3- Qualitative analysis of Bioactive Constituents

i- Qualitative chemical tests:

◼ Quality of pure active constituents are directly identified by
their melting point, refractive index absorption spectra,
chromatographic properties…… etc., or by preparing their
derivatives.
◼ Direct tests for the presence of specific chemical
compounds in crude drugs, cannot often be applied
properly because of the chemical complexity of the drug
constituents.
◼ So the specific chemical compounds should be extracted
or isolated and purified before the tests can be applied.
◼ Quite often micro-chemical techniques may be performed
on the drug using micro-extraction, micro-filtration and TLC
alongside with authentics for identification.
◼ Some qualitative color tests are specific for certain
substances. In some cases the nature of the colour
reaction is well understood, but this cannot be said for
most color tests. Hence, unless a test is known to be
highly specific, a positive reaction is never to be taken as
confirmatory, but should only be used in conjunction with
other tests.
◼ Glycosides: All give positive Molish’s test due to their
sugar moieties; then more specific tests are carried out
according to their types e.g.:
◼ Antrhaquinone glycosides: Tested by the modified
Borntrager’s test.
◼ Flavonoid glycosides: Tested by AlCl3, NaOH test.
◼ Cardiac glycosides: Tested by number of tests such as
Legals', Baljet, Raymond, and keller killiani tests.
◼ Saponins: These are tested by giving a permanent froth
when shaken with water and cause haemolysis of RBCs.
◼ Volatile oils: These usually give characteristic odour to
preparations containing them; they give colors with
Alkanna tincture and Sudan III.
◼ Tannins: Tannins give characteristic colors with FeCl3
due to their phenolic nature.
◼ Alkaloids: These are tested by 2 groups of chemical
reagents; the first group gives precipitate with most
alkaloids (e.g. Mayer's, Wagner's, Tannic acid,
Dragendorrff’s and such reagents); the other group of
reagents give characteristic colors with most alkaloids
(such as Mandalin's, Froehd's, Endman's Marquis (and
such reagents).
Standards Applicable to volatile & Fixed oils:
Certain standards are particularly appropriate to volatile
oils & fixed oils.
A- Refractive index
◼ The refractive index of a substance is the ratio between
the velocity of light in air & the velocity in the substance
under test for light of a given wavelength.
◼ The R. I. of a material is given by the sine of the angle of
incidence divided by the sine of the angle of refraction.
The R.I. varies with the temperature, and
pharmacopoeial determination are made at 20oC.
 ◼ A convenient instrument is the
Abbè refractometer.
◼ Measurements of R.I. are
particularly valuable for purity
assessments of volatile & fixed
oils, and many values can be
found in E.p. & other
pharmacopia.
R.I. Oils of
1- Cassia bark Cinnamon bark Cinnamon leaf
1.61 1.573-1.6 1.53

2- Lemon oil Terpeineless

1.474-1.476 lemon oil
1.475-1.485
B- Optical rotation
◼ The optical rotation of a liquid is the angle
through which the plane of polarization of
light is rotated when the polarized light is
passed through a sample of the liquid, this
rotation may be either clockwise or
anticlockwise.
C- Quantitative chemical tests
◼ A number of quantitative chemical
tests-acid value, iodine value,
saponification value, ester value,
unsaponifiable matter, acetyl value,
volatile acidity are mainly applicable to
fixed oils.
 ii-Thin layer chromatography
TLC is particularly valuable for the qualitative
determination of small amounts of impurities. The
technique is easy to perform, effective & requires
inexpensive equipment, therefore, it is frequently used
for evaluating medicinal plant materials & their
preparations. The following specifications should be
stated for each determination:
a) The type of adsorbent & method of activation, if the
latter is not mentioned, heat at 110oC for 30 minutes
b) The method of preparation & the concentration of the
test & reference solutions.
c) The volume of the solutions to be placed on the
plate.
d) The mobile phase, the temperature & time of
development, distance of migration of solvent (mobile
phase).
e) The drying method & the temperature to be used & the
method of detection
f) For the results obtained observe the spots:, number &
approximate position, the Rf values if necessary and
fluorescence and/or colour
Preparation of the samples
Prior carrying out the test, the sample should
undergo the following simple & rapid extraction
process:
◼ To 0.1 - 1.0 g of the powdered plant material add 1-
10 ml of solvent, extract by stirring or shaking the
mixture for 3-30 minutes or by heating to boiling
point & allowing to cool.
◼ Remove the insoluble matter by centrifugation, or
filter through small funnel with filter paper or cotton
plug if necessary, evaporate the filtrate on a water
bath. using just the time required to remove the
solvent & dissolve the residue in small volume of
solvent (e.g. 0.1 - 0.2 ml).
Observation & interpretation of the
chromatograms:
◼ In the first instance, observe the spots produced in
daylight, then under short wave and/or long wave
Ultraviolet light.
◼ Mark the centre of each spot with a needle.
◼ Measure & record the distance from the centre of
each spot to the point of the origin, and indicate for
each spot the wavelength under which it was
observed. If indicated in the specification, spray
the spots with the indicated reagent, observe &
compare the spots with these of reference material
if required calculate the Rf or Rsta values as
follows:
 a a
◼ Rf = _____ Rsta = _____
b c
a = the distance between the point of origin & the center of
the spot of test material
b = the distance between the point of origin & solvent front
c = the distance between the point of origin & the center of the
spot of reference material
◼ Rf values are only given for information & they may differ
slightly with each experiment due:
➢ To different saturation condition of the chromatographic
chamber,
➢ Or the activity of the adsorbent layer
➢ Or the composition of the mobile phase.
Micro method for TLC
◼ A new thin layer chromatography method has now been
developed that holds certain advantages over the classical test.
The advantage of the micro method:
◼ It is capable to give rapid information, since the process takes an
average of 3.5 minutes at a higher resolution, and uses very
small quantities of standard materials.
◼ Furthermore, volumes of only 1 ml or at most 2 ml of solvent need
to be used as opposed to 20 - 25 ml in the classical method.
◼ Detection is generally carried out by spraying the developed plate
with 10 - 15 ml of a rather aggressive reagent in the classical
method, but the new method allows the chromatograms to be
dipped in specified reagent solutions providing a very even
distribution of the reagent on the surface thus increasing the
sensitivity of the reactions.
◼ Where laboratories have no air conditions & are situated in
countries with high temp., the method can be carried out using a
refrigerator which is a suitable method for developing countries.
 Main differences between micro -TLC and
Classical TLC are shown in the following table
Classical TLC Micro - TLC
Glass plate Up to 20 x20 cm 10 x 10 cm

Amount of sample Relatively large Very small amount

Diameter of spot Up to 4 mm Not exceeding 2 mm.

Solvent volume 20 – 25 ml 1, rarely 2 ml.

Development distance 10 – 15 cm up to 6 cm

Time of running 30 - 45 mm. 3.5 mm.

Method of spraying Sprayed with 10 - 15 ml of spray Chromatogram can be dipped

reagent using a sprayer; It in the spray reagent.
provides less even distribution It provides a very even
and less sensitive reaction distribution, thus increasing
sensitivity of the reaction
Sensitivity
Less sensitive More sensitive
2D-TLC Finger prints (single and mixed
components) principle:
◼ 2D-TLC chromatogram of the marketed
herbal medicine is compared with 2D-TLC
chromatograms of authentic herbal mixture
prepared from authentic medicinal herbs
and markers mixtures representing each
herbal components under the same
analytical conditions.
The comparison should include:
◼ The number of the spots in the diagonal.
◼ The colour of each spot sprayed with
specific spray reagents.
◼ The Rf values of the spots corresponding to
the marketed herbal mixture and the
authentic herbal mixture as well as
authentic markers mixture.
 Example : Analysis of marketed renal herbal tea

1) Marketed renal herbal tea: 2) Authentic renal herbal tea

Rx (prepared from authentic
herbs)*
Rx
Liquorice root 20 g Liquorice root 20 g
Peppermint leaves 20 g Peppermint leaves 20 g
Achillea leaves 5g Achillea leaves 5g
Ammi visnaga fruits 20 g Ammi visnaga fruits 20 g
Cymbopogen leaves 20 g Cymbopogen leaves 20 g
Ambrosia leaves 10 g Ambrosia leaves 10 g
Chicory leaves 5g Chicory leaves 5g
Markers compounds selected to represent each
herb:

No. Herbal components Markers

1 Liquorice root Glycyrrhizin
2 Peppermint leaves Menthol
3 Achillea leaves Azulene
4 Ammi visnaga fruits Khellin
5 Cymbopogen leaves Proximadiol
6 Ambrosia leaves Damsin
7 Chicory leaves -
Procedure:
◼ A solvent system is developed to resolve the markers mixture
representing each herb in the mixture.
◼ One and two-dimensional TLC chromatograms are developed
separately under the same analytical conditions for:
➢ Marker mixture.
➢ Marketed herbal tea.
➢ Authentic herbal tea.
◼ Each developed chromatogram is sprayed separately with specific
spray reagent. (selective spray reagent (1% p-anisaldehyde).
◼ The three chromatograms should coincide (Finger print) regarding:
➢ Number of the spots on the diagonal.
➢ The colour of spots representing each herb (along with the
markers).
➢ Rf values.
Markers separation
◼ The result of TLC analysis, as well as, separation
of markers mixture using chloroform/methanol
(9.9:0.1) as solvent system and p-anisaldehyde as
spray reagent:
One-dimensional TLC of markers mixture

Colour after
Spot Comparable
Rf spraying with p-
No. with
anisaldehyde
1 0.96 Red Azulene
2 0.72 Blue Menthol
3 0.37 Yellow Damsin
4 0.29 Yellow Khellin
5 0.067 Bluish violet Proximadiol
6 0.056 Rose ted Glycyrrhizin
 Two-Dimensional TLC of markers
mixture

Colour after spraying

Spot Comparable
Rf with p-anisaldehyde
No. with
reagent
1 0.99 Red Azulene
2 0.91 Blue Menthol
3 0.38 Yellow Damsin
4 0.33 Yellow Khellin
5 0.12 Bluish violet Proximadiol
6 0.11 Rose red Glycyrrhizin
 Two-Dimensional TLC of alcoholic extracts of authentic
and marketed renal teas comparable with markers
Colour after spraying with
Rf
p-anisaldehyde reagent
Spot No. Comparable with
Authentic Marketed Authentic Marketed
mixture mixture mixture mixture
1 0.91 0.92 Blue Blue Menthol
2 - 0.72 - - -
3 0.59 0.6 Violet Violet -
4 0.51 0.51 Violet Violet -
5 0.37 0.36 Yellow Yellow Damsin
6 0.33 0.33 Green Green -
7 0.32 0.32 Yellow Yellow Khellin
8 0.28 0.28 Violet Violet -
9 0.25 0.25 Violet Violet -
10 0.14 0.17 Dark blue Green -
11 0.12 0.12 Bluish violet Bluish violet Proximadiol
12 0.1 0.1 Rose red Rose red Glycyrrhizine

Conclusion: Both 2D-TLC chromatograms of authentic and

marketed herbal mixture coincide with that 2D-TLC
chromatogram of the markers.