Polarity-based sequential extraction as a simple tool to reveal the structural complexity of humic acids

A sequential chemical extraction with a defined series of eluotropic organic solvents with an increasing polarity (trichloromethane < ethyl acetate < acetone < acetonitrile < n-propanol < methanol) was performed on peat-bog humic acid. Six organic fractions were obtained and subjected to a physicochemical characterization utilizing methods of structural and compositional analysis. Advanced spectroscopic techniques such as Attenuated Total Reflectance (ATR-FTIR), total luminescence, and liquid-state 13C NMR spectrometry were combined with elemental analysis of the organic fractions. In total, the procedure extracted about 57% (wt.) of the initial material; the individual fractions amounted from 1.1% to 19.7%. As expected, the apolar solvents preferentially released lipid-like components, while polar solvents provided organic fractions rich in oxygen-containing polar groups with structural parameters closer to the original humic material. The fraction extracted with acetonitrile shows distinct structural features with its lower aromaticity and high content of protein-like structural motifs. The last two—alcohol extracted—fractions show the higher content of carbohydrate residues and their specific (V-type) fluorescence suggests the presence of plant pigment residues. The extraction procedure is suggested for further studies as a simple but effective way to decrease the structural complexity of a humic material enabling its detail and more conclusive compositional characterization.

The elemental composition, atomic ratios and spectroscopic coefficients of SBPHA are presented in Table 1 and 2. As already emphasized in the paper, the elemental composition is expressed in atomic percent (at. %, equivalent to molar %), because, it better reflects the proportion of hydrogen in the structure of humic substances. The elemental composition and/or atomic ratios of peat HA were partially similar to that of peat humic acids from other regions of the world [1][2][3].
In the present study, the value of the EET/EBz ratio for SBPHA was determined to ~ 0.7 (see Table 2). This result suggests that the substitution of aromatic rings with oxygen-containing functional groups was comparable with humic acids isolated from other caustobioliths (i.e. lignite and leonardite) [4,5]. Another absorption coefficient described in the literature is the E2/E4 ratio (also referred to as A265/A465) which is frequently used as an indicator of the degree of aromaticity and average molecular weight, was utilized in this study for characterization of peat HA. We assume that the lower value of the E2/E4 ratio for SBPHA can be attributed to the higher average molecular weight rather than be related to highly differences in the aromaticity of the studied samples. In this study, the SUVA254 obtained for original HA was 5.68. This value of the SUVA254 is characteristic of humic acids from terrestrial origin [4,6].

FTIR characterization of SBPHA
The FTIR spectra of SBPHA were obtained by means of an Attenuated Total Reflectance (ATR-FTIR) and, for comparison, also by Diffuse Reflectance Infrared Fourier Transform (DRIFT) technique using a Nicolet iS50 spectrometer. Method of ATR analysis is described in Materials and Methods. For the DRIFT analysis, approximately 2 mg of powdered HA sample was homogenized with 200 mg of KBr in an agate bowl. DRIFT spectrum was recorded over the 4000-400 cm −1 range at a resolution of 4 cm −1 and was the average of 512 scans. The infrared grade spectrum of annealed KBr was used as the background for DRIFT measurement.
Both FTIR spectra of SBPHA are presented in Figure S1. The comparison illustrates typical features of ATR spectra -linear dependence of penetration depth on frequency (affecting apparent intensity) and a slight shift of infrared bands. As far as the refractive index of SBPHA (and organic fractions) was not determined directly, the ATR spectra were not corrected and compared in the as-recorded form. Figure S1: ATR-FTIR and DRIFT spectrum of SBPHA The spectrum contains characteristic absorption bands typical for humic acids isolated from peat and coals [5,7]. The DRIFT spectrum contains a sharp and intensive band at 1610 cm −1 corresponding to stretching C=C groups in aromatic rings. Another aromatic band is apparent at 1509 cm −1 , due to the aromatic C=C stretching of lignin residues. Another significant band occurring at 840 cm −1 can be assigned to out-of-plane C-H deformation in aromatic rings with tri-substitution per ring. Absorption band at 1720 cm −1 corresponds to carboxylic groups, which are also indicated also by the broad band centered at about 2650 cm −1 resulting from the O-H stretching vibration modes of the hydrogen-bonded -COOH which forms dimers. The broad band centred at about 3225 cm −1 corresponds to the O-H stretching of various functional groups such as carboxylic, phenolic and alcoholic functional groups. The fingerprint region is characterized by an intensive bands at 1222 cm −1 and 1180 cm −1 corresponding to the C-O stretching and O-H bending of carboxylic groups, phenoxy structures and ethers, the C-O stretching of phenols and ethers. The spectrum contains an intensive band at 1080 cm −1 which can be attributed to the C-O-C vibration in aliphatic ethers, and to the C-O stretching of secondary alcohol moieties. The appearance of hydrocarbons is revealed in the 3000-2800 cm −1 zone of valence vibration. The absorption band at 2930 cm −1 was ascribed to asymmetric C-H stretching in methylene groups. From the ATR-FTIR spectra, the ICH2/ICH3 and IAr/ICOOH ratios were calculated similarly to the extracted organic fractions (see Table 2). The values of both parameters were comparable to the last extracted organic fraction (MET6). This suggests that from the viewpoint of the structure of alkyl chains and the substitution of aromatic rings with carboxylic groups was practically identical for the parental HA and its last extracted fraction.

Fluorescence spectroscopy
The EEM contour map of the SBPHA is shown in Figure S2. The fluorescence domain is only located at excitation wavelengths in the ultraviolet region in the range 250-260 nm and at emission wavelengths in the range 480-500 nm. This fluorophore lies within the region referred to as the fulvic-like region, which is marked to as the A. Similarly to the two most polar organic fractions (PRO5 and MET6), A-type fluorophores were restricted to wavelengths in the range 255-265/495-505 nm, suggesting that all of them have similar structure of this fluorescence domain.