No

Group tests
Akaloids
Dragendoffs test

Procedure
0.2 g sample diluted in 2% H2SO4 warmed (2 m) filtered filtrate +
Dragendoffs reagent orange red precipitate (+)
1 ml sample solution + 2 ml Dragendoffs reagent orange red precipitate (+)
Prepare reagent

Sources
Uddin, et al., 2011
Madppa and Bapaih, 2012

Solution A : Bismuth nitrate (0.17g) in AcOH (2 mL) and H 2O (8 mL), solution B: KI (4 g) in AcOH (10
mL) and H2O (20 mL) Mix Solutions A and B and dilute to 100 mL with H2O
We need 12 ml (2 sample x 3 replications x 2 ml = 12 ml) ~ 25 ml
Bismuth nitrate = 0.17 g / 4 = 0.425 g
Acetic acid = 12 ml / 4 = 3 ml
Potassium iodide = 4 g / 4 = 1 gram
Mayers test

Sample solution + Mayers reagent (Potassium Mercuric Iodide) yellow colored

precipitate (+)

Tiwari, et al., 2011

Prepare reagent
created by dissolving 1.358 grams of HgCl2 in 60 ml of water, and pouring that solution into a solution of 5
grams of KI in 10 ml of water

We need 25 ml
Mercuric(II) chloride = (1.358 g / 80 ml) x 25 ml = 0.485 g

Potassium iodide = (5 g / 80 ml) x 25 ml = 1.5625 ~ 2 g

Wagners test

Sample solution + Wagners reagent reddish brown precipitate/coloration (+)

Sample solution + 3-5 drops of Wagners reagent [1.27g of iodine and 2g of potassium
iodide in100 ml of water] reddish brown precipitate/coloration (+)

Tiwari, et al., 2011

Ugochukwu, et al., 2013

Prepare reagent
1.27g of iodine and 2g of potassium iodide in100 ml of water

Anthraquinones

We need 10 ml
Iodine = 1.27 g / 10 = 0.127 g
Potassium iodide = 2 g / 10 = 0.2 g
0.5 g sample boiled in 10% HCl (few m in water bath) filtered cooled Uddin, et al., 2011
filtrate + CHCl3 (equal volume) + few drops 10% NH3 heated rose pink

(+)

Antocyanin
3
4

Tannins
FeCl3 test

Gelatin test

Vanilin- hydrochloric
acid test

1 ml sample + 10 ml benzene filtered filtrate + 5 ml 10% NH 3 shaken Madppa and Bapaih, 2012
pinkish (+)
Prepare reagent
Benzene = 2 sample x 3 replications x 10 ml = 60 ml
Conc. NH3 (25%) = (30 ml x 10%) / 25% = 12 ml, volume of 10% NH3 = 30 ml (2 sample x 3 replications x 5 ml =
30 ml)
1 ml sample solution + 5 ml diluted HCl pink colour (+)
Madppa and Bapaih, 2012
Prepare reagent
1% HCl, 2 sample x 3 replications x 5 ml = 30 ml ~ 50 ml
Conc. HCl (37%) = (50 ml x 1%) / 37% = 1.35 ml
Sample (a small quantity) diluted in H2O heated in water bath filtered
filtrate + few drops FeCl3 dark green solution (+)
1 ml sample solution + 2 ml FeCl3 dark green solution (+)
Prepare reagent
5% FeCl3, 2 sample x 3 replications x 2 ml = 12 ml ~ 25 ml
Iron(III) chloride = (5/100) x 25 ml = 1.25 g
Iron(III) chloride hexahydrate = (1.25 g x 270.3) / 162.2 = 2.08 g
Sample solution + gelatin solution white precipitate (+)
Sample solution + 1% gelatin containing NaCl white precipitate (+)
Prepare reagent
1% gelatin, 25 ml
Gelatin = (1/100) x 25 ml = 0.25 g
Sodium chloride = a few ~ 1 g
Sample solution + vanillin-hydrochloric acid reagent a pink or
red color (+)
Prepare reagent
Vanillin 1 g, alcohol 10 mL, concentrated hydrochloric acid 10 mL
Vanillin = 1 g
Ethanol = 10 ml

Uddin, et al., 2011

Madppa and Bapaih, 2012

Bhandary, et al., 2012

Tiwari, et al., 2011

Devmurari, 2010

Conc. HCl = 10 ml
Cadiac glycosides
Lieberman buchard test
Keller Kellianis test

Modified Borntragers test

FeCl3 reagent

Saponins (foam test)

6
7

Flavonoids
Alkaline reagent test

Journal in Thai language

5 ml sample solution + 2 ml Acetic glacial + a drop FeCl 3 + 1 ml conc. H2SO4 (addition
acid should be carefully to keep the mixture in 2 layers) brown ring at the interface
indicated the presence of deoxysugar characteristic of cardenolides. A violet ring may
appear below the ring while in the acetic acid layer, a greenish ring may form
Prepare reagent
Acetic glacial = 2 sample x 3 replications x 2 ml = 12 ml
Conc. Sulphuric acid = 2 sample x 3 replications x 1 ml = 6 ml
FeCl3, use tannins FeCl3 test
Sample solution + diluted HCl heated + FeCl3 immersed in boiling water for 5
m + benzene (equal volume) benzene layer + NH3 solution rose pink (+)
Prepare reagent
Conc. HCl (37%), use antocyanin test
FeCl3, use tannins FeCl3 test
Benzene = 2 sample x 3 replications x 2 ml = 12 ml
1 ml sample solution + FeCl3 reagent (5% FeCl3 : Ac. Glacial = 1:99) + Few drops conc.
H2SO4 greenish blue colour (+)
Prepare reagent
Iron(III) chloride = (5/100) x 1 ml = 0.05 g
Acetic glacial = 99 ml
Sample solution + H2O shaken stable froth for 15 m (+)
1 ml sample solution + 2 ml H2O shaken allowed 10 m eternal foam (+)
2 ml sample solution + 6 ml H2O shaken persistent foam (+)
0.2 mg sample in 5 ml H2O shaken boiled Frothing (+)
Sample solution + few drops diluted NaOH yellow mixture + few drops diluted
HCl colorless (+)
2 ml sample solution + few drops 20% NaOH yellow mixture + diluted HCl
colorless (+)
1 ml sample solution + 2 ml diluted NaOH golden yellow (+)

Ugochukwu, et al., 2013

Tiwari, et al., 2011

Madppa and Bapaih, 2012

Bhandary, et al., 2012

Madppa and Bapaih, 2012
Ugochukwu, et al., 2013
Uddin, et al., 2011
Uddin, et al., 2011 and
Bhandary, et al., 2012
Ugochukwu, et al., 2013
Madppa and Bapaih, 2012

FeCl3 test

Pb-Ac test

Steroids
Lieberman Burchard test

8
Salkowski test

Phenol
Phenol-flavonoids (Pb-Ac)

FeCl3 test

sample solution + few drops NaOH solution yellow + HCl solution colorless
(+)
Prepare sample
Sodium hydroxide = (20/100) x 10 ml = 2 g, Volume of 20% NaOH = 10 ml
Conc. HCl (37%), use antocyanin test
Sample solution + few drops FeCl3 blackish red (+)
Prepare reagent
FeCl3, use tannins FeCl3 test
Sample solution + Pb-Acetate yellow precipitate (+)
Sample solution + few drops 10% Pb-Acetate yellow precipitate (+)
Prepare sample
Lead acetate = (10/100) x 10 ml = 1 g, Volume of 10% Pb-Acetate = 10 ml

Khanam, et al. 2013

Bhandary, et al., 2012

Tiwari, et al., 2011

Bhandary, et al., 2012

Sample + acetic anhydride boiled cooled mixture + conc. H2SO4 (addition acid Bhandary, et al., 2012
should be carefully to keep the mixture in 2 layers) brown ring at the junction of two
layers, green coloration of the upper layer and the formation of deep red color in the
lower layer (+)
0.5 g sample + 2ml H2SO4 + 2 ml acetic anhydride violet to blue/green (+)
Uddin, et al., 2011
Prepare reagent
Acetic anhydride = (2 sample x 3 replications x 1 ml) = 6 ml
Conc. Sulphuric acid = few ~ 3 ml
Mixture of 1 ml sample solution and 10 ml CHCl 3 + 10 ml conc. H2SO4 (addition acid Madppa and Bapaih, 2012
should be carefully to keep the mixture in 2 layers) upper layer, red and H2SO4 layer,
yellowish green (+)
Prepare reagent
Look terpenoids test
1 ml sample solution + 2 ml 10 % Pb-Acetate brown precipitate (+)
Prepare reagent
Look Flavonoids test
Sample solution + 5% FeCl3 blue or black color (+)
Sample solution + FeCl3 bluish black color (+)

Madppa and Bapaih, 2012

Ugochukwu, et al., 2013

Tiwari, et al., 2011

Folin Ciocalteu
Terpenoids
(Salkowski test)

10

5 ml of 10 mg/ml sample solution + few drops 5% FeCl3 Bluish black color (+)
Prepare reagent
FeCl3, use tannins FeCl3 test

Khanam, et al. 2013

0.2 g sample diluted in 2 ml CHCl 3 mixture + 3 ml conc. H 2SO4 (addition acid

should be carefully to keep the mixture in 2 layers) reddish brown at interface (+)
1 ml sample solution + 3 ml CHCl 3 mixture + few drops conc. H2SO4 (addition acid
should be carefully to keep the mixture in 2 layers) red colour (+)
2 ml sample solution + 1 ml CHCl3 + few drops conc. H2SO4 dark pink/red color (+)
0.5 mg extract + few ml of CHCl3 + few ml of conc. H2SO4 (addition acid should be
carefully to keep the mixture in 2 layers) Reddish brown color at interface (+)
Prepare reagent
Chloroform = 2 samples x 3 replications x 2 ml = 12 ml
Conc. Shulpuric acid = 2 samples x 3 replications x 3 ml = 18 ml

Uddin, et al., 2011

Madppa and Bapaih, 2012
Ugochukwu, et al., 2013
Khanam, et al. 2013