Physico-Chemical Laboratory, Kharkov
Spot Chromatographic Adsorption Analysis and its Application in Pharmacy
Communication
(Translation from the Russian Journal Farmatsiya. 1936 No.3. P. 1-7.) Izmailov TLC KIT
The fundamentals of chromatography were originally developed by Russian botanist
M.S. Tswett during an investigation of plant pigments. The method consist in
passing a solution for analysis through a tube packed with an adsorbent. During
passage through the tube the compounds are separated into zones depending on
adsorption potential. ”Development” is used, to make a separation more
complete, i.e. a pure solvent is passed through the tube. Zones are separated by
solvent. Since a set of zones is obtained, each zone contains either one
component or simpler mixture. The so-called chromatogram is obtained.
If
colored, the zones can be identified in daylight. If the components are
fluorescent, they can be detected under ultraviolet light. The result of the
investigation were published by Tswett in 1910 [1, 2]. His discovery was not,
however widely used until 1931, when chromatography attracted the attention of
researchers. They came to the conclusion that chromatography could be of
practical importance to numerous areas of investigation. A detailed list of
references concerning the application of chromatography to the analysis of plant
substances, hydrocarbons, products of benzene coking, pigments, vitamins, etc.,
can be found in reviews by W. Stix[3] and E. Lederer [4,5].
In 1935,
chromatographic analysis was first used in pharmacy [6]. Valentin and Franck [7]
used chromatography to analyze Spanish fly infusion(tincture). They isolated
canth-aridin. In 1937 Franck [8] analyzed a wine Kondorango, infusion of
wormwood, foxglove, strophanthus, henbane oil, and other galenical preparations.
Different chromatograms were obtained from infusions prepared in different ways.
Adulteration of saffron was also identified.
In 1937 Merz and Franck used
chromatography for quantitative determination of components of the following
drugs: belladonna and cinchona infusions, cinchona extract, ipecacuanha and
strychnine infusions [9]. They mentioned that in comparison with the
traditionally used pharmacopeia procedure chromatography enabled the use of less
sample, and gave better quantitative results more rapidly.
In 1937 Ernst an
Weiner [10] used chromatography to analyze pharmaceutical preparations
containing antrax-quinones. Kondo et all [11] separated compounds in mixtures,
namely pyramidon-veronal, lupanin-spartein, morphine-thebaine, etc.
Chromatography is usually performed as follows. Glass tubes (sometimes of uviol
glass) of various length and diameters are packed with adsorbent. Then by means
of a Bunsen flask, the tube is connected to a pump. The adsorbent layer is
thoroughly compressed. The solution to by analyze is poured onto the layer and
sucked through it. To develop the sample into separated zones the adsorbent is
washed with different solvents (most simply with water). Various fine powders
are used as adsorbents, the most widely used being aluminum oxide. The
separation characteristics of the mixture analyzed depend on the adsorbent and
mobile phase selected. Preliminary tests are therefore necessary to select the
optimum conditions for chromatographic separation. The preliminary test require
both sufficient material and a lot of time.
Using chromatography in pharmacy, we
came to the conclusion concerning the simplification of the preliminary test
procedure.
We developed a spot chromatography technique consisting in
application of samples (as drop) on to a layer of adsorbent and separation of
the sample into zones.
The planar adsorbent layer was prepared as follows:
chalk, talc, magnesium oxide, calcium oxide, aluminum oxide, and other
adsorbents here mixed with water. The suspension formed was applied to the glass
plate. After drying, a thin layer of sorbent was obtained. In the course of the
experiments, we found that the appropriate layer thickness was 2 mm; thicker
layers were inclined to crack whereas thinner layers did not coat the surface
uniformly.
A drop of the mixture for analysis was applied to the prepared layer.
Concentric circles were formed and were visible owing to fluorescence under the
light of a mercury discharge lamp. The so called ultra-chromatogram was
obtained. This chromatogram can be developed by applying several spot of
solvent, the results being the same as in conventional development. We performed
comparative chromatography of belladonna, digitalis, and rhubarb infusions using
both conventional chromatography and our method. All samples met pharmacopeia
requirements.
Experimental
A drop of belladonna infusion was applied to a plate
coated with a thin layer of aluminum oxide. At the same time 5 ml of the sample
was passed through the absorption tube. Two zones were obtained. When the plate
and the tube were exposed to UV light it appeared that the zones separated were
totally similar both with respect to zone color and arrangement (Fig.1).
Izmailov TLC KIT To
elucidate the question of chromatographic identity, after development the column
was gradually washed with drops of alcohol (as far it was dried by alcohol). The
washing procedure was ceased when the spot separated. The washing procedure was
ceased when the spots were separated. The chromatograms obtained on the plate
and in the tube were identical and looked like Franck’s chromatograms.
Similar
comparative experiments with column and plate, with and without washing with
alcohol, were performed for samples of digitalis and rhubarb infusions (Fig.
2-4).
Izmailov TLC KIT Ultrachromatograms obtained on the layer coincided com pletely with that
in the adsorption tube. Zone number, color intensity, and arrangement were
identical. Spot chromatography can therefore be used for complete replacement of
conventional chromatography in pre-estimation and in all cases when
chromatography is needed for qualitative analysis.
Spot chromatography without
development was then used to test all infusions mentioned in the USSR
pharmacopeia (7 edition).
Testing different adsorbents, namely calcium oxide,
magnesium oxide and aluminum oxide we came to the conclusion that the best
result were obtained with aluminum oxide. Aluminum oxide prepared by various
techniques was used, the results being different only in respect of fluorescence
intensity.
Ultrachromatograms of different infusions differed greatly, enabling
rapid identification of galenical preparations even without zone development.
If, however, spot chromatograms were developed with alcohol there was an
increase in the numbers of zones. Such developed planar chromatograms were
obtained for some infusions.
Table 1 shows the arrangement of the colored zones
of the ultrachromatogram in consecutive order from center to periphery. Data
obtained by Franck (using conventional chromatography) are given for comparison.
Taking into account planar ultrachromatograms coincidence with Franck’s
chromatograms as well as individuality of chromatograms of infusions it can be
concluded that spot chromatography is worth using to identify galenical
preparation and their composition; characterization of galenical preparations is
based on the number of zones, their arrangement, and their color. The latter
test is to some extent uncertain, especially when fluorescence is used, whereas
the previous two are beyond any doubt.
Published data on the fluorescence color
of different alkaloids do not agree (table2).
Izmailov TLC KIT Some papers on chromatography [14]
describe colored zones as follows: cream-colored with pink tint, pistachio
colored transforming into cream-colored, etc. Such types of description do not
give exact characteristics. It is clear that exact colored nomenclature is
necessary for characterization of colored zones.
Our further investigation will
be aimed at experimental verification of the objective color of the fluorescent
zone and further development of the application of spot chromatographic
analysis.
Using spot chromatography planar ultrachromatograms of different
infusions were obtained, namely wormwood, belladonna, capsicum, cinchona,
foxglove, ipecacuanha, convallarie, Spanish fly, and cinnamon.
Conclusion
1.
Spot chromatographic development consist in zone separation on a thin adsorbent
layer, using one drop of sample.
2. The same quantitative results were obtained
using both spot chromatographic and conventional chromatographic methods.
3.
Advantages of spot chromatographic analysis consist in the possibility of using
one drop of sample under test, the very small amount of adsorbent and minimal
expenditure of time.
4. Spot chromatography can be used for preliminary testing
of adsorbent properties as well as development methods
5. Spot chromatography
can be used for qualitative evaluation galenical preparation and their
identification.
References
1. Tswett M.S. Chromophils in the plant and animal
world. Warsaw, 1910(in Russian).
2. M.S. Tswett, Chromatographic Adsorption
Analysis: Selected Works/Compiler V.G.Berezkin; translation editor Mary R.
Masson. Chichester: Ellis Horwood Ltd., 1990. 112 p.
3. Stix W. //Uspekhi
khimii. 1936. V. 5, N 1P. 4 (in Russian).
4. Lederer E. // Zhurnal Obschey
khimii. 1936. V. 5, N1. P. 4(in Russian).
5. Lederer E. // Chimie at Industrie.
1935. T th May. P.33.
6. Valentine //Pharm. Ztg. 1935. V. 80. P.469.
7.
Valentine, Franck // Pharm. 1937 V. 275. P.345.
8. Franck //Arch. Pharm. 1937.
V. 57. P. 218.
9. Merz, Franck// Arch. Pharm. 1937. V. 57. P. 218.
10. Ernst,
Weiner// Scientia Pharmaceutica. 1937. V. 8. P. 45.
11. Kondo H. //J. Pharm.
Soc. Japan. 1937. V. 57. P. 218.
12. Radley J. Fluorescence Analysis in
Ultraviolet Light. 1933.
13. Danckworth P.W. Lumineszenz Analyse im
ultravioletten Licht. 1931
14. Urazovskiy S.S., Rozum Yu.S.// Ukrainskiy
Khemiczniy Zhurnal. 1937. V. 11. P. 525(in Ukrainian)
