Reagen Penampak Noda KLT

Bagi mereka yang bekerja dalam sintesis organik dan ataupun isolasi senyawa-senyawa metabolit
sekunder bahan alam sangat terbantu sekali dengan kromatografi lapis tipis (KLT). Namun demikian
terkadang noda KLT tidak semuanya terlihat dengan lampu uv (254; 366 nm). Maka reagen penampak
noda dibawah sangat bermanfaat.
Stain

p-Anisaldehyde (PAA)

Used for

General purpose
(especially
nucleophiles)

Recipe

Comments

12.5 mL p-anisaldehyde
5 mL AcOH
465 mL EtOH
17.5 mL conc. H2SO4

Requires heating
Range of colors on
light pink background
Medium shelf life

To a cold (0 C) mixture of AcOH (5 mL) and absolute EtOH (465 mL) was added p-anisaldehyde (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.

Bromocresol Green

Acidic groups (pKa

< 5)
Carboxylic acids

0.04 g Bromocresol green

Typically doesn't
100 mL EtOH
require heating
0.1 M solution of NaOH

(added until blue)

Bright yellow spots

on blue background

5 g Ce(SO4)2
25 g (NH4)6Mo7O244H2O
50 mL conc. H2SO4
450 mL H2O

Requires heating
Dark blue spots on
General Purpose
white or light blue
background
Very long shelf life
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.

Ceric Ammonium
Molybdate (CAM) Hanessian's Stain

Ceric Sulfate

8 g Ce(SO4)22H2SO4
General purpose
100 mL 15% aq. H2SO4
(good for alkaloids)

Black spots on
yellow-white
background

Difficultly
2.5 g potassium chromate (K2Cr2O7)
stainable
100 mL 20% aq. H2SO4
compounds
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.

Chromic acid (K2Cr2O7)

 12 g 2,4-

dinitrophenylhydrazine
60 mL conc. H2SO4
80 mL H2O
200 mL 95% EtOH

2,4-Dinitrophenylhydrazine Aldehydes
Ketones
(DNP)

Requires heating
Yellow to red spots
on light yellow
background
Long shelf life

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.

Dragendorff

Amines
Organic base

Dragendorff-Munier

Amines

Ehrlich Reagent

Amines

Ferric chloride

Solution A:
1.7 g Bi5O(OH)9(NO3)4
80 mL H2O
20 mL AcOH
Solution B:
40 g KI
100 mL H2O
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 dimethylaminobenzaldehyde
10 mL conc. H2SO4
90 mL 95% EtOH
1 g FeCl3
50 mL MeOH
50 mL H2O

Phenols

Doesn't require heating

Unsaturated and
Orange to brown spots on light
aromatic
orange background
Iodine (I2)

compounds
Spots fade away very rapidly
Very long shelf life
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.

Morin hydrate

General Purpose

A few Iodine
crystals
Silica Gel

0.1 g C15H10O7xH2O
100 mL MeOH

 0.3 g Ninhydrin
3 mL conc. H2SO4
100 mL n-butanol

Ninhydrin

Amino acids
Amines

Phosphomolybdic
acid (PMA)

7g
phosphomolybdic
acid
General purpose
100 mL EtOH

Requires heating
Dark green/black spots on light
green background
Long shelf life

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.
Alkenes/alkynes/aromatics usually
stain without heating
Potassium
Other oxidizable groups require
Permanganate
heating
(KMnO4)
Yellow spots on purple background
Very long shelf life
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.

Olefins
Readily oxidized
groups

Vanillin

General
purpose

3 g KMnO4
20 g K2CO3
5 mL 5% aq NaOH
300 mL H2O

15 g vanillin
250 mL EtOH
2.5 mL conc. H2SO4

Requires heating
Range of colors
Medium shelf life

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.