TLC Stains
The following table lists TLC stains, along with their general usage, the components and a few
notes on the stains. For detailed preparation procedures along with example TLCs scroll to the
bottom.
Stain
Used for
Recipe

p-Anisaldehyde
(PAA)
General
purpose
(especially
nucleophile
s)
Bromocresol Green
Acidic
groups (pKa
< 5)
Carboxylic
acids

General
Purpose


Ceric Ammonium
Molybdate (CAM)







Ceric Ammonium
Sulfate
Vinca
alkaloids


Ceric Sulfate
Chromic acid
2,4Dinitrophenylhydrazi
ne (DNP)
12.5 mL panisaldehyde
5 mL AcOH
465 mL EtOH
17.5 mL conc. H2SO4
Requires heating
Range of colors on light
pink background
Medium shelf life
0.04 g Bromocresol
green
100 mL EtOH
0.1 M solution of
NaOH (added until
blue)
Typically doesn't require
heating
Bright yellow spots on
blue background
5 g Ce(SO4)2
25 g
(NH4)6Mo7O24·4H2O
50 mL conc. H2SO4
450 mL H2O
Requires heating
Dark blue spots on white
or light blue background
Very long shelf life
1g
(NH4)4Ce(SO4)4·2H2O
100 mL 50% H3PO4
General
purpose
(good for
alkaloids)
Difficultly
stainable
compounds


8 g Ce(SO4)2·2H2SO4
100 mL 15% aq.
H2SO4

2.5 g potassium
chromate (K2Cr2O7)
100 mL 20% aq.
H2SO4
Aldehydes
Ketones



Comments
12 g 2,4dinitrophenylhydrazin
e
60 mL conc. H2SO4
Black spots on yellowwhite background
Requires heating
Yellow to red spots on
light yellow background
Long shelf life
Dragendorff
Amines
Organic
base


80 mL H2O
200 mL 95% EtOH



1.7 g
Bi5O(OH)9(NO3)4
80 mL H2O
20 mL AcOH


40 g KI
100 mL H2O
A:
B:
Dragendorff-Munier
Amines
Ehrlich Reagent
Amines
5 mL A + 5 mL B in 20 mL
AcOH + 70 mL H2O
 10 g KI
 1.5 g Bi(NO3)3
 20 g Tartaric acid
 120 mL H2O



0.5 g
dimethylaminobenzald
ehyde
10 mL conc. H2SO4
90 mL 95% EtOH
Ferric chloride
Phenols



1 g FeCl3
50 mL MeOH
50 mL H2O
Iodine
Unsaturated
and
aromatic
compounds


A few Iodine crystals
Silica Gel
Morin hydrate
General
Purpose


0.1 g C15H10O7·xH2O
100 mL MeOH
Ninhydrin
Amino
acids
Amines



0.3 g Ninhydrin
3 mL conc. H2SO4
100 mL n-butanol
Doesn't require heating
Orange to brown spots
on light orange
background
Spots fade away very
rapidly
Very long shelf life
Phosphomolybdic
acid (PMA)
General
purpose


7 g phosphomolybdic
acid
100 mL EtOH
Potassium
Permanganate
(KMnO4)
Olefins
Readily
oxidized
groups




3 g KMnO4
20 g K2CO3
5 mL 5% aq NaOH
300 mL H2O
Vanillin
General
purpose



15 g vanillin
250 mL EtOH
2.5 mL conc. H2SO4
Requires heating
Dark green/black spots
on light green
background
Long shelf life
Alkenes/alkynes/aromati
cs usually stain without
heating
Other oxidizable groups
require heating
Yellow spots on purple
background
Very long shelf life
Requires heating
Range of colors
Medium shelf life
Procedures and examples
p-Anisaldehyde (PAA)
To a cold (0 °C) mixture of AcOH (5 mL) and absolute EtOH (465 mL) was added panisaldehyde (12.5 mL) followed by slow addition of sulfuric acid (17.5 mL). The resulting clear
solution was warmed to rt and used as is. The excess was stored in the fridge.
This stain is light and oxidation sensitive and will gradually turn pink/orange. The stain should
be kept in an aluminum-covered jar while in use, and the excess should be kept cold and in the
dark. Once the stain turns dark red, it should be discarded and made fresh again.
Ceric Ammonium Molybdate (CAM) - Hanessian's Stain
In a dark hood, ceric sulfate (5 g) was added to a cold (0 °C) clear colorless stirring solution of
ammonium molybdate (25 g) in water (450 mL). The resulting bright yellow cloudy solution was
vigorously stirred and sulfuric acid (50 mL) was slowly added over 90 min resulting in a very
exothermic reaction. The resulting clear gold solution was warmed to rt, poured in a jar for
immediate use and the excess was stored in the fridge.
This stain can be kept for months on the bench and in the fridge. Decoloration to very pale
yellow usually occurs but does not affect the stain.
Chromic acid (K2Cr2O7)
To a cold (0 °C) solution of sulfuric acid (100 mL, 20% v/v aq.) was slowly added potassium
chromate (2.5 g). The resulting clear bright red/orange solution was warmed to rt and used as is.
The excess was stored in the fridge.
2,4-Dinitrophenylhydrazine (DNP)
To a cold (0 °C) mixture of water (80 mL) and absolute EtOH (200 mL) was added 2,4dinitrophenylhydrazine (12 g). To the resulting cloudy bright orange solution was slowly added
sulfuric acid (60 mL) resulting in a clear orange solution that was warmed to rt and used as is.
The excess was stored in the fridge.
Iodine (I2)
In a mortar, grind a few crystals of iodine with some silica gel. Transfer to a jar with a plastic
screw cap.
Spots are visualized by leaving the plate in the chamber for a couple minutes until spots turn
brown. Once taken out of the chamber, spots will rapidly fade away.
This stain can be kept for months on the bench. Once it fades to white (by I2 sublimation) it
should be made fresh again.
Phosphomolybdic acid (PMA)
Phosphomolybdic acid (10 g) was added to cold (0 °C) absolute EtOH (100 g) and then warmed
to rt. The resulting cloudy yellow suspension was filtered over filter paper to give a clear bright
yellow solution that was used as is. The excess was stored in the fridge.
This stain can be kept for months on the bench and in the fridge. PMA stain will gradually turn
green but does not affect the stain.
Potassium Permanganate (KMnO4)
In a 1000 mL erlenmeyer flask was added sequentially water (600 mL), 5% aq. NaOH (10 mL)
and K2CO3 (40 g). Once the solution became clear, KMnO4 (6 g) was added resulting in a dark
purple solution. The solution was stirred 1 h at rt. The resulting purple solution was poured in a
jar for immediate use and the excess was stored in the fridge.
This stain can be kept for months on the bench and in the fridge.
Vanillin
To a cold (0 °C) clear colorless solution of vanillin (15 g) in absolute EtOH (250 mL) was
slowly added sulfuric acid (2.5 mL). The resulting clear solution was warmed to rt and used as is.
The excess was stored in the fridge.
This stain is light and oxidation sensitive and will gradually turn dark. The stain should be kept
in an aluminum-covered jar while in use, and the excess should be kept cold and in the dark.
Once the stain turns black, it should be discarded and made fresh again.
Example: TLC plates stained with CAM, PMA, DNP, Vanillin, KMnO4, p-anisaldehyde and I2