Detection of Alkaloids

Only a few alkaloids are directly visible on the chromatogram as coloured spots and visualization methods have to be applied to detect them. In order to detect the compounds under UV light, fluorescing indicators are added to the adsorbent.

Alkaloids become visible in short wavelength UV light (X = 254 nm), where they appear as dark zones on a fluorescent background. This is considered to be a nonselective method of detection because, on the layer containing a fluorescent indicator, the emission is quenched in regions where all aromatic organic compounds absorb the UV light with which the plates are irradiated.

Some alkaloids, such as indoles, quinolines, isoquinolines and purines, cause pronounced quenching of fluorescence, but some (e.g. tropine alkaloids) only weakly quench UV light. Sometimes compounds can be detected under a UV lamp due to their own luminescence. Excitation is usually performed using long wavelength radiation of X = 365 nm. Alkaloids absorb radiation and then usually emit it in the visible region of the spectrum, where they appear as bright-coloured luminous zones of blue, blue-green or violet, for example, Rauwolfiae radix, Chinae cortex, Ipecacuanhae radix, Boldo folium, and of yellow, e.g. colchicine, sanguinarinae, berberine.

Other methods of physical detection make the most of the chemical properties of alkaloids. As basic compounds, these properties can be detected using pH indicators applied to the chromatogram by dipping it or spraying it with 0.01-1% indicator solutions.

Bromocresol Green with pH transition from 3.8 to 5.4 is applied for many alkaloids; Bromocresol Purple (pH = 5.2-6.8) is predominantly applied for ephed-rine.

Another nonselective detection method for alkaloids as lipophilic substances is the treatment of a chromatogram with iodine vapour or dipping into or spraying with 0.5-1% iodine solutions. Molecular iodine is enriched in the chromatogram zones and colours them brown. Emetine and cephae-line, the two major alkaloids of ipecacuanha, begin to glow after treatment with iodine vapour. In this case, the molecular iodine which acts as a quencher must be removed by heating, before the yellow (emetine) and blue (cephaeline) fluorescent zones become visible.

Although the methods described are usually fairly sensitive and allow a detection limit of less than 1 |g, sometimes they are insufficient. That is why they have to be supplemented by specific reactions with a number of detection reagents (Table 3).

The most popular reagents which react with tertiary and quaternary nitrogen atoms present in alkaloid molecules are Dragendorff's reagent and potassium iodoplatinate. Alkaloids containing primary and secondary amino groups treated with dimethyl sulfate give quaternary nitrogen atoms, permitting effective detection with these reagents too.

Dragendorff's and iodoplatinate reagents exists in various modifications. The replacement of water in these reagents by acetic acid or ethyl acetate, diethyl ether-methanol or hydrochloric acid increases the sensitivity of the reaction and significantly improves the sharpness of spots. Spraying 10% sodium nitrate solution after the use of Dragendorff's reagent causes

Table 3 Selection of detection reagents for postchromatographic derivatization of alkaloids

Reagent

Substances detected

Reaction

Method

Result

Ammonia vapour

2,4-Dinitrophenyl-hydrazine

2,6-Dichloro-

quinone-4-

chloroimide

Alkaloids, e.g. morphine, heroin, 6-mono-acetylmorphine

Morphine and heroin form fluorescent oxidation products

Formaldehyde reagent (1,2-naphthoquinone-4-sulfonic acid)-perchloric acid

2-Methoxy-2, 4-diphenyl-3(2H)-furanone (MDPF)

Alkaloids, e.g. The reaction mechanism codeine, morphine, has not been elucidated.

heroin, 6-mono-acetylmorphine

Alkaloids from Colchicum autum-nale (Colchicine)

Alkaloids

Isoquinoline alkaloids o-Phthal-aldehyde (OPT, OPA)

Phosphomolybdic acid

Ergot alkaloids

Morphine

It is possible that formaldehyde reacts by oxidation, as in Marquis reaction

Heat the chromatogram in the drying cupboard to 110-120°C for 25 min and place it for 15 min in a twin-trough chamber, whose second trough contains 10 mL of 25% ammonia solution. Then immerse for 2 s in a solution of liquid paraffin-n-hexane (1 : 2)

Dry the chromatogram in a stream of warm air for 5 min, immerse in the reagent solution for 4 s and heat to 70°C for c. 10 min

MDPF reacts directly with primary amines to form fluorescent products

In the presence of 2-mercaptoethanol, o-phthalaldehyde reacts with primary amines to yield fluorescent isoindole derivatives

Morphine can be oxidized with phosphomolybdic acid, whereby a portion of the Mo(VI) is reduced to Mo(IV) which forms blue-grey oxides with the remaining Mo(VI)

Free the chromatogram from mobile phase in a stream of warm air (45 min), immerse in the reagent solution for 4 s and then heat chromatogram zone. The

Morphine, 6-monoacetylmorphine and heroin appear as blue fluorescent zones on a dark background under UV light (1 = 365 nm). In each case the detection limits are 2 ng of substance per chromatographic zone. The fluorimetric determination is carried out in UV light 1exc = 313 nm, = 390 nm

Morphine alkaloids yield blue chromatogram zones on a pale blue background. The detection limits are 10-20 ng of substance per chromatogram zone. The absorption photometric analysis can be performed at reflectance 1 = 610 nm

Colchicine appears as a yellow fluorescent zone on a dark background in UV light (365 nm). The detection limit is 10 ng per to 110°C for 20 min fluorimetric analysis is carried out with excitation at 1exc = 313 nm, and evaluation at A, > 390 nm

Reagent reacts with carbonyl groups with the elimination of water to yield hydrazone and with aldoses or ketoses to yield coloured osazones

Reagent reacts with phenols or anilines which are not substituted in the p-position

Immerse the chromatogram Substances yield yellow to in the dipping solution for 2 s or spray and then dry in a stream of warm air (10-20 min at 110°C)

Dry the chromatogram for 5 min in a stream of warm air, immerse in the dipping solution for 5 s and then heat to 110°C for 2 min

Immerse the dried chromatogram for 1 s in the reagent solution and then heat to 40-50°C in the dry cupboard for 10 min

Dry the chromatogram in a stream of warm air and immerse for 2-3 s in the reagent solution, or spray the layer with it orange-yellow chromatogram zones on an almost colourless background

Cephealine produces a blue colour immediately on immersion, while emetine only does so on heating. On storage this colour slowly changes to brown (background light brown). The detection limits are c. 10 ng per chromatogram zone. The absorption photometric analysis was made at 1= 550 nm

Substance zones are produced that mainly yield blue (or yellow) fluorescence under long wavelength light (1 = 365 nm)

Blue zones appear on a yellow background immediately or after a few minutes

Table 3 Continued

Reagent

Substances detected

Reaction

Method

Result

Trichloroacetic acid

Alkaloids from, e.g. Veratrum colchicum

Sulfuric acid

Alkaloids

7-Chloro-4-

nitrobenzo-2-

oxa-1,3-diazole

(NBD-chloride reagent)

Alkaloids tert-Butyl hypochlorite

Alkaloids

Formaldehyde-sulfuric acid (Marquis reagent)

The reaction mechanism has not yet been elucidated

NBD reacts with nucleophilic compounds to yield the corresponding

7-substituted

4-nitrobenzofurazan derivatives

Alkaloids, e.g. Morphine reacts with morphine, codeine, formaldehyde in heroin,

6-monoacetyl-morphine acidic solution to yield a cyclic ketoalcohol, which is transformed into the coloured oxonium or carbenium ion in acidic conditions

Dry the chromatogram in a stream of cold air and immerse for 1 s in the reagent solution or spray with it and then heat at 120°C for 10 min

The reaction mechanism has not yet been elucidated

The reaction mechanism has not yet been elucidated

Dry the chromatogram in a stream of warm air for 10 min, immerse in the dipping solution for 1-2 s or spray with the spray solution, dry in a stream of warm air and then heat to 95-140°C for 1-20 min

Dry the chromatograms. Immerse in dipping solution of sodium acetate in methanol-water for 1 s. Dry in a stream of warm air and dip after cooling in NBD-chloride reagent in ethanol and then heat to 100°C for 2-3 min. Alternatively the chromatogram can be sprayed with the appropriate spray solutions

Dry the chromatogram, immerse in dipping solution of reagent in carbon tetrachloride or cyclohexane for 1 s (or spray or expose to its vapours) then immerse in dipping solution of chloroform, paraffin oil and triethanolamine (6 : 1 : 1) for 1 s and dry in hot air

Dry the chromatogram in a stream of warm air for 5 min, immerse in the dipping solution for 6 s and heat to 110°C for 20 min

Light blue fluorescent zones appear mainly under long wavelength UV light (1 = 365 nm). The fluorescence can be stabilized and intensified by dipping the plate into a solution of liquid paraffin-n-hexane (1 : 2)

Under long wavelength UV light (1 = 365 nm) characteristic substance-specific yellow, green, red and blue fluorescent chromatogram zones usually appear, and are often recognizable in visible light

Under UV light (1 = 365 nm) the chromatogram zones fluoresce greenish-yellow, olive brown or violet. The plate background also fluoresces, but appreciably less. The detection limits are 100-800 ng substance per chromatogram zone

The analysed compounds appear in long wavelength UV light (365 nm), yellow to violet fluorescent zones, on a dark background. The detection limit for morphine is 10 ng and for papaverine 1 ng per chromatogram zone

Morphine alkaloids yield reddish chromatogram zones (codeine yielded blue on a pale pink background). If a quantitative fluorimetric analysis is to follow, the chromatogram is exposed to ammonia vapour for 20 min and immersed for 2 s in 20% dioctyl sulfosuccinate in chloroform. After drying, morphine alkaloids appear as pink to red flourescent zones on a blue fluorescent background under UV light (1 = 365 nm). The fluorimetric analysis is carried out at 1exc = 313 nm, 1f = 560 nm

Table 3 Continued

Reagent Substances Reaction Method Result detected

Iron (III) chloride- Indole alkaloids, The reaction mechanism Free the chromatogram perchloric acid (FCPA reagent)

Hydrochloric acid vapour e.g. from

Rauwolfia,

Tabernaemontana,

Mitragyna,

Strychnos,

Synclisia,Cinchona has not yet been elucidated

Alkaloids, e.g.

papaverubines

The reaction mechanism has not yet been elucidated from mobile phase in a stream of warm air (45 min), immerse in the dipping solution for 4 s. Dry and heat to 110°C for 20 min

Free the chromatogram from mobile phase (first in a stream of cold air for a few minutes, than at 100°C for 5 min), place in the free trough of the prepared twin-trough chamber for 5 min and then evaluate

Variously coloured chromatogram zones are produced on a colourless background. For instance, strychnine appears as a red and brucine as a yellow chromatogram zone on a colourless background. The detection limit for both is 10 ng per chromatogram zone. The light absorption in reflectance was measured at X = 450 nm

Alkaloids are visible after irradiation with unfiltered UV light from a mercury lamp

Figure 4 (See Colour Plate 54). The chromatogramsofthe separated alkaloids developed on silica gel or alumina in solvent systems 1-4, detected with different reagents. Solvent systems: 1, toluene-ethyl acetate-diethylamine (70: 20 : 10); 2, chloroform-diethyl-amine (90 : 10); 3, toluene-chloroform-ethanol (28.5 : 57 : 14.5); 4, 1-propanol-water-formic acid (90 : 9 : 1). For identification of compounds, reagents used and obtained results, see Table 4. (Reproduced with permission from Wagner H and Bladt S (1996) Plant DrugAnalysis. Thin-layerChromatographyAtlas. Berlin: Springer.)

Figure 4 (See Colour Plate 54). The chromatogramsofthe separated alkaloids developed on silica gel or alumina in solvent systems 1-4, detected with different reagents. Solvent systems: 1, toluene-ethyl acetate-diethylamine (70: 20 : 10); 2, chloroform-diethyl-amine (90 : 10); 3, toluene-chloroform-ethanol (28.5 : 57 : 14.5); 4, 1-propanol-water-formic acid (90 : 9 : 1). For identification of compounds, reagents used and obtained results, see Table 4. (Reproduced with permission from Wagner H and Bladt S (1996) Plant DrugAnalysis. Thin-layerChromatographyAtlas. Berlin: Springer.)

Table 4 Symbols used in Figure 4

Symbol Detection

Solvent Reference comPounds system

Result

Marquis reagent pvis

Natural products, polyethylene glycol reagent (NP/PEG) p UV 365 nm Sulfuric acid reagent p UV 365 nm

Dragendorff reagent pvis

Dragendorff reagent 1 followed by sodium nitrite pvis

Iodine/CHCI3 reagent p 1 UV 365 nm pvis 1

10% H2SO4 followed 2 by iodoplatinate reagent pvis

I van URK reagent pvis 3

J UV254nm 1

K UV365 nm 4

MorPhine (1), codeine (2), PaPaverine (3), noscaPine (4), oPium extract (5)

SerPentine (1), quinine (2), cinchonine (3), quinidine (4), cinchonidine (5), cePhaaeline (6), emetine (7), yohimbine (8), noscaPine (9), hydrastine (10), berberine (11), sanguinarine (12) Strychnine (1), yohimbine (2), Physostigmine (3), nicotine (4), veratrine (5), emetine (6), PaPaverine (7), lobeline (8), aconitine (9), narcotine (10) CePhaelis accuminata (1), cePhaeline: Rf&0.2; emetine: Rf & 0.4 (2).

CePhaelis iPecacuanha (3) China alkaloid mixture (1) Cinchona succirubra (2)

Ergocristine (1), Secale cornutum (2), ergotamine (3), ergometrine (4) Strychnine (1), Strychni semen (2), Ignatii semen (3), brucine (4) Chelidoniiherba different trade samPles (1-3), sanguinarine (4)

Morphine and codeine are immediately stained violet; papaverine: weak violet; noscapine: weak yellow brown Morphine, papaverine, noscapine give a blue fluorescence in UV 365 nm; codeine does not fluoresce

The fluorescence of quinine and quinidine is a radial blue; cinchonine and cinchonidine: deep violet, berberine and sanguinarine: bright yellow

Alkaloids give orange-brown, stable colours The zones become dark brown

Cephaeline fluoresces bright blue and emetine: yellow-white Cephaeline gives red and emetine weak yellow zones

The violet-brown zone of quinine is followed by the grey-violet zone of cinchonidine, a weak red-violet zone of quinidine and brown-red cinchonine (1) In Cinchona succirubra extract additionally three red-violet zones appear in the Rf range 0.4-0.6 (2)

Secale alkaloids appear as blue zones in the Rf range of 0.05-0.4

Strychnine and brucine are characterized in UV 254 nm by their strong quenching zones The major alkaloid coptisin at Rf & 0.15 (bright-yellow) is followed by berberine, chelerythrine, sanguinarine (broad yellow) and chelidonine (weak yellow-green) in the Rf range of 0.75-0.85

the colour of alkaloid zones to be intensified or stabilized and increases the sensitivity to 0.01-0.1 |g.

Modification, where a chromatogram is sprayed with 10% sulfuric acid after the use of Dragendorff's reagent, also causes an increase in the sensitivity of the reaction. Potassium iodoplatinate reagent gives preliminary identification, due to the fact that different colours are obtained with different alkaloids.

Table 5 Examples of PrechromatograPhic derivatization of alkaloids

PrechromatograPhic

Reagent used

Special applications

derivatization

Oxidation

10% Chromic acid in glacial acetic acid

Strychnine and brucine

Potassium dichromate

Dehydration by heating the applied sample on silica layer

Reduction

Sodium borohydride solution

Not specified

Iodination

Iodine vapour saturated chamber (18 h)

Quinoline, isoquinoline, indole alkaloids

Nitration

Concentrated nitric acid

Brucine

Dansylation

Dansyl chloride and twice bigger volume of 8%

Morphine, 6-monoacetylmorphine,

sodium bicarbonate solution

morphine-6-nicotinate

Table 6 Systematic analysis of alkaloids on TLC plates

Chemical Plant drug skeleton

Botanical origin

Major alkaloid

Fluorescence Colour with in UVlight iodoplatinate (366 nm) reagent

S2 S3

S4

S5

S6

S7

S8

Tropane Fol. Belladonnae

Atropa

Rad. Belladonnae

belladonna L,

Solanaceae

Atropine

Violet-blue

38

40

16

5

12

0

10

17

Fol. Hyoscyami

Hyoscyamus

Homatropine

Violet-blue

37

45

15

5

23

4

24

15

niger L,

Solanaceae

Fol. Stramonii

Datura

Apoatropine

Violet-blue

54

67

40

20

26

15

40

16

stramonium L,

Solanaceae

Rad. Scopoliae

Scopolia

Scopolamine

Violet

56

60

19

3

34

30

0

52

carniolica Jacq.

Scopoline

White

60

90

44

20

44

46

50

37

Solanaceae

Fol. Duboisiae

Duboisia

Tropacocaine

Violet

65

90

56

34

45

58

78

35

myoporoides R.

Br., Solanaceae

Fol. Cocae

Erythroxylon coca

Cocaine

Violet

73

90

65

36

58

84

77

62

Lamarck

Erythroxylaceae

Indole Semen Calabaris

Physostigma

Physostigmine

Pink

65

>90

32

4

44

59

50

46

venenosum

Balfour

Papilionaceae

Rad. Rauwolfiae

Rauwolfia

Reserpine

Green-yellow White

72

80

20

0

46

63

35

69

serpentina

Rad. Serpentinae

Bentham,

Serpentinine

Dark brown

Red-brown

24

15

0

0

4

0

0

0

Apocynaceae

Semen Strychni

Strychnos nux

Serpentine

Yellow-green Yellow-brown

53

56

8

0

10

0

3

12

vomica L,

Ajmaline

Blue

Beige

47

42

12

3

30

6

13

56

Loganiaceae

Strychnine

Yellow

53

76

28

5

38

57

60

22

Cortex

Pausinystalia

Brucine

Violet-brown

42

63

18

0

19

50

54

12

Yohimbehe

Yohimbe Pierre,

Yohimbine

Green-blue

Light yellow

63

62

18

3

37

33

15

60

Rubiaceae

Ergocristinine

Violet-blue

Light brown

61

57

13

0

20

0

27

70

Secole cornutum

Claviceps

Ergotamine

Violet-blue

Pink

24

16

0

0

3

10

5

59

purpurea Tulasne

Ergometrine

Violet-blue

White

14

6

0

0

2

3

0

64

Clavicipitaceae

Ergometrinine

Violet-blue

Violet-blue

42

25

3

0

8

12

10

62

Ergocristine

Violet-blue

Beige-light

51

38

14

5

13

46

15

70

brown

Ergotaminine

Violet-blue

Pink

24

51

0

0

14

42

15

68

Dihydroergotamine

Violet-blue

Brownish

21

12

0

0

3

7

0

61

Dihydroergocristine

Violet-blue

Brownish

12

30

3

0

7

15

7

69

Isoquinoline Opium

Papaver

Thebaine

Red-brown

65

90

51

16

50

71

76

40

somniferum L,

Narceine

Deep-blue

3

0

0

0

3

0

0

0

Papaveraceae

Morphine

Deep-blue

10

8

0

0

3

3

0

34

Papaverine

Yellowish

Yellow

67

90

42

3

47

85

84

70

Codeine

Pink-violet

38

53

16

4

26

12

27

35

Noscapine

Blue

Light-yellow

72

90

51

10

57

81

79

72

Hydrastinine

Steel blue

Violet-blue

66

90

58

41

50

0

25

0

Dihydromorphinone

Brownish

24

23

8

1

11

5

8

16

yellow

Dihydrocodeine

Blue

Violet-blue

38

54

18

6

28

10

30

25

Dihydrocodeinone

Violet

51

65

21

4

30

48

43

18

Fol. Boldo

Peumus boldus

Boldine

Violet

Beige

16

16

3

0

5

24

6

58

Monimiaceae

Quinoline Cortex Chinae

Cinchona

Quinidine

Blue

Light yellow

34

40

15

0

25

12

18

50

Succirubra,

Quinine

Blue

Yellow-white

19

26

7

0

17

9

18

43

Pavon, Rubiceae

Cinchonine

Beige-brown

38

44

17

7

27

0

22

40

Imidazole Fol. Jaborandi

Pilocarpus

Pilocarpine

Light brown

41

52

9

0

13

32

25

55

microphyllus

Stapfe.a.;

Rutaceae

Chemical Plant drug Botanical Major alkaloid Fluorescence Colour with hRF values skeleton origin in UVlight iodoplatinate -

(366 nm) reagent S1 S2 S3 S4 S5 S6 S7 S8

Pyridine

Quinolizidine

Dihydroindole Aporphine

Isoquinoline

Miscellaneous alkaloids Derivatives of diterpene

Xanthine

Colchicine

Semen Arecae Areca catechu L., Arecoline Herba Lobeliae Palmae

Lobelia inflata L., Lobeline Lobeliaceae

Sarothamnus Sparteine

Scoparius;

Leguminosae

Fol. Catharanti Catharantus roseus Aspidospermine Apocynaceae

Rhizoma Corydalidis Corydalis cava L.

Schweigg et Koerte

Papaveracae, Fumariaceae Rad. Ipecacuanhae Cephaelis ipecacuanha Rubiaceae

Aconiti Tuber

Herba Ephedrae

Semen Colchici

Aconitum napellus L., Ranunculaceae Ephedra sinica Stapf.

Ephedraceae Colchicum autumnale L, Liliaceae

Bulbocapnine

Emetine Cephaeline

Aconitine

Ephedrine

Colchicine

Blue

Blue

Violet-blue

Violet 66 90 56 34 48 0 0 0

Red-brown 68 90 48 14 48 55 60 55

Violet 70 90 68 68 55 0 55 5

White 65 90 54 20 49 50 60 65

Red-brown 67 90 40 6 45 38 58 50 White 56 63 19 2 23 25 17 37

Light-grey 47 41 4 0 4 11 0 57

Light brown

TLC systems

51, Silica gel G, activated: chloroform-acetone-diethylamine (5:4: 1).

52, Silica gel G, activated: chloroform-diethylamine (9 : 1).

53, Silica gel G, activated: cyclohexane-chloroform-diethylamine (5 : 4 : 1).

54, Silica gel G, activated: cyclohexane-diethylamine (9:1).

55, Silica gel G, activated: benzene-ethyl acetate-diethylamine (7:2:1).

56, Aluminium oxide G, activated: chloroform.

57, Aluminium oxide G, activated: cyclohexane-chloroform (3:7)+ 0.05 diethylamine.

58, Silica gel G, impregnated with 0.1 mol L_1 sodium hydroxide, activated: methanol.

(Reproduced with permission from Svendsen AB and Verpoorte R (1983) ChromatographyofAlkaloids. Journal of Chromatography Library. Amsterdam: Elsevier.)

For particular alkaloids, specific reagents can be used; for instance, Marqui's reagent (formalde-hyde-sulfuric acid) or Frohde's reagent (sul-fomolybdic acid-sulfuric acid) for morphine. Konig's reaction can be used to detect nicotine and related alkaloids; Wachtmeister's reagent (bis-diazatized benzidine-sulfuric acid) is applied for alkaloids belonging to the protoberberine and protopine group.

The Vitaly reaction is specific for the tropane alkaloids, and reaction with 4-dimethylaminobenzal-dehyde for indole alkaloids. Some examples of applications of different reagents are illustrated in Figure 4 and Table 4.

The use of re-acceptor reagents producing colour spots (TCNQ: 7,7,8,8-tetracyano-quinodimenthane; TNF: 2,4,7-trinitrofluorenone; TetNF: 2,4,5,7-tetranitro-9-fluorenone; DDQ: 2,3-dichloro-5,6-di-cyanoquinone; DNFB: 2,4-dinitrofluorobenzene) for the detection of alkaloids has been employed.

Initial derivatization during sample preparation or in situ on the layer after the application of the sample is called prechromatographic derivatization and comprises oxidation, reduction, iodination, nitration and dansylation (Table 5).

Starting chromatographic separation with sample derivatization allows better-quality results to be obtained, especially as far as reproducibility and lowering the detection limits are concerned. Morphine as a dansyl derivative is an example of fluorescence stabilization and intensity augmentation as a result of treatment of the chromatogram with a 20% solution of liquid paraffin in n-hexane.

A similar phenomenon is observed for codeine, morphine, monoacetylmorphine and heroin with the aid of hydrophilic liquids, such as a 20% solution of dioctyl sulfasuccinate in ethanol as a fluorescence intensifies

Enhanced sensitivity can be achieved by impregnating the layer, by adding the reagent to the solvent or by spraying the plate after development. In addition to the reagents mentioned above, fluorescence intensifies such as triethanolamine, glycerol and Triton X-100 are quite popular.

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