Separation and Quantification of Humic Acid and Fulvic Acid using Column Chromatography


Humic substances are complex, heterogeneous, mixtures of organic compounds with different functional groups. Their composition and structural formula are still controversial. Humic substances can be operationally defined into fulvic acid (FA; soluble at all pH values), humic acid (HA; soluble in alkaline media and insoluble at pH 1.0) and humin (insoluble at all pH values) according to their water solubility. Among these three groups, FAs have the least molecular masses and are the most mobile fraction of humic substances. Some researchers have reported that FAs have potential effects on the bioavailability and transport of nutrients, heavy metals, polycyclic aromatic hydrocarbons, and other chemicals. HAs and FAs play a pivotal role in soil fertility and plant nutrition.

The size of fulvic acids (HAs) are smaller than humic acids (HAs), with molecular weights which range from approximately 1,000 to 10,000. Fulvic acids (FAs) have an oxygen content twice that of humic acids (HAs). They have many carboxyl (- COOH) and hydroxyl (-OH) groups, thus fulvic acids (FAs) are much more chemically reactive.Because of the relatively small size of fulvic acid (FA) molecules they can readily enter plant roots, stems, and leaves. As they enter these plant parts, they carry trace minerals from plant surfaces into plant tissues. Due to this reason FAs act as indicator of pollution.


Our aim of work is to quantify both humic acid and fulvic acid simultaneous as humic and fulvic acids are both essential for healthy plants and both of the acids are prime constituent of soil and thus we may conclude the soil quality also in term of percentage of both acids are present in particular soil. The method may also applicable to quantify humic acid and fulvic acid in the fertilizer that contains both .


A modified form of the International Humic Substances Society (IHSS) was executed.


  • a) Substrate : Humic Acid (Make: Loba Chemie)
  • b) NaOH & HCl
  • c) Nitrogen gas- 99.99%
  • d) Supelco Superlite DAX-8 resin
  • e) Amberlite IR 120 strong cation exchange resin


  • a) Erlenmeyer flasks- 500 ml
  • b) Glass chromatography columns – 2 x 35
  • b) Ceramic combustion crucibles


  • a) Analytical balance- Sartorious
  • b) Hot Air Oven
  • c) Centrifuge tubes- 50 ml capacity (tarson)
  • d) Polyethylene Centrifuge tubes – 50 ml
  • e) Rotary Evaporator
  • f) TCLP Agitator
  • g) Electical Conductivity meter – EI
  • h) pH meter – Toschon
  • i) Desiccator

Analytical Procedure

Step 1: Alkaline Extraction

A petri plate was weighed (Wt1) and approximately 2 grams of substrate was transferred. The mass was recorded (W1). It was kept in drying oven for 24±2 hrs at 90 °C. Then it was removed from air oven and placed in a deciccator to cool for 1 hr. The mass was recorded along with the petri plate (W2). The moisture ratio was determined (equation 1).

Next, approximately 2.5 g of HA was weighed and the test mass was noted (W3). After that it was transferred to a 500 ml Erlenmeyer flask and 0.1 M NaOH was added to make the volume up to 500ml. The dry weight of the test portion was determined (equation 2).

The solution was purged with N2 and mixed with the help of TCLP agitator for 6hrs to extract the substances. After removing from TCLP Agitator the supernatant alkaline solution containing HA and FA was decanted and the insoluble particles were discarded.

To accumulate HA, the test solution was put under a pH electrode and 1:1 HCl was added dropwise until pH 1.0±0.05 was reached. Once the pH is stable the material was covered with parafilm and kept for 6 hrs. The HA portion is precipitated.

Step 2: Separation of HA

Once the HA has completely precipitated, the FA fraction was decanted carefully into a clean 500 ml Erlenmeyer flask. It should not contain any HA part. After decanting off the supernatant part, the semi-solid part was centrifuged at 10,000 rpm for 30 mins. The FA containing part was added to the acidified fulvic acid portion.

Step 3: Determination of HA Concentration

The centrifuge tubes containing the precipitated HA were placed in a drying oven set at 90°C, and the HA was dried (typically 24h) to constant weight.

After drying, the tubes from the drying oven and placed in a desiccator to cool to room temperature. After cooling, the residue was quantitatively transferred from the tube by scraping it from the sides and bottom of the tube with a spatula and transferred to a tared weigh boat, and the mass (W4HA) was recorded. This residue is the “Extracted HA” 

Step 4: Determination of Ash Content

The extracted HA was transferred to a pre-weighed (Wt2) ceramic dish that had been previously dried in a drying oven set at 90°C and then cooled in a desiccator to room temperature. After recording

the combined mass of the extracted HA and dish (W5), it was combusted in a muffle oven for 4 hrs at 500°C. While still warm, the dish was removed from the muffle oven and placed in a desiccator to cool. Once cool, weigh the dish with ash (W6) and the ash ratio was calculated (Equation 3). The final mass of the extracted HA was determined by correcting for ash content using Equation 4. 

Step 5: Separation of Fulvic Acid

FA was separated from the other acid-soluble compounds in the test solution by using a 2×35 mm glass column prepared with a nonionic macroporous acrylic ester resin (i.e., Supelite DAX-8).

The fulvic fraction was passed through the column via the top of the column. It is critical that the top of the resin in the column remains covered with solution until all the extract has been added to prevent drying of the resin.

Once the fulvic fraction has been completely loaded onto the resin, the resin was washed with deionized water. The eluent was discarded. The column was washed until the absorbance at 350nm of the column eluent was equal (e.g., within 0.015 absorbance units) to that of the deionized water used to wash the column, using deionized water to zero (i.e., blank) the spectrophotometer.

The FA was desorbed back by elution (i.e., influent introduced into the bottom of column) by adding 0.1 M NaOH. Most of the FA was adsorbed to the very top of the DAX-8 resin. Desorption from the column bottom uses a minimal amount of 0.1 M NaOH to fully desorb the FA. All the FA has been desorbed when the absorbance of the column effluent is equal to the absorbance of influent at 350 nm. 0.1 M NaOH was used as the spectrophotometric blank. The eluent was added back to the desorbed FA solution.

The FA was protonated and de-ashed by passing repeatedly (by gravity feed) through Amberlite IR120 hydrogen form ion exchange resin contained in a 2× 35 cm column until the electrical conductivity of the eluent is <120 mS/m as measured with a conductivity meter. To ensure that all the FA is removed from the resin after the final pass, the column was washed with deionized water until the absorbance of effluent at 350nm is the same (e.g., within 0.015 absorbance units) as the deionized water used to wash the column. Deionized water was used as the spectrophotometric blank. The wash and any effluent portions were added back to the purified FA solution.

The FA was concentrated to a volume of approximately 15 ± 2 mL by using a rotary evaporator at 55°C. 15mL fulvic acid concentrate was transferred to a 50 mL plastic centrifuge tube and dried at 90°C to constant dryness in a drying oven. After drying, as described for the HA above under Step 4 the tube was placed in a desiccator to cool. FA was removed from the tube by complete scraping of the tube sides and bottom with a spatula, and it was weighed on pre-tared weigh paper (W4FA). This material is the “Extracted FA”. The residual ash content of extracted FA was determined as described under Step 4 for HA and the ash ratio was calculated (Equation 3). Finally, the weight of the extracted FA without ash was determined using Equation 5.


Step 6: Calculations

Moisture ratio = [(W1– W2)/ (W1– Wt1)]                                                      (1)

Dry test portion dry weight = (W3) (1 – moisture ratio)                          (2)

Ash ratio = [(W5–W6)/ (W5 – Wt2)]                                                               (3)

Weight of extracted HA without ash (g) = (W4HA) (1 – Ash Ratio)         (4)

Weight of extracted FA without ash (g) = (W4FA) (1-Ash Ratio)             (5)

where W1= weight of test portion taken for moisture plus ceramic dish before drying, g; W2 = weight of test portion taken for moisture plus ceramic dish after drying, g; Wt1 = weight of ceramic dish, g; W3 = test portion weight, g; W4HA= weight of extracted HA, g; W4FA= weight of extracted FA, g; Wt2= weight of ceramic dish used for ashing, g; W5= weight of extracted HA or FA taken for ash plus ceramic dish before combusting, g; and W6= weight of ash plus ceramic dish after combusting, g.

For solid materials, determine the percentages of FA and HA (dry weight basis) as follows:

FA, % = [Ashless FA (g)/test portion dry weight)] × 100

HA, % = [Ashless HA (g)/test portion dry weight)] × 100


The quality and fertility of any soil can be determined excluding the moisture content and ash ratio calculated.

Table 1: Determination of Moisture Ratio – 

Initial wt. of boat(wt1) Wt. of HA(g) Wt. of boat+HA(w1) Final wt. of boat after drying(w2) Moisture Ratio Moisture Ratio Avg
44.6242 2.5032 47.1274 47.0105 0.0467 0.0546
48.8189 2.5116 51.3305 51.1733 0.0625

Dry weight of the standard is applied to determine the fractions of HA and FA.

Table 2: Calculation of Dry weight of the sample- 

Weight of substrate taken (W3,g) 1-moisture ratio  Dry weight of sample 
1.8004 0.9451 1.70155804

Combustion gives the ash ratio value.

Table 3: Calculation of Ash Ratio-

Fraction Weight of crucible (Wt2, g) Weight of Extracted HA (W4HA,g)/ FA (W4FA,g)/ Weight of crucible+HA/FA (W5,g) Weight of crucible+ HA and FA after combustion (W6,g) Ash Ratio
HA 30.4632 0.9206 31.3838 31.3553 0.032
FA 30.294 0.1602 30.4542 30.4509 0.021

Deducting the ash ratio value HA content was found.

Table 4: Calculation of % HA and % FA- 

Fraction Dry weight of sample Weight of Extracted HA (W4HA,g) & FA (W4FA,g Ash Ratio 1-Ash Ratio Ashless HA (g) &

Ashless FA (g)

%HA & %FA
HA 1.7 0.9206 0.032 0.968 0.94 55.27
FA 1.7 0.1602 0.021 0.979 0.157 9.23

The % HA was found to be ~ 55% and the % FA was ~9.23 %


The possible mechanisms of stepwise elution for FA adsorbed DAX-8 resin were still unknown. In order to improve the separation methods, these mechanisms need to be investigated. The standard used has a declaration of containing min 60-70% HA and min 15% FA. Approximately 92% recovery was found in case of Humic Acid whereas FA showed 62% recovery. Hence further modification can be done during the procedure.