Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products

The new standard method for quantitation of humic acids provides a more accurate and precise analysis compared to the existing regulatorily accepted methods, and also provides a standard method for pure hydrophobic fulvic acid quantitation. The advantage of this protocol is that it provides a gravimetric analysis of humic and hydrophobic fulvic acid concentrations on an ash-free basis, and the extraction process has been optimized to obtain the highest recoveries of both humic and fulvic acids from samples. Demonstrating the procedure will be Ryan Fountain, an analytical chemist from our humic chemistry laboratory.

To prepare a solid sample, use a mortar and pestle to crush approximately five grams of the sample to be analyzed until 100%of the well-mixed sample can pass through a U.S.standard sieve, mesh size number 200. To gravimetrically determine the moisture content of the powder, transfer approximately two grams of the sample powder into a pre-weighted aluminum weigh boat, and record the mass of the boat and the sample. Next, place the weigh boat in a drying oven at 102 degrees Celsius for 24 hours.

The next day, place the boat in a desiccator to cool for at least one hour before weighing and recording the mass of the weigh boat and dried sample, then use the formula to determine the moisture content as indicated. To perform an extraction from a solid sample, weigh approximately 2.5 grams of the remaining sample and record the weight to four decimal places. Then, load the sample into a one liter Erlenmeyer flask and rinse the weigh boat with deionized water to remove all of the ore.

Fill the beaker to one liter with 0.1-molar sodium hydroxide and add a five-to seven-centimeter magnetic stir bar to facilitate a rapid stirring of the sample at 300 to 400 revolutions per minute on a stir plate. When the sample has been thoroughly mixed, evacuate the head space of the flask with nitrogen gas, then seal the flask opening with an airtight cover and place the flask on a stir plate for mixing at 300 to 400 revolutions per minute for 16 to 18 hours. For liquid material extraction, thoroughly shake the sample to ensure that the test liquid is homogeneously mixed, including any residue that may have fallen to the bottom of the container.

Weigh approximately five grams of the test liquid and record the weight to four decimal places. Then, add the liquid to a one-liter graduated cylinder and fill the cylinder with 0.1-molar sodium hydroxide to a final volume of one liter. Use a magnetic stir bar to rapidly mix the sample as demonstrated until the test sample is completely mixed before transferring the mixture into a one-liter Erlenmeyer flask, then evacuate the head space with nitrogen gas and stir the flask contents at 300 400 revolutions per minute for one hour as demonstrated.

To remove non-soluble materials from alkaline extracts, at the end of the stirring incubation, transfer the mixture to suitable centrifuge tubes and centrifuge the entire volume of sample at 4, 921x G for 30 minutes at room temperature. At the end of the centrifugation, collect the alkaline supernatant containing the humic acid and fulvic fraction, which contains the hydrophobic fulvic acid, in a clean, one-liter Erlenmeyer flask with a magnetic stir bar. For humic acid precipitation from the fulvic fraction, while stirring the collected alkaline extract at 300 to 400 revolutions per minute on a stir plate, insert a pH probe into the middle portion of the solution and add concentrated hydrochloric acid drop wise until a stable pH of pH 1.0 plus or minus 0.1 is reached.

Once the pH is stable, remove the probe and stir bar, rinse them with deionized water, and seal the flask with an air-tight cover. Let the flask sit for one to six hours until the precipitated humic acid has settled to the bottom before centrifuging the extract and precipitated humic acid at 4, 921x G for one hour. At the end of the centrifugation, pour the fulvic fraction-containing supernatants into a clean, one-liter Erlenmeyer flask and seal the flask with an airtight cover.

Place the humic acid-containing centrifuge tubes in a 100-degree-Celsius drying oven for 24 hours and make sure lids are loose or removed to ensure drying. After drying, cool the tubes in a desiccator until they reach room temperature. After cooling, use a spatula to transfer the residue from each tube into a single tared weigh boat to determine the mass of the extracted humic acid sample.

To determine the ash content within the sample, transfer approximately 30 milligrams of the dried humic acid to a clean, pre-weighed ceramic dish to determine the mass of the weigh sample and dish. Next, combust the humic acid in a muffle oven for two hours at 600 degrees Celsius before cooling the dish in the desiccator. Once cool, weigh the dish and use the formula to calculate the ash ratio.

To determine the final mass of the pure humic acid, use the formula to correct for the ash content. Then use the formulas to determine the pure humic acid concentration. To prepare a new, low-pressure polymethyl methacrylate DAX8 resin-packed chromatography column, soak the resin in the methanol for two hours in a beaker before rinsing the resin thoroughly with deionized water until all of the methanol is removed.

Then remove any small resin particles floating on the water. Once thoroughly rinsed, pour the rinsed or regenerated resin into a 5 by 25 centimeter glass chromatography column fitted with an end piece with a 10-micron frit for resin bed support, leaving 2.5 to 5 centimeters at the top of the column and replace the top piece onto the column. To refurbish a previously-used resin, or prepare a freshly-washed new resin, use the peristaltic pump to pump 0.1-molar hydrochloric acid at a 35-to 40-milliliter flow rate through the bottom of the column until the pH of the effluent is equal to the pH of the influent, then pump deionized water into the top of the column until the pH of the effluent is equal to the pH of the influent.

Once the resin bed is packed, use a peristaltic pump to load the fulvic fraction onto the column under low pressure via the top of the column at a 35-to 40-milliliter flow rate. Once the fulvic fraction has been completely loaded onto the resin, wash the resin with deionized water to remove the non-adsorbed hydrophilic fulvic fraction. Wash the column with deionized water until the absorbance of the column effluent at 350 nanometers is equal to that of the deionized water used to wash the column.

To desorb the HFA, pump 0.1-molar sodium hydroxide via the bottom of the column and capture the pump sodium fulvate effluent in a clean, sufficiently-sized container. All of the HFA has been desorbed when the absorbance of the column effluent is equal to the absorbance of 0.1-molar sodium hydroxide influent at 350 nanometers. To de-ash the HFA by protonation, first pour proton/cation exchange resin into a large beaker and cover and mix the resin with fresh deionized water several times, pouring off the water after each rinse until the red color has been completely removed.

After the last wash, cover the resin with one molar hydrochloric acid and let the mixture stand for at least two hours, with stirring every 30 minutes. At the end of the acidification treatment, replace the acid with deionized water and stir vigorously with a stirring rod for 15 seconds before letting the resin drop to the bottom of the flask and pouring off the water. Repeat the process until the electrical conductivity of the rinse water is less than or equal to 0.7 microSiemens per centimeter and load the regenerated resin into a 5 by 50 centimeter column with a glass frit for retaining the resin.

Then, cover the resin with fresh deionized water and repeatedly pass the sodium fulvate through the strong cation/proton-exchanged resin by gravity feed until the electrical conductivity of the effluent is less than 120 microSiemens per centimeter. To ensure that all of the FA is removed from the resin, after the final pass, wash the resin with deionized water until the absorbance of the effluent at 350 nanometers is the same as the deionized water used to wash the column. Add the wash and any effluent used to check the absorbance to the purified FA solution.

To help with removal of the FA, the resin can be agitated several times. Use a rotary evaporator at 55 degrees Celsius to concentrate the FA to an approximately 15 plus or minus two-milliliter volume and completely transfer the entire volume of FA concentrate to a 50-milliliter plastic centrifuge tube. Dry the sample to 60 plus or minus three degrees Celsius to constant dryness in the drying oven and transfer the tube to the desiccator to cool.

When the sample has cooled, use a spatula to scrape the extracted FA onto a piece of pre-weighed weigh paper and determine the ash ratio, extracted FA weight, and percent pure FA in the original sample as demonstrated for the humic acid. In this table, precision data for the method of extraction of HA and HFA from liquid commercial samples with very different concentrations of HA and HFA are shown. The residual standard deviation for HA were lower than those for HFA, but the average HFA residual standard deviation over three liquid samples was 6.83%indicating a high degree of precision.

The Horowitz ratio was also greater than two for only one of the HFA analyses. In this table, precision data for the extraction of HA and HFA from three humic ore samples are shown. Similar to the previous analysis, with the exception of the HFA extracted from ore two and the HA from ore three, all of the Horowitz ratios were below two, demonstrating the high degree of precision of this method for the extraction of HA and HFA from humic ore samples.

In this analysis, no plant biostimulant additives were observed to significantly affect the recovery of HA or HFA. In these tables, the recoveries of HA and HFA from liquid samples that stimulated commercial products with very low concentrations are shown. The recoveries were excellent, ranging between 88%and 97%for HA, and 92%and 104%for HFA, but also indicating the need to perform laboratory replicates.

Following this procedure, the dry humic and fulvic acids obtained can be used for characterization purposes, such as the carbon-13 and the proton NMR electron resonance, and the ultrahigh resolution mass spectrometry, among others useful techniques. And this can be used for the characterization of the humus chemistry, as well as useful tool to dig deep into the structure-activity relationship with plant fitness and the underlying plant defense mechanisms.