Thin Layer Chromatography

Authored by: J. Bruno Thomas , D. N. Svoronos Paris

Handbook of Basic Tables for Chemical Analysis Third Edition

Print publication date:  December  2010
Online publication date:  December  2010

Print ISBN: 9781420080421
eBook ISBN: 9781420080438
Adobe ISBN:

10.1201/b10385-4

 

Abstract

The following table contains the common solvents used in thin layer chromatography, with a measure of their “strengths” on silica gel and alumina. The solvent strength parameter, ε°, is defined as a relative energy of adsorption per unit area of standard adsorbent [13]. It is defined as zero on alumina when pentane is used as the solvent. This series is what was called the eluotropic series in the older literature. For convenience, the solvent viscosity is also provided. Note that the viscosity is tabulated in cP for the convenience of most users. This is equivalent to mPas in the SI convention. Additional data on these solvents may be found in the tables on high performance liquid chromatography.

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Thin Layer Chromatography

Strength of Common TLC Solvents

The following table contains the common solvents used in thin layer chromatography, with a measure of their “strengths” on silica gel and alumina. The solvent strength parameter, ε°, is defined as a relative energy of adsorption per unit area of standard adsorbent [13]. It is defined as zero on alumina when pentane is used as the solvent. This series is what was called the eluotropic series in the older literature. For convenience, the solvent viscosity is also provided. Note that the viscosity is tabulated in cP for the convenience of most users. This is equivalent to mPas in the SI convention. Additional data on these solvents may be found in the tables on high performance liquid chromatography.

References

Snyder, L. R. Principles of Adsorption Chromatography. New York: Marcel Dekker, 1968.
Willard, H. H. , L. L. Merritt , J. A. Dean , and F. A. Settle .Instrumental Methods of Analysis. 7th ed. New York, Belmont: Van Nostrand, 1988.
Hamilton, R. , and S. Hamilton .Thin Layer Chromatography. Chichester: John Wiley and Sons (on behalf of “Analytical Chemistry by Open Learning,” London), 1987.

  Strength of Common TLC Solvents

Solvent

ε° (Al2O3)

Viscosity cP, 20 °

ε° (SiO2)

Fluoroalkanes

–0.25

n-Hexane

0.00

0.23

0.00

n-Pentane

0.001

0.23

0.00

2,2,4-Trimethylpentane (isooctane)

0.01

0.54

n-Heptane

0.01

0.41

n-Decane

0.04

0.92

Cyclohexane

0.04

1.00

–0.05

Cyclopentane

0.05

0.47

Carbon disulfide

0.15

0.37

0.14

Tetrachloromethane (carbon tetrachloride)

0.18

0.97

1-Chloropentane (n-pentylchloride)

0.26

0.43

Diisopropyl ether

0.28

0.37

2-Chloropropane (isopropyl chloride)

0.29

0.33

Methylbenzene (toluene)

0.29

0.59

1-Chloropropane (n-propyl chloride)

0.30

0.35

Chlorobenzene

0.30

0.80

Benzene

0.32

0.65

0.25

Bromoethane (ethyl bromide)

0.37

0.41

Diethyl ether (ether)

0.38

0.23

0.38

Trichloromethane (chloroform)

0.40

0.57

Dichloromethane (methylene chloride)

0.42

0.44

Tetrahydrofuran

0.45

0.55

1,2-Dichloroethane

0.49

0.79

Butanone (methyl ethyl ketone)

0.51

0.43

Propanone (acetone)

0.56

0.32

0.47

1,4-Dioxane

0.56

1.54

0.49

Ethyl ethanoate (ethyl acetate)

0.58

0.45

0.38

Methyl ethanoate (methyl acetate)

0.60

0.37

1-Pentanol (n-pentanol)

0.61

4.1

Dimethyl sulfoxide (DMSO)

0.62

2.24

Aminobenzene (aniline)

0.62

4.4

Nitromethane

0.64

0.67

Cyanomethane (acetonitrile)

0.65

0.37

0.50

Pyridine

0.71

0.94

2-Propanol (isopropanol)

0.82

2.3

Ethanol

0.88

1.20

Methanol

0.95

0.60

Ethylene glycol

1.11

19.9

Ethanoic acid (acetic acid)

large

1.26

Water

large

1.00

Modification of The Activity of Alumina by Addition of Water

The following table describes five different activity grades of commercial alumina used in chromatography [13]. The activity grades are defined by the degree of adsorption of azobenzene (called azobenzene number) on the types of hydrated alumina. Those types are prepared by heating commercial alumina to redness, giving grade I, and then adding controlled amounts of water and allowing equilibration in a closed vessel. The azobenzene number decreases with the amount of water added. The Rf value is the ratio of distance traveled by the solute spot to that traveled by the solvent.

References

Randerath, K. Thin Layer Chromatography. New York: Verlag Chemie-Academic Press, Weinheim Bergstr., 1968.
Gordon, A. J. , and R. A. Ford .The Chemist’s Companion: A Handbook of Practical Data, Techniques, and References. New York: John Wiley and Sons, 1972.
Brockmann, H. , and H. Schodder . “Aluminum Oxide with Buffered Adsorptive Properties for Purposes of Chromatographic Adsorption.” Berichte der Deutschen Chemischen Gesellschaft 74B (1941): 73.

  Modification of the Activity of Alumina by Addition of Water

Water Added (wt/wt%)

Activity Grade

Azobenzene Number [maximum adsorption of azobenzene (10−5 mol/g)]

Rf (p-Aminoazobenzene)

0

I

26

0.00

3

II

21

0.13

6

III

18

0.25

10

IV

13

0.45

15

V

0

0.55

Stationary and Mobile Phases

The following table provides a comprehensive guide to the selection of thin layer chromatography media and solvents for a given chemical family. Mixed mobile phases are denoted with a slash, /, between components, and where available, the proportions are given. Among the references are several excellent texts [13,60], review articles [424], and original research papers and reports [25–59,61–98]. A table of abbreviations follows this section.

References

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  Stationary and Mobile Phases

Abbreviations/Solvent Table

Abbreviation

Solvent Name

Abbreviation

Solvent Name

Ac

acetone

Et2O

diethylether

Ace

acetate

Foram

amylformate

AcOH

acetic acid

HCl

hydrochloric acid

n-AmOH

n-amyl alcohol

H3BO3

boric acid

t-AmOH

t-amyl alcohol

Hex

hexane

AmSO4

ammonium sulfate

HForm

formic acid

i-BuAc

isobutylacetate

MeCl

methylene chloride

BuFor

n-butylformate

MeCN

acetonitrile

i-BuOH

isobutanol

MEK

methylethylketone

n-BuOH

n-butanol

MeOH

methanol

i-Bu2O

diisobutylether

NaAc

sodium acetate

CCl4

carbon tetrachloride

NH3

ammonia, aqueous

C2HO3

trichloroethene

Petet

petroleum ether

CHCl3

chloroform

Ph

phosphate

(CH2)6

cyclohexane

PhOH

phenol

C6H6

benzene

PrAc

propylacetate

n-C6H14

n-hexane

PrFor

propylformate

n-C7H16

n-heptane

Progl

propylene glycol

i-C8H18

isooctane

i-PrOH

isopropanol

(ClCH2)2

dichloroethane

n-PrOH

n-propanol

DEAE

diethyl aminoethyl

i-Pr2NH

diisopropylamine

Diox

dioxane

i-Pr2O

diisopropylether

DMF

dimethylformamide

Py

pyridine

EtFor

ethylformate

THF

tetrahydrofuran

EtOAc

ethylacetate

Tol

toluene

EtOH

ethanol

w

water

Et2NH

diethylamine

m-X

m-xylene

  Stationary and Mobile Phases

Family

Stationary Phase

Mobile Phase

Ref.

Adrenaline and derivatives

alumina (two dimensional)

C6H6/EtOAc (60:40) CHCl3/EtOH/Tol (90:6.5:3.1)

25

Adrenochromes

cellulose

AcOH(2 %)/w

26,27

Whatman #1 (descending)

AcOH(2 %)/w

26–29

Alcohols

silica gel (G-coated)

EtOAc/Hex

30

Alcohols, polyhydric

alumina or Kieselguhr (impregnated with polyamide) or silica gel

CHCl3/Tol/HForm or n-BuOH/NH3 or CHCl3

31

Aldehydes

silica gel (G-coated)

EtOAc/Hex

30

Aldehydes, 2,4-dinitro-phenylhydrazones

alumina

C6H6 or CHCl3 or Et2O or C6H6/Hex

3

alumina IB

MeCl or Tol/THF (4:1)

32

silica gel

Hex/EtOAc (4:1 or 3:2)

3

silica gel IB

MeCl or Tol/THF (4:1)

32

Alkaloids

alumina

i-BuOH/AcOH or i-BuOH/NH3 or i-PrOH/AcOH

33

alumina

3

cellulose (impregnated with formamide)

CHCl3 or EtOH or (CH2)6/CHCl3 (3:7)

3 34

paper (S&S #2043b) paper electrophoresis silica gel

C6H6/n-C7H16/CHCl3/Et2NH (6:5:1:0.02) n-BuOH/HCl(25 %)/w (100:26:39) i-BuOH/AcOH or i-BuOH/NH3 or i-PrOH/HOAc

35 3

C6H6/EtOH (9:1) or CHCl3/Ac/Et2NH (5:4:1)

Amides

Kieselguhr (adipic acid impregnated)

i-Pr2O/Petet/CCl4/HForm/w

36

i-Pr2O/Petet/CCl4/HForm/w

36

silica gel

Amines

alumina

Ac/n-C7H16 (1:1)

3

alumina G

i-BuAc or i-BuAc/AcOH

37

Keiselguhr G

Ac/w (99:1)

3

silica gel

EtOH (95 %)/NH3 (25 %) (4:1)

3

silica gel (aromatic only)

38

Amino acids

alumina

n-BuOH/AcOH/w (3:1:1) or Py/w

3

cellulose

n-BuOH/AcOH/w (4:1:1)

3

cellulose (two-dimensional)

n-BuOH/Ac/NH3/w (10:10:5:2) followed by i-PrOH/HForm/w (20:1:5)

3

silica gel

3

n-BuOH/AcOH/w (3 or 4:1:1) or PhOH/w (3:1) or n-PrOH/NH3 (34 %) (2:1)

Aminochromes

Whatman #1 (acid washed)

w or AcOH/w or MeOH/w or EtOH/w or n-BuOH/AcOH/w or i-PrOH/w

26,39

Barbiturates

silica gel

CHCl3/n-BuOH/NH3 (25 %) (14:8:1)

3

Benzophenones, hydroxy

alumina or cellulose or Kieselguhr (impregnated with adipic acid triethylene glycol polyester) or silica gel

HForm/m-X

40

Bile acids

silica gel

C6H6/Et2O (4:1) or Et2O/AcOH (99.6:0.4) or CHCl3/MeOH (9:1)

41

Caffeine

chromatography paper

n-BuOH/NH3 or n-BuOH/HForm

42

Carboxylic acids

Keiselguhr/polyethylene glycol

i-Bu2O/HForm/w (90:7:3)

43

Polyamide powder

i-Pr2O/Petet/CCl4/HForm/w (50:20:20:8:1) or MeCN/EtOAc/HForm or BuForm/EtOAc/HForm

43

silica gel (G-coated)

EtOH/NH3/THF

30

silica gel (CaSO4 impregnated)

n-PrOH/NH3 or EtOH/CHCl3/NH3

44

silica gel/polyethylene glycol M-1000

i-Pr2O/HForm/w (90:7:3)

45,46 43

Carboxylic acids, unsaturated

silica gel

CHCl3/MeOH

47

Catecholamines

alumina

48

boric acid gel (neutral pH)

HCl (0.025N)

48

Kieselguhr

phenylboronate

49

phosphocellulose

Dilute acids

50

Ph buffer (pH = 6.2)/EDTA

51

Catecholamines, dansyl derivatives

alumina (two dimensional) Amberlite IRC50

C6H6/EtOAc (60:40) or CHCl3/EtOH/Tol (90:6.5:3.5)

25 52

Catecholamines, o-methyl derivatives

silica gel (sodium borate impregnated)

53

Corticosteroids

silica gel

EtOH(5 %)/MeCl or EtOH/CHCl3

54,55

Coumarins

polyamide

MeOH/w (4:1 or 3:2)

3

silica gel G

Petet/EtOAc (2:1)

3

silica gel G (impregnated with NaAc)

Tol/EtFor/HForm (5:4:1)

3

EtOAc/Skellysolve B

3

silicic acid (starch bound)

Dicarboxylic acids

Kieselguhr/polyethylene glycol

i-Pr2O/HForm/w (90:7:3)

43

polyamide powder

i-Pr2O/Petet/CCl4/HForm/w (50:20:20:8:1) or MeCN/EtOAc/HForm (9:1:1) or BuFor/EtOAc/HForm (9:1:1)

43

polyamide Woelm DC powder

MeCN/PrFor/PrAc/HForm (45:45:10:10) or i-Pr2O/Petet/CCl4/HForm/w (50:20:20:8:1) or n-AmOH/CCl4/HFor (3:2:1)

56

silica gel

i-Pr2O/HForm/w (90:7:3)

43

silica gel (G-coated)

EtOH/NH3/THF

30

Diols (see Alcohols, polyhydric)

Disulfides

alumina

Hex

57

Disulfides, 3,5-dinitro-benzoates

Whatman #3 (impregnated with 10 % paraffin oil in cyclohexane), (CH2)6

DMF/MeOH/w or Foram/MeOH/w

58

Flavinoids

paper

n-BuOH/AcOH/w or EtOAc/w or AcOH/w or C6H6/AcOH/w

60

polyamide

3

silica gel

MeOH/H2O

59

silica gel (impregnated with NaAc)

C6H6/Py/AcOH (36:9:5) Petet/EtOAc (2:1)

3 3

silicic acid (starch bound)

Tol/EtForm/HFor (5:4:1) EtOAc/Skellysolve B

Glycerides

silica gel G

CHCl3/C6H6 (7:3)

3

silica gel G (impregnated with silver nitrate)

CHCl3/AcOH (99.5:0.5)

3

Glycolipids

silica gel G

n-PrOH/NH3 (12 %) (4:1)

3

Glycols, polyethylene

paper

n-PrOH/EtOAc/w (7:1:2) or n-BuOH/AcOH/w (4:1:5) or t-AmOH/n- PrOH/w (8:2:3) or EtOAc/AcOH/w (9:2:2)

60

silica gel

Ac or n-BuOH/AcOH/w

61

Hydroxamates

silica gel

i-Pr2O or i-Pr2O/EtOAc (1:4) or i-Pr2O/i-C8H18

62

Hydroxamic acids

Kieselguhr G (impregnated with diethy-lene glycol or triethylene glycol adipate polyesters)

i-Pr2O/Petet/CCl4/HForm/w (50:20:20:8:1)

98

Indoles

acetylated (ascending)

CHCl3/MeOH/w (10:10:6)

63

cellulose (thin-layer)

w or HCl (0.005N) or n-BuOH/AcOH/w (12:3:5) or C6H6/AcOH/w (125:72:3)

64

α-Ketoacids

silica gel (CaSO4 impregnated)

EtOH/CHCl3/NH3

44

Ketones, 2,4-dinitro-phenyl hydrazones

alumina IB silica Gel IB

MeCl or Tol/THF (4:1) MeCl or Tol/THF (4:1)

32 32

Lactams

silica gel

i-Pr2O or i-Pr2O/EtOAc (1:4) or i-Bu2O/i-C8H18

62

Lactones

silica gel

i-Pr2O or i-Pr2O/EtOAc (1:4) or i-Bu2O/i-C8C18

62

Lipids

alumina

Petet/Et2O (95:5)

3

silica gel G

Petet/Et2O/AcOH (90:10:1)

3

silicic acid

CHCl3/MeOH/w (80:25:3)

3

Mercaptans (see Thiols)

Nitrosamines

silica gel

Hex/Et2O/MeCl

65

Kieselgel

MeCl/Hex/Et2O (2:3:4) (aliphatic, aromatic); MeCl/Hex/Et2O (5:7:10) (cyclic)

66

Nucleotides

cellulose cellulose (on DEAE)

AmSO4 (sat’d)/NaAc(1 M)/i-PrOH (80:18:2) HCl (aq)

3 3

Oximes

silica gel G

C6H6/EtOAc or C6H6/MeOH (abs)

67

Peroxides

silicone filter paper

w/EtOH/CHCl3

68

Phenols

alumina

Et2O

3

alumina/AcOH

C6H6

3

silica gel A

CHCl3/AcOH (5:1) or CHCl3/Ac/AcOH (10:2:1) or C6H6/AcOH (5:1) or Petet (80 °)/CCl4/AcOH (4:6:1) or CHCl3/Ac/Et2NH (4:2:0.2)

69

silica gel G

C6H6/Diox/AcOH (90:25:4)

3

silica gel/oxalic acid

C6H6

70

silica gel/potassium carbonate

MeCl/EtOAc/Et2NH (92:5:3 or 93:5:2)

70

Phosphates, esters

alumina Kieselgel

Hex/C6H6/MeOH (2:1:1) or Hex/MeOH/Et2O

71 71

Hex/C6H6/MeOH (2:1:1) or Hex/MeOH/Et2O

Phospholipids

silica gel G

CHCl3/MeOH/w

3

Polynuclear aromatics

alumina

CCl4

3

alumina

C6H/(CH2)6 (15:85)

72

silica gel

Hex or CH3CHCl2 or C2HCl3 or CCl4

73,74

Polypeptides

Sephadex G-25

w or NH3 (0.05 M)

3

silica gel G

CHCl3/MeOH (9:1) or CHCl3/Ac (9:1)

3

Pyridines

Whatman #1 (descending)

n-BuOH/w or n-BuOH/w/NH3 or Ac or i-BuOH/w or MEK/AcOH/w

75

Pyridines, quaternary salts (descending)

Whatman #1

Ac/w or AmSO4/Ph buffer (pH = 6.8)/n-PrOH(2 %) or n-PrOH

75

Purines

silica gel

Ac/CHCl3/n-BuOH/NH3 (25 %) (3:3:4:1)

3

Pyrrole, tri-carboxylic acid

silica gel

n-BuOH/EtOH/NH3/w (10:10:1:1)

64

Skatoles, hydroxy

silica gel G

i-Pr2O or (ClCH2)2/i-Pr2NH (6:1)

76

Steroids

alumina

CHCl3EtOH (96:4)

3

paper paper (impregnated with kerosene)

Petet/Tol/MeOH/w or Petet/C6H6/MeOH/w n-PrOH/w

60 77 78,79

silica gel G

EtOAc/(CH2)6/EtOH(abs) or EtOAc/(CH2)6 or CHCl3/EtOH (abs) or C6H6/EtOH or n-C6H14/EtOAc or EtOAc/n-C6H14/EtOH(abs)/AcOH or EtOAc/n-C6H14/AcOH

80,81

Sugars

cellulose

n-BuOH/Py/w (6:4:3) or EtOAc/Py/w (2:1:2)

3

Kieselguhr G (buffered with 0.02N NaAc)

EtOAc/i-PrOH/w

3

silica gel (buffered with H3BO3)

C6H6/AcOH/MeOH (1:1:3)

3

silica gel (impregnated with sodium bisulfite)

EtOAc/AcOH/MeOH/w (6:1:5:1) or n-PrOH/w (85:15) or i-PrOH/EtOAc/w (7:1:2) or MEK/AcOH/w (6:1:3)

3

silica gel G

n-PrOH/conc NH3/w (6:2:1)

3

Whatman #1 (descending-two dimensional)

PhOH or n-BuOH/AcOH

82

Sugars, aldoses

paper

EtOAc/Py/w (2:1:2) or n-BuOH/AcOH/w (4:1:5) or n-BuOH/EtOH/H2O (5:1:4) or EtOAc/AcOH/w (9:2:2) or EtOAc/AcOH/HForm/w or EtOAc/Py/NaAc (sat’d)

60

Whatman #1

PhOH or n-BuOH/AcOH

82

Sugars, carbamates

silica gel

n-BuOH/H3BO3 (0.03 M) (9:1)

Sugars, deoxy

Whatman #1

PhOH or n-BuOH/AcOH

82

Sugars, ketoses

paper

EtOAc/Py/w (2:1:2) or n-BuOH/AcOH/w (4:1:5) or n-BuOH/EtOH/H2O (5:1:4) or EtOAc/AcOH/w (9:2:2) or w/PhOH (pH = 5.5)

60

Whatman #1

PhOH or n-BuOH/AcOH

82

Sulfides

alumina

Hex

75

alumina

CHCl3/MeOH

96

silica gel

CCl4 or C6H6

83

silica gel DF-5

Ac/C6H6 or Tol/EtOAc

95

Sulfilimines, p-nitrosobenzene sulfonyl

Whatman #4 (impregnated with formamide)

C6H6 or C6H6/(CH2)6

84

Sulfonamides

Kieselguhr silica gel

CHCl3/MeOH (9:1) or CHCl3/MeOH/NH3

85 86

silica gel (neutral)

Et2O or CHCl3/MeOH (10:1)

87

silica gel (G)

n-BuOH/MeOH/Ac/Et2NH (9:1:1:1) CHCl3/EtOH/n-C7H16

3

Sulfones

alumina

Et2O or Hex/Ac (1:1)

57

silica gel DF-5

Ac/C6H6 or Tol/EtOAc

95

Sulfones, esters

alumina

Et2O or Hex/Ac (1:1)

57

Sulfones, hydroxyethyl

alumina

Hex/w (1:3)

57

Sulfoxides

alumina

C6H6/Py (20:1) and Diox

88

alumina

Ac/CCl4 (1:4)

97

silica gel

Ac or EtOAc or CHCl3/Et2O

83

silica gel DF-5

Ac/C6H6 or Tol/EtOAc

95

Whatman #1

PhOH/w (8:3) or n-BuOH/AcOH/w (9:1:2.5)

89

Sulfoxides, hydroxy-ethyl

alumina

Et2O or Hex/Ac (1:1) or Hex/Et2O (1:3)

57

Terpenes

alumina

C6H6 or C6H6/Petet or C6H6/EtOH

3

silica gel G

i-Pr2O or i-Pr2O/Ac

3

silica gel/gypsum

CHCl3/C6H6 (1:1)

90

silicic acid (starch bond)

n-C6H14/EtOAc (85:15)

3

Thiobarbiturates

paper

n-AmOH/n-BuOH/25 % NH3 (2:2:1)

91

Thiolactones

silica gel

i-Pr2O or i-Pr2O/EtOAc (1:4) or i-Bu2O/i-C8H18

62

Thiols

alumina

Hex

57

alumina (activated)

AcOH/MeCN (3:1)

96

alumina (5 % cetane impregnated)

AcOH/MeCN (3:1) EtOAc or CHCl3

96 83

silica gel

Thiophenes

alumina G

Petet (40–60 °C)

92

silica gel

MeOH or C6H6/CHCl3 (9:1)

92

Thiophosphate, esters

Petet or C6H6/CHCl3 or Ac or EtOH or EtOAc or MeOH

93

Ureas

acetylated plates

CCl4/EtOAc/EtOH (100:5:2)

94

silica gel

CCl4/MeCl/EtOAc/HOAc (70:50:15:10)

94

Urethanes (See ureas)

Typical Stationary and Mobile Phase Systems Used in The Separation of Various Inorganic Ions

The following table lists a series of stationary and mobile systems that are used in the separation of various inorganic ions [18]. The list is far from detailed and the reader is advised to consult the given references for details.

References

Kirchner, J. G. Thin Layer Chromatography. 2nd ed. New York: Wiley-Interscience, 1978.
Bobbitt, J. M. Thin Layer Chromatography. New York: Reinhold, 1963.
Randerath, K. Thin Layer Chromatography. New York: Academic Press, 1963.
Randerath, K. Thin Layer Chromatography. 2nd ed. Weinheim: Verlag, Chemie, 1975.
Gagliardi, E. , and B. Brodar .Chromatographia 2 (1969): 267.
Gagliardi, E. , and B. Brodar .Chromatographia 3 (1970): 7.
Gagliardi, E. , and B. Brodar .Chromatographia 3 (1970): 320.
MacDonald, J. C. , ed.Inorganic Chromatographic Analysis. New York: John Wiley and Sons, 1985.

  Typical Stationary and Mobile Phase Systems Used in the Separation of Various Inorganic Ions

Stationary Phase

Mobile Phase

Solvent Ratio

Separated Ions

Silica Gel G

butanol/1.5N HCl/2,5-hehanedione

100:20:0.5

hydrogen sulfide group

Silica gel G

acetone/conc. HCl/2,5-hexanedione

100:1:0.5

ammonium sulfide group

Silica gel G

water sat’d ethyl acetate/tributyl phosphate

100:4

U, Ga, Al

Silica gel G

ethanol/acetic acid

100:1

alkali metals

Silica gel G

acetone/1-butanol/conc. NH4OH/water

65:25:10:5

halogens

Silica gel G

methanol/conc. NH4OH/10 % trichloroacetic acid/water

50:15:5:30

phosphates

Dowex 1-cellulose (1:1)

1 M aqueous sodium nitrate

halogens

Cellulose

HCl (or HBr)/alcohol mixtures

variable

Groups IA, IIA, IIIB, IVB, VB, VIB, transition metals

Cellulose

1-butanol/water/HCl

8:1:1

Fe, Al, Ga, Ti, In

Cellulose

acetic acid/pyridine/conc. HCl

80:6:20

ammonium sulfide group

DEAE cellulose

sodium azide/HCl

variable

Cd, Cu, Hg

Amberlite CG 400 and CG 120

HCl/HNO3

variable

Pb, Bi, Sn, Sb, Cu, Cr, Hg

Sat’d = Saturated.

Conc. = Concentrated.

Spray Reagents in Thin Layer Chromatography

The following table lists the most popular spray reagents needed to identify organic compounds on chromatographic plates. These reagents have been thoroughly covered in several books [13], and reviews [423]. Due to the aerosol nature of the spray and the chemical hazards associated with several of these chemicals, the use of a fume hood is highly recommended. The original references of the spray reagents are given in order to provide information about their results with individual compounds [24138]. A list and description of some complicated protocols follows this section of the chapter.

Note: 1γ = 1 μg/cm2 on a TLC plate.

References

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Bobbitt, J. M. “Visualization.” In Thin Layer Chromatography. New York: Reinhold, 1963.
Touchstone, J. C. “Visualization Procedures.” In Techniques and Application of Thin Layer Chromatography. New York: John Wiley and Sons, 1985.
Pataki, G. “Paper, Thin-Layer, and Electrochromatography of Aminoacids in Biological Material.” Z. Klin. Chem. 2 (1964): 129; Chemical Abstracts 64 (1966): 5425c.
Padley, F. B. Thin-Layer Chromatography of Lipids, Thin-Layer Chromatography, Proceedings Symposium, Rome 1963, 87 (Pub. 1964).
Honjo, M. “Thin-Layer Chromatography of Nucleic Acid Derivatives.” Kagaku No Ryoiki, Zokan 64 (1964): 1.
Kazumo, T. “Thin-Layer Chromatography of Bile Acids.” Kagaku No Ryoiki, Zokan 64 (1964): 19.
Nakazawa, Y. “Thin-Layer Chromatography of Compound Lipids.” Kagaku No Ryoiki, Zokan 64 (1964): 31.
Nishikaze, O. “Separation and Quantitative Analysis of Adrenocortical Hormone and Its Metabolite (C21) by Thin-Layer Chromatography.” Kagaku No Ryoiki, Zokan 64 (1964): 37.
Shikita, M. , H. Kazikazi , and B. Tamaoki . “Thin-Layer Chromatography of Radioactive Substances.” Kagaku No Ryoiki, Zokan 64 (1964): 45.
Mo, I. , and Y. Hashimoto . “Method of Thin-Layer Zone Electrophoresis.” Kagaku No Ryoiki, Zokan 64 (1964): 61.
Kinoshita, S. “Thin-Layer Chromatography of Sugar Esters.” Kagaku No Ryoiki, Zokan 64 (1964): 79.
Okada, M. “Thin-Layer Chromatography of Cardiotonic Glycosides.” Kagaku No Ryoiki, Zokan 64 (1964): 103.
Omoto, T. “Thin-Layer Chromatography of Toad Toxin.” Kagaku No Ryoiki, Zokan 64 (1964): 115.
Furnya, C. , and H. Itokawa . “Thin-Layer Chromatography of Triterpenoids.” Kagaku No Ryoiki, Zokan 64 (1964): 123.
Zenda, H. “Thin-Layer Chromatography of Aconitine-Type Alkaloids.” Kagaku No Ryoiki, Zokan 64 (1964): 133.
Hara, S. , and H. Tanaka . “Thin-Layer Chromatography of Mixed Pharmaceutical Preparations.” Kagaku No Ryoiki, Zokan 64 (1964): 141.
Katsui, G. “Thin-Layer Chromatography of Vitamins.” Kagaku No Ryoiki, Zokan 64 (1964): 157.
Fujii, S. , and M. Kamikura . “Thin-Layer Chromatography of Pigments.” Kagaku No Ryoiki, Zokan 64 (1964): 173.
Hosogai, Y. “Thin-Layer Chromatography of Organic Chlorine Compounds.” Kagaku No Ryoiki, Zokan 64 (1964): 185.
Takeuchi, T. “Thin-Layer Chromatography of Metal Complex Salts.” Kagaku No Ryoiki, Zokan 64 (1964): 197.
Yamakawa, H. , and K. Tanigawa . “Thin-Layer Chromatography of Organic Metal Compounds.” Kagaku No Ryoiki, Zokan 64 (1964): 209.
Ibayashi, H. “Thin-Layer Chromatography of Steroid Hormones and Its Clinical Application.” Kagaku No Ryoiki, Zokan 64 (1964): 227.
Beckett, A. H. , M. A. Beavan , and A. E. Robinson . “Paper Chromatography: Multiple Spot Formation by Sympathomimetic Amines in the Presence of Acids.” Journal of Pharmacy and Pharmacology 12 (1960): 203T; Chemical Abstracts 55 (1961): 9785c.
Heacock, R. A. , and B. D. Scott . “The Chemistry of the ‘Aminochromes’: Part IV. Some New Aminochromes and Their Derivatives.” Canadian Journal of Chemistry 38 (1960): 516.
Matthews, J. S. “Steroids (CCXXIII) Color Reagent for Steroids in Thin-Layer Chromatography.” Biochimica et Biophysica Acta 69 (1963): 163; Chemical Abstracts 58 (1963): 14043d.
Wasicky, R. , and O. Frehden . “Spot-Plate Tests in the Examination of Drugs (I) Aldehyde and Amine Tests for the Recognition of Ethereal Oils.” Mikrochimica Acta 1 (1937): 55; Chemical Abstracts 31 (1937): 5944.
Lane, E. S. “Thin-Layer Chromatography of Long-Chain Tertiary Amines and Related Compounds.” Journal of Chromatography 18 (1965): 426; Chemical Abstracts 63 (1965): 7630f.
Neu, R. “A New Color Method for Determining Alkaloids and Organic Bases with Sodium Tetraphenylborate.” Journal of Chromatography 11 (1963): 364; Chemical Abstracts 59 (1963), 12181d.
Zinser, M. , and C. Baumgartel . “Thin-Layer Chromatography of Ergot Alkaloids.” Arch. Pharm. 297 (1964): 158; Chemical Abstracts 60 (1964): 13095f.
Ashworth, M. R. F. , and G. Bohnstedt . “Reagent for the Detection and Determination of N-Active Hydrogen.” Talanta 13 (1966): 1631.
Whittaker, V. P. , and S. Wijesundera . “Separation of Esters of Choline.” Biochemistry Journal 51 (1952): 348; Chemical Abstracts 46 (1952): 7940g.
Heacock, R. A. , and M. E. Mahon . “The Color Reactions of the Hydroxyskatoles.” Journal of Chromatography 17 (1965): 338; Chemical Abstracts 62 (1965): 13824g.
Micheel, F. , and H. Schweppe . “Paper chromatographic separation of hydrophobic compounds with acetylated cellulose paper.” Mikrochimica Acta 53 (1954); Chemical Abstracts 48 (1954): 4354i.
Smyth, R. B. , and G. G. Mckeown . “Analysis of Arylamines and Phenols in Oxidation-Type Hair Dyes by Paper Chromatography.” Journal of Chromatography 16 (1964): 454; Chemical Abstracts 62 (1963): 8930e.
Kawerau, E. , and T. Wieland . “Aminoacids Chromatograms.” Nature 168 (1951): 77; Chemical Abstracts 46 (1952): 382h.
Sturm, A. , and H. W. Scheja . “Separation of Phenolic Acids by High Voltage Electrophoresis.” Journal of Chromatography 16 (1964): 194; Chemical Abstracts 62 (1965): 6788b.
Feigl, F. Spot Tests in Organic Analysis. 7th ed. Amsterdam: Elsevier Publishing Co., 1966.
Curzon, G. , and J. Giltrow . “A Chromatographic Color Reagent for a Group of Aminoacids.” Nature 172 (1953): 356.
Heacock, R. A. , C. Nerenberg , and A. N. Payza . “The Chemistry of the ‘aminochromes’: Part I. The Preparation and Paper Chromatography of Pure Adrenochrome.” Canadian Journal of Chemistry 36 (1958): 853.
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  Spray Reagents in Thin Layer Chromatography

Family/Functional Group

Test

Result

Ref.

Adrenaline (and derivatives)

2,6-dichloroquinonechloroimide (0.5 % in absolute ethanol) potassium ferricyanide (0.6 % in 0.5 % sodium hydroxide

Variety of colors

1

Red spots

25

Adrenochromes

4-N,N-diemthylaminocinnamaldehyde

Blue-green to grey-green spots

26

Ehrlich reagent

Blue-violet to red-violet spots

26

zinc acetate (20 %)

Blue or yellow fluorescent spots

26

Alcohols

ceric ammonium sulfate (or nitrate)

Yellow/green spots on red background

1,3

2,2-Diphenylpicrylhydrazyl (0.06 % in chloroform)

Yellow spots on purple background after heating (110 °C, 5 min)

3

vanillin (1 % in conc. sulfuric acid)

Variety of spots after heating (120 °C); good only for higher alcohols

27

Aldehydes

o-dianisidine (saturated solution in acetic acid)

Variety of spots

28

2,4-dinitrophenylhydrazine

Blue colors (saturated ketones); olive green colors (saturated aldehydes); slow developing colors (unsaturated carbonyl compounds)

1

2,4-diphenylpicrylhydrazyl (0.06 % in chloroform)

Yellow spots on a purple background after heating (110 °C, 5 min)

3

hydrazine sulfate (1 % in 1N hydrochloric acid)

Spots under UV (especially after heating)

3

Aldehydes, carotenoids

Tollens reagent

Dark spots

1

Rhodamine (1–5 % in ethanol)

Variety of spots after treatment with strong alkali (sensitivity 0.03 μg)

1

Alkaloids

Bromcresol green (0.05 % in ethanol)

Green spots, especially after exposure to ammonia

3

Chloramine-T (10 % aqueous)

Rose spots after exposure to hydrochloric acid and heat

4

cobalt (II) thiocyanate

Blue spots on a light pink background

29

p-N,N-dimethylaminobenzaldehyde (4 % in 1:3 hydrochloric acid/methanol)

Characteristic spots for individual alkaloids

4

iodine/potassium iodide (in 2N acetic acid)

Variety of spots

3

Kalignost test

Orange/red spots fluorescing under long-wave UV

30

Sonnenschein test

Variety of spots

1

Alkaloids (ergot or fungal)

p-N,N-dimethylaminobenzaldehyde/sulfuric acid

Blue spots

31

Amides

chlorine/pyrazolinone/cyanide

Red spots turning blue (detection limit 0.5 μg)

32

hydroxylamine/ferric chloride

Variety of spots

33

Amines (all types unless specified)

alizarin (0.1 % in ethanol)

Violet spots on yellow background

3

chlorine/pyrazolinon/cyanide

Red spots turning blue (aromatic only)

32

cobalt (II) thiocyanate

Blue spots on white/pink background

29

diazotization and α-naphthol coupling

Variety of spots (1 ° aromatic amines only)

1

Ehrlich reagent

Yellow spots for aromatic amines

34

Fast Blue B Salt

Variety of spots (only for amines that can couple)

1

Glucose/phosphoric acid (4 %)

Variety of spots (aromatic amines only) especially after heating

35

malonic acid (0.2 %)/salicylaldehyde (0.1 %) (in ethanol)

Yellow spots after heating (120 °C, 15 min)

3,4

Amines (all types unless specified; cont.)

1,2-naphthoquinone-4-sulfonic acid, sodium salt (0.5 % in 1N acetic acid)

Variety of colors after 30 min (aromatic amines only)

36

ninhydrin

Red colors when exposed to ammonium hydroxide

37

p-nitroaniline, diazotized

Variety of colored spots

38

nitroprusside (2.5 %)/acetaldehyde (5 %)/sodium carbonate (1 %)

Variety of spots (2 ° aliphatic only)

39

picric acid (3 % in ethanol)/sodium hydroxide (10 %) (5:1)

Orange spots

4

potassium iodate (1 %)

Variety of spots for phenylethylamines (after heating)

3

vanillin-potassium hydroxide

Variety of colors

40

Amino acids

dehydroascorbic acid (0.1 % in 95 % n-butanol)

Variety of colored spots

3

2,4-dinitrofluorobenzene

Variety of spots

1

Isatin-zinc acetate

Variety of colors

1

Folin reagent

Variety of colors

1

ninhydrin

Red colors when exposed to ammonium hydroxide

36

vanillin/potassium hydroxide

Variety of colors

40

Amino alcohols

alizarin (0.1 % in ethanol)

Violet on yellow background

3

Aminochromes

p-N,N-dimethylaminocinnamaldehyde

Variety of colors

41

Ehrlich reagent

Violet spots

26,41

ferric chloride (3 %)

Gray-brown spots

41

p-nitroaniline, diazotized

Red/brown spots

26,41

sodium bisulfite, aqueous

Yellow fluorescence under UV

41,42

Aminosugars

ninhydrin

Red colors when exposed to ammonium hydroxide

37

Ammonium salts, quaternary

cobalt (II) thiocyanate

Variety of spots

29

Anhydrides

hydroxylamine/ferric chloride

Variety of spots

33

Arginine

Sakaguchi reagent

Orange/red spots

1

Azulenes

EP reagent

Blue spots (room temperature) that fade to green/yellow shades and can be regenerated with steam

1

Barbiturates

cobalt (II) nitrate (2 %)/lithium hydroxide (0.5 %)

Variety of colors

1

cupric sulfate/quinine/pyridine

Variety of colors (white, yellow, violet)

1

s-diphenylcarbazone (0.1 % in ethanol)

Purple spots

ferrocyanide/hydrogen peroxide

Yellow/red colors

1

fluorescein (0.005 % in 0.5 M ammonia)

Variety of spots under long or short-wave UV

43

mercurous nitrate (1 %)

Variety of spots

1

Zwikker reagent

Variety of spots

1

Bile acids

anisaldehyde/sulfuric acid

Variety of spots

4

antimony trichloride (in chloroform)

Variety of spots

44

perchloric acid (60 %)

Fluorescent spots (long wave UV) after heating (150 °C, 10 min)

44

sulfuric acid

Variety of spots

44,45

Bromides

fluorescein/hydrogen peroxide

Nonfluorescent spots

1

Caffeine

chloramine-T

Pink-red spots

1

silver nitrate (2 % in 10 % sulfuric acid)

Carmine-red spots (limit 2γ)

46

Carboxylic acids

Bromcresol blue (0.5 % in 0.2 % citric acid)

Yellow spots on blue background

3

Bromothymol blue (0.2 % in ethanol, pH = 7)

Yellow spots upon exposure to ammonia

47

2,6-dichlorophenol/indophenol (0.1 % in ethanol)

Red spots on blue background after heating

48

hydrogen peroxide (0.3 %)

Blue fluorescence under long-wave UV

49

Schweppe reagent

Dark brown spots

1

Carboxylic acids, ammonium salts

Catechins

p-toluenesulfonic acid (20 % in chloroform)

Fluorescent spots under long-wave UV

50

Catecholamines

ethylenediamine (50 %)

Spots under short/long wave UV after heating (50 °C, 20 min)

1

Chlorides, alkyl

2,6-dichlorophenol indophenol (0.2 %)/silver nitrate (3 %) in ethanol

Variety of spots

1

silver nitrate (0.5 % in ethanol)

Dark spots upon UV irradiation

51

silver nitrate/formaldehyde

Dark grey spots

1

silver nitrate/hydrogen peroxide

Dark spots

1

Chlorinated insecticides

diphenylamine (0.5 %)/zinc chloride (0.5 %) in acetone

Variety of colors upon heating (200 °C)

1

and pesticides

2-phenoxyethanol (5 %) in 0.05 % silver nitrate

Variety of spots

4

silver nitrate/formaldehyde

Dark grey spots

1

o-toluidine (0.5 %) in ethanol

Green spots under UV (sensitivity 0.5 μg)

4

Choline derivatives

dipicrylamine (0.2 % in 50 % aqueous acetone)

Red spots on yellow background

1

Corticosteroids

Blue Tetrazolium (0.05 %)/sodium hydroxide (2.5 M)

Violet spots (limit 1γ/cm2)

1,52

2,3,5-Triphenyl-H-tetrazolium chloride (2 % in 0.5 NaOH)

Red spots after heating (100 °C, 5 min)

1

Coumarins

Benedict reagent

Fluorescent spots under long-wave UV

1

potassium hydroxide (5 % in methanol)

Variety of spots under long-wave UV

1

Dextrins

iodine/potassium iodide

Blue-black spots (α-dextrins); brown-yellow spots (β- or γ-dextrins)

53

Dicarboxylic acids

bromocresol purple (0.04 % in basic 50 % ethanol, pH = 10)

Yellow spots on blue background

4,54

Diols (1,2-)

lead tetraacetate (1 % in benzene)

White spots after heating (110 °C, 5 min; limit 2 μg)

55

Disulfides

iodine (1.3 % in ethanol)/sodium azide (3.3 % in ethanol)

White spots on brown iodine background

3

nitroprusside (sodium)

Red spots

56

Diterpenes

antimony (III) chloride/acetic acid

Reddish yellow to blue-violet

57

Esters

hydroxylamine/ferric chloride

Variety of spots

33

Flavonoids

aluminum chloride

Yellow fluorescence on long-wave UV

58

antimony (III) chloride (10 % in chloroform)

Fluorescence on long-wave UV

59

Benedict’s reagent

Fluorescence on long-wave UV (only for o-dihydroxy compounds)

59

lead acetate (basic, 25 %)

Fluorescent spots

4,50

p-toluenesulfonic acid (20 % in chloroform)

Fluorescent spots under long-wave UV after heating (100 °C, 10 min)

Fluorescamines

perchloric acid (70 %)

Blue fluorescent spots

60

Glycols, polyethylene

quercetin/sodium tetraphenylborate

Orange-red spots

61

Glycolipids

diphenylamine (5 % in ethanol) dissolved in 1:1 hydrochloric acid/acetic acid

Blue-grey spots

1

Glycosides, triterpene

Liebermann-Burchard reagent

Fluorescence under long-wave UV

1

Hydroxamates

ferric chloride (10 % in acetic acid)

Brown spots

62

Hydroxamic acids

ferric chloride (1–5 % in 0.5N hydrochloric acid)

Red spots

1

Imidazoles

p-anisidine/amyl nitrite

Red/brown spots

3

Indoles

chlorine/pyrazolinone/cyanide

Red spots turning blue after a few minutes (limit 0.5 μg)

32

cinnamaldehyde/hydrochloric acid

Red spots

1

p-N,N-dimethylaminocinnamaldehyde

Variety of colored spots

63

Ehrlich reagent

Purple for indoles; blue for hydroxyindoles

9,34,64

ferric chloride (0.001 M) in 5 % perchloric acid

Red spots

3

naphthoquinone/perchloric acid

Orange spots

65

perchloric acid (5 %)/ferric chloride (0.001 M)

Variety of colored spots

66

Prochazka reagent

Fluorescent (yellow/orange/green) spots under long wave UV

1

Salkowski reagent

Variety of colored spots

67

van Urk (or Stahl) reagent

Variety of colored spots

1

xyanthydrol (0.1 % in acidified ethanol)

Variety of colored spots after heating (100 °C)

68

Iodides

Sonnenschein test

Variety of spots

1

α-Ketoacids

2,6-dichlorophenol/indophenol (0.1 % in ethanol)

Pink spots upon heating

4,48

o-phenylenediamine (0.05 % in 10 % trichloroacetic acid or 0.2 % in 0.1N H2SO4/ethanol)

Green fluorescence under long wave UV after heating (100 °C, 2 min)

1

Ketones

o-dianisidine (saturated solution in acetic acid)

Characteristic spots

28

2,4-dinitrophenylhydrazine

Yellow-red spots

3

Lactones

hydroxylamine/ferric chloride

Variety of colors

33

Lipids

α-cyclodextrin

Variety of spots (for straight chain lipids)

53

2’,7’-dichlorofluorescein (0.2 %) in ethanol

Spots under long-wave UV

1,69

fluorescein

Spots after treatment with steam

1

Rhodamine 6G (1 % in acetone)

Spots under long-wave UV

70

tungstophosphoric acid (20 % in ethanol)

Variety of colored spots after heating

71

Mercaptans (see Thiols)

Nitrocompounds

p-N,N-dimethylaminobenzaldehyde/stannous chloride/hydrochloric acid

Yellow spots

3

Nitrosamines

diphenylamine/palladium chloride

Violet spots after exposure to short-wave UV (limit 0.5γ)

1,72

sulfanilic acid (0.5 %)/a-naphthylamine (0.05 %) in 30 % acetic acid

Spraying is preceded by short-wave UV irradiation (3 min); aliphatic nitrosamines yield red/violet spots, while aromatic ones green/blue spots (limit 0.2–0.5γ)

1,72,73

Oximes

cupric chloride (0.5 %)

Immediate green spots (β-oximes); green-brown spots after 10 min (α-oximes)

74

Peroxides

ammonium thiocyanate (1.2 %)/ferrous sulfate (4 %)

Brown-red spots

74

N,N-dimethyl-p-phenylene diammonium dichloride

Purple spots

76

ferrous thiocyanate

Red-brown spots

1,75

iodide (potassium)/starch

Blue spots

1

Persulfates

benzidine (0.05 % in 1N acetic acid)

Blue spots

77

Phenols

anisaldehyde/sulfuric acid

Variety of colors

1,78

p-anisidine/ammonium vanadate

Variety of spots on pink background

3

benzidine, diazotized

Variety of colors

79

ceric ammonium nitrate (46 % in 2 M nitric acid)

Variety of spots

80

α,α’-dipyridyl (0.5 %)/ferric chloride (0.5 %) in ethanol

Variety of spots

4,81

emerson

Red-orange to pink spots

1

fast Blue B salt

Variety of spots

1

ferric chloride (1–5 % in 0.5N HCl)

Blue-greenish spots

1

Folin-Denis reagent

Variety of spots

82

Gibbs reagent

Variety of colors

1

Millon reagent

Variety of colors after heating

1

naphthoquinone/perchloric acid

Yellow spots (phenol, catechol); dark blue spots (resorcinol)

65

p-nitroaniline, diazotized

Variety of colored spots

38

p-nitrobenzenediazonium fluoroborate

Variety of spots

84

silver nitrate (saturated in acetone)

Pink to deep green colors

84

stannic chloride (5 %) in equal volumes of chloroform/acetic acid

Variety of spots after heating (100 °C, 5 min)

1

tetracyanoethylene (10 % in benzene)

Variety of colors

85

Tollen’s (or Zaffaroni) reagent

Dark spots

86

vanillin (1 % in sulfuric acid)

Variety of colors after heating

27

Phenols, chlorinated

Folin-Denis reagent

Variety of spots

82

Phenothiazines

ferric chloride (5 %)/perchloric acid (20 %)/nitric acid (50 %; 1:9:10)

Variety of colors

4,87

formaldehyde (0.03 % in phosphoric acid)

Variety of spots

88,89

palladium (II) chloride (0.5 % pH < 7)

Variety of spots

1

Phosphates, esters

cobalt (II) chloride (1 % in acetone or acetic acid)

Blue spots upon warming the plate at 40 °C

90

Polynuclear aromatics

formaldehyde (2 %) in conc. sulfuric acid

Variety of colors

91

tetracyanoethylene (10 % in benzene)

Variety of colors

85

Purines

fluorescein (0.005 % in 0.5 M ammonia)

Variety of spots under long- or short-wave UV

43

Pyrazolones

ferric chloride (5 %)/acetic acid (2N; 1:11)

Variety of colors

4

Pyridines

König reagent

Variety of spots (for free α-position pyridines)

92,93

Pyridines, quaternary

König reagent

Blue-white fluorescence under UV

93

Pyrimidines

Fluorescein (0.005 % in 0.5 M ammonia)

Variety of spots under long- or short-wave UV

43

Pyrones (α- and γ-)

Neu reagent

Fluorescent spots under long-wave UV

1

Quinine derivatives

formic acid vapors

Fluorescent blue spots

3

Sapogenins

Komarowsky reagent

Yellow/pink spots

94

paraformaldehyde (0.03 % in 85 % phosphoric acid)

Variety of spots

88

zinc chloride (30 % in methanol)

Fluorescent spots after heating (105 °C, 1 h) in a moisture-free atmosphere

94

Steroids

anisaldehyde/sulfuric acid

Variety of colors

95,96,97

antimony (III) chloride (in acetic acid)

Variety of colors

57,96

Carr-Price reagent

Variety of colors

1

chlorosulfonic acid/acetic acid

Fluorescence under long-wave UV

78,95

Dragendorff reagent

Variety of spots

88,89

formaldehyde (0.03 % in phosphoric acid)

Variety of spots

96

Hanes and Isherwood reagent

Variety of spots (only for 3-hydroxy-A5-steroids)

1

Liebermann-Burchard reagent

Fluorescence under long-wave UV

1,44

perchloric acid (20 %)

Fluorescent spots (long-wave UV) after heating (150 °C, 10 min)

96

phosphomolybdic acid

Blue color

95,96,98

phosphoric acid (50 %)

Fluorescent spots after heating (120 °C) (limit 0.005γ)

99

phosphotungstic acid (10 % in ethanol)

Variety of spots

1, 100

stannic chloride (5 %) in equal volumes of chloroform/acetic acid (1:1)

Variety of spots after heating (100 °C, 5 min)

1

sulfuric acid

Variety of spots

50

p-toluenesulfonic acid (20 % in chloroform)

Fluorescent spots under long-wave UV

96

trichloroacetic acid (50 % aqueous)

Variety of colors

95,96

Zimmerman reagent

Variety of colors

Sterols

antimony (III) chloride (50 % in acetic acid)

Variety of spots

99

bismuth (III) chloride

Fluorescence under long-wave UV

1

chlorosulfonic acid/acetic acid

Fluorescence under long-wave UV

1

Liebermann-Burchard reagent

Fluorescence under long-wave UV

1

1,2-naphthoquinone-4-sulfonic acid/perchloric acid

Pink spots that change to blue upon prolonged heating (cholesterol limit 0.03γ)

65,101

phosphoric acid (50 %)

Fluorescent spots after heating (120 °C, 15 min)

98,102

phosphotungstic acid (10 % in ethanol)

Variety of spots

99

stannic chloride (5 %) in equal volumes of chloroform/acetic acid

Variety of spots after heating (100 °C, 5 min)

1

sulfuric acid

Variety of spots

102

Sugars

o-aminodiphenyl (0.3 %)/orthophosphoric acid (5 %)

Brown spots after heating

103

aniline/phosphoric acid

Variety of colors

104

anisaldehyde/sulfuric acid

Variety of colors

1,78

Anthrone test

Yellow spots

105

benzidine/trichloroacetic acid

Red-brown/dark spots

106

carbazole/sulfuric acid

Violet spots on blue background

103

Lewis-Smith reagent

Brown spots

107

naphthoquinone/perchloric acid

Pink-brown spots (glucose, mannose, lactose, sucrose)

65

naphthoresorcinol (0.2 % in ethanol)/phosphoric acid (10:1)

Variety of spots after heating (100 °C, 5–10 min)

1

naphthoresorcinol (0.1 %)/sulfuric acid (10 %)

Variety of spots after heating (100 °C, 5–10 min)

1

orcinol reagent

Variety of spots

1

permanganate, potassium (0.5 % in 1N sodium hydroxide)

Variety of spots after heating (100 °C)

108

phenol (3 %)/sulfuric acid (5 % in ethanol)

Brown spots after heating (100 °C, 10 min)

103

silver nitrate (0.2 % in methanol)/ammonia (saturated)/sodium methoxide (2 % in methanol)

Variety of spots after heating (110 °C, 10 min)

1

silver nitrate/sodium hydroxide

Variety of spots

1

sulfuric acid

Variety of spots

108

thymol (0.5 %) in sulfuric acid (5 %)

Pink spots after heating (120 °C, 20 min)

103

Sugars, deoxy

metaperiodate/p-nitroaniline

Fluorescent (long-wave UV) yellow spots

109

Sugars, ketoses

Anthrone test

Bright purple (pentoses); orange-yellow (heptoses); blue fluorescence (aldoses)

110

dimedone (0.3 %)/phosphoric acid (10 % in ethanol)

Dark-grey spots (white light); dark-pink fluorescing spots (UV) after heating (110 °C, 15 min)

1,111

Sugars, reducing

4-aminohippuric acid

Fluorescence under long-wave UV

112

aniline/diphenylamine/phosphoric acid

Variety of colors

113,114,115

aniline hydrogen phthalate

Variety of colors (limit 1 μg)

116

p-anisidine phthalate

Variety of colors

1

3,5-dinitrosalicylic acid (0.5 % in 4 % sodium hydroxide)

Brown spots (sensitivity 1 μg)

3

Sulfides

ceric ammonium nitrate (in 2 M HNO3)

Colorless spots (limit < 100 μg/spot)

117

chloranil (1 %) in benzene

Yellow-brown spots

119

2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2 %) in benzene

Purple-blue spots changing to orange upon ammonia exposure

119

Gibbs reagent

Yellow-brown spots changing to blue-orange upon exposure to ammonia

119

iodine vapors

Brown spots

118

tetracyanoethylene (2 %) in benzene

0range spots

119

N,2,6-trichloro p-benzoquinoneimine (2 %) in ethanol

Brown spots

119

Sulfilimines

potassium permanganate

Colorless spots

120

Sulfilimines, p-nitro-benzene-sulfonyl

tin chloride/4-N,N-dimethylaminobenz-aldehyde

Yellow spots

121

Sulfites

malachite green oxalate

White spots on blue background

4

Sulfonamides

chlorine/pyrazolinone/cyanide

Red spots changing to blue

32

diazotization and coupling

Variety of spots (limit 0.25γ)

122,123

Ehrlich

Variety of colors

124

chloranil (1 %) in benzene

Pink turning to violet or green after heating

119

2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2 %) in benzene

Lilac-violet turning to yellow-green upon ammonia exposure

119

Gibbs reagent

Violet turning to tan upon exposure to ammonia and heat

119

Sulfones

iodine vapors

Brown spots

118

tetracyanoethylene (2 %) in benzene

Pink to yellow upon exposure to ammonia and heat

119

Sulfonic acids

Pinacryptol yellow (0.1 %)

Yellow-orange spots under long-wave UV

124

silver nitrate/fluorescein

Yellow spots under long-wave UV

125,126

Sulfoxides

acetyl bromide

Yellow-orange spots

127

ceric ammonium nitrate (40 %) in 2 M nitric acid

Brown spots after heating (especially good for α-polychlorosulfoxides); limit 80 μg/spot

117

chloranil (1 %) in benzene

Yellow-blue spots

119

2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2 %) in benzene

Orange-crimson spots

119

Dragendorff reagent

Orange-brown-red spots (limit 30–150γ)

128

Gibbs reagent

Yellow turning to brown upon ammonia exposure

168

Iodide (sodium)/starch

Brown spots (limits 0.01 μmol/20 μl solution)

129

iodine vapors

Brown spots

118,130

tetracyanoethylene (2 %) in benzene

Yellow or crimson turning to white or tan upon exposure to ammonia

119

N,2,6-trichloro-p-benzoquinoneimine (2 %) in ethanol

Yellow spots

119

Terpenes

anisaldehyde/sulfuric acid

Variety of colors

1

antimony (V) chloride

Variety of colors

1

Carr-Price reagent

Variety of colors

1

diphenylpicrylhydrazyl in chloroform

Yellow spots on purple background after heating (110 °C) (limit 1 γ/0.5 cm diameter)

131

phenol (50 % in carbon tetrachloride)

Variety of spots upon exposure to bromine vapors

3

vanillin (1 % in 50 % H3PO4)

Variety of spots after heating (120 °C, 20 min)

4

Tetracyclines

ammonium hydroxide

Yellow fluorescence under long-wave UV

132

Thioacids

silver nitrate/ammonium hydroxide/sodium chloride

Yellow-brown spots

1

Thiobarbiturates

cupric sulfate (0.5 %)/diethylamine (3 % in methanol)

Green spots (limit 15 γ)

3,133

Thiolactones

nitroprusside (sodium), basic

Red spots

62

Thiols (Mercaptans)

ceric ammonium nitrate (in 2 M nitric acid)

Colorless spots on yellow background (limit < 100 μg/spot)

117

iodine (1.3 % in ethanol)/ethanol

White spots in brown iodine background

3

nitroprusside (sodium; 3 %)

Red spots

134

Thiophenes

Isatin (0.4 % in conc. sulfuric acid)

Variety of colors

135

Thiophosphates, esters

ferric chloride/sulfosalicylic acid

White spots on violet background

136

palladium (II) chloride (0.5 % in acidified water)

Variety of spots

1,136

periodic acid (10 % in 70 % perchloric acid)

Variety of spots

3

Unsaturated compounds

fluorescein (0.1 % in ethanol)/bromine

Yellow spots on a pink background upon exposure to bromine vapors

1

osmium tetroxide vapors

Brown/black spots

3,95

Ureas

p-N,N-dimethylaminobenzaldehyde (1 % in ethanol)

Characteristic spots after exposure to hydrochloric acid

4

Vitamin A

antimony (V) chloride

Variety of colors

1

Carr-Price reagent

Variety of colors

1

sulfuric (50 % in methanol) followed by heating

Blue spots that turn brown

1

Vitamin B1

dipicyrlamine

Characteristic spots

3

Thiochrome

Variety of spots under long-wave UV

137

Vitamin B6

N,2,6-trichloro-p-benzoquinoneimine (0.1 % in ethanol)

Blue spots after exposure to ammonia

3

Vitamin B6, acetal

2,6-dibromo-p-benzoquinone-4-chlorimine (0.4 % in methanol)

Characteristic spots

138

Vitamin C

cacotheline (2 % aqueous)

Purple spot after heating (100 °C)

3

iodine (0.005 %) in starch (0.4 %)

White spot on blue background

3

methoxynitroaniline/sodium nitrite

Blue spots on orange background

3

Vitamin D

antimony (V) chloride

Variety of colors

139

Carr-Price reagent

Variety of colors

1

trichloroacetic (1 % in chloroform)

Variety of spots after heating (120 °C, 5 min)

1

Vitamin E

2’,7’-dichlorofluorescein (0.01 % in ethanol)

Spots under long-wave UV light

1

α,α’-dipyridyl (0.5 %)/ferric chloride (0.5 % in ethanol)

Variety of colors

1

Protocol For Reagent Preparation

The following section gives a summary for the preparation of the major spray reagents listed in the previous section (Spray Reagents in Thin Layer Chromatography). Reference to the original literature is recommended for any reagents not listed here [14].

acetic anhydride-sulfuric acid

See Liebermann-Burchard reagent.

alizarin

A saturated solution of alizarin in ethanol is sprayed on the moist plate, which is then placed in a chamber containing 25 % ammonium hydroxide solution to yield a variety of colors.

aluminum chloride

A 1 % aluminum chloride solution in ethanol is sprayed on the plate, which is then observed under long-wave UV light.

4-aminoantipyrine-potassium ferricyanide

See Emerson reagent.

4-aminobiphenyl-phosphoric acid

See Lewis-Smith reagent.

4-aminohippuric acid

A 0.3 % 4-aminohippuric acid solution in ethanol is sprayed on the plate, which is then heated at 140 °C (8 min) and observed under long-wave UV light.

ammonium hydroxide

The chromatogram is placed in a chamber containing 25 % ammonium hydroxide, dried, and then observed under long-wave UV light.

aniline-diphenylamine-phosphoric acid

An aniline (1 g)/diphenylamine (1 g)/phosphoric acid (5 mL) solution in acetone (50 mL) is sprayed on the plate, which is then heated at 85 °C (10 min) yielding a variety of colors.

aniline-phosphoric acid

A 20 % aniline solution in n-butanol, saturated with an aqueous (2N) orthophosphoric acid solution is sprayed on the plate, which is then heated at 105 °C (10 min) yielding a variety of colors.

aniline phthalate

An aniline (1 g)/o-phthalic acid (1.5 g) solution in n-butanol (100 mL; saturated with water) is sprayed on the plate, which is then heated at 105 °C (10 min) yielding a variety of colors.

anisaldehyde-sulfuric acid

A 1 % anisaldehyde solution in acetic acid (acidified by conc. sulfuric acid) is sprayed on the plate, which is then heated at 105 °C to yield a variety of colors.

p-anisidine phthalate

A 0.1 M solution of p-anisidine and phthalic acid in ethanol is sprayed on the plate, which is then heated at 100 °C (10 min) to yield a variety of colors.

anthrone

A 1 % anthrone solution in 60 % aqueous ethanol solution acidified with 10 mL 60 percent phosphoric acid is sprayed on the plate, which is then heated at 110 °C (5 min) to yield yellow spots.

antimony (III) chloride

See Carr-Price reagent.

antimony (III) chloride-acetic acid

A 20 % antimony (III) chloride solution in 75 % chloroform-acetic acid solution is sprayed on the plate, which upon heating at 100 °C (5 min) yields a variety of colors.

antimony (V) chloride

A 20 % antimony (V) chloride solution in chloroform or carbon tetrachloride is sprayed on the plate yielding a variety of colors upon heating.

Benedict’s reagent

A solution that is 0.1 M in cupric sulfate, 1.0 M in sodium citrate and 1.0 M in sodium carbonate is sprayed on the plate, which is then observed under long-wave UV light.

benzidine diazotized

A 0.5 % benzidine solution in 0.005 % hydrochloric acid is mixed with an equal volume of 10 % sodium nitrite solution in water; the mixture is sprayed on the plate to yield a variety of colors.

benzidine-trichloroacetic acid

A 0.5 % benzidine in (1:1:8) acetic acid/trichloroacetic acid/ethanol is sprayed on the plate to yield red-brown spots upon heating (110 °C) or exposure to unfiltered UV light (15 min).

bismuth (III) chloride

A 33 % ethanol solution of bismuth (III) chloride is sprayed on the plate, which upon heating (110 °C) yields fluorescent spots under long-wave UV light.

carbazole-sulfuric acid

A 0.5 % carbazole in ethanol/sulfuric acid (95:5) is sprayed on the plate, which yields violet spots (on blue background) after heating at 120 °C (10 min).

Carr-Price reagent

A 25 % antimony (III) chloride solution in chloroform or carbon tetrachloride is sprayed on the plate, which is heated at 100 °C (10 min) to yield a variety of colors.

ceric ammonium sulfate

A 1 % solution of ceric ammonium sulfate in strong acids (phosphoric, nitric) is sprayed on the plate to yield yellow/green spots on a red background, after heating at 105 °C (10 min).

chloramine-T

A 10 % chloramine-T solution is sprayed on the plate, followed by 1 N hydrochloric acid. The chromatogram is dried and exposed to 25 % ammonium hydroxide and warmed.

chlorine-pyrazolinone-cyanide

An equal volume mixture of 0.2 M 1-phenyl-3-methyl-2-pyrazolin-5-one solution (in pyridine) and 1 M aqueous potassium cyanide solution is sprayed on the plate that has been previously exposed to chlorine vapors. The resulting red spots turn blue after a few minutes.

chlorosulfonic acid-acetic acid

A 35 % chlorosulfonic acid solution in acetic acid is sprayed on the plate, which is then heated at 130 °C (5 min) to produce fluorescence under long-wave UV.

cinnamaldehyde-hydrochloric acid

A 5 % cinnamaldehyde solution in ethanol (acidified with hydrochloric acid) is sprayed on the plate, which is then placed in a hydrochloric acid chamber to yield red spots.

cobalt (II) thiocyanate

An ammonium thiocyanate (15 %)/cobalt (II) chloride (5 %) solution in water is sprayed on the plate yielding blue spots.

cupric sulfate-quinine-pyridine

A solution that is 0.4 % in cupric sulfate, 0.04 % in quinine hydrochloride and 4 % in pyridine in water is sprayed on the plate followed by a 0.5 % aqueous potassium permanganate solution. A variety of colors (white, yellow, violet) is detected on the chromatogram.

α-cyclodextrin

A 30 % α-cyclodextrin solution in ethanol is sprayed on the plate, which is further developed in an iodine chamber.

diazonium

See Fast Blue B salt.

diazotization and coupling reagent

A 1 % sodium nitrite solution (in l M hydrochloric acid) is sprayed on the plate, followed by a 0.2 % α-naphthol solution in l M potassium hydroxide and drying.

4-N,N-dimethylaminobenzaldehyde-sulfuric acid

A 0.125 % solution of 4-N,N-dimethylaminobenzaldehyde in 65 % sulfuric acid mixed with 5 % ferric chloride (0.05 mL per 100 mL solution) is sprayed on the plate giving a variety of spots.

4-N,N-dimethylaminocinnamaldehyde

A 0.2 % solution of 4-N,N-dimethylaminocinnamaldehyde in 6N HCl/ethanol (1:4) is sprayed on the plate, which is then heated at 105 °C (5 min) revealing a variety of colored spots. Vapors of aqua regia tend to intensify the spots.

2,4-dinitrofluorobenzene

A 1 % sodium bicarbonate solution in 0.025 M sodium hydroxide is sprayed on the plate followed by a 2,4-dinitrofluorobenzene (10 %) solution in methanol. Heating the plate in the dark (40 °C, one hour) and further spraying it with diethyl ether yields a variety of spots.

2,4-dinitrophenylhydrazine

A 0.4 % solution of 2,4-dinitrophenylhydrazine in 2N hydrochloric acid is sprayed on the plate followed by a 0.2 % solution of potassium ferricyanide in 2N hydrochloric acid yielding orange/yellow spots.

Dragendorff reagent

A 1.7 % aqueous solution of basic bismuth nitrate in weak acids (tartaric, acetic) mixed with an aqueous potassium iodide or barium chloride solution is sprayed on the plate to yield a variety of spots.

Ehrlich reagent

A 1 % 4-N,N-dimethylaminobenzaldehyde solution in ethanol is sprayed on the plate, which is dried and then placed in a hydrochloric acid chamber to yield various spots.

Emerson reagent

A 2 % 4-aminoantipyrine solution in ethanol is sprayed on the plate, followed by an 8 % aqueous potassium ferricyanide solution. The chromatogram is then placed in a chamber containing 25 % ammonium hydroxide.

EP

A 0.3 % solution of 4-N,N-dimethylaminobenzaldelyde in acetic acid/phosphoric acid/water (10:1:4) is sprayed on the plate to yield a variety of spots.

Fast Blue B Salt (diazonium)

A 0.5 % aqueous solution of Fast Blue B Salt is sprayed on the plate followed by a 0.1 M sodium hydroxide.

ferric chloride-perchloric acid

A solution made out of 5 mL 5 % aqueous ferric chloride, 45 mL 20 % perchloric acid and 50 mL 50 % nitric acid is sprayed on the plate to yield a variety of spots.

ferric chloride-sulfosalicylic acids

The plate is first exposed to a bromine atmosphere then sprayed with a 0.1 % ethanolic solution of ferric chloride. After air drying (15 min) the chromatogram is sprayed with a 1 % ethanolic solution of sulfosalicylic acid to yield a variety of spots.

ferrocyanide-hydrogen peroxide

0.5 g ammonium chloride is added to a 0.1 % potassium ferrocyanide solution in 0.2 % hydrochloric acid and the resulting solution is sprayed on the plate, which is then dried (100 °C). The chromatogram is further sprayed with 30 % hydrogen peroxide, heated (150 °C, 30 min) and sprayed with 10 % potassium carbonate to yield yellow/red spots.

ferrous thiocyanate

A 2:3 mixture of a 4 % aqueous ferrous sulfate and 1.3 % acetone solution of ammonium thio-cyanate is sprayed on the plate yielding red-brown spots.

fluorescein-hydrogen peroxide

A 0.1 % fluorescein solution in 50 % aqueous ethanol is sprayed on the plate followed by a 15 % hydrogen peroxide in glacial acetic acid and heated (90 °C, 20 min) yielding nonfluorescent spots.

Folin reagent

A 0.02 % sodium 1,2-naphthoquinone-4-sulfonate in 5 % sodium carbonate is sprayed on the plate, which is then dried to yield a variety of colors.

Folin-Denis reagent

A tungstomolybdophosphoric acid solution is sprayed on the plate, which is then exposed to ammonia vapors.

Gibbs reagent

A 0.4 % methanolic solution of 2,6-dibromoquinonechloroimide is sprayed on the plate followed by a 10 % aqueous sodium carbonate yielding a variety of spots.

glucose-phosphoric acid

A 2 % glucose solution in phosphoric acid/water/ethanol/n-butanol (1:4:3:3) is sprayed on the plate followed by heating (115 °C, 10 min) to yield a variety of spots.

hydroxylamine-ferric chloride

A 1:2 mixture of a 10 % hydroxylammonium chloride/10 % potassium hydroxide in aqueous ethanol is sprayed on the plate followed by drying. The chromatogram is then sprayed with an ether solution of ferric chloride in hydrochloric acid to yield a variety of spots.

iodide (potassium) starch

A 1 % potassium iodide solution in 80 % aqueous acetic acid is sprayed on the plate followed by a 1 % aqueous starch solution. A pinch of zinc dust is recommended as an addition to the potassium iodide solution.

iodide (sodium) starch

A solution made by mixing a 5 % starch/0.5 % sodium iodide solution with an equal volume of concentrated hydrochloric acid is sprayed on the plate, which is then exposed to dry sodium hydroxide (desiccator) and evacuated (30–60 min) to yield brown spots.

isatin-zinc acetate

An isatin (1 %)/zinc acetate (1.5 %) solution in isopropanol acidified with acetic acid is sprayed on the plate, which is then heated to yield a variety of spots.

Kalignost reagent

A 1 % solution of sodium tetraphenylborate in aqueous butanone is sprayed on the plate, followed by a 0.015 % methanolic solution of fischtin or quercetin to yield orange-red spots that fluo-resce under long-wave UV.

Komarowski reagent

A 2 % methanolic solution of p-hydroxybenzaldehyde that is 5 % in sulfuric acid is sprayed on the plate, which is then heated (105 °C, 3 min) to yield yellow or pink spots.

König reagent

A 2 % p-aminobenzoic acid in ethanolic hydrochloric acid (0.6 M) is sprayed on the plate that has been exposed (1 hr) to vapors of cyanogen bromide.

Lewis-Smith reagent

o-Aminobiphenyl (0.3 g dissolved in 100 ml of a 19:1 ethanol/phosphoric acid mixture) is sprayed on the plate, which is then heated at 110 °C (15 min).

Liebermann-Burchard reagent

A freshly prepared mixture of 5 mL acetic anhydride/5 mL conc. sulfuric acid in 50 mL cold absolute ethanol is sprayed on the plate, which is heated at 100 °C (10 min) and observed under long-wave UV light.

malachite green oxalate

A 1 % ethanolic potassium hydroxide solution is sprayed, the plate heated (150 °C, 5 min), and further sprayed with a buffered (pH = 7) water/acetone solution of malachite green oxalate to yield white spots on blue background.

metaperiodate (sodium)-p-nitroaniline

A 35 % saturated solution of sodium metaperiodate is sprayed on the plate, which is left to dry (10 min). The chromatogram is then sprayed with a 0.2 % p-nitroaniline solution in ethanol/hydro-chloric acid (4:1) to yield fluorescing (long-wave UV) yellow spots.

methoxynitroaniline - sodium nitrite

A 0.02 M 4-methoxy-2-nitroaniline solution in 50 % aqueous acetic acid/5N sulfuric acid is sprayed on the plate, which is dried and re-sprayed with 0.2 % sodium nitrite to yield blue spots on an orange background.

Millon reagent

A solution of mercury (5 g) in fuming nitric acid (10 mL) diluted with water (10 mL) is sprayed on the plate to yield yellow/orange spots that are intensified by heat (100 °C).

1,2-naphthoquinone-4-sulfonic acid/perchloric acid

A 0.1 % 1,2-naphthoquinone-4-sulfonic acid solution in ethanol/perchloric acid/40 % formaldehyde/water (20:10:1:9) is sprayed on the plate, which is then heated (70 °C) to yield pink spots that turn to blue on prolonged heating.

Neu reagent

A 1 % methanolic solution of the β-aminoethylester of diphenylboric acid is sprayed on the plate to yield fluorescent spots under long wave UV light.

ninhydrin

A ninhydrin solution (0.3 % in acidified n-butanol or 0.2 % in ethanol) is sprayed on the plate, which is then heated (110 °C). The resulting spots are stabilized by spraying with a solution made of 1 mL saturated aqueous cupric nitrate, 0.2 mL 10 % nitric acid and 100 mL 95 % ethanol, to yield red spots when exposed to ammonium hydroxide (25 %).

p-nitroaniline, diazotized

A solution made by mixing 0.1 % aqueous p-nitroaniline/0.2 % aqueous sodium nitrite/10 % aqueous potassium carbonate (1:1:2) is sprayed on the plate to yield colored spots.

p-nitroaniline, diazotized (buffered)

A solution of 0.5 % p-nitroaniline (in 2N hydrochloric acid), 5 % aqueous sodium nitrite and 20 % aqueous sodium acetate (10:1:30) is sprayed on the plate to yield a variety of colored spots.

nitroprusside (sodium)

A solution made by mixing sodium nitroprusside (1.5 g), 2N hydrochloric acid (5 mL), methanol (95 mL), and 25 % ammonium hydroxide (10 mL) is sprayed on the plate to yield a variety of colors.

nitroprusside (sodium), basic

A 2 % sodium nitroprusside solution in 75 % ethanol is sprayed on the plate, which has already been treated with lN sodium hydroxide to yield red spots.

orcinol

A mixture consisting of 0.6 % ethanolic orcinol and 1 % ferric chloride in dilute sulfuric acid is sprayed on the plate, which is further heated (100 °C, 10 min) to yield characteristic spots.

Prochazka reagent

A 10 % formaldehyde solution in 5 % hydrochloric acid solution in ethanol is sprayed on the plate, which is then heated to yield fluorescent spots (yellow/orange/green) under long-wave UV.

quercetin-sodium tetraphenylborate

A mixture of quercetin (0.015 % in methanol) and sodium tetraphenyl-borate (1 % in n-butanol saturated with water) is sprayed on the plate to yield orange/red spots.

quinaldine

A 1–1.5 % solution of 3,5-diaminobenzoic acid dihydrochloride in 30 % phosphoric acid is sprayed on the plate, which is then heated (100 °C, 15 min) to yield fluorescent (green/yellow) spots under long-wave UV or (in case of high concentrations) brown spots in daylight.

Sakaguchi reagent

A 0.1 % acetone solution of 8-hydroxyquinoline is sprayed on the plate followed by a 0.2 % 0.5N sodium hydroxide solution to yield orange/red spots.

Salkowski reagent

A 0.01 M aqueous ferric chloride/35 % perchloric acid solution is sprayed on the plate, which is then heated (60 °C, 5 min) to yield a variety of colors intensified when exposed to aqua regia.

Schweppe reagent

A mixture of 2 % aqueous glucose/2 % ethanolic aniline in n-butanol is sprayed on the plate, which is heated (125 °C, 5 min) to yield a variety of spots.

silver nitrate-ammonium hydroxide-sodium chloride

A mixture of silver nitrate (0.05 M)/ammonium hydroxide (5 %) is sprayed on the plate, followed by drying and further spraying with 10 % aqueous sodium chloride to yield yellow/brown spots.

silver nitrate-fluorescein

A mixture of silver nitrate (2 %)/sodium-fluorescein (0.2 %) in 80 % ethanol is sprayed on the plate to yield yellow spots on pink background.

silver nitrate-formaldehyde

The plate is consecutively sprayed with 0.05 M ethanolic silver nitrate, 35 % aqueous formaldehyde, 2 M potassium hydroxide and, finally, a solution made of equal volumes of hydrogen peroxide (30 %) and nitric acid (65 %). Each spraying is preceded by a 30 min. drying and at the end the plate is kept in the dark for 12 hr before exposing to sunlight to yield dark grey spots.

silver nitrate-hydrogen peroxide

A 0.05 % silver nitrate solution in water/cellosolve/acetone (1:10:190; to which a drop of 30 % hydrogen peroxide has been added) is spayed on the plate, which is then treated under unfiltered UV to yield dark spots.

silver nitrate-sodium hydroxide

A saturated silver nitrate solution is sprayed on the plate followed by a 0.5 M aqueous/methanol solution. Subsequent drying (100 °C, 2 min) yields a variety of spots.

Sonnenschein reagent

A 2 % ceric sulfate solution in 20 % aqueous trichloroacetic acid (that has been acidified with sulfuric acid) is sprayed on the plate. A variety of colors appears upon heating (110 °C, 5 min).

Stahl

See van Urk reagent.

sulfanilic acid-1-naphthylamine

A mixture of 2 % sulfanilic acid/1-naphthylamine in 30 % acetic acid is sprayed on the plate to yield a variety (violet/green/blue) of colors.

thiochrome

A 0.3 M aqueous potassium ferricyanide solution that is 15 % in sodium hydroxide is sprayed on the plate yielding a variety of spots under long-wave UV.

Tollen’s reagent

See Zaffaroni reagent.

vanillin-potassium hydroxide

A 2 % solution of vanillin in n-propanol is sprayed on the plate, which is heated (100 °C, 10 min) and sprayed again with 1 % ethanolic potassium hydroxide. Reheating yields a variety of colors observed under daylight.

van Urk (Stahl) reagent

A 0.5 % solution of 4-N,N-dimethylaminobenzaldehyde in concentrated hydrochloric acid/ethanol (1:1) is sprayed on the preheated plate, which is then subjected to aqua regia vapors to yield a variety of colors.

Zaffaroni (Tollen’s) reagent

A mixture of silver nitrate (0.02 M)/ammonium hydroxide (5 M) is sprayed on the plate, which is then heated (105 °C, 10 min) to yield black spots.

Zwikker reagent

A 1 % cobaltous nitrate in absolute ethanol is sprayed on the plate, which is dried (at room temperature) and exposed to a wet chamber containing 25 % ammonium hydroxide.

References

Krebs, K. G. , D. Heusser , and H. Wimmer .Thin Layer Chromatography, A Laboratory Handbook, edited by E. Shahl . New York: Springer-Verlag, 1969.
Bobbitt, J. B. Thin Layer Chromatography. New York: Reinhold, 1963.
Touchstone, J. C. , and M. F. Dobbins .Practice of Thin Layer Chromatography. New York: John Wiley and Sons, 1983.
Randerath, K. Thin-Layer Chromatography. 2nd ed. Verlag Chemie, GmbH. (in the United States, Academic Press, New York, 1968).
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