This method provides a gravimetric quantification of humic substances (e.g., humic and fulvic acids) on an ash-free basis, in dry and liquid materials from soft coals (i.e., oxidized and non-oxidized lignite and sub-bituminous coal), humate ores and shales, peats, composts and commercial fertilizers and soil amendments.
The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and shales, peats, composts and humic substance-containing commercial products. The method is based on the alkaline extraction of test materials, using 0.1 N NaOH as an extractant, and separation of the alkaline soluble humic substances (HS) from nonsoluble products by centrifugation. The pH of the centrifuged alkaline extract is then adjusted to pH 1 with conc. HCl, which results in precipitation of the HA. The precipitated HA are separated from the fulvic fraction (FF) (the fraction of HS that remains in solution,) by centrifugation. The HA is then oven or freeze dried and the ash content of the dried HA determined. The weight of the pure (i.e., ash-free) HA is then divided by the weight of the sample and the resulting fraction multiplied by 100 to determine the % HA in the sample. To determine the FA content, the FF is loaded onto a hydrophobic DAX-8 resin, which adsorbs the FA fraction also referred to as the hydrophobic fulvic acid (HFA). The remaining non-fulvic acid fraction, also called the hydrophilic fulvic fraction (HyFF) is then removed by washing the resin with deionized H2O until all nonabsorbed material is completely removed. The FA is then desorbed with 0.1 N NaOH. The resulting Na-fulvate is then protonated by passing it over a strong H+-exchange resin. The resulting FA is oven or freeze dried, the ash content determined and the concentration in the sample calculated as described above for HA.
Humic substances (HS) are dynamic residues that result from the microbial decomposition and transformation of dead plant tissues1,2,3 augmented with microbial by-products and biomass3,4,5 through a process that is termed humification6. HS are present in soils, natural waters, lake sediments, peats, soft coals and humic shales and represent an estimated 25% of total organic carbon on the earth7. These substances are complex mixtures of thousands of unique molecules that are fractionated into three main fractions based on their different solubilities in strongly basic and acid aqueous solutions. These fractions are humic acids (HAs), which comprise the alkali-soluble but acid-insoluble fraction; fulvic acids (FAs), the fraction soluble in both alkali and acid; and the humin fraction, which is insoluble at all pH values6,8. The fulvic fraction (FF) is further subdivided into the hydrophobic FA (HFA) and hydrophilic (HyFA) fractions. These fractions are defined as the part of the FF that binds to a hydrophobic DAX-8 resin (HFA) and the part that does not bind to the resin (HyFA).
HS are increasingly being used in agriculture, where they are widely used as crop biostimulants, in animal husbandry, in particular as a livestock feed additive, in mining in drilling muds, and environmental remediation as electron shuttles. Research in the use of HS in human medical applications is also increasing.
Many methods for the quantitation of HA and FA exist. However, most of these methods are neither accurate nor precise. For example, the two most widely used methods for the determination of HA in the USA are the colorimetric method9 and the California Department of Food and Agriculture (CDFA) method, both of which were shown to overestimate the amount of HA in a range of ore and extract sources from the western US and Canada10.The colorimetric or spectrophotometric method is inaccurate because it relies on the absorbance of alkaline extracts that include, in addition to HA, FA and other chromophores that all absorb at the wavelength used and the standard is not representative of the materials being tested10.The CDFA method is not accurate because it does not provide HA concentrations on an ash-free basis. Because different ores have different amounts of ash, some of which is carried with the extraction and the extraction process itself adds ash, this method does not provide an accurate value for HA concentrations10. In response to the need for an accurate and precise method, a standardized gravimetric procedure based on the one detailed by11 was published in 2014 to address quantitation of both HA and FA on an ash free basis12. This method was then adapted, with minor modifications, by the International Organization for Standardization (ISO) in 2018 under Fertilizers and soil conditioners as “Determination of humic and hydrophobic fulvic acids concentrations in fertilizer materials”13.
This paper outlines the protocol for extraction and quantitation of humic and hydrophobic fulvic acids and gives details on the accuracy and precision of the data produced from the method.
1. Solid sample preparation
2. Extraction procedure
3. Removal of nonsoluble materials from alkaline extracts
4. Precipitation and separation of HA from FF
5. Determination of ash content
NOTE: The procedure for determination of ash content of dried HA and FA samples is the same. The procedure using notation for HA is shown.
6. Determination of the percentage of purified extracted HA
7. Determination of the concentration (%) of pure HA in the original source sample
8. Column preparation for HFA purification
9. Isolation of HFA
10. HFA de-ashing by protonation
11. DAX-8 resin regeneration
12. H+-cation exchange resin regeneration
Performance data for the method are provided in Tables 1 – 5. The precision of method for extraction of HA and FAH from liquid commercial samples with very different concentrations of HA and FHA are given in Table 1.
The relative standard deviations (RSDs) for HA were lower than those for HFA, but the average HFA RSD over the three liquid samples was on 6.83% which indicates a high degree of precision. The Horwitz ratio (HorRat) is a normalized performance parameter that indicates the suitability of a methods of analysis regarding among laboratory precision. Here it was used for intra-laboratory precision. Value < 0.5 may indicate undisclosed averaging or a high level of experience with the method. Values > 2.0 indicate heterogeneity of test samples, a need for method optimization or more extensive training, operating below the limit of detection or an inapplicable method. For analysis of liquid samples, the HorRat was only > 2 for one of the HFA analyses (Table 1).
Precision data for the extraction of HA and HFA from three humic ore samples is given in Table 2. Again, with the exception of the HFA extracted from Ore 2 and the HA from Ore 3, all of the HorRat’s were below 2. This demonstrates a high degree of precision of this method for extraction of HA and HFA for humic ore samples.
Manufacturers of plant biostimulants often formulate products that contain HS in addition to other ingredients like seaweed, inorganic fertilizers, coals or molasses. Table 3 gives the results of an analysis of the inclusion of these types of additives on the precision of the method. None of the additives effected the recovery of HA or HFA significantly (Table 3).
Table 4 and Table 5 report the recoveries of HA and HFA, respectively, from liquid samples that simulated commercial products with very low concentrations. Recoveries were excellent and ranged between 88% and 97% for HA (Table 4) and 92% and 104% for HFA (Table 5). Mean recoveries for HA and HFA were 93% and 97%, respectively and % RSD for both HS were less than 5%. While precision is excellent, these data indicate the need to perform laboratory replicates. The method detection limit (MDL) and method quantitation limit were 4.62 and 1.47 mg/L for HA and 4.8 and 1.53 mg/L for HFA.
Humic substances, % | |||||||
Material | L16 | L17 | L2 | ||||
HFA | HA | HFA | HA | HFA | HA | ||
Rep 1 | 1.44 | 17 | 6.59 | 7.76 | 0.36 | 4.46 | |
Rep 2 | 1.39 | 16.03 | 6.25 | 7.79 | 0.42 | 4.93 | |
Rep 3 | 1.34 | 16.44 | 6.02 | 7.55 | 0.4 | 4.46 | |
Rep 4 | 1.54 | 16.75 | 6.2 | 7.69 | 0.33 | 4.53 | |
Mean | 1.43 | 16.56 | 6.27 | 7.7 | 0.38 | 4.6 | |
SD | 0.09 | 0.42 | 0 | 0.11 | 0.04 | 0.23 | |
24 | |||||||
RSD, % | 6.29 | 2.53 | 3.8 | 1.39 | 10.4 | 4.91 | |
Hor Rat(r) | 1.58 | 0.72 | 1.25 | 0.47 | 2.31 | 1.55 | |
aExtraction conditions were 1 g in 1 L 0.1 M NaOH. |
Table 1. Precision of the method in extraction and quantitation of HA and HFA from liquid commercial samples. Extraction conditions were 1 g in 1 L 0.1 M NaOH.
Humic substances, % | ||||||
Ore 1 | Ore 2 | Ore 3 | ||||
Material | HFA | HA | HFA | HA | HFA | HA |
Rep 1 | 1.75 | 67.4 | 1.31 | 27.01 | 1.55 | 8.95 |
Rep 2 | 1.69 | 67.63 | 1.25 | 27.48 | 1.41 | 7.2 |
Rep 3 | 1.63 | 67.1 | 1.27 | 27.34 | 1.47 | 8.35 |
Rep 4 | 1.77 | 67.59 | 1.55 | 26.89 | 1.51 | 7.98 |
Mean | 1.71 | 67.53 | 1.35 | 27.18 | 1.49 | 8.12 |
SD | 0.06 | 0.94 | 0.14 | 0.28 | 0.06 | 0.73 |
RSD, % | 3.7 | 1.39 | 10.33 | 1.02 | 4.02 | 9.02 |
HorRat(r) | 0.99 | 0.66 | 2.71 | 0.42 | 1.07 | 3.09 |
Table 2. Precision of the method in extraction and quantitation of HA and HFA from humic ores. Extraction conditions were 1 g sample in 1 L 0.1 M NaOH. (Data taken from Lamar et al., 2014)
Replicate | Adulterant | HA, % | FA, % | Relative Recovery HA, % | Relative Recovery HFA, % |
1 | None | 81.61 | 12.86 | ||
2 | None | 80.16 | 12.78 | ||
1 | Seaweed | 80.21 | 12.85 | ||
2 | Seaweed | 80.72 | 12.79 | 99.5 | 99.6 |
1 | Fertilizer | 80.25 | 12.98 | ||
2 | Fertilizer | 79.57 | 123.77 | 98.8 | 101.6 |
1 | Coal | 78.79 | 12.92 | ||
2 | Coal | 81.27 | 12.84 | 98.9 | 101.8 |
1 | Molasses | 79.38 | 12.99 | ||
2 | Molasses | 81.02 | 12.72 | 99.2 | 100.9 |
Mean | 80.3 | 12.85 | |||
SD | 0.885 | 0.09 | |||
a Final concentration of FA + HA of 2.5 g/L added to 0.1 M NaOH. (data taken from Lamar et al., 2015) |
Table 3. Effect of adulterants on quantitation of HA and HFA from a Gascoyne leonardite. (Data taken from Lamar et al., 2015)
HA | |||
Sample ID | Extracted, mg | Recovered, mg | Recovered, % |
1 | 24.6 | 23.7 | 96.3 |
2 | 22.6 | 19.9 | 88.1 |
3 | 25.2 | 23.6 | 93.7 |
4 | 22.5 | 21.5 | 95.6 |
5 | 23.9 | 21.8 | 91.2 |
6 | 23.2 | 20.8 | 89.7 |
7 | 24 | 23.2 | 96.7 |
Mean | 23.7 | 22.1 | 93 |
SD | 1.01 | 1.52 | 3.43 |
RSD, % | 4.35 | 6.88 | 3.67 |
(data taken from Lamar et al., 2014) |
Table 4. Recovery of HA from spiked blanks. (Data taken from Lamar et al., 2014)
FA | |||
Sample ID | Extracted, mg | Recovered, mg | Recovered, % |
1 | 19.9 | 19 | 95.48 |
2 | 23.1 | 22.9 | 99.13 |
3 | 20.7 | 19.4 | 93.72 |
4 | 20.5 | 19.8 | 96.39 |
5 | 20.8 | 21.6 | 103.85 |
6 | 21.9 | 20.1 | 91.78 |
7 | 22.7 | 22.3 | 98.24 |
Mean | 21.37 | 20.73 | 96.94 |
SD | 1.21 | 1.53 | 3.95 |
RSD, % | 5.64 | 7.36 | 4.07 |
(Data taken from Lamar et al., 2014) |
Table 5. Recovery of HFA from spiked blanks. (Data taken from Lamar et al., 2014)
The initial steps of extraction and isolation of the HA in this method are relatively straightforward. Because the isolation of the HFA involves column chromatography, obtaining repeatable results comes with strict adherence to the details of each step and practice. In particular, correct preparation of the resins is of primary importance. It is extremely important that the polymethylmethacrylate DAX-8 resin is prepared and packed properly. Correct packing of the resin affects both the yield and quality of the HFA. If channeling exists, then neither pretreatment (i.e. acidification) or adsorption of HFA will be complete, and the separation will lead to inaccurate results. If channels or spaces in the resin are observed prior to sample loading the column should be removed and shaken to redistribute the resin beads, by allowing them to settle without channels, and then re-packed by pumping clean DI H2O through the resin. In addition, as mentioned in the protocol, maintaining a volume of liquid above the resin when loading the FF onto the resin, will allow the FF to mix prior to entering the resin and result in more effective adsorption. For the strong cation H+-exchange resin (Table of Materials), complete regeneration cannot be rushed. The Na+/H+ exchange takes time and therefore this is best done in a bulk treatment so that the resin can be mixed while being re-acidified. Mixing the resin while rinsing with DI H2O helps remove the excess HCl. When rising the acidified resin to remove excess acid, mixing the resin help removing the HCl. It is extremely important to remove the acid to the point where an electrical conductivity of ≤ 0.7 µS/cm is reached. If not, the HCl will be carried over with the HFA.
Finally, when desorbing the HFA from the DAX-8 resin, once the absorbance of the influent equals the absorbance of the effluent, it is a good practice to let the column sit for a couple hours to see if any additional HFA will be released. If so, it will be seen as a yellowing of the liquid above the resin. If this occurs, the additional HFA can be removed by continued desorption until influent/effluent absorbances are equal again.
One of the disadvantages of the HFA isolation is that the entire process is time consuming. The complete desorption of HFA from the DAX-8 resin and complete removal from the H+-exchange resin both result in a significant volume of HFA that has to be reduced by rotary evaporation. This is definitely a bottleneck in the analysis. In an effort to reduce this time, desorbing the HFA from the DAX-8 resin using acetone rather than 0.1 M NaOH has been suggested14. The authors claimed that by using 50% acetone as desorbent in place of NaOH, a similar HFA result was obtained and the DAX-8 was adequately regenerated and thus the H+-exchange step could be eliminated. This modification resulted in a greatly reduced analysis time as a result of decreased volume produced and quicker rotary evaporation of acetone compared to water. This modification deservers further study.
This method is limited to the analysis of organic matter that has undergone the process of humification, and for the case of peat and soft coals, the further processes of peatification and both peatification and coalification, respectively. Humification is the process whereby dead, primarily plant material, is decomposed by a sequence of microbes that consume and modify increasingly recalcitrant substrates. Abiotic processes also participate in decomposition and re-synthesis reactions. Humification ultimately results in the production of relatively recalcitrant materials comprising heterogeneous mixtures of thousands of molecules that form a range of molecular weight and carbon, oxygen and hydrogen contents that form HS. HS are further modified by peatification and coalification. Therefore, this method is not appropriate for plants materials that have been modified by chemical processes. For example, lignosulfonate is widely used as an HFA adulterant. Lignosulfonate is a by-product of the sulfite pulping process. Therefore, this material has not been produced by the process of humification. In addition, there are many substances that bind to the DAX-8 resin. For example, DAX-8 resin has been used to adsorb pesticides from solution15. Obviously, pesticides are not HS. Thus, binding of a material to DAX-8 resin does not justify a claim that it is an HFA. The prerequisites are both production by humification and binding to DAX-8 resin.
As more is learned about the contribution of the various components of HS in different applications, it may become advantageous to further fractionate HS and thus modify the method accordingly. As it exists, the method does not quantify the HYFA. However, this fraction might also have activity e.g. in plant biostimulation, where the whole FF is generally applied in agricultural treatments rather than purified HFA.
The authors have nothing to disclose.
The authors would like to acknowledge the Humic Products Trade Association (HPTA) for their support in funding the work that resulted in the standardization of the methods described in this paper and also Lawrence Mayhew and Drs. Dan Olk and Paul Bloom for technical support during the standardization work.
Amberlite IR 120 H+-exchange resin | Sigma-Aldrich | 10322 | H+ form |
Analytical Balance | Ohaus | PA214 | w/ glass draft shield |
Centrifuge | Beckman Coulter | Allegra X-14 | minimum 4300 rpm |
Centrifuge tubes | Beckman Coulter | To fit rotor selected | |
Ceramic Combustion Crucibles | Sigma | Z247103 | |
Chromatography column for DAX-8 | Diba | Omnifit 006EZ-50-25-FF | |
Chromatography column for IR 120 | Chemglass | CG-1187-21 2 in. by 24 in. | |
Dessicator | Capitol Scientic | Kimax 21200-250 | Vacuum type |
Drying Oven | Fisher Scientific | Isotemp | Precision±3˚C |
Electrical conductivity meter | HM Digital | EC-3 | |
Erlenmeyer Flasks | Amazon | 1L, 2L | |
HCl concentrated | Sigma-Aldrich | 320331 | |
Magnetic Stir Plate | Barnstead-Thermolyne | Dataplate 721 | |
Magnetic Stir bars | These can be obtained at many outlets | ||
Muffle Furnace | Fisher scientific | Thermolyne Type 47900 | |
NaOH | Sigma-Aldrich | 795429 | |
Nitrogen gas | Praxair | UNI1066 | 99.99% purity |
Peristaltic pump | Cole Parmer | Masterflex 7518-00 | |
Perstaltic tubing | Cole Parmer | Masterflex Pharmed 06508-17 | |
pH meter | Oakton Instruments | WD-35618–03 | |
Rotary Evaporator | Buchi | R-210/R-215 | |
Spectrophotometer | Healthcare SCiences | Ultrospec II | Dual beam 200 to 900 nm with wavelength accuracy of ±1 nm and reproducibility of ±0.5 nm. |