar solution then add excess NaOH a permenant

violet color is formed.

- Resorcinol test: As under fructose.

(B) Reducing disaccharides:

1- Lactose
Physical characters:

White, crystalline, slightly sweet, soluble in water and insoluble in alcohol.

Tests for identity:

- Molisch' s test: positive reaction.

- Reduction of Fehling's solution: positive reaction.

- Reduction of Barfoed's solution: negative reaction.

.
- Osazone test: gives characteristic crystals in the form of tufts of needles.

2- Maltose

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Physical characters:

White, crystalline, slightly sweet, soluble in water and insoluble in alcohol.

Tests for identity:

- Molisch' s test: positive reaction.

- Reduction of Fehling's solution: positive reaction.

- Reduction of Barfoed's solution: negative reaction.

.
- Osazone test: gives characteristic crystals in the form radiating plates or broad needles.

- Reduction of Barfoed's solution: positive reaction..

- Osazone test: it gives crystals of osazone in the form of radiating plates or broad needles.

14
 Laboratory # 2
Work sheet # 1
Identify the sucrose sample you are provided with and write down full identification scheme

15
 Laboratory # 2
Work sheet # 2
Identify the lactose sample you are provided with and write down full identification scheme

16
 Laboratory # 2
Work sheet # 3
Identify the maltose sample you are provided with and write down full identification scheme

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 Qualitative tests of polysaccharides
A) Holosaccharides:

1- Starch

Physical characters:
Starch occurs in a fine white powder, it is odorless, taste

less, insoluble in water and alcohol.

Tests of identity:

- Molisch's test: positive reaction.

- Reduction of Fehling's solution: negative reaction (positive after acid hydrolysis).

- Boil small amount of starch with 15 times its weight of water and cool. A translucent viscous
fluid, jelly or past is formed which is colored blue upon addition of solution of iodine.
-The blue color disappears on warming and reappears on cooling.
- Add 2 mI of strong alcohol to a few ml of the starch paste solution, the starch is precipitated.
-Add few mI of basic lead acetate to another portion of the starch solution, the starch is

precipitated.

- Boil on a water-bath a little starch paste solution with few drops of 25% sulfuric acid. From time
to time take a little of the solution, cool and test with iodine solution, for partial and finally for
complete hydrolysis.

First  blue color

After 10 min  red color (dextrin, i.e. partial hydrolysis).

After 30 min  no color (dextrose, i.e. complete hydrolysis)

2- Dextrins (Artificial gum)

Physical characters:

White or yellowish white, amorphous, starchy taste, insoluble in cold, soluble in hot water,

forming a gummy solution.

Tests for identity:

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- Molisch's test: positive reaction.

- Reduction of Fehling's solution: negative reaction.

- Add few drops of iodine solution to about 2 mI of dextrin solution, a red or violet color is formed.

- Add 1 mI of lead acetate solution to about 2 mI of dextrin solution, no ppt. is formed.

- Mix 2 mI of dextrin solution with 1 mI of alcohol, a white ppt. is formed.

3- Inulin
Physical characters:

White, amorphous, slightly soluble in cold water, more soluble in hot water, and insoluble in alcohol.
Tests for identity:
- Molisch's test: positive reaction.

-Reduction of Fehling's solution: negative reaction before hydrolysis, positive after acid

hydrolysis.

- Add I ml of lead acetate solution to about 2 ml of inulin solution, no ppt. is formed.

- Mix few drops of iodine solution with about 2 ml of inulin solution, a brown color is formed.
- Add 1 ml of alcohol to about 2 ml of inulin solution, a white ppt. is formed.
-Resorcin test: Boil 0.2 g inulin powder with few crystals of resorcin and few ml of HCI, a deep red
color is formed which changes into dark brown and finally gives a red ppt.

B- Heterosaccharides

1- Gum arabic (Gum acacia)

Physical characters:
Gum arabic occurs in rounded or ovoid tears of variable size, nearly colorless or pale amber,
opaque due to the presence of numerous minute fissures, brittle, almost, odorless with
mucilaginous taste, soluble in water and insoluble in alcohol.
Tests for identity:
- Molisch' s test: positive reaction.

- Reduction of Fehling's solution: negative reaction (positive after acid hydrolysis).

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 - Shake about 10 g of gum arabic with 50 mI of water. On the resulting solution, carry out the

following tests:

a- In a test tube add 5 drops of lead sub acetate solution to about 3 mI of gum arabic solution, a

flocculent white ppt. is formed.

b- In a test tube mix 3 mI of gum arabic solution with 5 drops of lead acetate solution, a faint

turbidity is formed.

- Mix about 0.1 g powdered gum arabic with few drops of N/50 iodine solution, a yellowish-brown

color is formed (not blue or red color).

- Test for oxidase enzyme:

- To few mI conc. aqueous solution of gum arabic add, few drops of hydrogen peroxide and few

drops of I % solution of benzidine in alcohol, shake and allow to stand for 2 min, a deep blue or

greenish-blue color is formed.

Notes:

* Tincture guaiacum can be used instead of the benzidine solution.

* If the gum is bleached or heated, it fails to give positive results.

2- Gum tragacanth
Physical characters:
It occurs as translucent strips, or creamy powder with mucilaginous taste, insoluble in cold,
but soluble in hot water.

Tests for identity:

- Molisch' s test: positive reaction.
- Reduction of Fehling's solution: negative reaction (positive after hydrolysis).
- Prepare 10% solution of gum tragacanth in distilled water. On this solution carry out the following
tests:-

- To about 5 mI of gum tragacanth solution, add 5 drops of lead acetate solution, a heavy white ppt.

is formed.

20
- To about 5 mI of the gum tragacanth solution add 5 drops of lead subacetate solution, a faint
turbidity is formed.

- Mix a small quantity of powdered gum tragacanth with few drops of N/50 iodine solution, the
particles acquire an olive green color.

3- Agar
Physical characters:

It occurs as translucent strips or creamy powder with mucilaginous taste, insoluble in cold

but soluble in hot water.

Tests for identity:

- Molisch's test: positive reaction.

- Reduction of Fehling's solution: negative reaction.

- Boil 1 g of powdered agar for 10 minutes with 100 mI of water, the solution yields a stiff jelly on
cooling.

- Dissolve 0.1 g of agar by boiling for 5 minutes with 10 mI of water; add 1 mI of HCI and heat for 30

minutes on a boiling water bath, then add 1 mI of BaCl2 solution, a white ppt is

formed. This ppt. is due to the sulfate ions present in agar.

- Add few drops of N/50 iodine solution to a little of powdered agar, the particles are colored red.

21
 Scheme for Identification of carbohydrates

I - Physical characters:
1- State: solid
2- Color: white or creamy
3- Odor: odorless
4-Taste: sweet (sugars)

tasteless (polysaccharides)

5- Solubility in cold water:

soluble (sugars)

insoluble (polysaccharides)

II- Chemical Characters:

1- Molisch's test:

It gives positive reaction (violet ring between the two layers) with all carbohydrates and

carbohydrates-containing compounds, and negative with non carbohydrate compounds.

2- Reduction of Fehling's Solution:

a. If the compound gives positive reaction (red ppt.), it may be monosaccharide or reducing
disaccharide.

b- If the compound gives negative reaction (no red ppt.), it may be a non reducing disaccharide

or a polysaccharide.

(A) In case of positive reduction of Fehling's solution

Carry out the following tests:-

3- Barfoed's test:
a. A positive reduction of Barfoed's reagent within 3 minutes, indicates a monosaccharide
(pentoses or hexoses).

b- A negative reduction of Barfoed's reagent within 3 minutes, indicates a reducing disaccharide.

4- Resorcinol test:

The appearance of red color within 20 seconds of heating, indicates the presence of fructose,

22
 or oligo or polysaccharides with fructose units.

5- Bial's test:

The positive reaction (green color), indicates the presence of pentoses.

Notes:

Carry out special tests for each carbohydrate suspected.

6- Osazone reaction:

- If the compound gives needles (raphides) during boiling in the W.B. it may be glucose or fructose

or mannose.

- Clusters of needles (tufts) it may be lactose.

- Plates (rosettes) it may be maltose.

- Crystals of indefinite shape it may be arabinose.

(B) If Fehling's test from the beginning is negative.

-The compound may be non reducing disaccharide (sucrose) or polysaccharide (starch
dextrin, gums or agar).
-Carry out the following tests:-

7- Resorcinol test:
The appearance of red color within 20 seconds of heating, indicates the presence of sucrose or
inulin (sucrose is water soluble, while inulin is water insoluble).

- If the resorcinol test gives negative results, the compound may be polysaccharides (starch,
dextrin, gums or agar).
*Differentiation between them is carried out by solubility in hot water (dextrin is soluble) and by
the color produced with iodine solution.

8- Test with N/50 iodine solution:

Starch gives… ........................... blue color
Dextrin gives….............................reddish violet color
23
Gum tragacanth gives… ............olive green color
Agar gives… .............................. crimson color
Gum Arabic gives… .................. no change in color
Note:

For further differentiation between them, carry out special test for each carbohydrate suspected

as follows:

* Gum acacia…............... oxidaze enzyme and lead subacetate test.

* Gum tragacanth ........... lead acetate test.

* Agar… ............................ test for SO4 ion after hydrolysis.

24
 Laboratory # 3
Work sheet # 1
Identify the starch sample you are provided with and write down full identification scheme

25
 Laboratory # 3
Work sheet # 2
Identify the dextrin sample you are provided with and write down full identification scheme

26
 Laboratory # 3
Work sheet # 3
Identify the inulin sample you are provided with and write down full identification scheme

27
 Laboratory # 3
Work sheet # 4
Identify the gum acacia sample you are provided with and write down full identification

scheme

28
 Laboratory # 3
Work sheet # 5
Identify the gum trgacanth sample you are provided with and write down full identification

scheme

29
 Laboratory # 3
Work sheet # 6
Identify the agar sample you are provided with and write down full identification scheme

30
 Laboratory # 4
General revision on carbohydrate unknown
1. Identify the unknown carbohydrate sample you are provided with and write down full

identification scheme in separate sheet of paper.

2. Discuss the scheme and method of identification with your instructor to manage any

problem you are encountering in identifying the unknown.

31
 Laboratory # 5
Carbohydrate Unknown Exam
Identify the unknown carbohydrate sample you are provided with and write down full

identification scheme in the specified examination sheet.

32
 Quantitative analysis of carbohydrates

Estimation of sugars:
The copper reduction method and the iodometric method are the most widely used chemical
methods for the estimation of sugars.

Estimation of reducing sugars by the copper reduction method

This method could be applied for all reducing sugars e.g. all monosaccharides (glucose,

fructose, galactose mannose. etc.) and the reducing disaccharides (e.g. lactose and maltose, etc.).

Principle:
This method is based on the reduction of a known volume of standardized Fehling's solution
by titrating it directly against the reducing sugar solution. Such sugars are capable of reducing

copper sulfate in hot alkaline medium to cuprous oxide which forms a red precipitate and the blue

color of Fehling's solution will disappear.

Procedure:

1- Measure accurately 10 ml of freshly prepared standard Fehling's solution in a porcelain dish by

means of a pipette or a burette. Add 40 ml of distilled water and boil very gently over a small flame.
2- Fill the burette with the sugar solution and titrate the boiling Fehling's solution (without removing
the flame).
3- Continue addition of the sugar solution (2 ml at a time) until the blue color of Fehling's solution
disappears. The ppt. of cuprous oxide is allowed to settle before the next addition of the sugar
solution.
4- Add 4 drops of methylene blue indicator to the mixture and add the sugar solution in drops. The
end point is reached when the color of the indicator disappears. Measure the number of mI of the
sugar solution consumed in this titration; considering it as a preliminary or an approximate end
point.
5- Carry out a second titration by adding the volume of the sugar solution used in the first titration
(-1 mI) at once to the boiling Fehling's solution. Allow the solution to boil and continue titration

33
 without removing the flame, until the end point is reached (the color of methylene blue indicator
disappears).

This exact titration should be completed in a total boiling time of 3 minutes (to avoid re-

oxidation of Fehling's solution by air).

6- Repeat the process until two successive readings do not differ by more than 0.2 ml. Take the
average of the last two readings.
7- Calculate the percentage of reducing sugars as follows:

% of reducing sugars = [0.05 X 100] ÷ [ml of sugar solution consumed - 0.2]

Notes:
1- Continuous boiling of solution during titration is important to expel atmospheric oxygen and to
prevent reoxidation of cuprous oxide.
2- 10 ml standadized Fehling's solution is reduced by 0.05 g of monosaccharides.
3- 0.2 m1 are subtracted from the volume of sugar solution taken (end point) to correct for the
amount consumed for reducing the indicator (4 drops of methylene blue).

Iodometric estimation of aldoses

Principle:
The iodometric method of assay of aldoses is based on the fact that iodine can oxidize aldoses in
weak alkaline medium, whereas it has no effect on ketoses.

Procedure:

1- Measure accurately 25 mI of the sugar solution in a stoppered flask, add a considerable known

excess of 0.1 N iodine solution (20 mI by pipette) and 5 mI of normal sodium carbonate solution or

40 mI of 0.1 N KOH. Allow to stand in a dark place for 20 minutes.

2- Acidify with 7 mI of 2 N H2SO4 and back titrate immediately the excess iodine with 0.1 N
Na2S2O3 using starch as indicator. The starch indicator should be added only when the solution

becomes faint yellow in color.

34
3- Carry out a blank experiment using 25 mI of water instead of the sugar solution.
4- Calculate the percentage of aldose in the solution. The difference between the volume of
thiosulfate solution taken in the experiment and that of the blank, represents the volume of
thiosulfate equivalent to the excess iodine.

% of aldose (w/v) = [(B - E) X 0.009 X 100]/ml of sugar.

1 ml 0.1 N iodine Ξ 0.009 g glucose.

(B) is the reading of blank experiment.

(E) is the reading of the experiment.

Notes on the procedure:

I2 + Na2CO3  Nal + NaOI + CO2

NaOI + C5H11O5CHO  C5H11O5COOH + 2NaI

(gluconic acid)
I2 + 2 Na2S2O3  Na2S4O6 + 2NaI.

Estimation of non-reducing sugars

Principle:
Non-reducing sugars and polysaccharides do not reduce Fehling's solution, and therefore are
not assayed as such, but:

They are first hydrolyzed (by an acid) and the liberated monosaccharides are estimated by the

copper-reduction method, as mentioned before.

Estimation of sucrose (Cane sugar)

Procedure:
1- Transfer 20 ml of the sugar solution by means of a pipette into a conical flask. Add 5 ml of 2
N HCI. Heat at 70°C on a water-bath for 15 minutes and then cool.
2- Neutralize the excess acid with sodium carbonate till effervescence ceases.

3- Transfer the neutralized solution to a volumetric flask (100 ml). Wash the conical flask several
times with distilled water. Add the washing to the volumetric flask. Complete to volume with
35
distilled water.

4- Fill the burette with the resulted inverted sugar solution, then apply the copper-reduction

method and calculate the total reducing sugars.

Note:

10 ml of Fehling's solution Ξ 0.05 g of invert sugar (glucose + fructose).

Percentage of total reducing sugars (after hydrolysis) calculated as glucose

= [0.05 X 100 X 100] ÷ [ml of sugar consumed (- 0.2) X 20] w/v

Percentage of sucrose in the original solution = % of total reducing sugars X 0.95

Explanation:

C12 H22O11 + H2O  C6H12O6 + C6H12O6

Sucrose Glucose Fructose

Inverted sugar

342 180 + 180

342 g sucrose  360 g inverted sugar

xg sucrose  I g inverted sugar

1 g inverted sugars = 342/360 = 0.95 g sucrose

i.e. Each 1 g of invert sugar originates from 0.95 g of sucrose.

36
 Estimation of a mixture of glucose, fructose and sucrose

Principle:

(I) To estimate the percentage of the total reducing sugars (glucose and fructose) apply the

aforementioned copper reduction method.

(2) To estimate the percentage of glucose (aldose) only. Apply the aforementioned iodometric

method to another portion of the mixture.

(3) Calculate the percentage of fructose (ketose) by subtracting the result obtained in (2) from

that obtained in (1).

(4) For estimation of the percentage of sucrose, apply the method mentioned before for non-

reducing sugars. In this case the result corresponds to the original glucose and fructose in
addition to the inverted sugar (resulting from the hydrolysis of sucrose). Subtracting the result
obtained in (I) from that obtained in (4) gives the invert sugar only.

(5) To find the percentage of the original sucrose, the percentage of invert sugar is multiplied

by (0.95).

Estimation of a mixture of glucose and maltose

Principle:
Each of glucose and maltose reduces Fehling's solution before hydrolysis. The reducing power
of maltose is less than that of glucose as equimolar amounts of both contain equivalent number of
reducing functions (Mol. of glucose = 180 and mol. of maltose = 342). After hydrolysis, each
molecule of maltose yields two molecules of glucose and hence its reducing power is markedly
increased. The increase in the reducing power of maltose, after hydrolysis is used to calculate its
percentage when present in combination with glucose.
So
37
-The result of titration following the copper reduction method gives the percentage of glucose and
maltose before hydrolysis, calculated as glucose (A).

- If we hydrolyze a known volume of the mixture and a second titration is made, the result obtained

will represent the amount of glucose corresponding to the glucose originally present plus the

glucose obtained from the hydrolysis of maltose (B).

The difference between (A) & (B) represents the increase in the reducing power resulting

from hydrolysis of maltose.

Procedure:

1- Carry out the copper reduction method (as mentioned before) for the original mixture of

glucose and maltose, and calculate the percentage of reducing sugars present (A).

2- To 25 mI of the sugar mixture add 25 mI of conc. HCl and reflux for 90 minutes on a boiling

water bath.

3- Cool and neutralize the excess acid with sodium carbonate till effervescence ceases.

4- Transfer the above solution to a 100 mI volumetric flask, Wash the flask several times with
water. Transfer the washings to the volumetric flask and complete the volume with water. Fill
the burette with this solution and carry out the copper reduction method (B).
Calculation:

25 ml of solution (1 vol.)  hydrolysis  100ml (4 vol.) of maltose (1 reducing unit) 

Hydrolysis  additional glucose.

Additional glucose in volume (B) = [0.05 – (B/4A x 0.05)]…….(I)

% of maltose = (I) x 100/B x 4 x 432/100...................................... (II)

% of original glucose = [0.05 – (I)] = 100/B x 4 ......................... (III)

38
 Estimation of starch

Principle:
Starch, being a polysaccharide, does not reduce Fehling's solution except after hydrolysis. It
can be determined by estimating the glucose resulting from complete hydrolysis of a known weight
of starch using the copper reduction method.

Procedure:

1- Transfer 1.5 g of starch to a conical flask. Add 90 m1 of distilled water and 10 ml of conc. HCl.

heat at 95 - 100°C under reflux condenser for 2.5 hours with frequent shaking.

2-Cool, neutralize the excess acid with sodium carbonate till effervescence ceases.

3- Transfer the solution to 250 ml volumetric flask. Wash the conical flask with distilled water and

add the washings to the volumetric flask. Complete to volume with distilled water.
4- Fill the burette with this solution, then determine the result in glucose by the copper reduction

Calculation:

Percentage of starch calculated as glucose:-

= [0.05 X 100 X 250]/[ mI of sugar soln. consumed - 0.2) X 1.5] w/w (A)

Percentage of starch = A X 0.9

Since starch is built up of a large number of glucose molecules (n), that needs (n-I) moles of

water for hydrolysis, then:

(C6H10O5), + (n - I) H2O  n (C6H12O6)

Starch Glucose

To simplify the calculation we can consider that it consumes (n) moles of water i.e.

(C6H10O5)n + n H20  n C6H12O6

162 Ξ 180
X g starch Ξ 1 g glucose
1 g glucose = 162/180 = 0.9 g starch.

39
 Colorimetric estimation of sugars by reaction with ortho-toluidine

Principle:

Various aromatic amines react with reducing sugars in hot acetic acid solution to produce

colored derivatives. Among those are aniline, benzidine, 2-aminobiphenyl, and o-toluidine. The

Latter condenses with reducing sugars to give a green colored product (schiff' s base) with an

absorption maximum at about 630 nm.

Note:
Pentoses react with o-toluidine to produce an orange color with a maximum absorption near 480
nm.

Reagents:
1- Ortho-toluidine reagent. 5% v/v glacial acetic acid containing 0.15 % thiourea.
2- Standard glucose solution, 200 mg/I 00 ml in acetic acid solution.

40
Procedure:

1- Pipette 5 mI of the sugar solution into 25 ml volumetric flask.

2- Add 5 ml of the reagent.

3- Heat on boiling W.B. for 15 min.

4- Complete to 25 mI with dist. H2O.

5- Measure the color produced at 630 nm.

Blank:

Use 5 ml dist. H2O instead of the sample and complete as above.

Calibration Curve:

Conc. (mg/I) A
112.5 mg/I 0.12
225 mg/l 0.24
450 mg/I 0.336
650 mg/l 0.404

41
 Volatile Oils

Volatile oils (also known as ethereal oils or essential oils) are generally complex products

composed of mixtures of compounds of widely variant chemical characteristics. The most

important chemical components of volatile oils are:

1. Hydrocarbons, occasionally of the acyclic series, such as heptane and myrcene, but more
often of the isocyclic series, e.g. pinene, camphene, limonene, phellandrene. etc.
2. Alcohols, present both in the free state and in combination with acids as esters.

The alcohols most generally found in volatile oils are linalol, geraniol, citronellol, terpineol,

bomeol, menthol, and santalol.

3. Aldehvdes, such as benzaldehyde, cinnarnic aldehyde, salicyl aldehyde, citral, and citronellal.

4- Ketones, the most important being camphor, carvone, fenchone, thujone, and menthone.

5- Phenols and phenolic ethers such as anethole,eugenol, carvacrol, safrol and thymol.

6- Peroxides, such as ascaridole.

7- Oxides, such as cineole.

8- Acids, sometimes are present in the free state in small quantities. Those occuring most

commonly being acetic, propionic, butyric, valeric, benzoic, cinnarnic and hydrocyanic acids.
9- Sulfur compounds, such as allyl isothiocyanate (mustard oil).

Physical methods of examination of volatile oils

The analysis of volatile oils for the purposes of determining their purity and value is based

largely on the measurement of certain physical characteristics, such as:

Color: Most oils are colorless when pure and fresh, or can be made colorless by redistillation

(rectification), upon exposure to air they acquire various colors; viz.; green; as in oil of wormwood;

yellow, as in oil of peppermint; red, as in oil of origanum; brown, as in oil of cinnamon. The blue

42
color of oil of chamomile is an inherent property of the oil even when freshly distilled, and is said to

be due to the highly unsaturated hydrocarbon chamazulene (C15H18).

Odor: The odor of volatile oils is extremely variable. It is their most characteristic feature. The odor

of many oils is sensibly modified by exposure to air.

Taste: The tastes of volatile oils are almost as variable as their odor. Some are sweet, others have

a mild, pungent, hot, acrid, caustic, or burning taste.

Specific gravity: The specific gravity of a volatile oil may be determined with the Westphal balance

or pycnometer, the latter being the official and more reliable method. Specific gravity is expressed

as "the ratio of the weight of a volume of oil to that of an equal volume of pure water when both are

determined at 25°C.

The sp. gr. of volatile oils vary approximately between 0.84 and 1.2. Those oils which are lighter

than water, such as orange, caraway, coriander, lemon, turpentine, and rosemary oils, are usually

rich in hydrocarbons, alcohols, esters, and ketones, Oils with specific gravities that approach or

exceed 1.0, e.g., anise, cinnamon, clove and sassafras oils, usually contain chiefly aldehydes,

phenols, phenolic derivatives or certain esters.

The specific gravity of any volatile oil is not absolutely constant, since it is influenced by such

factors as the maturity of the plant from which the oil is obtained and the age of the oil, as well as by

the methods of preparation and purification.

Optical rotatory power: The optical rotatory power of a volatile oil is generally measured with a

polarimeter, using sodium light and a tube 10 cm long, but for highly colored oils, tubes 5 or even

2.5 cm long may be used. The observation of the optical activities of volatile oils should be made at

25°C. Slight deviations from this temperature do not greatly affect the rotatory power of a volatile oil,

except in case of lemon oil and orange oil.

to
[α] =a/dxL
D
t°
where [α] = specific rotation at temperature t°, using sodium light.
D

43
 a = observed rotation in degrees at temperature to, using sodium light.

d = the specific gravity of the liquid at the temperature t°.

L = length of the polarimeter tube in decimeters.
The rotatory power of some of the volatile oils varies within relatively wide limits, this

determination should never be omitted in their examination, however, since it frequently

serves as a valuable mean of detecting adulteration with inactive

substances, such as alcohol, or with substances of different rotatory power nom that of the
oil being examined, e.g. The dextro-rotation of lemon oil (+ 57° to + 65.6°) is highly

affected when adulterated with turpentine oil (about + 25° to - 40°).

Refractive Index: The refractive index of a volatile oil is commonly determined by means of
an Abbe refractometer. The measurement of the refractive indices of volatile oils is required

to be performed at 20°C, except in the case of rose oil, which is measured at 30°C. The
index of refraction does not vary greatly with different official volatile oils, the values being
between about 1.46 and 1.61 at 20°C.
In some cases, however, this determination may serve for the detection of extraneous matter.
Note: The refractive index of the oil should be expressed as NtD, denotes the index of refraction
for the D line (sodium light) measured at temperature t°.
Congealing Temperature or point: Most essential oils solidify only at low temperatures.
Consequently, in practice, this determination is carried out with only a few oils, such as anise oil,
eucalyptus oil, and fennel oil, which contain large amounts of the readily crystallizable
constituents vis, anethole and eucalyptol. The higher the congealing temperature of these oils,
the more they are valued. An abnormally low congealing temperature of these oils indicates the
partial removal of the characteristic constituent for which the oil is valued or the addition of
extraneous matter, such as alcohol.
Oils with congealing temperature requirements
Oil Congealing temperature Co.
n.l.t = not lower than
Anise oil n.l.t. 15
Anethole n.l.t. 20
Eucalyptus oil n.l.t. -15.4
Eucalyptol n.I.t. O
Fennel oil n.I.t. 3
Menthol(dl) n.l.t. 30.5 - 32

44
Distillation range or limit: The distillation range of a volatile oil is determined by the general

method of distillation. Volatile oils that are composed of complex mixture of hydrocarbons,

alcohols, esters, etc., boil between certain limits of temperature, usually a wide range.

Consequently, the official standards usually designate the temperature or range of temperature at

which a definite percentage of the oil distills; e.g., not less than 90% of turpentine oil should distill

between 154°C and 170°C; not less than 95% of pine oil should distill between 200 and 225°C,

and less than 10% of dwarf pine needle oil should distill below 165°C.

Boiling or distillation range of certain oils

Oil Boiling-range (°C) Requirment %
Pine oil 200-225 n.l.t. 95
Turpentine oil 154-170 n.l.t. 90
Eugenol 250-255 n.l.t. 95
Eucalyptol 174-177 100%
Anethole 231-237 100%

Solubility: Volatile oils are generally soluble in organic solvents, such as absolute alcohol, ether,

chloroform, benzene, carbon disulphide, etc.

They dissolve more or less readily in dilute alcohols according to the nature of their components.

Oils containing a large percentage of oxygenated substances often produce turbid solutions with

light petroleum or carbon disulfide because of the separation of water, small quantities of which are

dissolved in such oils. Practically, all volatile oils exhibit almost constant solubilities in 90, 80 or

70% alcohol. Consequently, the test of the solubility of a volatile oil in a specified dilute alcohol

usually gives valuable data relative to its purity, since the commonly used adulterants (turpentine

oil, petroleum oils, and fatty oils) are less soluble in these dilute alcohols.

45
 Laboratory # 6
Assay of bitter almond oil (Hydroxylamine method)

Constituents: Benzaldehyde, n.l.t. 95% w/w.

Procedure:

1- Weigh accurately about 1 g of the volatile oil into a long tube with a stopper.

2- Add 5 m1 of benzene, 12 m1 of hydroxylamine hydrochloride solution (N/2 in 60% alcohol)

and 2 drops of methyl orange. The solution is colored red.

3- Titrate the liberated acid with N/2 alcoholic KOH, shaking frequently, till the red color changes

to yellow.
4- Continue shaking and neutralizing until the full yellow color of the indicator is permanent in the
aqueous layer, after shaking vigorously for 2 minutes and allowing the separation to take place.

5- Add 0.5-1 ml (N/2 alcoholic KOH), shake and leave aside as a color standard.

6- Carry out a second determination in exactly a similar manner using the first determination as

standard color.
Calculation: ml of N/2 KOH X 0.05305 X 1.008 X 100
Percentage of benzaldehyde (% w/w) = -----------------------------------------------------
Bitter almond oil assay wt. of oil in gm
Principle of the assay:
Tyhderocxoyrlaremcitnineghyfa
H drcotor lo1r.i0d0e8miesthnoedcessary in the calculation as the end point of the titration
ch

occBuernszaaldt eahypdH different from that of H

e + Hydroxylamine HCl
nColrm al hydroxylamine hydrochloride.
+ aldoxime

Explanation: H2NOH.HCl

Hydroxylamine hydrocEhquloivraildenet troebaenz

ctasldw
ehiytdhe b
enzaldehyde in the oil to form benzaldoxime, liberating
= N/2 KOH , M.O

equimolar amount of hydrochloric acid.

A standing period of at least 10 min is necessary to ensure completion of this reaction. A

somewhat longer period might, in fact, be desirable, if results are consistently too low. Since

benzaldehyde reacts mole for mole with hydroxylamine HCI. The HCN present in bitter almond oil
does not influence the accuracy of the assay. Hydrocyanic acid is a very weak acid, in the
presence of a strong acid, such as HCI, the common hydrogen ion represses the dissociation of
46
the weak acid, so that at the end point, which in this case is at a pH of about 3.45, none ofthe HCN

is involved in the reaction.

Addition of benzene to the reaction mixture serves as:

(I) A solvent for the oil, producing a nice dispersion of the globules in the mixture, and thus

promotes surface contact between the reacting solutions.

(2) A solvent for the non-aldehydic portion of the oil, thus prevents the inherent color of this from

interfering with the color of the indicator in the dilute alcoholic solution

(N.B. aldehydes tend to oxidize and to condense with other constituents inducing dark colors;

which is not the case with ketones).

Advantages of the hydroxylamine hydrochloride method:

The hydroxylamine method offers many advantages over the bisulfite process.

1- Relatively small amounts of the oil are required for a determination.

2- The reaction of hydroxylamine HCI with aldehydes is specific and rapid, shortening the time

required for a determination.

3- Water-soluble adulterants which do not contain a carbonyl group do not analyze as apparent

aldehydes or ketones.

4. The method have proved satisfactory for the determination of certain ketones (such as

menthone and thujone) which cannot be determined conveniently by the absorption method.

5- Furthermore; the hydroxylamine method proved exceptionally applicable to oils which contain

small amounts of aldehydes or ketones (e.g. lemon oil), and to oils containing large amount

of free acids.

47
48
 Laboratory # 6
Work sheet
Determine the percentage of benzaldehyde (% w/w) in bitter almond oil sample you are

provided with and write down your calculations in the specified work sheet

49
 Assay of caraway oil (Sulfite method)

Constituents: Carvone n.l.t. 55% w/v.

Procedures:
1- Introduce 10 ml of caraway oil, accurately measured, into a cassia flask.

2- Add 50 ml of a saturated solution of sodium sulfite which has been carefully rendered neutral

to 2 drops of phenolphthalein by means of a saturated sodium bisulfite solution.

. .
3- Heat the flask in a boiling water bath and shake repeatedly, neutralizing the mixture from time
to time by the addition of few drops of saturated sodium bisulfite solution.

4- When no coloration appears upon adding a few more drops of phenolphthalein and heating for
15 minutes, cool the mixture to room temperature, and when the liquids separated completely,
add sufficient sodium sulfite solution to raise the lower limit of the oily layer within the graduated
portion of the neck of the flask.

5- Note the volume of the residual oily liquid. This volume should not exceed 5 ml, indicating the

presence in caraway oil of not less than 50 per cent, by volume of carvone (C10H14O).

Calculation:
Percentage of carvone = (10 - volume of residual liquid) x 100

10

Explanation:

Carvone, the principal ketone present in the oil, reacts with sodium bisulfite, forming a water-

soluble addition product as follows:

Sodium sulfite is hydrolyzed in part by water with the forma Jon of sodium hydroxide, viz:

Na2SO3 + H2O  Na HSO3 + NaOH

50
 + NaHSO3 




Carvone Water soluble addition product
The presence of free alkali in the aqueous layer renders other components of the oil, such as

phenols, water-soluble and also tends to reverse the addition reaction. To prevent this any liberated

sodium hydroxide is neutralized by the addition of sodium acid sulfite:

NaHSO3 + NaOH  Na2SO3 + H2O

The difference between the initial volume of oil (used as the sample) and the volume of the

oily layer that remains insoluble, represents the volume of carvone which dissolved in the

aqueous layer. If drops of oil adhere to the walls of the flask, they may be made to rise into

the neck by gently tapping and rotating it.

51
 Laboratory # 7
Work sheet
Determine the percentage of carvone (% v/v) in caraway oil sample you are provided

with and write down your calculations in the specified work sheet

52
 Separation of a mixture of menthol and thymol

The principle of the separation of the components of this mixture. is based on the fact that

thymol forms a water soluble phenate with alkali hydroxides (being a phenol) while menthol,

which is a terpene alcohol, is not affected by alkali hydroxides.

Procedure:

1- Transfer 2 ml of the mixture (dissolved in chloroform or ether to a separator).

2- Shake with successive portions sodium hydroxide solution (5, 2 and 2 ml)

3- Separate each alkaline layer ill another separator (the chloroform contains menthol while

the aqueous sodium hydroxide layer, contains thymol as the sodium salt)

4- Wash the chloroform with water, reject the aqueous layer, evaporate the chloroform and

test for menthol in the residue.

5- Acidify the alkaline solution which contains thymol with cone HCI (Litmus), then shake

successively with 10, 5, 5 ml portions of chloroform (or ether).

6- Wash the combined chloroform with water (reject the aqueous layer), evaporate the

chloroform and test for thymol in the residue.

Test for menthol

OH

Dissolve about 10 mg of menthol in I ml conc. H2SO4. (in a porcelain dish) warm slightly,

add I ml of vanillin/H2SO4. reagent, an orange yellow color is produced which on the addition

of I ml of water changes to purple or violet.

53
 Test for thymol

1- Heat 10 mg of thymol with 5 ml NaOH T.S., in a test tube on a water bath, a clear colorless

or pink solution is formed which becomes darker on standing and no oily drops separate out.

On the addition of few drops of chloroform and shaking the mixture, a violet color is produced.

2. In a porcelain dish dissolve about 5 mg in 1 ml glacial acetic acid, add 3 drops of conc.

H2SO4 and 1 drop of conc. Nitric acid, a green color is produced.

3. With alcoholic ferric chloride solution a blue color is produced.

4. In a dry clean test tube, fuse about 10 mg of thymol with phthalic anhydride, in the presence
of conc. H2SO4, a violet to red color is produced. Upon the addition of dilute alkali solution, an
intense blue color is produced (thymol phthalein).

54
 Assay of clove oil

Constituents: Eugenol 76-90 % acetyl eugenol 7-17 %.

Procedures:

1- Place 5 ml of clove oil, measured by a pipette, into a 100 mI cassia flask.

2- Add 75 ml N/1 KOH (aqueous) and shake for 5 minutes, heat on a water bath with frequent

shaking for 15 minutes, then set the flask aside to cool to room temperature.

3- When the liquids have separated completely, add sufficient N/1 KOH to raise the lower limit

of the oily layer into the graduated portion of the neck.

Calculation:
Percentage of phenols (% v/v) =
(vol. of oil)-(vol. of non-phenolic)
X 100
vol. of oil

Explanation:
KOH reacts with the phenol eugenol, and acetyl eugenol, as follows:-

Clove oil assay

Principle of the assay:
Clove oil contains:
Eugenol + Acetyleugenol + Non-phenolic part

Remains

Alkali 1. Heat
(KOH) 2. Alkali

Phenate salt
(water soluble)

55
 The mixture is heated on a water bath for 10 min to saponify the eugenol acetate

content.

Potassium eugenol ate dissolves in water leaving non-phenolic content un-dissolved

(hydrocarbons, ketones ... etc.). The volume of the latter is then measured in the

graduated neck of the flask.

If strong alkali solutions are employed, the results obtained are too high, since strong

alkali exercises some solvent action on the non-phenolic constituents, particularly on

oxygenated compounds. The volume of the un-dissolved liquid is subtracted from the tota!

volume of the oil to give the volume of total phenols that dissolved in the alkali (eugenol

and acetyleugenol).

56
 Laboratory # 8
Work sheet
Determine the percentage of phenolic contents (eugenol and acetyl eugenol) (% v/v) in clove

oil sample you are provided with and write down your calculations in the specified work

sheet

57
 Assay for peroxide content
Volatile oil of chenopodium

Constituents: Ascaridole n.l.t. 60% w/w.

Various chemical and biological methods of evaluating the oil have been proposed, but the

official method, which is based on the oxidative properties and the solubility of ascaridole (the

principal constituent of chenopodium oil) in acetic acid, is probably as good as any; although it

gives variable results and should be regarded as approximation (As it is a non-stoichiometric

reaction).

Procedures:

1 - Dissolve about 2.5 g of the oil, accurately weighed in sufficient acetic acid (90%) to produce 50

ml.

2- Into a stoppered tube introduce 3 ml of potassium iodide solution (83%), 5 ml conc. HCI and 10

ml of glacial acetic acid.

3- Immerse the tube in a freezing mixture until the temperature is reduced to - 3°C.

4- Add 5 ml of the acetic acid solution of the oil to the cooled mixture, mix quickly and set aside in a

cool place for 5 minutes.

5-Titrate the liberated iodine (without dilution) with N/IO sodium thiosulphate, using starch as
indicator, only near the end point.
6- At the same time carry out blank experiment (without the oil), diluting the reagent mixture with 20
ml of water before the titration, The difference between the two titrations is equivalent to the iodine
liberated by ascaridole.
Each ml of NIIO sodium thiosulphate Ξ 0.00665 g of ascaridole

Calculation:
Percentage of ascaridole (% w/w) =

(E.P. - B) x 0.00665 x 50/2.5 x 100/5

Explanation: Ascaridole dissolves in 90% acetic acid; and any variation in the strength of the

acetic acid employed would results in the dissolution of variable amounts of the constituents
58
 of the oil (decomposition and reduction products of ascaridole); consequently, the acetic acid
concentration must be exactly 90%.

When solution of the oil is added to the reaction mixture, iodine

is liberated as a result of the interaction of ascaridole (an organic peroxide) with potassium iodide,

and hydrochloric acid.

The reaction is exothermic and at usual room temperature, the reaction is rapid and

vigorous, and considerable heat is generated. So, the cooling in freezing mixture is to delay the

reaction, and to guard against the loss of iodine through volatilization.

The mixture of reagent and solution of oil is set aside for 5 minutes before titration to make
59
sure that sufficient time has elapsed to ensure complete reaction between ascaridole and the

reagent. During this time, the temperature is kept down (between 5 and 10°C) for the reasons

stated above and also to guard against explosion of the organic peroxide.

Some iodine is liberated in the reagent mixture because of dissolved atmospheric oxygen

and other oxidizing contaminants. It is therefore necessary to run a blank experiment. The

difference between the two titrations represents the number of milIiliters of 0.1 N sodium thiosulfate

equivalent to the peroxide content of the oil.

60
 Laboratory # 9
Work sheet
Determine the percentage of peroxide contents (ascaridole) (% w/w) in chenopodium oil

sample you are provided with and write down your calculations in the specified work sheet

61
 Assay for oxide content
(Volatile oil of eucalyptus)

Constituents: Cineole 50-80% v/v.

The method of determination of cineole in eucalyptus oil is based on the formation of a solid,
loose molecular compound of cineole and phosphoric acid, from which cineole may be regenerated
by the action of water.

Procedures:
1- Transfer 5 ml of the oil, accurately measured by a pipette, to a clean dry evaporating dish and
place over a freezing mixture. 2- Measure 5 ml of syrupy phosphoric acid in a measure or test tube
and cool it in the freezing mixture.
3- When sufficiently cooled add the acid gradually to the oil and stir continuously until a solid
product is obtained.

4- Transfer to a piece of calico (lint), fold well, wrap in 2 sheets of filter paper and press strongly for

5 minutes.

5- Remove from the press, change the filter paper and press again for 2 minutes.
6- The pressed cake which is the cineole phosphoric acid coupled compound is broken into small
pieces and introduced into a cassia flask containing warm water.

7- Heat on a water bath and when decomposition is complete (oily layer is clear), raise the oily

layer into the graduated portion of the neck of the flask by gradual addition of water.

8- Allow to cool to 15.5°C and read the volume of cineole.

Calculation:

volume of cineole

Percentage of cineole (% v/v) = ------------------------- x 100

volume of the oil

Explanation:
With phosphoric acid, cineole forms a crystalline additive compound which can be separated
from the non-cineole part of the oil, and then decomposed with water to liberate cineole again.

62
The method gives an accurate determination of cineole if the following points are noted.

a- The oil must be dry.

b- The syrupy phosphoric acid must be pure and dry, with a minimum freezing point.

c- The volumes of oil and syrupy phosphoric acid should be as recomended in the

procedure.

63
 Laboratory # 10
Work sheet
Determine the percentage of oxide contents (cineole) (% v/v) in eucalyptus oil sample you

are provided with and write down your calculations in the specified work sheet

64
 Laboratory # 11
Volatile Oil Exam
Determine the percentage of the main active constituent (aldehyde, ketone, phenol, oxide,

peroxide, etc..) in the volatile oil sample you are provided with and write down your

calculations in the specified examination sheet

65
 Grade Sheet

# Date Grade Signature

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

66