Isolation of IHSS Samples
Disclaimer: The procedures used by the IHSS to isolate standard and reference humic and fulvic acids are simply the methods which were chosen by a working group of scientists in 1981. On the basis of information that was available at that time, these methods were considered as being most suitable for the specific purpose of establishing a collection of standard and reference samples from the selected bulk source materials. IHSS does not endorse or recommend these methods as the best methods for extracting humic substances. It is hoped, in fact, that the study of humic substances will be furthered through the development of better methods of isolation of humic and fulvic acids.
When the first IHSS standard samples were isolated from the selected source materials, the state-of-the-art method for isolation of humic substances from solid-phase source materials such as soils, peat, and leonardite was alkaline extraction with aqueous NaOH, followed by precipitation of humic acid at low pH and a series of desalting steps involving cation exchange, dialysis, etc. to obtain fulvic acid. Through these procedures, all organic acids that are extracted from a solid-phase source material are ultimately found in either the humic acid or fulvic acid.
The state-of-the-art method for isolation of humic substances from natural waters was the XAD-8 resin adsorption method. In this method, the dissolved organic matter (DOM) was fractionated initially into hydrophobic and hydrophilic fractions through preferential adsorption of the hydrophobic fraction on the XAD-8 resin. Subsequently, the hydrophobic fraction was eluted from the resin by alkaline extraction with aqueous NaOH, followed by precipitation of humic acid at low pH and a desalting step involving cation exchange to obtain fulvic acid. The humic acid and fulvic acid that are isolated from aqueous samples thus contain only hydrophobic organic acids, but samples extracted from solid-phase materials include both hydrophobic and hydrophilic acids.
The IHSS has adopted the XAD-8 resin adsorption method to isolate humic and fulvic acids from natural waters, so IHSS humic and fulvic acids from natural waters contain only hydrophobic organic acids. In contrast, the IHSS has used a hybrid procedure to isolate humic and fulvic acids from solid-phase source materials. In this procedure, humic acids are isolated directly from the alkaline extract, without removal of hydrophilic acids. The IHSS humic acids from solid-phase source materials are thus expected to contain both hydrophobic acids and hydrophilic acids that are co-precipitated at pH 1, so they are not operationally equivalent to IHSS humic acids that are isolated from natural waters. In contrast, fulvic acids are first separated from hydrophilic acids by adsorption on XAD-8 resin, then eluted and purified. The fulvic acids from solid-phase source materials contain only hydrophobic organic acids, so they are operationally equivalent to fulvic acids from natural waters.
Starting in the late 1990's, IHSS has also employed reverse osmosis to concentrate natural organic matter (NOM) from selected water sources. These reference NOM samples contain not only the hydrophobic and hydrophilic acids but also other soluble organic solutes that are present in natural waters.
In 2013 an IHSS team collected NOM from the Upper Mississippi River using of reverse osmosis (RO) combined with electrodialysis (ED) to separate the NOM from a hard water, high in calcium and magnesium. They were able to produce a sample that contains only 8% ash.
IHSS humic acids, fulvic acids, and NOM samples are stored as desalted (H+-saturated), freeze-dried solid powders.