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midterms pcog lab

Fixed Oils
termed as acyl lipids plant oils

building blocks or main constituents are fatty acids (can be saturated or

unsaturated)

Different Methods to extract fixed oils:

the most ideal way of extracting fixed oils is through Expression

whether by hot compression or cold compression methods

Maceration of Fixed Oils

Ideal solvents: polar solvents

Non-polar solvents: hexane, petroleum ether, benzene

Non-polar solvents: flammable and mutagenic solvents

Hexane: most common and safest; non-polar; good extractor of fats and
fixed oils

1st: weigh the sample

2nd: immerse the sample with the solvent

midterms pcog lab 1

 → there are times, the seeds need to be triturated before immersing it to non-
polar solvent
→ After 24 hours, filter the plant material from the solvent → dry afterwards

→ fixed oils= syrupy, viscous matter

→ After attaining the fixed oils, do the spot test (to test the quality) → get 3
drops of the fixed oil sample

→ Positive result of spot test (true fixed oil): permanent translucent spot

→ glycerol: causes the permanent stain

→ After the spot test, subject the fixed oil sample into solubility test

→ Select one representative of polarity of solvents; 1 non-polar, 1 semi-polar, 1

polar

Solubility test:

3 test tubes)

1 mL Benzene (non-polar)

1 mL Chloroform (semi-polar)

1 mL Ethanol (polar)

1 drop of fixed oil on each test tube

forms heterogeneous mixture or lumps inside the test tube= indication of

insolubility

the oil dissolved in the solvent = indication of solubility

Positive result: fixed oils are soluble in Benzene (non-polar solvent)

Fixed oils are not soluble in Chloroform (semi-polar) and Ethanol (polar)

Fixed Oils: Color and Appearance

Appearance: viscous liquid

Color: yellow

USP/NF test for fixed oils

 Acid value

 Saponification value

 Iodine value

midterms pcog lab 2

 → most important

→ measures saturation

→ basis on the classification of fixed oils

4. Ester value
5. Hydroxyl value

6. Peroxide and Anisidine value

Characterization of Sucrose and Starch

identification of carbohydrates

Plant materials used:

For sucrose extraction:

Saccharum officinarum → locally known as sugar cane

For starch extraction:

Ipomoea batatas → locally known as sweet potato

Carbohydrates

→ are both simple and complex type of compounds

→ their classification depends on the number of monosaccharide unit present

on each type per classification of carbohydrates

Monosaccharides

→ one sugar molecule

→ glucose

→ fructose

→ galactose

Disaccharides

→ two sugar molecules

→ sucrose
→ lactose

→ maltose

Oligosaccharides

midterms pcog lab 3

 → two to ten sugar molecules

→ raffinose
→ stachynose

Polysaccharides
→ most complex form
→ starch

→ glycogen
→ cellulose

3 sources of sucrose

 Saccharum officinarum → sugar cane

 Beta vulgaris → sugar beets

 Acer saccharum → sugar maple plant

Procedure for Sucrose:

Prepare 1020 grams of freshly chopped Saccharum officinarum (sugar

cane).

Once chopped, moist it with amounts of distilled water

Place the chopped material in a mesh cloth

Squeeze out the juice/ liquid that would come out from the plant material

💡 Extra care must be observed because the sugar cane material might
injure the hands of the one holding it or the one directly exposed to
it.

Remove the scum that would arise from the top

💡 Presence of scum → plant material is not totally separated from the

expressed liquid (it’s like some of the wood or sugar cane bits that
usually floats on the top of the expressed liquid so, that must be
removed, because it might interfere the chemical tests)

midterms pcog lab 4

 Heat the mixture for 10 minutes at 50 degrees Celsius

💡 ideal methods in order to get sucrose = crystallization process, but

for this experiment we just heat the mixture in order for it to be
viscous as much as possible

Chemical Test

Molisch Test

one of the general tests for the presence of carbohydrates

aka Alpha-naphthol test (main reagent)

0.5 to 1 mL of the sample + 1 gtt of Molisch reagent (placed in a test tube)

Incline position, carefully add 1 mL conc. Sulfuric acid

observe the middle part (junction) between the two layers→ must be
color purple, red, dark blue/purple

For starch extraction:

contains complex mixtures of polysaccharides

sweet potato is a good example of a plant product that is rich in

polysaccharides

cut into small or fine pieces and mix it with water

filter using mesh cloth to get the filtrate then do the expression method
again. Squeezed out as much as liquid would come out of the cloth

Filter again using mesh cloth to remove bigger particles of the plant
material then collect again the filtrate.

Stand the mixture and container where the mixture was placed. The
container used was beaker.

Decant the liquid to separate it from the starch particles that settle at the
lower part of the medium.

Collect the starch aggregates

Dry it for a certain time by placing it on a filter paper and allow the
moisture that came from the aggregates to be absorbed by the filter paper

midterms pcog lab 5

 then cover it with another filter paper like making a sandwich. In which the
starch particles are at the center.

Place the starch particles on an evaporating dish, this is the time to dry it.
In order to remove excess moisture in an oven.

💡 In drying, temperature must be controlled and must not be more than

50 degrees celsius Why? Because there are tendencies that
excessive heat might destroy polysaccharide that are present on the
starch particles)

Iodine Test/Lugol's Test

One pinch of the starch product

5 mL distilled water

Mix

Add 2 drops of Lugol’s solution

Positive result: Red, purple or dark blue color of the mixture.

Tannins
are polyphenolic compounds

→ most of them could be polar compounds, subclasses of tannins can be

polar compounds or they have miscibility with water and at the same time
they possess a lot of hydroxyl groups
→ one of the major classes of polyphenolic compounds

pharmaceutical uses:

→ astringent
→ hemostatic

→ direct effect on blood vessels

→ other commercial use: used to be dyes for leather products

→ binds to proteins

they usually bind to proteins or they are used as precipitating

agents for proteins

midterms pcog lab 6

 types of tannins:

 hydrolysable

 condensed

→ one of the most beneficial type of tannins are hydrolysable

→ condensed tannins are toxic or they have mutagenic effects

Methods:
Plant specie of interet: tea, Camellia sinesis

25 grams of dried leaves tea

air dry it for a number of days, in order that it would be away from any
moisture or moisture content

Triturate it into powdered form we need to ground it into a pulverized

form.

Weigh and transfer to an Erlenmeyer flask

💡 Once that it is inside the Erlenmeyer mask, that’s the time to

macerate it with ethanol, 95% ethanol.

Add enough ethanol to cover the powdered leaves

The amount of the ethanol that would be used would be enough to

cover or immersed the powdered leaves, then allow it to be soaked for
24 hours.

Macerate for 24 hours

After 24 hours, drain the liquid extract.

Filter and collect the liquid

Collect the filtrate, and subject it to rotary evaporation.

After subjecting it to rotary evaporation, there would be a viscous

liquid.

If that viscous liquid contains aggregates or crystals is a

representation of the tannin compound.

midterms pcog lab 7

 Color - Usually dark, like green to brown
Appearance
Physical State

Chemical tests
→ We need to dissolve tannin crystals or aggregates with a diluent like water
or alcohol

Ferric chloride – classification

can classify what type of tannins are present on your sample mixture

after dropping ferric chloride reagent, once that it would appear green or
brown means
it contains condensed tannins

if in case it would appear blue, dark blue or near black, it is an indication

of the presence
of hydrolysable tannins

10% Gelatin

→ one of the most basic or general test for characterizing tannins, we are
after for a jelly precipitate.

Bromine TS

→ resultant should haw precipitation

Lime water

→ resultant should have precipitation because metallic elements or

compounds present in lime water will form chelates or metal complexes with
your tannins

one of the major manifestations of formation of precipitates is by metal

complexation

Iodine solution

→ expected to have a negative result in iodine solution because iodine

solution is
only indicative for the presence of polysaccharides or starch in the sample

Lugol’s solution is only indicated for the presence of complex

carbohydrates

midterms pcog lab 8

 Extraction of Glycosides
saponin glycosides→ froth test

frothing (bubbles on top in coffee, chocolate drinks; last for 10 minutes)

plant: Entada phaseoloides → gugo → 10 grams of bark

1 cut into 5 pieces and mix with 100 mL water

2 heat the mixture for 5 minutes

3 filter the mixture → add 10 mL magnesium acetate

4 Magnesium acetate: precipitating agent for saponin glycosides

5 Ideal precipitant: Lead acetate

6 Filter again

7 Get the precipitate from the filter paper and dissolve in 10 mL ethanol

Resultant → Chemical tests

Froth Test

5 mL or 1 mL of the solution + 10 mL water or 5 mL water

cannot be added with more than 10 mL water for it can cause congestion

agitate the test tube vigorously

observe frothing that will last 10 minutes

if the frothing will last for > 10 minutes → great amounts of saponin
glycosides are present in the sample

Baryta water

3 mL sample + 1 mL baryta water → note the color of the solution; if there

is precipitation, note the color of the precipitate

Reaction with Magnesium acetate or Lead acetate:

3 ml sample + 1 ml magnesium acetate or lead acetate → note for the

resulting precipitation

Glycosides

contains glycone and aglycone

Baryta water test, froth test, fehlings test, hemolytic test

midterms pcog lab 9

 Fehlings test → detects glycone or the sugar moiety present in the sample
mixture

Hemolytic test → essential because saponins ruptures blood cells

specifically red blood cells RBC

Manifestation of disruption of red blood cells: by hemolysis

when hemolysis occurs → the sample contains saponin glycosides

Magnesium acetate or Lead acetate test and Baryta water test →

complexation reaction occurs

Manifestation of complexation reaction: precipitation

midterms pcog lab 10