⚗️ Phenol index in water — flow analysis (FIA/CFA) with 4-aminoantipyrine and potassium peroxodisulfate, in-line extraction into chloroform (470–475 nm)
In short
The sample is introduced into the carrier stream and mixed with solutions of 4-aminoantipyrine (in a buffer of about pH 10. The test is performed according to PN-EN ISO 14402:2004 (ISO 14402:1999); Range: 0.01–1 mg/l (range II 0.01–0.1; range I 0.1–1 mg/l), a separate calibration with at least 5 standards; with detergents only above 0.1 mg/l. The procedure comprises 6 steps; it is used for: Industrial wastewater (coking plants, refineries, the chemical industry, tanneries) and municipal wastewater — the phenol index as a limited parameter (0.1 mg/l of volatile phenols), Surface water and groundwater, leachate from landfills — monitoring of phenols from 0.01 mg/l, High-throughput laboratories — automatic FIA/CFA analysis with in-line extraction into chloroform (without distillation).
At a glance
- Standard: PN-EN ISO 14402:2004 (ISO 14402:1999)
- Category: Physicochemistry
- Procedure steps: 6
- Range: 0.01–1 mg/l (range II 0.01–0.1; range I 0.1–1 mg/l), a separate calibration with at least 5 standards; with detergents only above 0.1 mg/l
- Detection: 470–475 nm in the chloroform phase; FIA cell 10 mm; CFA cell 0.5–5 cm; peak heights
- In-line reagents: R1: 0.5 g of 4-aminoantipyrine per 100 ml of buffer of pH 10.3 (NaHCO₃ 23 g, H₃BO₃ 27 g, KOH 35 g/l); R2: 5 g of K₂S₂O₈ per 100 ml at pH 11; both freshly prepared every day and degassed
Overview
ISO 14402:1999 specifies two methods for determining the phenol index in waters of various origin (groundwater, surface water, leachate, wastewater) at concentrations of 0.01–1 mg/l in the undiluted sample (the range can be adjusted by the working conditions): clause 3 — the phenol index without distillation, after extraction; clause 4 — the phenol index without extraction, after distillation. The 2019 regulation (Dz.U. 2019 poz. 1311 [Journal of Laws 2019, item 1311]) names PN-EN ISO 14402 (flow analysis, CFA and FIA) alongside PN-ISO 6439 (spectrophotometry) as the reference methods for the phenol index in wastewater, for which the limit for volatile phenols is 0.1 mg/l. In Poland it is in force as PN-EN ISO 14402:2004 (Polish version; it replaced PN-EN ISO 14402:2002).
Interferences: aromatic amines form condensation products with 4-aminoantipyrine (results too high); after the reagents have been added the sample has to reach a pH of 10.0–10.5 — strongly acid, strongly alkaline and buffered samples are brought beforehand to pH 5–7; turbidity does not interfere; with coloured samples it is checked whether the colour is extracted by chloroform, and a blank is measured without the reagents R1 and R2; detergents in wastewater interfere strongly (foam disturbs the distillation of the volatile phenols and the segmentation/separation of the phases) — where the detergent content is considerable, the standard applies only above 0.1 mg/l of phenol. The classical manual method (PWr textbook "Chemia wody" [Water chemistry], PN-ISO 6439) is based on the same reaction: after steam distillation the volatile phenols react at pH 10.0 ± 0.2 in the presence of potassium ferricyanide with 4-aminoantipyrine, the product being extracted with chloroform and measured at 510 nm (0.002–5.0 mg/dm³).
Method principle
The sample is introduced into a continuously flowing carrier stream and mixed with the likewise continuously flowing solutions of 4-aminoantipyrine and potassium peroxodisulfate. The phenolic compounds are oxidised by the peroxodisulfate, and the quinones formed react with the 4-aminoantipyrine, giving coloured condensation products which, in a flow extraction unit, pass from the aqueous phase into chloroform; the chloroform phase is separated by a phase separator (e.g. a hydrophobic semi-permeable PTFE membrane, thickness 150–200 µm, pores 0.5–2 µm, porosity 75 %), and the absorbance of the organic phase is measured by a flow spectrometer at 470–475 nm. Reagents (of analytical grade, the solutions degassed, best under reduced pressure): grade 1 water, KOH, NaHCO₃, 4-aminoantipyrine, K₂S₂O₈, phenol (colourless crystals), boric acid, ethanol 96 %, 2-propanol, H₂SO₄ 1.84 g/ml, HCl 50 %, KOH 1.0 mol/l; buffer (23 g of NaHCO₃ + 27 g of H₃BO₃ + 35 g of KOH per 1000 ml; about pH 10.3; 1 month); carrier C — degassed water; R1 — 0.5 g of 4-aminoantipyrine in 100 ml of the buffer (freshly prepared every day, degassed); R2 — 5 g of K₂S₂O₈ in about 90 ml of water, pH 11 (KOH 1 mol/l), made up to 100 ml (every day); Org — degassed chloroform (membrane or 3 min of ultrasound); phenol stock solution 1000 mg/l (1.000 g/l; 1 month at 2–5 °C), standards of 10 mg/l (a week) and 1 mg/l; calibration solutions (at least 5 per range, freshly prepared every day): range I 0.1–1 mg/l (1, 3, 5, 6, 8, 10 ml of the 10 mg/l standard made up to 100 ml), range II 0.01–0.1 mg/l (the same volumes of the 1 mg/l standard). FIA apparatus: reagent reservoirs, a low-pulsation pump, a displacement bottle for the chloroform, an injector (600 µl for the range 0.01–0.1 mg/l, 200 µl for 0.1–1.0 mg/l), an extraction cell with a segmenter and a phase separator, tubing and coils of 0.5–0.8 mm (reaction coils of 60 and 80 cm, extraction coil 160 cm × 0.7 mm), a detector with a 10 mm cell at 470–475 nm, a recorder (peak heights), an autosampler; CFA: an autosampler, a pump with inert tubing, a manifold with air segmentation (e.g. internal diameter 2 mm; coils of 40, 40 and 150 cm), a phase separator, a detector with a cell of 0.5–5 cm at 470–475 nm. In addition: flasks of 100 and 1000 ml, pipettes of 1–10 ml, membrane filters 0.45 µm, a pH meter. Samples: glass or PTFE vessels, rinsed before use with sulfuric acid of about pH 2; analysis immediately after sampling, or acidification to about pH 2 (H₂SO₄ or HCl), storage in the dark at 2–5 °C and analysis within 24 h (exceptionally, after acidification and pressure filtration, for up to 2 weeks after verification); filtration where there is a risk of blocking. Preparation for the measurement: pass C, R1, R2 and Org through until the baseline is stable, then zero it; a poor signal-to-noise ratio usually results from a damaged separator membrane or traces of water on the walls of the cell (rinse with ethanol or 2-propanol). Check of the system: in range II a standard of 0.05 mg/l has to give an absorbance of at least 0.01 cm⁻¹; reagent blank: water instead of R1 and R2 — a drop of absorbance above 0.05 cm⁻¹ means self-condensation products (repeat the preparation of the solutions). Calibration separately for each range (FIA: 200 µl for I, 600 µl for II; CFA: choice of the cell length and of the flow rate for the highest response).
Applications
- Industrial wastewater (coking plants, refineries, the chemical industry, tanneries) and municipal wastewater — the phenol index as a limited parameter (0.1 mg/l of volatile phenols)
- Surface water and groundwater, leachate from landfills — monitoring of phenols from 0.01 mg/l
- High-throughput laboratories — automatic FIA/CFA analysis with in-line extraction into chloroform (without distillation)
- Turbid samples (which do not interfere) and coloured samples (with a blank measured without the reagents)
- The variant with distillation (clause 4 of the standard) for samples with non-extractable substances or where volatile phenols are required
Key parameters
| Parameter | Value |
|---|---|
| Range | 0.01–1 mg/l (range II 0.01–0.1; range I 0.1–1 mg/l), a separate calibration with at least 5 standards; with detergents only above 0.1 mg/l |
| Detection | 470–475 nm in the chloroform phase; FIA cell 10 mm; CFA cell 0.5–5 cm; peak heights |
| In-line reagents | R1: 0.5 g of 4-aminoantipyrine per 100 ml of buffer of pH 10.3 (NaHCO₃ 23 g, H₃BO₃ 27 g, KOH 35 g/l); R2: 5 g of K₂S₂O₈ per 100 ml at pH 11; both freshly prepared every day and degassed |
| Extraction | degassed chloroform; phase separator — a PTFE membrane 150–200 µm, pores 0.5–2 µm, porosity 75 % |
| Injection volume (FIA) | 600 µl (0.01–0.1 mg/l), 200 µl (0.1–1.0 mg/l); coils of 60/80 cm × 0.5 mm, extraction coil 160 cm × 0.7 mm |
| Control | standard of 0.05 mg/l → A ≥ 0.01 cm⁻¹; reagent blank: drop ≤ 0.05 cm⁻¹; pH of the sample after the reagents 10.0–10.5; samples: pH 2, 2–5 °C, 24 h |
Standard
- Standard number
- PN-EN ISO 14402:2004 (ISO 14402:1999)
- Title (PL)
- Jakość wody — Oznaczanie indeksu fenolowego za pomocą analizy przepływowej (FIA i CFA)
- Title (EN)
- Water quality — Determination of phenol index by flow analysis (FIA and CFA)
Step-by-step procedure
-
Sample
Rinse the glass or PTFE vessels with sulfuric acid of about pH 2. Analyse at once or acidify to about pH 2 (H₂SO₄ or HCl), store in the dark at 2–5 °C and examine within 24 h (exceptionally for up to 2 weeks after acidification and pressure filtration, once verified); filter where there is a risk of blocking the tubing; bring strongly acid, strongly alkaline or buffered samples to pH 5–7.
-
Reagents
Prepare fresh R1 (0.5 g of 4-aminoantipyrine in 100 ml of the buffer) and R2 (5 g of K₂S₂O₈ in 100 ml, pH 11) every day, degas them (membrane), degas the chloroform (membrane or 3 min of ultrasound) and the carrier water (under reduced pressure); prepare at least 5 calibration solutions of the chosen range (I: 0.1–1.0 mg/l; II: 0.01–0.1 mg/l).
-
Starting up the system
Pass C, R1, R2 and Org through the system until the baseline is stable and zero it; check the signal-to-noise ratio (a damaged separator membrane or water on the walls of the cell — rinse with ethanol/2-propanol).
-
Check of the system and of the blank
In range II a standard of 0.05 mg/l has to give an absorbance of at least 0.01 cm⁻¹ (otherwise the system is not suitable). Instead of R1 and R2 pass water through until the signal is constant — a drop of absorbance above 0.05 cm⁻¹ means self-condensation products: prepare the solutions again and repeat the checks; then restore R1 and R2.
-
Calibration
Calibrate separately for each range: FIA — an injection of 200 µl (range I) or 600 µl (range II); CFA — choose the cell length and the flow rate for the highest response of the highest standard; record the peak heights.
-
Measurement and result
Measure the samples under the same conditions; for coloured samples check whether the colour is extracted by chloroform and measure a blank without R1 and R2, taking the difference into account in the calculations; read the phenol index (mg/l as phenol) from the calibration; where detergents are present report only results above 0.1 mg/l; record the variant (extraction/distillation), the preservation and any deviations.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| FIA analyser (low-pulsation pump, injector of 200/600 µl) or CFA analyser (autosampler, manifold with air segmentation) | tubing of 0.5–0.8 mm (FIA) or about 1–2 mm (CFA) of inert plastics | — |
| Displacement bottle for feeding the chloroform | the Org stream | — |
| Extraction cell with a phase segmenter and a PTFE membrane separator | in-line extraction of the condensation products into chloroform | — |
| Flow spectrometer at 470–475 nm (10 mm cell in FIA; 0.5–5 cm in CFA) and a recorder | measurement of peak heights | — |
| Volumetric flasks of 100 and 1000 ml, pipettes of 1–10 ml, membrane filters 0.45 µm, pH meter | standards and preparation of the samples | — |
| Ultrasonic bath or a membrane degassing set | degassing of the chloroform and of the reagents | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| 4-aminoantipyrine (solution of 0.5 g per 100 ml in the borate-hydrogen carbonate buffer of pH 10.3) | — | reagent coupling with the quinones (R1) |
| Potassium peroxodisulfate 5 g per 100 ml, pH 11 | — | oxidant for the phenols (R2) |
| Buffer: NaHCO₃ 23 g + H₃BO₃ 27 g + KOH 35 g per 1000 ml | — | pH of about 10.3 for the reaction |
| Degassed chloroform | — | extraction phase (Org) |
| Phenol (colourless crystals) — 1000 mg/l, standards of 10 and 1 mg/l | — | calibration of ranges I and II |
| Sulfuric acid / hydrochloric acid 50 %, KOH 1 mol/l, ethanol 96 %, 2-propanol | — | preservation of the samples (pH 2), pH adjustment, rinsing of the cell |
Health and safety (OHS)
- Phenol is toxic and is readily absorbed through the skin — gloves, goggles; chloroform is toxic and carcinogenic — work in a fume hood, chloroform and phenol waste to be disposed of
- Potassium peroxodisulfate — an oxidant; potassium hydroxide and concentrated sulfuric acid — corrosive
- Ethanol and 2-propanol — flammable
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 14402:2004 (ISO 14402:1999) — “Water quality — Determination of phenol index by flow analysis (FIA and CFA)”.
How does this method work?
The sample is introduced into a continuously flowing carrier stream and mixed with the likewise continuously flowing solutions of 4-aminoantipyrine and potassium peroxodisulfate. The phenolic compounds are oxidised by the peroxodisulfate, and the quinones formed react with the 4-aminoantipyrine, giving coloured condensation products which, in a flow extraction unit, pass from the aqueous phase into chloroform; the chloroform phase is separated by a phase separator (e.
What is the measuring range and accuracy?
Range: 0.01–1 mg/l (range II 0.01–0.1; range I 0.1–1 mg/l), a separate calibration with at least 5 standards; with detergents only above 0.1 mg/l; Detection: 470–475 nm in the chloroform phase; FIA cell 10 mm; CFA cell 0.5–5 cm; peak heights; In-line reagents: R1: 0.5 g of 4-aminoantipyrine per 100 ml of buffer of pH 10.3 (NaHCO₃ 23 g, H₃BO₃ 27 g, KOH 35 g/l); R2: 5 g of K₂S₂O₈ per 100 ml at pH 11; both freshly prepared every day and degassed; Extraction: degassed chloroform; phase separator — a PTFE membrane 150–200 µm, pores 0.5–2 µm, porosity 75 %.
How long does the test take?
The procedure comprises 6 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
FIA analyser (low-pulsation pump, injector of 200/600 µl) or CFA analyser (autosampler, manifold with air segmentation), Displacement bottle for feeding the chloroform, Extraction cell with a phase segmenter and a PTFE membrane separator, Flow spectrometer at 470–475 nm (10 mm cell in FIA; 0.5–5 cm in CFA) and a recorder, Volumetric flasks of 100 and 1000 ml, pipettes of 1–10 ml, membrane filters 0.45 µm, pH meter, Ultrasonic bath or a membrane degassing set.
Where is this test used?
Industrial wastewater (coking plants, refineries, the chemical industry, tanneries) and municipal wastewater — the phenol index as a limited parameter (0.1 mg/l of volatile phenols); Surface water and groundwater, leachate from landfills — monitoring of phenols from 0.01 mg/l; High-throughput laboratories — automatic FIA/CFA analysis with in-line extraction into chloroform (without distillation); Turbid samples (which do not interfere) and coloured samples (with a blank measured without the reagents); The variant with distillation (clause 4 of the standard) for samples with non-extractable substances or where volatile phenols are required.
What safety precautions apply?
Phenol is toxic and is readily absorbed through the skin — gloves, goggles; chloroform is toxic and carcinogenic — work in a fume hood, chloroform and phenol waste to be disposed of; Potassium peroxodisulfate — an oxidant; potassium hydroxide and concentrated sulfuric acid — corrosive; Ethanol and 2-propanol — flammable.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 14402.