Separation of humate into humic and fulvic acids into their fractions and their biological activity

Based on research, it has been firmly established that the main structural units of the humic acid molecule are linked together by simple ether bonds. The latter can be hydrolyzed.

As a result of hydrolysis, as is now commonly believed, the humic acid molecule splits into its constituent structural elements. Aliphatic substances, which represent the peripheral part of the acid, connected to the core by simple ether bonds, pass into the solution. The cores of humic acids, according to most researchers, are not subject to hydrolysis and therefore, freed from peripheral chains, should remain in the sediment.

In our study, we took advantage of this provision and isolated individual fractions of humic acids according to the scheme presented in Fig. 1.

Research methodology

The dry humate preparation , which was subsequently subjected to hydrolysis, was obtained from peat. Humic and fulvic acids were extracted from peat by boiling 3% NaOH, after which they were precipitated with 10% HCl. The humic acid gel was collected on a filter, washed with water until a negative chlorine test was obtained, then reprecipitated, dried at a temperature of 45-60°C, ground into powder and used in experiments in this form.

The hydrolysis of humic acid was carried out as is customary for proteins. A 1 g sample of the acid was placed in a test tube, filled with 10 ml of 6 N HCl, the test tube was sealed and placed in a drying cabinet for 6 hours at a temperature of 125-130°C.

Cleaning of fractions

After hydrolysis, the decay products were extracted from the ampoule. The humic acid nuclei (residue after hydrolysis) were separated from the hydrolysate by filtration. Since both fractions contained a lot of chlorine and were supposed to be tested in a vegetation experiment, they required preliminary purification. The purification method was different for the fractions obtained.

Faction No. 1

The residue after hydrolysis, which, unlike the initial humic acid, was completely black and poorly soluble in alkali, was washed on a filter until a negative chlorine test was obtained. Then sodium humate was prepared from it. 2 ml of 0.1 N NaOH were taken per 100 mg of non-hydrolyzable residue. In the form of such a compound, this fraction was used in the experiment and, according to our assumption, characterized the physiological properties of the humic acid core.

Fractions #2 and #3

Purification of hydrolysate from hydrochloric acid turned out to be more complicated. We tried a method known in the literature for purifying hydrolysate from chlorine by repeated evaporation under vacuum (fraction No. 2). However, since some mineral salts passed into the hydrolysate, resulting in the formation of chlorides, it was impossible to completely get rid of chlorine. Therefore, other purification methods were tested, which turned out to be more preferable. One of them is dialysis of the hydrolysate in a cellophane bag against the flow of water until the chlorine sample disappeared from the wash water. After dialysis, a colored solution remained in the bag, which was fed to the experiment (fraction No. 3), and a small sediment, which was removed. The sediment was especially noticeable when the hydrolysate was neutralized with caustic soda before dialysis (fraction No. 3-a).

Chromatographic separation

An original approach to studying the composition of humate substances is their chromatographic separation on activated carbon with subsequent elution of adsorbed compounds with various solvents. Forsyth (1947) was the first to propose this method for the substances in question, and then Dragunov and Khan successfully used it.

Later, Drozdova's work appeared, which developed a method for purifying fulvic acids by fractionation on carbon. We used this method, considering that the purification of fulvic acids from an acidic solution and the purification of an acidic hydrolysate have much in common. The hydrolysate, like fulvic acids, is orange in color and soluble in alkalis and acids. When neutralized at the point of color transition of congorot, a precipitate partially falls out, which, upon further alkalization or acidification, again passes into the solution.

Fractions #4, #5 and #6

Compounds of non-aromatic nature, such as amino acids, amino sugars, sugar alcohols, carbohydrates, uronic acids, etc., pass into the hydrolysate of humic acids, the activity of which also had to be tested. Using the Forsyth method as modified by Drozdova, it was possible to separate the hydrolysate into at least two fractions and study each of them separately.

For this purpose, the hydrolyzate was passed through a layer of activated carbon. The solution passing through the carbon was colorless and contained, according to Drozdova, inorganic salts, hydrochloric acid, amino acids, nitrogen bases, simple carbohydrates, etc. This solution, as far as possible, was concentrated and purified from chlorine under vacuum and then entered into the experiment (fraction No. 4). The colored substances of the hydrolyzate, adsorbed on the carbon, were removed with a mixture of acetone and alkali. Then acetone was distilled off from the eluate, the residue was dialyzed in a cellophane bag until a negative sample of the washing waters for phenolphthalein and entered into the experiment (fraction No. 5). This fraction had a lemon-yellow color in an acidic medium, and an orange color in an alkaline medium. By analogy with the purification of fulvic acids, it can be assumed that the fraction adsorbed on carbon contains a substance of a quinoid nature, since it did not give a positive reaction with α-naphthol, Millon's reagent and ferric chloride, but changed color depending on the reaction of the medium and had a sharp chlorine-like odor.

It should be noted that in order to avoid possible losses during desorption and dialysis, the said fraction of hydrolysate was also tested directly in the form adsorbed on carbon. In this case, the activated carbon after passing the hydrolysate through it was washed from chlorine, dried, crushed and used in the experiment (fraction No. 6).

Research results

The biological activity of the fractions obtained by the above method and the original humic acid in the form of potassium humate was studied in water cultures. Distilled water was used as a control.

4-6 mg of humic acid preparations were added to a 0.5 liter vessel. Adsorbed substances on activated carbon were added on the same basis. Table 1 shows the effect of different humic acid fractions on the growth of barley, wheat, and tomato roots.

These data indicate that the fragments of the humic acid molecule are biologically active. Potassium humate prepared from the non-hydrolyzable residue is not inferior to the standard potassium humate solution in stimulating root growth. Consequently, the humic acid core obviously plays a leading role in stimulating root growth. At the same time, the experiment also shows quite strong stimulation by substances that are adsorbed on carbon after hydrolysis. If we take into account that these substances are polyphenolic and quinone compounds, the effect obtained in the experiment can be explained by their direct influence on the plant.

We have also noticed that activated carbon itself as an adsorbent also causes increased root growth. Some researchers explain this effect by the ability of carbon to adsorb hydrogen ions released by the root system, due to which carbon promotes the process of respiration and mineral nutrition of plants. P. A. Vlasyuk, for example, recommends adding brown coal to the rows as an adsorbent, which reduces the concentration of salts in the root zone and improves crop nutrition.

Therefore, in the experiment with tomatoes we used activated carbon, which before the experiment was washed with strong hydrochloric acid, then washed from chlorine, part of it went to adsorb the hydrolyzate, and the other was introduced in pure form into the experiment. When comparing the growth of wheat, barley and tomato roots in length, it can be seen that there are impurities in activated carbon that can distort the results of the research, since they promote root growth. The adsorbed substance on the carbon in this case is also biologically active.

From this experiment, one can also be convinced that humic acid itself in the form of a finely ground gel does not produce any effect as an adsorbent. These adsorbents do indeed have different properties, since they have different absorption capacities (Table 2).

A comparison of the absorption capacities of carbon and humic acid gel shows that if the positive effect of carbon on root growth in our experiments was explained by adsorption capacity, humic acid gel would also have been effective, while in fact it even slightly inhibited root growth.

Below (Table 3) are the results of an experiment in which humic acid fractions were tested after desorption from activated carbon.

Conclusions

From these experiments it is evident that the mixture of hydrolysis products (fraction No. 2) is more active than the fraction of substances that is not adsorbed on carbon and which presumably includes the peripheral chains of humic acids. Consequently, the biological activity of humic acids obviously depends on the fraction that is adsorbed on carbon and which, according to a number of features, has a polyphenolic structure.

Pre-neutralization of the hydrolysate with alkali before dialysis leads to a decrease in the stimulating effect, apparently due to the fact that as a result of neutralization, the active substances pass into the sediment, which is removed after dialysis.