Thinlayer Chromatography

Thin-layer chromatography (TLC) is widely used as a versatile method in the analysis of alkaloids. It offers the advantage of a broad range of polarities being separated in one single analysis, which is of interest in plant materials and metabolism studies. The most widely used stationary phase is silica; alumina plates are rarely employed nowadays. Rever-sed-phase materials, such as chemically bonded C18 on silica, are also applied but silica is still used most widely.

Strongly basic alkaloids will show severe tailing on silica gel plates, due to the acidic properties of silica. The use of mobile phases which contain

Table 2 Some common thin-layer chromatography systems for the analysis of alkaloids

Solvent system (all with silica plates)

Commonly used ratios

Polarity range

Cyclohexane-chloroform-diethylamine

5:4:1-

(0) : 9

:1

lp-mp

Chloroform-acetone-diethylamine

5:4:1

mp

Chloroform-methanol-ammonia

8:1:1

mp

Chloroform-methanol/ethanol

99 : 1 - 1

:1

lp-mp, wb

Ethyl acetate-isopropanol-25% ammonia

100:2

1, 80

15

5, 45 : 35 : 5

lp-mp

Ethyl acetate-methanol

9:1-1:

1

lp-mp, wb

Toluene-ethyl acetate-diethylamine

7:2:1

lp-mp

Toluene-acetone-ethanol-25% ammonia

20 : 20 :

3 : 1

mp

Dicholoromethane-diethyl ether-diethylamine

20 : 15 :

5

mp

Acetone-methanol-25% ammonia

40 : 10 :

2, 95 :

(0)

:5

mp-p

Methanol-25% ammonia

95 : 5

lp-p

n-Butanol-acetic acid-water

4:1:1

lp-p

Methanol-1 mol L~1 aq. M NH4NO3-2 mol L~1 aq. ammonia

7:1:2

lp-p

Methanol-0.2 mol L_1 aq. M NH4NO3

3:2

lp-p

Ip, Low polarity compounds; mp, medium polarity compounds; p, polar compounds; wb, weakly basic compounds.

Ip, Low polarity compounds; mp, medium polarity compounds; p, polar compounds; wb, weakly basic compounds.

a base such as ammonia or diethylamine will overcome this problem. A more elaborate method is the use of TLC plates impregnated with a basic solution.

For the analysis of highly polar quaternary alkaloids and N-oxides, solvent systems consisting of methanol and aqueous salt solutions are useful. In Table 2 some widely used TLC systems are summarized. For the detection of alkaloids a large number of methods have been reported. Besides quenching ultraviolet (UV) light on fluorescent plates and fluorescence, general reagents for selectively detecting alkaloids are Dragendorff's reagent (orange-brown spots) and potassium iodoplatinate (brown-violet-purple spots; Table 3). Dragendorff's reagent may cause false-positive reactions with, for example, compounds containing conjugated carbonyl or lactone functions. The iodoplatinate reagent has less risk of false-positive reactions and is more selective due to a broader spectrum of colours observed for individual alkaloids.

Highly selective reagents have been reported for the visualization of various classes alkaloids (Table 4). These are based on different colorations under strongly oxidative conditions.

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