Thin Layer Chromatography

For Detection Group

Phytochemistry
Antonius Padua Ratu, M.Farm., Apt.
STTIF Bogor

TLC/KLT 1
 TLC of Alkaloid
Solvent system:
Toluene-ethyl acetate-diethylamine (70:20:10),
is suitable for the major alkaloids of most
drugs.
Stationary phase:
The principal alkaloids of the most common
alkaloid drugs can be identified by Silica gel
60F254 precoated TLC plates Adsorbent.
TLC/KLT 2
Detection of Alkaloid:
❑ UV-254nm some alkaloid types such as
indoles, quinolines, isoquinolines,
purines.
❑ UV-365 nm
❑ Blue, blue-green or violet fluorescence
of alkaloids,e.g: Boldo folium.
❑ Yellow fluorescence, e.g. colchicine.
TLC/KLT 3
 TLC of Flavanoids
Solvent System:
Different solvent system can be used,
ethyl acetate-formic acid-glacial acetic acid-water
(100:11:11:26 ) or formic acid - water – ethyl acetate
mixed in different proportion with or without ethyl methyl
ketone are suitable for the TLC screening of polar
flavonoids glycosides.
For less polar flavonoids aglycones we would use a
mobile phase composed of
Toluene - ethyl formiate -formic acid (50:40:10 ) or
Toluene - dioxane - glacial acetic acid (90:25:4 ).
Stationary phase: Silica gel,polyamide
TLC/KLT 4
Detection of Flavanoids:
The solvent must be thoroughly removed from silica gel
layer before detection UV-254 nm
All flavonoids cause fluorescence. UV-365 nm , Depending
on the structure type, flavonoids shows dark yellow,
green or blue fluorescence, which is intensified and
changed by the use of various spray reagent.
Spray Reagents Fast Blue Salt reagent (FBS)
Detection of phenolic compounds.
Natural products reagents (NP/PEG)
The plate is sprayed with 1% methanolic diphenylboric
acid, β - ethylamino ester (diphenylboryloxyethylamine ,
NP), followed by 5% ethanolic polyethylene glycone-4000
TLC/KLT 5

spray.
 TLC of Anthracene Derivatives
❑ Solvent System:
Aloin, frangulin A/B, glucofrangulin A/B, rhein, aloe-
emodin and rliaponticoside are applied as 0.1%
methanolic solutions.
Solutions Sennasides A and B are prepared as a 0.1%
solution in methanol-water (1:1).
A total of 10 L of each reference solution is used for
TLC.
❑ Stationary Phase:
Chromatography is performed on silica gel 60 F254
TLC/KLT 6
precoated
Detection Anthracene Derivatives:
• UV 254 nm All anthracene derivatives
quench fluorescence.
• UV 365 nm All anthracene derivatives give
yellow or red-brown fluorescence.
Spray reagents:
Potassium hydroxide
After spraying with 5% or 10% ethanolic KOH,
anthraquinones appear red in the visible and
show red fluorescence in UV-365 nm.
TLC/KLT 7
 TLC of Cardiac Glycoside
❑ Solvent System:
▪ Ethyl acetate-methanol-water (100:13.5:10)
solvents.
▪ A generally applicable solvent system for
cardiac glycosides Ethyl acetate-methanol-
ethanol- water (81 : 11 :4: 8).
▪ The addition of ethanol increases the Rf
values of strongly polar compounds.
❑ Stationary System:
Adsorbent Silica gel 60 F254 precoated.
TLC/KLT 8
Detection of Cardiac Glycoside :
Without chemical treatment UV-254 nm very weak
fluorescence quenching of all cardiac glycosides UV-
365 nm no fluorescence at all.
Spray reagents:
Specific detection of the -lactone ring of
cardenolides: Kedde reagent Immediately on
spraying, cardenolides generate a pink or blue-violet
(vis) colour.
The colour fades after a few minutes, but can be
regained by repeated spraying.
Raymond reagent also give red, red-orange or violet
9
(vis) cardenolide-specifics colors. TLC/KLT
 TLC of Coumarin
Solvent System:
For coumarin Aglycones solvent :
Toluene-ether
(1:1, saturated with 10% acetic acid)
For glycosides :
Ethyl acetate-fortnic acid-glacial acetic acid-water
(100:11:11:26).

Stationary Phase:
Adsorbent Silica gel 60 F254 precoated TLC plates.
TLC/KLT 10
Detection of Coumarin :
• UV-254 nm distinct fluorescence quenching of all
coumarins.
• UV-365 nm run intense blue or blue-green fluorescence
(simple coumarins) yellow, brown, blue or blue-green
fluorescence (furano and pyranocoumarins).
• The non-substituted coumarin fluoresces yellow-green
in UV-365 nm only after trearment with KOH- reagent or
ammonia vapour.
Spray reagents:
The fluorescence of the coumarins are intensified by spraying
with 5%-10% ethanolic KOH. Concentrated ammonia vapour
has the same effect. TLC/KLT 11
 TLC of Saponin
Solvent System:
The solvent which is suitable for separation of the
saponin mixtures
Chloroform-glacial acetic acid- methanol-water
( 64:32:12:8) solvents.
Chloroform-methanol-water (70:30:4) ginsenosides
(Ginseng radix).
Stationary Phase:
Adsorbent Silica gel 60 F254 precoated TLCplates.
TLC/KLT 12
Detection of Saponin:
Without chemical treatment With the exception of
glycyrrhizin and glycyrrhetic acid (Liquiritiae
radix),no saponins are detectable by exposure to
UV-254 or UV-365 nm.
Spray reagents:
Hemolytically active saponins are detected as white
zones on a reddish background.
Hemolysis may occur immediately, after allowing the
TLC plate to stand or after drying the plate in a warm
airstream. TLC/KLT 13
 TLC of Triterpenes

Solvent System:
Ethyl formiate-toluene-formic acid (50:50: 15)
Toluene-chloroform-ethanol (40:40:10)

Stationary Phase:
Silica gel 60 F254 precoated plates

TLC/KLT 14
Detection of Triterpenes:
• UV-254 nm calfeic acid, its derivatives and
isoflavones show quenching.
• UV-365 nm caffeic acid, its derivatives and
isoflavones fluorescence blue.
Spray Reagents:
Anisaldehyde-sulphuric acid reagent Tile sprayed
TLC is heated for 6 min at 100°C; evaluation in vis.:
triterpenes blue-violet (Cimicifugae rhizoma) and
red to red-violet (Ononidis radix).
TLC/KLT 15
 TLC of Lignans

Solvent System:
Chloroform-methanol-water (70:30:4)
Chloroform-methanol( 90:10)
Stationary Phase:
Silica gel 60 F254 -precoated plates

TLC/KLT 16
Detection of Lignans:
• UV-254 nm all lignans show prominent
quenching.
• UV-365 nm e.g. eleutheroside E, gives
blue fluorescence.
Spray reagents:
50% ethanolic sulphuric acid for Cubebae
fructus Vanillin-phosphoric acid reagent for
Eleutherococci radix.
TLC/KLT 17
 TLC of Essential Oil

Solvent System:
Toluene-ethyl acetate (93:7).
This system is suitable for the analysis
and comparison of all important essential
oils.
Stationary Phase:
Silica gel 60 F254 precoated TLC plates.
TLC/KLT 18
Detection of essential oil:
• Without chemical treatment UV-254nm Compounds
containing at least two conjugated doulble bonds quench
fluorescence and appear as dark zones against the light-
green fluorescent background of the TLC plate.
• UV-365 nm No characteristic Fluorescence of terpenoids
and propylphenols is noticed.
Spray reagents:
Anisaldehyde-sulphuric acid 10 min/110°C;
Evaluation in vis.: essential oil compounds show strong
blue, green, red and brown colouration.
Most of the compounds develop fluorescence under UV-365
nm. TLC/KLT 19
THANK YOU

TLC/KLT 20