Biochemical Humic Acid vs Weathered Coal-derived Humic Acid

Laboratory Identification Methods: Biochemical Humic Acid vs Weathered Coal-derived Humic Acid

 

The core laboratory methods to distinguish biochemical humic acid (fermented/plant-sourced) and weathered coal-derived humic acid (mineral-sourced) rely on analyzing elemental composition, spectral characteristics, structural fragments, radiocarbon content, ash content and impurities to establish unique fingerprint differences.

1. Preliminary Screening: Appearance & Basic Physicochemical Properties

• Color & Water solubilityWeathered coal-derived: Black or dark brown; dissolves into deep brown solution.Biochemical sourced: Yellow or light brown; dissolves into bright yellow or orange-red solution.
• OdorWeathered coal-derived: Odorless or with faint coal smell.Biochemical sourced: Fermented sour aroma or wine lees odor.
• Ash content (calcination method)Weathered coal-derived: High ash content (20%-50%).Biochemical sourced: Low ash content (<10%).
• pH valueWeathered coal-derived: Neutral to weakly alkaline.Biochemical sourced: Acidic (pH 3-5).

2. Elemental Analysis (C/H/O/N/S & Ash Test)

Test via elemental analyzer and calcination method:

Index	Weathered Coal-derived Humic Acid	Biochemical Humic Acid
Carbon (%)	50-65% (High)	40-50% (Low)
H/C Ratio	< 1.0 (High aromaticity)	> 1.2 (More aliphatic chains)
O/C Ratio	0.5-0.7	0.7-1.0 (More hydroxyl & polysaccharide groups)
Nitrogen (%)	0.5-2%	3-6% (Rich in protein & amino acids)
Ash Content	20%-50%	< 10%

Judgment Criteria:H/C ＜1.0, high carbon and low nitrogen = mineral source (weathered coal);H/C ＞1.2, low carbon, high nitrogen and low ash = biochemical source.

3. UV-Visible Spectroscopy (UV-Vis)

Measure absorbance at 250/365/465 nm and calculate E4/E6 & E2/E3 ratios.

• Weathered coal-derived: E4/E6 = 3.0-4.5; high aromatic condensation and large molecular weight.
• Biochemical sourced: E4/E6 = 5.0-7.0; low aromaticity and small molecular weight.

Judgment Criteria:E4/E6 ＜4.5 = mineral source; E4/E6 ＞5.0 = biochemical source.

4. FTIR Spectroscopy (Core Structural Fingerprint)

• Weathered coal-derived humic acidWeak or no peak at 1720 cm⁻¹ (low carboxylic acid content);Strong peak at 1600 cm⁻¹ (aromatic skeleton);Strong absorption at 1380-1400 cm⁻¹; weak peak at 1050-1100 cm⁻¹ (low polysaccharide content).
• Biochemical humic acidStrong peak at 1720 cm⁻¹ (rich in carboxylic acid and ester groups);Medium-weak peak at 1600 cm⁻¹ (low aromatic content);Intense peak at 1050-1100 cm⁻¹ (polysaccharide characteristic);Visible peak at 1540 cm⁻¹ (amide from protein).

Judgment Criteria:Strong 1600 peak + weak 1720 & 1050 peaks = weathered coal source;Strong 1720 & 1050 peaks + 1540 characteristic peak = biochemical source.

5. Fluorescence Spectroscopy (Rapid Identification)

• Weathered coal-derived: Excitation 420-470 nm / Emission 500-530 nm (red-shifted long wavelength).
• Biochemical sourced: Excitation 300-350 nm / Emission 420-470 nm (blue-shifted short wavelength).

6. Solid-state ¹³C NMR (Gold Standard for Structure Analysis)

• Weathered coal-derived: Aromatic carbon (100-160 ppm): 50%-70% (main component); low aliphatic carbon content.
• Biochemical sourced: Aliphatic & oxygenated aliphatic carbon: 60%-80% (polysaccharide & cellulose); aromatic carbon ＜30%.

7. Radiocarbon Test (Most Authoritative Method)

• Weathered coal-derived: Almost no ¹⁴C activity (ancient fossil carbon).
• Biochemical sourced: ¹⁴C activity consistent with modern atmospheric level (contemporary plant/fermented carbon).

 

How to Distinguish Biochemical Humic Acid from Weathered Coal Humic Acid by Spectral Characteristics

1. UV-Visible Spectroscopy (UV–Vis) & E4/E6 Ratio (Most Common Quick Test)

1. Weathered coal-derived humic acid

• Higher aromatic condensation degree, large molecular weight
• E4/E6 ratio: 0 – 4.5 (low value)
• Solution color: dark brown / blackish brown, strong light absorption in long wavelength

1. Biochemical humic acid

• Low aromaticity, small molecular weight, rich in aliphatic chains & polysaccharides
• E4/E6 ratio: 0 – 7.0 (high value)
• Solution color: yellow / orange-red

Judgment rule:E4/E6 ＜ 4.5 → weathered coal sourceE4/E6 ＞ 5.0 → biochemical source

2. FTIR Infrared Spectroscopy (Structural Fingerprint Identification)

Weathered coal humic acid characteristic peaks

• 1600 cm⁻¹: Very strong absorption (aromatic ring skeleton dominant)
• 1720 cm⁻¹: Weak or almost absent (few carboxylic acid groups)
• 1050–1100 cm⁻¹: Weak peak (low polysaccharide content)

Biochemical humic acid characteristic peaks

• 1720 cm⁻¹: Strong peak (abundant carboxyl & ester groups)
• 1600 cm⁻¹: Medium or weak peak (low aromatic structure)
• 1050–1100 cm⁻¹: Extremely strong peak (rich polysaccharide C–O structure)
• 1540 cm⁻¹: Obvious absorption peak (protein amide characteristic)

3. Fluorescence Spectroscopy

• Weathered coal source:Excitation 420–470 nm / Emission 500–530 nm (red-shifted long wavelength)
• Biochemical source:Excitation 300–350 nm / Emission 420–470 nm (blue-shifted short wavelength)

4. Solid-state ¹³C NMR Spectrum

• Weathered coal: Aromatic carbon (100–160 ppm) accounts for 50%–70%
• Biochemical type: Aliphatic & oxygenated aliphatic carbon dominant; aromatic carbon less than 30%

 

Lab Testing Method process

SOP: Spectral Identification of Biochemical Humic Acid & Weathered Coal-derived Humic Acid

1 Sample Pretreatment

1. Dry the sample at a constant temperature of 45 °C until constant weight is achieved.
2. Crush the dried sample and sieve through a 100-mesh screen.
3. Purification procedure:

• Dissolve the powder with 0.1 mol/L NaOH solution.
• Allow standing, then centrifuge and collect the supernatant.
• Acidify with hydrochloric acid to precipitate crude humic acid.
• Centrifuge to collect the precipitate and wash with pure water until neutral pH.
• Dry at low temperature to obtain purified humic acid for testing.

2 UV‑Vis Spectroscopy Test Procedure

1. Accurately weigh the purified sample and prepare a dilute homogeneous solution with weak NaOH solvent.
2. Use blank alkaline solution for baseline calibration of the spectrophotometer.
3. Perform full-wavelength scanning from 200 nm to 700 nm.
4. Record absorbance values at 465 nm and 665 nm, then calculate the E4/E6 ratio.

Judgment Standard

• E4/E6 = 3.0–4.5: Weathered coal-derived humic acid
• E4/E6 = 5.0–7.0: Biochemical humic acid

3 FTIR Spectroscopy Test Procedure (KBr Pellet Method)

1. Fully dry KBr powder and test sample; mix at a mass ratio of 100:1 and grind finely.
2. Press the mixture into a transparent pellet with a professional mold.
3. Scan the full infrared range from 4000 cm⁻¹ to 400 cm⁻¹.

Spectrum Identification Criteria

• Weathered coal-derived humic acid: Strong absorption at 1600 cm⁻¹; weak peaks at 1720 cm⁻¹ and 1050 cm⁻¹.
• Biochemical humic acid: Strong peaks at 1720 cm⁻¹ and 1050 cm⁻¹; obvious characteristic peak at 1540 cm⁻¹.

4 Fluorescence Spectroscopy Test Procedure

1. Dilute the sample solution to an appropriate low concentration.
2. Instrument setting: Excitation wavelength 300–480 nm; Emission wavelength 400–550 nm.
3. Observe the maximum fluorescence peak position:

• Maximum peak Ex＞420 nm: Weathered coal-derived humic acid
• Maximum peak Ex＜360 nm: Biochemical humic acid

5 Solid-state ¹³C NMR Confirmation Procedure

1. Completely dry the purified sample to remove moisture.
2. Load the sample into a rotor and conduct NMR testing.
3. Integrate and calculate the proportion of carbon structures:

• Aromatic carbon content ≥ 50%: Weathered coal-derived humic acid
• Aromatic carbon content ＜ 30% (aliphatic & polysaccharide carbon dominant): Biochemical humic acid