FRUIT WINE PRODUCTION yeast.
Typically, 70% of the fermentation activity
Intended Learning Outcomes:
At the end of the exercise, the student should be able to:
1. Produce a fruit wine from different tropical or
sub-tropical fruits through natural fermentation
reaction.
2. Package a final bottled fruit wine with the
correct label.
Materials:
• Citric Acid - Food Grade
• Calcium Carbonate - Food Grade
• Clean Cheesecloth, Fruits, Knife
• Chopping Board, Plastic containers
• two 1 Liter Amber Bottle, Cotton, Gauze, Yarn, Foil
Preparation of the Must
1. Prepare two 1-L amber bottle. Wash and make sure that
there are no traces of dirt inside the bottle. Dry sterilize
for 1 hour at 160-170°C. After sterilizing, place a cotton
plug on both bottles. Set aside and make sure to keep
these bottles in a clean place.
2. Meanwhile, prepare 1.0 kg of chosen fruits. The best
fruits to use are those that are slightly overripe.
3. Wash and clean the fruits to used. Crush the fruit to
extract its juices, enough to produce 1.0 L. Remember to
remove the skin, pits and seeds as it may result to bitter
taste.
4. Filter the juice using a clean unbleached cheesecloth.
Place the must in a 1-L beaker. Do not throw the fruit
pulp yet. Let the fruit pulp remain in the cheesecloth.
5. To the must, add around 0.20 to 0.50 kg of sugar per liter
of the must. The amount of sugar to be added depends
on the fruit chosen. Remember that the sugar will
determine the amount of alcohol that could be present in
the wine. Then add enough mineral water to bring the
volume to 1.0 L, if necessary. Then measure the sugar
content using a hydrometer, if available. The specific
gravity should be around 1.08-1.09.
6. Heat the solution at 60-70°C for 30 minutes.
7. Remove from heat and set aside to cool. Then measure
the pH of the must. It must be around 3.2-3.8. Adjust the
pH using citric acid (if the must is basic) and calcium
carbonate (if the must is acidic), if necessary.
8. Transfer the adjusted must to a 2-L plastic bucket or
container. The container ideally must have a wide mouth.
9. Then on the adjusted must placed on the 2-L plastic
container, submerge the fruit pulp sealed inside the
cheesecloth. Make sure that the fruit pulp will not leak
out from the cheesecloth. This step will add more
character to the taste and bouquet or aroma of the wine.
10. Cover the container with a thin, clean towel. Make sure
to tie the towel around the container. Let it stand for 2
days. During this period, the sodium metabisulfite is
sterilizing the must with a mild sulfur gas. After 2 days,
the sulfur gas leaves the container.
After Two Days
1. After 2 days, sprinkle 2 g of active dry yeast to the
must inside the container.
2. Allow the mixture to ferment for 7 days. Foaming
becomes evident within 24 hours after adding the
occurs within 7 days.
After One Week
1. After 7 days, remove the cheesecloth containing the
fruit pulp. Wring out excess juice from the
cheesecloth. Do this step carefully so as not to leak
out the fruit pulp into the fermented must.
2. After removing the fruit pulp, siphon the fermented
must into the previously sterilized 1-L amber bottle.
This step is called as racking.
3. Get as much fermented juice as possible but make
sure that the mixture should not be stirred. During
this racking process, some sediments may still be
transferred to the bottle.
4. Let the mixture ferment for another 4 weeks. Make
sure to place the bottle with cotton plug in a dark
place with temperature ranging from 16-24°C.
After One Month
1. After 4 weeks, siphon again the wine into another
previously sterilized 1-L amber bottle with cotton
plug. If the volume is greatly reduced, sufficient
amount of mineral water could be added to bring up
the volume.
2. Place the wine in a cool place, around 8-15°C. Check
the wine weekly and make sure that the wine remains
clean.
3. Let it settle for 8 weeks.
After Two Months
1. After 8 weeks, transfer the wine into a 1-L beaker.
Then add one well-beaten egg white to the wine. This
process is called as clarifying.
2. Pasteurize the wine with egg white at 60°C for 30
minutes. Do not heat the wine too much as it may
evaporate the alcohol content of the wine. Remember
to seal the beaker during the pasteurization.
3. After pasteurization, filter the wine using a
cheesecloth into the final bottle for the wine. Take
care that there should be no sediments transferred to
the final bottle of the wine. Ideally, the volume is now
reduced at around 750 mL. Remember that in this
procedure, the wine should now be clear.
4. Let the wine age for another 6 weeks. Ideally, the fruit
wine should be aged for one to two years.
After One and Half Months
1. Package the wine with appropriate labels.
2. The wine can now be tasted.
IMPORTANT NOTE: Change the cotton plug now and
then to avoid mold formation
SCHEDULES:
1. Preparation of Must - At School
2. After Two days
3. After One Week
4. After two months - At School
5. After One and Half Months - Bring for Final presentation
 THE PHYTOCHEMICAL INVESTIGATION
1. INTENDED LEARNING OUTCOMES:
2. To identify the different primary and secondary
metabolites present in different medicinal plants
3. To acquire the skills in identifying different plant
constituents
DISCUSSION:
"Phyto" is a Greek word that means plant. Different methods
can be conducted to determine the different constituents
present in plants. One of the methods of identifying the
constituents is through phytochemical screening. A method
for use in phytochemical screening should be: (a) simple; (b)
rapid; (c) designed for a minimum of equipment; (d)
reasonably selective for the class of compounds under study;
(e) quantitative in so far as knowing the lower limit of
detection is concerned; and if possible (f) should give
additional information as to the presence or absence of
specific members of the group being evaluated.
Phytochemical investigation of plants involves the following:
1. Authentication and extraction of the plant material
2. Separation and isolation of the constituent of interest
3. Characterization of the isolated compounds
4. Investigation of the biosynthetic pathways of compounds
5. Quantitative evaluation
6. Pharmacological assessment of the separated
components
The proper time in the collection of plant parts for
phytochemical screening is an important matter to consider.
The plant part is best collected as follows
PLANT PART BEST TIME OF COLLECTION
Roots/Rhizomes After vegetative process
Stem/Bark Before vegetative process
Flowers When they are about to bloom
Seeds When matured
Leaves When photosynthesis is active
Fruits When ripe
Extraction of Plant Material
Extraction depends on the nature of the plant material and
the components to be isolated. Dried materials should be
powdered before a. Fresh materials can be homogenized or
soaked to a solvent such as alcohol. Alcohol general solvent
for extraction, light petroleum (essential, fixed oils, steroids),
ether and chloroform (Alkaloids), water immiscible solvent
(Alkaloids), and acidification (aromatic acids & phenols)
PROCEDURE
Collection and Preparation of Samples
Collect plant for phytochemical screening. Wash air-dried and
oven-dried at 40oC and grind.
Phytochemical Screening of Samples
Preparation and Extraction of the Samples
1. Weigh 100 g of the powdered sample; place it in a 500 mL
Erlenmeyer flask.
2. Macerate it with sufficient amount of 80% ethyl alcohol to
completely submerge the material.
3. Keep the material soaked for 24-48 hours. Reflux distillation
could be used to further extract the constituents.
4. Filter and rinse the flask with fresh portions of 80% ethyl
alcohol. Discard the residue.
5. Collect the filtrate and evaporate it using rotavapor to
separate the solvent from the extract.
6. Further concentrate the extract to about 50mL at a
controlled temperature of 40o
7. Measure the volume of the concentrated extract.
8. Compute for the concentration of the extracts in grams of
dried plant material per mL extract.
9. Store the extract in a tightly closed container in a cool
temperature. Label the container properly with the name of
the sample, concentration of the plant extract in grams per
mL, and the date of extraction.
Note: To prevent fungal growth, the researcher should add a
small amount of chloroform.
TEST FOR THE PRESENCE OF ALKALOIDS
Alkaloids are basic nitrogenous compounds of high
pharmacologic functions.
Alkaloids are present in plant tissues as
1. water-soluble salts of organic acids (tartaric, acetic, oxalic,
citric, malic, and lactic acids),
2. esters (e.g., atropine, scopolamine, cocaine, aconitine),
3. combined with tannins (Cinchona bark)
4. sugars (e.g., the glycoalkaloids of Solanum species)
rather than as free bases. Most alkaloids are isolated from
plant matrices in the form of crystalline, amorphous,
nonodorous, and nonvolatile compounds. Majority of
alkaloids are colorless with a bitter taste.
Free bases of alkaloids are soluble in nonpolar organic
solvents (chloroform, methylene chloride, ether), while their
solubility in water is low (exceptions include caffeine and
ephedrine). Salts of alkaloids are soluble in water or dilute
acids, whereas they are insoluble or sparingly soluble in
organic solvents.
These differences in the solubility of alkaloids,
depending on their form, are used in the pharmaceutical
industry for their purification from complex plant matrices
and to produce pharmaceutically acceptable products.
Preliminary test
1. Get an equivalent of 20 grams of the sample from the
stock extract and place it in an evaporating dish.
2. Evaporate to a syrupy consistency over a steam bath.
Add 5 mL of 2M hydrochloric acid (HCl) and stir and heat
it for about 5 minutes in a water bath and allowed to
cool. Add 0.5g sodium chloride, stir, and filter.
3. Wash the residue with enough 2M HCl to bring the
filtrate to a volume of about 6 mL.
4. Get one mL of the filtrate and test with 2 to 3 drops of
Mayer’s reagent, Wagner’s reagent and, Draggendorrf’s
reagent. Observe the relative amounts of precipitation as
follows:
(+) Slight turbidity
(++) Definite turbidity
(+++) Heavy precipitation
Confirmatory test
1. To the remaining 3 mL of the filtrate, add enough 28%
ammonia until the solution becomes alkaline to litmus
(Caution: ammonia causes burns, vapors extremely
irritating.)
2. Extract the alkaline solution three times with small
portions of 10 mL of chloroform (Caution: Carcinogenic).
3. Combine the lower chloroform extracts and reserve the
upper aqueous layer for the test for quaternary bases.
4. Evaporate the chloroform extract to dryness under the
hood and over a steam bath.
5. Add 5 mL of 2M hydrochloric acid.
6. Filter the solution and divide the filtrate into three
portions.
7. Test one portion with Mayer’s reagent, Dragendorff’s
reagent and Wagner’s reagent. Observe the result and
record as (+), (++) or (+++).
8. Positive results indicate the presence of primary,
secondary and tertiary alkaloids.
Test for quaternary bases and/or amine oxide
1. Acidify the alkaline aqueous layer obtain (refer to number
3 above) with 2M HCl.
2. Filter and divide the filtrate into three portions. Add
Mayer’s reagent to one portion, Dragendorff’s reagent
and Wagner’s reagent to the other two portions. Observe
and record the result.
3. A score of (++) or (+++) in both Mayer’s and
Dragendorff’s tests indicates the presence of Quaternary
and/ or amine oxide bases; a (+) score may be the result
of incomplete chloroform extraction; thus, it should be
considered negative quaternary bases.
SCREENING FOR GLYCOSIDES (HETEROSIDES)
Glycosides are organic compounds in which a hemiacetal
linkage usually connects the anomeric carbon of a sugar
(glycone) with an alcohol or phenolic hydroxyl of a second
non-sugar molecule (aglycone) this type of linkage rise to the
so-called o-glycosides (e.g.,salicin) the most common type of
glycosides found in plants.
TEST FOR STEROIDS: CARDENOLIDES AND
BUFADIENOLIDES
1. Take an equivalent of 10 g sample from the plant extract
stock solution previously prepared.
2. Evaporate to incipient dryness over a water bath and set
aside to cool at room temperature.
3. Defat the residue with the use of 6 mL of hexane and
water, 2:1 v/v; observe a partition by gently shaking the
mixture in a test tube; pipette out the upper hexane
layer; then, repeat the treatment with hexane until most
of the colored pigments had been removed.
4. Discard properly all the hexane extracts. Heat the
defatted aqueous layer over a water bath to remove the
residual hexane; cool at room temperature.
5. Divide into three portions for Keller-killiani test,
Liebermann-Burchard test and Salkowiski's test.
6. Positive result: A reddish-brown color, which may turn
blue or purple, indicates the presence of 2-deoxysugars.
Keller-killiani test: test for 2-deoxysugars
1. Add three mL of Ferric chloride to one portion of the
defatted aqueous layer free from hexane and stir.
2. Incline the test tube and add 1 mL of concentrated
sulfuric acid cautiously using a pipette, (Caution: very
corrosive), letting the acid flow down along the inside of
the test tube; allow the mixture to stand upright and
observe the coloration at the interface of the acid and the
aqueous layer.
Liebermann-Burchard test: test for Unsaturated steroids
1. Add 10 mL dichloromethane to a portion of the defatted
aqueous layer free from hexane, stir with a glass rod for a
few minutes and allow to stand. The lower
dichloromethane (CH2Cl2) extract was pippetted off.
2. Dry the dichloromethane (CH2Cl2) extract by passing the
extract through about 100 mg anhydrous sodium sulfate
(Na2SO4) place a dry filter paper in a funnel.
3. Divide the filtrate into two portions; one will be used as
the control and the other portion will be treated with 3
drops of acetic acid anhydride (Caution: Corrosive); then
add one drop of concentrated sulfuric acid (Caution:
Highly corrosive). Observe for any immediate color
change.
4. Allow to stand for an hour and observe for further color
change. Compare with the control.
5. Positive results: Observe colors ranging from blue to
green, red, pink, purple or violet because of the
steroid/triterpenoid skeleton.
Salkowiski's test
To the second portion of chloroform filtrate an equal volume
of sulfuric acid is added. The appearance of a red color
indicates the presence of unsaturated sterol and /or
triterpenes.
TEST FOR ANTHRAQUINONE GLYCOSIDES
The largest groups of naturally occurring quinine
substances are the anthraquinones. Although they have a
widespread use as dyes, their chief medicinal value is
dependent upon their cathartic action.
They are restricted distribution in the plant kingdom and are
found most frequently in members of the
Rhamnaceae,Polygonaceae, Rubiaceae, Leguminosae and
Liliaceae. As found in plant, they are usually carboxylated,
methylated or hydroxylated derivatives of the anthracenes,
anthrone, anthranol, anthraquinone, or dianthrone.
Borntrager’s test
1. Take an equivalent of 1 g sample from the prepared stock
extract and evaporate to incipient dryness over a steam
bath.
2. Take the residue with 10 mL distilled water, filter and the
discard the residue; extract the aqueous filtrate with 5
mL portions of benzene (Caution: Carcinogenic) twice
and combine the two portions of the benzene extracts.
3. Divide the combined benzene extracts into two portions.
One portion serves as the control. Treat the other portion
with 5 mL ammonia solution and shake. Compare the
results with the control tube.
4. Positive result: A red coloration in the lower ammoniacal
layer indicates the presence of anthraquinones.
Modified Borntrager’s test
1. Take an equivalent of 1 g sample from the prepared stock
extract and evaporate to incipient dryness over a steam
bath.
2. Add 10 mL 0.5 M Potassium hydroxide and 1 mL of 5%
hydrogen peroxide (H2O2) and stir. Heat the resulting
mixture over a steam bath for 10 minutes. Filter and
discard the residue.
3. Acidify the filtrate with glacial acetic acid. Collect the
filtrate and extract it with two 5 mL portions of benzene
(Caution: Carcinogenic); combine the benzene extracts
and divide into two portions, one portion serves as the
control and alkalinify the other portion with ammonia and
shake. Compare the result with the control.
4. Positive result: A pink color indicates a positive result.
TEST FOR FLAVONOIDS
The term flavonoids refers to a class of plant secondary
metabolites based around a phenyl benzopyrone structure.
Flavonoids are most commonly known for their antioxidant
activity. Flavonoids are also commonly referred to as
bioflavonoids in the media –these terms are equivalent and
interchangeable since all flavonoids are biological in origin.
The flavonoid synthetic pathway begins with a product of
glycolysis phosphoenolpyruvate entering into the shikimate
pathway to yield phenylalanine.
1. Take an equivalent of 10 g sample from the prepared
stock extract and evaporate to incipient dryness over a
steam bath.
2. Cool at room temperature; defat by taking up the residue
with 9 mL hexane and water (2:1) or with petroleum
ether (Caution: solvents are flammable).
3. Discard the hexane or petroleum ether extract.
4. Dilute the defatted aqueous layer with 10 mL of 80%
ethyl alcohol; filter and divide the filtrate into three test
tubes.
5. Use one portion as the control and the 2 test tubes for
test for leucoanthocyanin and test for g-benzopyrone
nucleus .
Test for Leucoanthocyanins: Bate-Smith and Metcalf Test
Method
1. Treat one portion of the above alcohol filtrate with 0.5
mL concentrated hydrochloric acid (12 M); and observe
for any color change.
2. Warm the solution for 15 minutes in a water bath.
Observe for further color change in color within an hour
and compare with the control. The results are recorded.
3. Positive result: A strong red or violet color indicates the
presence of leucoanthocyanins.
Test for y-benzopyrone nucleus: Wilstatter “cyanidin” test
1. Take another portion of the alcohol filtrate and treat it
with 0.5 mL concentrated hydrochloric acid (12 M).
2. Place three to four pieces of magnesium turnings and
observe any color change within 10 minutes; compare
the results with the control tube.
3. If definite coloration occurs, dilute it with an equal
volume of water and 1 mL octyl alcohol. Shake the
solution and allow to the solution to stand for few
minutes. Note the color in each layer. Record the results.
4. Positive result: Observe colors ranging from orange to
red, crimson, and magenta and occasionally to green or
blue.
SCREENING METHODS FOR SAPONINS:
Saponins are glucosides with foaming characteristics.
Saponins consist of a polycyclic aglycones attached to one or
more sugar side chains. The aglycone part, which is also called
sapogenin, is either steroid (C27) or a triterpene (C30). The
foaming ability of saponins is caused by the combination of a
hydrophobic (fat-soluble) sapogenin and a hydrophilic (water-
soluble) sugar part. Saponins have a bitter taste. Some
saponins are toxic and are known as sapotoxin.
Froth Test
Preparation of the “gugo” extract
1. Take about 1 g of gugo Entada phaseoloides (L.) Merr.
bark. Cut into strips and soak in 10 mL of 80% ethyl
alcohol.
2. Allow to stand for 30 minutes. Take a volume of the
prepared sample extract equivalent to 2 g sample and
transfer into a test tube.
3. In a separate test tube, take 1 mL of the “gugo” extract
and served as the standard; Add ten mL of distilled water
to each of the test tubes; place a stopper and shake both
test tubes vigorously for 30 seconds and allow to stand
for 10 minutes; observe for “honeycomb” froth; compare
the results of the extract with those of the standard.
4. Positive result: If the “honeycomb” froth greater than 2
cm height from the surface of the liquid persists after 10
minutes, consider the sample positive for saponins.
SCREENING FOR TANNINS
Two groups of phenolic constituents, hydrolysable and
condensed, comprise the tannins, substances which are
important economically as agents for the tanning of leather
and for certain medicinal purposes. More recently, evidence
has been presented in support of their potential value as
cytotoxic neoplastic agents.
Properties of tannins
Hydrolysable tannins are yellow-brown amorphous substances
which dissolve in hot water to form colloidal dispersions.
They are astringent and could be used in tanning industry.
They are esters which can be hydrolyzed by boiling with
diluted acid to yield a phenolic compound, usually a derivative
of gallic acid, and a sugar. These are often referred to as
pyrogallol tannins.
Condensed tannins are polymers of phenolic compounds
related to the flavonoids and are similar in general properties
to the hydrolyzed tannins but are not very soluble in water
and following treatment with boiling dilute acid red-brown
insoluble polymers known as phlobaphene or tannins-red are
formed.
Test for Tannins
1. Take an equivalent of 10 g sample from the prepared
extract and evaporate to incipient dryness over a steam
bath; extract the residue with 20 mL of hot distilled water
and add 5 drops of 10% sodium chloride solution.
2. Filter the solution and divide the filtrate into three test
tubes. One portion serves as the control; take an aqueous
solution of tannic acid as reference standard.
Gelatin test
1. Treat one portion of the filtrate of the sample extract
with three drops of gelatin salt solution (mix an equal
amount of 1% gelatin solution and 10% sodium chloride
solution) which serves as the reference standard. Then
compare the control and the reference standard.
2. Positive result: The formation of a jelly-precipitate
indicates the presence of tannins.
Ferric chloride test
1. Treat another portion, the sample extract with three
drops of ferric chloride reagent and likewise to the tannic
acid solution. Compare the results with the control and
the reference standard.
2. Positive result: A blue-black color indicates the presence
of hydrolyzable tannins, while a brownish-green color
may indicate the presence of
nonhydrolyzable/condensed tannins.
SCREENING FOR CARBOHYDRATES
Carbohydrates are carbon compounds that contain
large quantities of hydroxyl groups. The simplest
carbohydrates also contain either an aldehyde moiety (these
are termed polyhydroxyaldehydes) or a ketone moiety
(polyhydroxyketones). All carbohydrates can be classified as
either monosaccharides, oligosaccharides or polysaccharides.
Anywhere from two to ten monosaccharide units, linked by
glycosidic bonds, make up an oligosaccharide. Derivatives of
the carbohydrates can contain nitrogens, phosphates and
sulfur compounds. Carbohydrates also can combine with lipid
to form glycolipids or with protein to form glycoproteins
Procedure
Evaporate about 2 grams equivalent of 80% alcoholic extract;
take the residue with 10 mL distilled water and divide it into
three equal parts; then perform the following
tests:
Fehling’s Test
1. Boil about 5 mL of freshly prepared Fehling’s solution
(2.5 mL each of Fehling’s A & B) using a test tube. Add an
equal quantity of plant extract and boil again.
2. Positive result: brick red precipitate indicates the
presence of reducing sugars.
Molisch's test:
1. Mix two mL of the prepared filtrate with 0.2 mL of
alcoholic solution of α-naphthol 10% in addition to 2 mL
of sulfuric acid.
2. Positive result if: bluish violet zone is formed this
indicates the presence of carbohydrates and/or
glycosides.
Benedict's test:
1. Add to 1 mL of the filtrate, 5 mL of Benedict's reagent.
Heat the mixture.
2. Positive result: appearance of red precipitate indicates
the presence of reducing sugars.
SCREENING FOR PROTEINS
Proteins contain a wide range of functional groups. These
functional groups include alcohols, thiols, thioethers,
carboxylic acids, carboxamides, and a variety of basic groups.
When combined in various sequences, this array of functional
groups accounts for the broad spectrum of protein function.
For instance, the chemical reactivity associated with these
groups is essential to the function of enzymes, the proteins
that catalyze specific chemical reactions in biological systems
1. Evaporate 2 g equivalent of the plant extract.
2. Take up the residue with 10 mL of water. Perform the
following tests:
Millon’s Test
1. Add to 1 mL of the aqueous extract, 10 drops of Millon’s
reagent and place in a boiling water bath.
2. Positive result: Observe for the presence of flesh color.
Xanthoproteic Test
1. Add to 1 mL of the aqueous extract, 10 drops of nitric
acid.
2. Positive result: Formation of yellow precipitate.
SCREENING FOR LIPIDS
Lipids in biological systems include fats, sterols, fat soluble
vitamins, phospholipids, and triglycerides. They serve as
structural components of biological membranes. They provide
energy reserves, predominantly in the form of triglycerides
(TGs; also called triacyglycerols, TAGs). Lipids and lipid
derivatives serve as biologically active molecules exerting a
wide range of functions. Lipophilic bile acids aid in
emulsification, digestion and absorption of dietary lipids as
well as being a form of bioactive lipids
Procedure
1. Boiled about 2 g equivalent of plant sample and add 10
mL of petroleum ether or hexane.
2. A piece of white paper and on its surface, place two
drops petroleum ether extract.
3. Positive result: a permanent greasy stain on paper.
THIN LAYER CHROMATOGRAPHY
TLC is a type of planar chromatography. It is routinely used
by researchers in the field of phyto-chemicals, biochemistry,
and so forth, to identify the components in a compound
mixture, like alkaloids, phospholipids, and amino acids. It is a
semi quantitative method consisting of analysis.
Principle
Like other chromatographic methods, thin layer
chromatography is also based on the principle of separation.
The separation depends on the relative affinity of compounds
towards stationary and the mobile phase.
The compounds under the influence of the mobile phase
(driven by capillary action) travel over the surface of the
stationary phase. During this movement, the compounds with
higher affinity to stationary phase travel slowly while the
others travel faster. Thus, separation of components in the
mixture is achieved.
Once separation occurs, the individual components are
visualized as spots at a respective level of travel on the plate.
Their nature or character are identified by means of suitable
detection techniques.
System Components
TLC system components consists of:
1. TLC plates, preferably ready made with a stationary
phase: These are stable and chemically inert plates,
where a thin layer of stationary phase is applied on its
whole surface layer. The stationary phase on the plates is
of uniform thickness and is in a fine particle size.
2. TLC chamber. This is used for the development of TLC
plate. The chamber maintains a uniform environment
inside for proper development of spots. It also prevents
the evaporation of solvents, and keeps the process dust
free
3. Mobile phase. This comprises of a solvent or solvent
mixture The mobile phase used should be particulate-free
and of the highest purity for proper development of TLC
spots. The solvents recommended are chemically inert
with the sample, a stationary phase.
4. A filter paper. This is moistened in the mobile phase, to
be placed inside the chamber. This helps develop a
uniform rise in a mobile phase over the length of the
stationary phase.
Procedure
1. With a pencil, a thin mark is made at the bottom of the
plate to apply the sample spots.
2. Then, samples solutions are applied on the spots marked
on the line in equal distances.
3. The mobile phase is poured into the TLC chamber to a
leveled few centimeters above the chamber bottom. A
moistened filter paper in mobile phase is placed on the
inner wall of the chamber to maintain equal humidity
4. Now, the plate prepared with sample spotting is placed in
TLC chamber so that the side of the plate with the
sample line is facing the mobile phase. Then the chamber
is closed with a lid.
5. The plate is then immersed, such that the sample spots
are well above the level of mobile phase (but not
immersed in the solvent — as shown in the picture) for
development.
6. Allow enough time for the development of spots. Then
remove the plates and allow them to dry. The sample
spots can now be seen in a suitable UV light chamber, or
any other methods as recommended for the said sample.
 EXERCISE NO. 2
MOISTURE CONTENT DETERMINATION OF CRUDE
DRUGS
Intended Learning Outcomes:
At the end of the exercise, the student should be able to:
1. Determine the importance of moisture content
determination in the quality and purity of crude
drugs.
2. Analyze moisture content of crude drugs using IR
moisture analyzer and drying oven and analytical
balance.
3. Compute for the percentage moisture content
using the formula.
Discussion:
Moisture can be simply defined as water diffused in a
relatively minute quantity. Moisture content is relatively
difficult to accurately measure due to the complex
intermolecular bonding properties within the substance
matrix. This property is an important factor to identify among
crude drugs mainly because the moisture is largely
responsible for the decomposition of crude drugs through
chemical change, microbial or fungal growth.
Moreover, the presence of moisture may increase the
following physical properties of crude drugs: weight, density,
viscosity, refractive index and electrical conductivity.
Therefore, the moisture content determination is a crucial
component of quality of crude drugs and essentially a quality
control parameter in most production and laboratory
facilities. According to USP/NF, there are official two types of
water present in drugs: water of crystallization which is a
definite proportion of water as part of drug’s crystalline
structure, and water in the adsorbed form which is the water
present in the surface (whether internal or external) of the
drug.
Moisture content can be determined through the
following official methods:
1. Thermogravimetric Analysis. This method involves the
use of heating through convection with forced or
circulating hot air on the crude drug. The moisture
content is the computed based on the loss of mass of the
substance on drying through water vaporization after it
was heated. The sample is weighed before and after the
drying process and the moisture content is computed on
percentage basis. There are two methods available
utilizing this principle: the drying oven and balance
method and the use of IR moisture analyzer. The drying
oven and balance method is the official reference method
for some substances and is used for cross-referencing
alternative methods. This method is useful for very
heterogenous large sample size (e.g. 500 g) however, it is
very time consuming compared to alternative methods.
The IR moisture analyzer is highly convenient for fast and
reliable moisture content analysis to avoid lengthy delays.
This method has a simple process and minimal errors due
to automatic calculated results.
2. Volumetric Analysis. This method involves the use of
azeotropic distillation principle wherein the liquid mixture
is separated into pure components with the help of an
additional solvent known as entrainer (e.g. toluene). This
is usually applied for crude drugs with or without volatile
substances. During the process, the crude drug with
water, after the azeotrope is formed, is added with
toluene to separate the water and the organic
components. The water collected will be then directly
measured through its volume (mL or L).
3. Titrimetric Analysis. This method, also known as Karl
Fischer titration, utilizes the quantitative reaction of
water with iodine and sulfur dioxide in the presence of a
low molecular weight alcohol (e.g. methanol) and an
organic base (e.g. pyridine). During the process, once all
the water present in the crude drug is consumed, the
presence of excess iodine is determined volumetrically by
the indicator electrode of the titrator. The amount of
water present in the sample is calculated based on the
concentration of the excess iodine. There are two
methods of KR titration: the volumetric KF and
coulometric KF titrations. Volumetric KF titration
determines moisture content by measuring the amount of
iodine consumed as a result of reaction with water within
the sample, while the coulometric KF titration determines
the moisture content by measuring the quantity of
electricity which is required for the electrolysis.
In this exercise, the thermogravimetric analysis will be
employed in measuring the moisture content of the crude
sample. Both drying oven and balance method and the IR
moisture analyzer will be both demonstrated and compared
both on results and the process. The loss on drying will be
computed using the formula indicated below:
Procedures:
Loss on Drying: IR Moisture Analyzer
1. Prepare the plant sample assigned by manually
cutting the leaves into approximately 3 mm in
thickness.
2. Set-up the Denver IR Moisture Analyzer properly.
Plug the equipment in to the correct voltage
output socket (refer to the user manual).
3. Turn ON the power switch (I/O).
4. Set the temperature to 105°C, the time to 10
minutes and the result display to “by weight”.
5. Place the aluminum pan using the forceps and
accurately weigh 2 g of the sample.
6. Spread the sample evenly over the aluminum pan
then close the hood.
7. Wait for the reading after the desired time (10
minutes).
8. Open the hood and remove the pan using the
forceps.
9. Turn OFF the power switch and unplug the
instrument.
10. Clean the machine and the aluminum pan using a
clean tissue.
 11. Record the results obtained.
EXERCISE NO. 3
ASH CONTENT DETERMINATION OF CRUDE DRUGS
Intended Learning Outcomes:
At the end of the exercise, the student should be able to:
1. Analyze the importance of ash content determination
(total ash and acid insoluble ash) in the quality and
purity of crude drugs.
2. Conduct analysis of ash content determination using
the muffle furnace and other laboratory equipment.
3. Compute for the percentage total ash and percentage
acid-insoluble ash.
Discussion:
Ash refers to the inorganic residue left after the
moisture and other organic substances had been removed by
incinerating the sample in the presence of oxidizing agents.
Ash content determination is one of the most widely
employed quality control parameter, based on fact that
minerals are not destroyed by heating. This analytical test
provides a measure of the total amount of minerals in a
sample, furnishes a basis for judging the identity and purity of
a drug and gives relative information on adulteration with
inorganic matter.
The total ash content refers to the residue remaining on
the vessel after the incineration. This allows to easily identify
the physiological and non-physiological materials contained in
the drug. Physiological ashes are derived from the plant tissue
itself while non-physiological ashes are often derived from
environmental contaminations (e.g. sand, soil). The total ash
usually contains carbonates, phosphates, sulfates, chlorides,
oxides of calcium, magnesium, potassium, sodium, aluminum,
iron and other metals. Another index to measure the purity
and quality of plant samples is the acid-insoluble ash. This ash
refers to the part of the total ash that is insoluble in dilute
inorganic acids. The diluted hydrochloric acid, for example,
can dissolve calcium carbonate, alkali chlorides, and the like,
leaving an acid-insoluble residue that consists almost entirely
of silica from soil adhering to the drug.
In ash content determination, several methods can be
used. One of which is the use of direct incineration of the
sample placed in the crucible using open flame burner. The
sample is placed in a crucible and cover and is incinerated
over an open flame burner set-up using a clay triangle, tripod
and a Bunsen burner. A crucible and cover can be made of a
porcelain or a more sophisticated quartz used for high-
temperature requirements up to 1050°C. Another more
convenient method is to use a muffle furnace. The working
principle of this equipment is to heat the air inside the
chamber by heating a Nichrome (nickel-chromium) wires.
More often, a simple fan-based exhaust system supported by
a chimney operates to cool the unit. This simple exhaust
system takes out toxic gases inside the chamber which comes
out during the heating of the sample. The temperature inside
the furnace can be controlled and adjusted using an
electronic controller unit. The following are the approximate
temperatures used in incinerating the samples inside the
muffle furnace:
In doing the procedure, it is important to note that
before using the crucible, it is necessary to ignite it first to
dull redness until its weight is constant. This would remove
any moisture and adsorbed gases trapped or adhering into
the crucible which can affect the accuracy of the results.
Moreover, the drug sample must be incinerated at
temperature not more than dull redness.
In this exercise, muffle furnace will be employed in measuring
the ash content of the crude sample. Moreover, the acid-
insoluble ash will also be computed based from the obtained
total ash. The total ash and acid-insoluble ash will be
computed using the formula indicated below, respectively:
Procedures:
Determination of the Total Ash Content
1. Prepare the plant sample assigned by manually cutting the
leaves into small pieces.
2. Weigh a clean plain crucible using analytical balance.
3. Then accurately weigh 2 g of the plant sample in the tared
plain crucible. Cover the crucible and place it inside the
muffle furnace.
4. Set the temperature of the muffle furnace into 550°C then
start incinerating the plant samples for 5-6 hours or until the
sample turns to white ash.
5. Turn off the muffle furnace and allow the samples to cool
down (this might take few more hours).
6. Remove the crucibles from the muffle furnace then place
them into a desiccator to further cool down the sample.
7. Weigh the crucible containing the sample. Record the weight.
8. Compute the percentage of the total ash from the weight of
the sample taken.
Determination of Acid-insoluble Ash
1. Boil the ash obtained in procedure 1 with 25mL of dilute
hydrochloric acid for five minutes in a small beaker.
2. Filter the solution using Whatman filter paper then wash it
with hot water
3. Collect the residue left on the filter paper in a previously
weigh evaporating dish.
4. Ignite the residue to remove excess water. Cool and weigh.
5. Compute the percentage of acid-insoluble ash from the
weight of the sample taken.