Relative quantitative analysis of plant material for alkaloids.

adapted from
Quick test for Reed Canarygrass alkaloid concentration, Frelich and Marten, J. Crop Sci. #13:548-51, 1973.
and
Field test for alkaloids in plant material, Culvenor and Fitzgerald, J. Pharm. Sci. #52:303-4, 1963
(see quick test article)

Steps

1. Macerate 5 gm of fresh or frozen material in 50 ml ammoniacal chloroform (as outlined in the quick test article). The actual quantities can be different, though must remain standardised. Eg 10 gms of plant material, 100 ml of ammoniacal chloroform, 12 ml sulphuric acid, 12 drops of silicotungstic acid, 2 gm plant material, 20 ml ammoniacal chloroform, 3 ml suphuric acid, 3 drops of silicotungstic acid, etc. Dry powdered material can also be used instead of fresh or frozen samples. Being able to obtain relative quantitative analysis requires that different samples be treated in exactly the same fashion, and differences in the amounts used will mean that different samples can not be directly related.

2. Filter the plant extract into a separation funnel. Can wash with another 10 ml (this amount also must be standardised) of ammoniacal chloroform to obtain a more thorough extract, and filtered and added to the original chloroform.

3. Add 6 ml of sulfuric acid and shake vigorously, and allow the aqueous and solvent layers to separate. Or if using different amounts as outlined in step 1. eg 12 ml, 3 ml etc. Depending on the actual alkaloid yield, it might be better to use these lesser or greater volumes.

4. Drain the chloroform layer and discard. Drain the aqueous phase into test tube.

5. Place 2 ml (or 1ml or 3 ml if greater or lesser volumes are being used as commented on in step 1) of aq. phase in a test tube and add  6 drops of  Silicotungistic acid solution  (12 gm silicotungistic acid to 100 ml water). The amount of precipitate will depend on alkaloid content, from a cloudy to dense precipitate depending on amount.

6. Then various dilution's of the aqueous phase are made up,  2X, 4X, 8X, with further sulfuric acid soln. Eg in a series of test tubes put 2 ml, 1 ml, 0.5 ml and 0.25 ml, and top up with sulphuric acid to 2 ml the latter test tubes. This gives the dilutions as stated above.

7. These different dilution's are treated as for the original aqueous phase (2 ml in test tube, 6 drops of Silicotungstic acid) and the precipitate density noted. The table below gives an idea how the different precipitate densities can be used to give a rough quantitative analysis of the material. The different precipitate ratings of each dilution is recorded and added up, to give an overall precipitate rating, which can be extrapolated to a graph or table that will give a general idea of the samples alkaloid content, especially in relation to other samples. With practice it can be used to establish rough total alkaloid percentages of different samples.
 

 Precipitate ratings for purposes of these tests, a general guide.
 Relative analysis rather than exact quantitative analysis, most useful after some practice and experience
 0 - absolutely clear solution
 1 - transparent solution with faint traces of precipitate
 2 - mostly transparent solution with some precipitate
 3 - translucent solution with definite precipitate visible
 4 - immediate precipitate formation
 5 - large amount of precipitate formed immediately, indicative of large amount of alkaloid

1X, 2X etc. represent the various dilution's of the original aqueous phase.
In the original paper they were able to determine Gramine only races from DMT and 5MeoDMT dominant races, but not between DMT and 5MeoDMT themselves (in Phalaris grasses) with the colour of the precipitate. This once already being aware of what the alkaloids were though, and with experience in the plants and the process.
The authors of the in the Crop Science journal article commented on why they felt Silicotungstic acid was a better indicator than Mayers reagent, due to density of the precipitate and the colour reactions with alkaloids. Also they felt that whilst other solvents and acids could be used instead of chloroform and sulphuric acid, this combination produced the best results, other mixtures tended to form emulsions and foams that didn't seperate effectively.
Numbers are merely representative and not to be taken as exact correlation's between percentage alkaloid content of all plant material or species. Also apart from some differences in colour with different types of alkaloids when reacting with Silicotungstic acid, this process has no great value in qualitative analysis of the alkaloids.
 
sample no
1X
2X
4X
8X
prec. rating
% alkaloid
1
1
0
0
0
1
0.11
2
2
0
0
0
2
0.13
3
4
1
0
0
5
0.16
4
5
1
0
0
6
0.18
5
5
3
1
0
8
0.21
6
5
5
3
1
14
0.48
7
5
5
5
1
16
0.60