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 Used for Recipe Comments

12.5 mL p-anisaldehyde
Requires heating
General purpose 5 mL AcOH
Range of colors on
p-Anisaldehyde (PAA) (especially 465 mL EtOH
light pink background
nucleophiles) 17.5 mL conc. H2SO4 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.

0.04 g Bromocresol green

Bromocresol Green
Typically doesn't
Acidic groups (pKa 100 mL EtOH
require heating
< 5) 0.1 M solution of NaOH
Bright yellow spots
Carboxylic acids (added until blue)
on blue background

5 g Ce(SO4)2 Requires heating

Ceric Ammonium 25 g (NH4)6Mo7O244H2O Dark blue spots on
Molybdate (CAM) - General Purpose 50 mL conc. H2SO4 white or light blue
Hanessian's Stain 450 mL H2O 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.

8 g Ce(SO4)22H2SO4 Black spots on

General purpose
Ceric Sulfate 100 mL 15% aq. H2SO4 yellow-white
(good for alkaloids)
background

Difficultly
Chromic acid (K2Cr2O7) 2.5 g potassium chromate (K2Cr2O7)
stainable
compounds 100 mL 20% aq. H2SO4
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.
 12 g 2,4-
Requires heating
dinitrophenylhydrazine
Yellow to red spots
2,4-Dinitrophenylhydrazine Aldehydes 60 mL conc. H2SO4
on light yellow
(DNP) Ketones 80 mL H2O
background
200 mL 95% EtOH
Long shelf life
To a cold (0 C) mixture of water (80 mL) and absolute EtOH (200 mL) was added 2,4-
dinitrophenylhydrazine (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.

Solution A:
1.7 g Bi5O(OH)9(NO3)4
80 mL H2O
Amines 20 mL AcOH
Dragendorff Organic base 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
Dragendorff-Munier Amines 20 g Tartaric acid
120 mL H2O

0.5 g dimethylaminobenzaldehyde
Ehrlich Reagent Amines 10 mL conc. H2SO4
90 mL 95% EtOH

1 g FeCl3
50 mL MeOH
Ferric chloride Phenols
50 mL H2O

Doesn't require heating

A few Iodine
Unsaturated and Orange to brown spots on light
crystals
Iodine (I2) aromatic
Silica Gel
orange background
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.

0.1 g C15H10O7xH2O
Morin hydrate General Purpose
100 mL MeOH
 0.3 g Ninhydrin
Amino acids
Ninhydrin Amines 3 mL conc. H2SO4
100 mL n-butanol

7g
Requires heating
phosphomolybdic
Phosphomolybdic Dark green/black spots on light
General purpose acid
acid (PMA) green background
100 mL EtOH
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.

3 g KMnO4 Alkenes/alkynes/aromatics usually

stain without heating
Potassium Olefins 20 g K2CO3
Other oxidizable groups require
Permanganate Readily oxidized 5 mL 5% aq NaOH
heating
(KMnO4) groups 300 mL H2O 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.

15 g vanillin
Requires heating
General 250 mL EtOH
Vanillin purpose 2.5 mL conc. H2SO4
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.