SPECIFIC TEST:

ALKALOIDS

Name of Test: Chemicals Procedures Positive Principles References

Involved Results Involed
1. Hager’s Test Hager’s Preparation of Hager’s Crystalline Formation of Raaman, N. (2006).
reagent: Reagent: Yellow microcrystalline Phytochemical Techniques.
Saturated 1. Precipitate complexes Shahdara, Delhi: New India
Solution of Publishing Agency
Picric Acid Hager’s Test:
1. 50 mg of the plant Retrieved from
extract is stirred http://www.scribd.com/doc/
with a few mL of 25959116/ Section-1-
dilute Phytochemistry. Retrieved
Hydrochloric acid August 14, 2010
and filtered.
2. In a test tube,
place a few mL of
the filtrate and
add 1 to 2 mL of
Hager’s reagent.

2. Wagner’s Test Wagner’s Preparation of Red or Alkaloids are Retrieved from

reagent: Iodine Wagner’s reagent: Brown precipitated from http://www.scribd.com/doc/
Potassium 1. 1.3 grams of iodine Precipitate their neutral or 25959116/ Section-1-
iodide and 3 grams of acidic solution Phytochemistry. Retrieved
Water potassium iodide is by reagents August 14, 2010
dissolved in 5 mL contain heavy
Diluted of water. metals Raaman, N. (2006).
Hydrochloric 2. Add enough Phytochemical Techniques.
Acid distilled water to Shahdara, Delhi: New India
make 100 mL. Publishing Agency
Wagner’s test:
 1. 50 mg of the 1 mL alkaloidal
plant extract is solution + 1 mL
stirred with Diluted HCl
reagent
diluted
hydrochloric acid Ppt.
and filtered.
2. Transfer a few
mL of filtrate to a
test tube and add
a few drops of the
wagner’s reagent.
3. Erdmann’s Erdmann’s Preparation of Morphine: Color reaction Welcher, F. (1942). Chemical
Test Reagent: Erdmann’s Reagent: Reddish solely dependent Solutions. Philidelphia, Pa:
HNO3 1. Add 1 mL of Papaverine: upon either the George S. Ferguson Co.
H2SO4 dilute nitric acid Violet dehydration or
Water to 60 mL of the oxidation of Kar, A. (2003). Pharmacognosy
concentrated the alkaloid and Pharmacobiotechnology.
sulphuric acid. Daryaganj, New Delhi: K.K.
Mix well. Gupta for New Age
International
4. Thin Layer Marqui’s Preparation of Yellow to Solubility and Kar, A. (2003). Pharmacognosy
Chromatograp Reagent: Marqui’s Reagent: Purple strength of and Pharmacobiotechnology.
hy: Marqui’s 40% 1. Two to Three Spots adsorption to the Daryaganj, New Delhi: K.K.
Test Formaldehyde drops of 40% adsorbent. Gupta for New Age
Solution formaldehyde International
Conc. H2SO4 solution is mixed
with 3 mL of Harborne, J.B. (1998).
n-butanol concentrated Phytochemical Methods: A
aqueous citric H2SO4 Guide to Modern Techniques of
acid Marqui’s Test: Plant Analysis. London, UK:
1. Chromatograph Chapman & Hall.
an aliquot on
sodium citrate Retrieved from
 buffered paper in http://orgchem.colorado.edu/
n-butanol aqueous hndbksupport/TLC/TLC.html.
citric acid. Retrieved August 14, 2010.
2. Chromatograph
another aliquot on
silica gel G plates
in methanol-conc.
NH4OH(200:3)
3. Detect the
presence of
alkaloids by
spraying Marqui’s
reagent.
4. Measure Rf Value
General Test

Alkaloids

Name of Test Chemicals Procedures Positive Principles References

Involved Results Involed
Culvenor-Fitzgerald Ammonial Preparation of Kar, A. (2003).
Method: Chloroform Ammonial Chloroform: Pharmacognosy and
Sulfuric Acid 1. Add 3.6 mL of 28% Pharmacobiotechnolog
ammonia to 1 liter y. Daryaganj, New
of Chloroform. Add Delhi: K.K. Gupta for
enough anhydrous New Age International
sodium sulphate to
take up the water. Welcher, F. (1942).
2. Filter or decant. Chemical Solutions.
Preparation of Sulfuric Philidelphia, Pa:
Acid: George S. Ferguson Co.
1. Add 2.8 mL of the
concentrated H2SO4 Guevara, B. (2005). A
to sufficient distilled Guidebook to Plant
water to make 100 Screening:
mL. Phytochemical and
Culvenor-Fitzgerald Biological. Manila:
Method: UST Publishing House
1. With the aid of a
mortar and pestle
and clean sand,
triturate a 2-4 g of
the fresh, finely cut
plant material with
sufficient
chloroform. Then
add 10ml of
ammoniacal
 chloroform and stir
the mixture. Filter
into test tubes,
pressing the
material to recover
the extract.
2. Add 1ml 1M
sulfuric acid, shake
well and let it stand.
Pipette off the clear
upper layer into two
small test tubes
3. Pipette of the clear
upper layer in two
test tubes
Preparation of Mayer’s
Reagent:
1. Mayer’s test Mayer’s 1. Dissolve 1.36 grams White Formation of
reagent: of Mercuric Precipitate microcrystalline
Mercuric Chloride and 3.00 complexes
Chloride grams Potassium (salts)
Potassium iodide in sufficient
Iodide water to make 100
mL.
Mayer’s test:
1. Get one of the test
tubes containing the
plant sample and
add 2-3 drops of the
mayer’s reagent

Preparation of
2. Dragendorffs’s Dragendroff’s Reagent:
 test Dragendorffs’s 1. Solution A: Orange Formation of
reagent: Dissolve 0.85 g Precipitate microcrystalline
Bismuth(III) bismuth (III) nitrate complexes
Nitrate in a mixture of 10 (salts)
Acetic Acid mL acetic acid and
Potassium 40 mL water
Iodide 2. Solution B:
Dissolve 8 g
potassium iodide in
20 mL water
3. Stock Solution: mix
equal parts of
Solution A and B.
4. Mix 1 mL of stock
solution with 2 mL
of acetic acid and 10
mL water
Dragendorff’s test:
1. Get one of the test
tubes containing the
plant sample and
add 2-3 drops of the
dragendorff’s
reagent

Laboratory Test tube Hydrochloric Preparation of 2M HCl: (Kar, 2003)

Method: Acid 1. Add 17 mL of (Guevara, 2005)
1. Preliminary Assay Dragendorff’s conc. HCl to
Reagent enough distilled
Mayer’s water to make 100
reagent mL solution
Preliminary Assay: (+) Slight
1. Take an equivalent Turbidity
 of 20g plant (+)(+)Definite Formation of
material from the Turbidity double salts of
stock plant extract. alkaloids and
2. Evaporate it to a (+)(+)(+) heavy metals.
syrupy consistency Heavy The resulting
over a steam bath. Precipitation double salts are
3. Add 5ml of 2M found to be
hydrochloric acid, Orange possessing
then heat with Precipitate with characteristics
stirring to about 5 Dragendorffs’s of
minutes and cool. reagent microcrystalline
Add 0.5g sodium structures
chloride then stir White
and filter. Precipitate with
4. Wash the residue Mayer’s
with enough 2M Reagent
HCl to bring the
filtrate to a volume
of 5ml.
5. Take 1ml of the
filtrate and test
with 2 to 3 drops of
Dragendorff’s
reagent.
6. Take another 1 ml
of the filtrate and
test with 2 to 3
drops of Mayer’s
reagent.
7. Record the relative
amount of
precipitation
observed
2. Confirmatory Test Ammonia Confirmatory Test: (+) slight Formation of
HCl 1. Add drop wise turbidity double salts of
Chloroform enough 28% (++) definite alkaloids and
Mayer’s ammonia to the turbidity heavy metals.
Reagent remaining 3ml of (+++) heavy The resulting
Dragendorff’s the filtrate used precipitation double salts are
reagent in the found to be
preliminary possessing
assay until Orange characteristics
solution is Precipitate with of
alkaline to a Dragendorffs’s microcrystalline
litmus paper. reagent structures
2. Extract the
alkaline solution White
three times with Precipitate with
small portions of Mayer’s
less than 10ml of Reagent
chloroform.
Then combine
the lower
chloroform
extracts and
reserve the upper
aqueous layer for
the test for
quaternary bases.
3. Evaporate the
chloroform
extracts to
dryness under
the hood and
over a steam bath
for about 2
 minutes then
cool.
4. Filter and divide
the filtrate into
two portions.
5. Test one portion
with Mayer’s
reagent and to
the other with
Dragendorff’s
reagent.
6. Record the
relative amount
of precipitation
observed.