Infrared Spectrometer characteristics and how to use

The functional groups were determined by the generation of spectrograms using an IR spectrometer (Nicolet IR Series, FTIR Spectrometer, USA). The solid sample extracted from the compounds and mixed with potassium bromide (KBr) to a fine powder in an agate mortar. The sample: KBr ratio was 1:100 and a portion of these compounds was separated and placed on a sample holder to prepare subsamples as pellets using a press, the pellets should be homogeneous and thin, placed on a steel plate for samples to read in the IR. Sample contamination by dust must be avoided and instructions followed on how to use the press to make pellets (subsamples).

Subsamples were placed in the spectrometer and analyzed in the same band for 32 scans for the best spectrum which should be in the fine and intense band according to its transmittance percentage. The optimal spectrum is based on sample/solvent ratio (KBr), gain value and chart rate. Once the optimal spectrum for each sample was obtained, the spectrum scale was calibrated on paper, obtaining the wave number of the different components.

Measurement of humic and fulvic acids percentage and functional groups

From the solution, 50 ml were taken, the pH adjusted to 4 with acetic acid, separating humic from fulvic acids. Percentages of humic and fulvic acids were measured by reverse titration with potassium permanganate (K₄MnO₇) (Table 1).

Table 1 Percentage of humic and fulvic acids. 

They were measured for the total acidity (TA), i.e., the content of oxygenated functional groups, such as the carboxyls (-COOH) and hydroxyls (- OH), the methods described here are based on measurements of total and carboxylic acidity from ^{Wright and Schnitzer (1959)}, and ^{Schnitzer and Gupta (1965)}.

For total acidity measurement, the sample is treated with 2N barium hydroxide for 24 h. The Ba(OH)2 remaining in solution after reaction, is titrated with a standard acid solution.

For carboxylic group titration, humic materials are stirred for 24 h with a calcium acetate solution in excess causing the release of acetic acid according to this type of reaction:

The acetic acid released is then titrated with a standard bicarbonate solution.

The proportion of phenolic groups was calculated by the difference between total and carboxylic acidity.

Generally, fulvic acids are more oxidized than humic ones, i.e. they are more polymerized. In particular, fulvic acids are the most oxidized, since they show higher value of total acidity.

The above indicates that fulvic acids contain more negative electric charges.

^{Canellas et al. (2008)} defined the HS as relatively small supramolecular associations basically grouped by hydrophobic interactions and hydrogen bonds. ^{Schulten and Schnitzer (1993)} developed a two-dimensional (2D) molecule, the HA model structure with alkyl aromatic rings which play an important role. Oxygen is present as carboxyls, phenolic and alcoholic hydroxyls, esters and ketones, while nitrogen is produced in nitriles and heterocyclic structures. The resulting carbon skeleton exhibits high microporosity with pores of various sizes, which can trap and bind other organic and inorganic soil components, as well as water.

Table 2 Total acidity (TA) and carboxyl (-COOH) and hydroxyls (-OH) functional groups of humic substances extracted from leonardite. 

AH= ácido húmico; AF= ácido fúlvico.

Calcium humate and iron fulvate extraction by regulating pH in the solution.

Following the criteria established by ^{Iakimenko, 2005} and ^{Rady, 2012}, who comment that a humic acid binding a mineral, is called here calcium humate and iron fulvate where addition of these minerals in our study was as follows:

Calcium humate: to 100 mL of HA, originally at pH 8, 125 g of Ca(NO3)2 were added, reaching pH 7 then 20 g were added i.e. overall 145 g of Ca(NO₃)₂ reaching pH 6.

Iron fulvate: to 100 mL of FA originally at pH 4, 0.5 mL of KOH were added, increasing pH to 8, then 10 g of FeSO₄ were added, reaching pH 7, then 1.75 g more were added to a final pH 6.

Solidification of humic and fulvic acids, calcium humates and iron fulvates

The solidification was performed by a rotary evaporator (Yamato, SE 500 Model, Japan), placing 500 mL of humic acid (pH 8) and its fractions prepared at pH 6 and 7, also for fulvic acid (pH 4) and its derivatives at pH 6 and 7, overall six treatments, which were deposited in a 1 L flask, heated on water bath at 60 ^oC, rotating at 80 rpm until water is removed, the solidification required 8 h. The solvent was not completely removed so it was transferred to a porcelain dish and heated at 40 °C for 2 h on an electric grill to remove the remaining water and thereby obtaining the solid.

The Figure 1 shows the spectrogram of humic acid enriched with calcium nitrate (Ca (NO3)2) at pH 6, 7 and the original pH 8 after extraction.

Figure 1 Infrared spectra of humic acids enriched with calcium nitrate extracted from leonardite. 

The blue line represents the HA at pH 8 in which major signals or functional groups involved in ion exchange are noticeable: the -OH in the extractant or extraction vehicle appears in the 1 375 cm^-1 band, NH amines or amides at 1 620 cm^-1, the -CH₃ and -CH₂ at 2 922cm^-1 and -OH free functional groups at 3 400 cm^-1, the HA at pH 7 and 6, red and green lines, respectively, show additional peaks after addition of the mineral element, thus the signal description is in greater proportion, favoring its description, differentiating peaks by purity and concentration, in the functional group at the lowest wavelength range an aromatic-N-R group was found, this represents a tertiary amine at the 760 and 631 cm^-1 bands for pH 7 and 6, the secondary -OH for HA at both pH were found at 1 130 and 1 120 cm^-1, the CH-OH, -OH groups in the extraction vehicle were found at 1 390 cm^-1, amines or amides N-H or with carboxyl-C= O at 1 620 and 1 600 cm^-1, respectively, the -CH₃ showed a similar value of 1 930 cm^-1, the CH2-CH3 was located at2 870 and 2 840 cm^-1, the free -OH appeared at 3 390 cm^-1 in both organic-mineral complexes.

The HA at pH 6 shows a higher secondary -OH content followed by the HA at pH 7, however the latter shows more N-H, C= O and free -OH, this indicates less light absorption or higher individual frequency of this type of compounds, and thus higher Ca (NO₃)₂ absorption in HA at pH 7.

The fulvic acid spectrogram (Figure 2) at the original pH, plus the two levels generated after mixing with ferrous sulfate (FeSO₄) yielded the following results.

Figure 2 Infrared spectra of fulvic acids enriched with ferrous sulfate extracted from leonardite. 

The FA at pH 4 (blue line) represents the original pH at the time of extraction, in this spectrum, the reading shows the following functional groups, methylene -CH2-CH3 has the best signal at 1 162 cm^-1, for the -OH as extraction vehicle 1 439cm^-1, N-H amines or amides group at 1 620cm^-1 and free -OH at 3 445cm^-1, the FA at pH 7 and 6, red and green lines respectively, showed different peaks after mixing with FeSO₄, for both pH the primary -OH is located at 1 050 cm^-1, just as the -CH2-CH3 at 1 360 cm^-1, the -CH2-(C=O)- , -CH₂-(C=N) groups at 1 380 and 1 390 cm^-1 respectively, for the extraction -OH 1 440 and 1 450 cm^-1, -N-H amines or amides at 1640 cm^-1 for both mixtures, an overtone caused by an -NH- amines or amides overlap at 1 810 and 1 830 cm^-1 for pH 6 and 7, the -CH₃ group is at 1 860 cm ^-1 for both pH, just as the -CH₂- CH₃ for both pH is at 2 930 cm^-1, and free -OH at 3 570 cm^-1 and 3 430 cm^-1 for pH 6 and 7 respectively. The FA at pH 6 and 7 show many similarities, however FA at pH 7 shows higher frequency for the (-OH) free functional group and thus higher iron adsorption.

These results agree with those obtained by ^{Chassapis et al. (2010)}, regarding the use of a typical infrared spectrum showing interactions and differences in humic substances, absorption, adsorption, or associations of different relevant phenolic groups distributed in the regions 1 050, 1 650 and 3 400 cm^-1, this coincides with ^{Sivakova et al. (2010)} who showed IR spectra with characteristic absorption bands for humic acids due to the presence of carboxyl and carbonyl groups (1 720-2 700 cm^-1), hydroxyl groups (3 400-1 256 cm^-1), oxygen-ether (1650 cm^-1), -CH₃ -CH₂ (2 930 cm^-1).

It is also noted that the physicochemical properties of humic substances vary according to their origin (^{Fukushima et al., 2006}), they are amorphous multifunctional biopolymers, comprising hundreds of organic compounds including carbohydrates and condensed aromatic rings, which may be substituted by phenol groups, carboxyls, hydroxyls and methyls, and these results are consistent with those obtained by ^{Peuravuori et al. (2006)} and ^{Evangelou et al. (1999)}. These groups act like chelating agents by complexing and chelating cations in the soil solution, transferring them to the root cell wall, then these nutrients are translocated by the xylem stream to the growing points.

The metal-ion bond (Ca and Fe) has been shown to vary with the solute concentration of cations, pH, mineral and bond type (^{Puls and Bohn, 1988}). Organo-mineral complexes allow plants to overcome adverse effects of salinity, improve soil aggregation, aeration, permeability, water holding capacity, micronutrients absorption, and decreased absorption of some toxic elements (^{Ryabova, 2010}). Results from many authors suggest that the number and strength ofion groups are not related to atomic weight, atomic number, ionic radius, or hydrated radius of retained metal, since humic substances contain different types of functional groups with a wide range of ion complexing capacities.

One promising way to improve the quality of humic substances is their combination with inorganic products, conferring mechanical strength and resistance to acid and alkaline hydrolysis and improved sorption properties. This is a low-cost and accessible procedure to develop organic-mineral adsorbents since these raw materials are abundant, cheap, and can be chemically modified.