Metodo di Determinazione Dell'acido Beta-Indolacetico Nei Vegetali Per Cromatografia e Diluizione Isotopica (Riassunto)

Abstract

Method of determination of beta-indolacetic acid in plants by chromatography and isotopic dilution. — The quantitative determination of beta-indolacetic acid content in plants is a very complex problem still unsolved because of the difficulty of separation and the scarce specificity and sensitivity of thè analytical methods. The separation of IAA from plant tissues extracts, which can be obtained by chromatography, is not satisfactory; especially for the interfering of substances, the elimination of which is very difficult and which often influence the colorimetric and spectrofotometric determination of the fractions in which we find the IAA after separation. An analysis with biological methods on the same fractions might have perhaps a greater probability to be free from interferences (but that is obviously difficult to control). As far as the specificity of the fluorimetric method in the IAA determination is concerned, the literature data are too scarce to give a judgement about it. According to the method of colorimetric determination it is known that the Salkowsky's reactive and the Herlich's one are aspecific as they both give coloured products with all the indolic compounds; the reaction is altered by the presence of a large number of substances (Nichols, Gordon, Linser). It is clear that all those substances which absorb in the wave length range of the IAA (from 300 to 270 mμ) will influence the spectrophotometric determination.

Considering the uncertainty of the measures carried out by the several mentioned authors and by others we have thought of using both the chromatographic separation technique and the method of isotopic dilution.

So, if we add to the plant tissues extract a known quantity of IAA — C¹⁴ we can make all the operations of separation and identification with greater safety. An attempt to use IAA-C¹⁴ in order to facilitate the measure was made in 1961 by Hamilton and coll. but, perhaps, because of the used technique of separation the recovery of the IAA was not more than the 28%.

With our technique, on the contrary, we can obtain the 98–100% of recovery. The following method has been used: to the plant tissues extracts liofilized we added 0,0126 μc of IAA-C¹⁴ with specificic activity 1,94 mc/mmol. Then we proceeded with the ethereal extraction according to the classic methods (Bennet-Clark and Kefford); the extracts were purified by NaOH (or NaHCO₃) (Bennet-Clark and Kefford, Rakitin-Povo-Lotskaya, Haagen-Smith e coll., Avery e coll.) and were extracted again with ethyl ether after acidification at pH 3 with HCl (or CH₃COOH).

The ethereal purified extract was vacuum evaporated and the residue extracted with the organic phase of petroleum ether and n-butyl alcohol mixture saturated with formic acid 0,5 M (97:3). Then it was chromatographated on silica gel column (⊘ mm 0,05–0,2). hydrated by H-COOH 0,5 M, in accordance with the method described by Loyd E. Powell in Plant Physiology 1960 and slightly modified by us.

In fact if we perform the separation of the acid indolderivates by petroleum ether containing 0,2 and 3% of butyl alcohol saturated in H-COOH 0,5 M, (as foreseen by the original method) with the second solvent IAA is eluted with an impurity which absorbs in UV in the same wave-length of the IAA. The use of three eluents at 0,2–1,5 and 3% in butyl alcohol permits, on the contrary, to obtain IAA free from impurity interfering with spectrophotometric reading in the fractions eluted by the solvent at 3%.

Recovery values of the 98–100% were obtained by adding known quantities of IAA and IAA-C¹⁴ to the liofilized; moreover the ratio IAA-C¹⁴/IAA remained constant for all the collected fractions. By appling the known formula of the isotopic dilution it is possible in theory to go back from the radiochemical and spectrophotometric measurement of a given fraction to the total value of IAA extracted from plant tissue, knowing the radioactivity of the IAA-C¹⁴ added to the liofilized.

Trials carried out in this way beginning from 25 gr of peas apices or from the same quantity of maize seeds, after 40 hours from germination, did not show presence of IAA. Afterwards, we will try the extraction and the determination beginning from highter quantitatives of plant material.