Sequential extraction

Dispensettes

Dispensettes

Image: Nico Köppner

Sequential extraction according to Zeien & Brümmer allows the differentiation of heavy metals in moderately carbonate soils according to their mobility and binding form.

Equipment

For extraction according to Zeien and Brümmer, overhead shakers of the type Rotoshake RS12 (Gerhardt, Königswinter, Germany) and ELU (Edmund Bühler, Hechingen, Germany), a centrifuge of the type Multifuge 3L (Heraeus, Langenselbold, Germany) for tubes with a maximum volume of 50 ml, and a vacuum filtration apparatus (home-made, Lippmann Labortechnik, Dresden, Germany) are available.

Sample preparation

For Zeien & Brümmer extractions, samples must be air dried and sieved to < 2 mm (minimum sample mass 2.0 g, 5.0 g is recommended).

Execution

Sequential extraction to determine the heavy metal binding forms according to Zeien & Brümmer, 1989 (slightly modified). Usually two blanks and up to 14 samples are run in parallel.

Equipment and materials used

Devices

  • Centrifuge: ‘Multifuge 3L, Heraeus’
  • Centrifuge tubes 15 ml and 50 ml, material PP, Greiner company
  • Overhead shaker: Rotoshake RS12, Gerhardt company
  • Vacuum filtration apparatus: Lippmann-Labortechnik Dresden
  • Dispensing flasks: BRAND
  • Analytical balance: Sartorius BP210S
  • Ultrapure water system: ThermoScientific 2GenPure ‘UV-TOC’
  • Sample bottles: acid-rinsed with diluted HNO3, 100 ml, material PP, Kautex or VWR
  • Membrane filter: Cellulose acetate (Sartorius 11106-47, 0.45 µm)

Chemicals

(unless otherwise stated, then purity ‘for analysis’ p.a.)

  • Ammonium nitrate NH4NO3 (Roth, No. K299.1)
  • Ammonium acetate CH3COONH4 (NH4OAc) (Merck, No. 101116)
  • Hydroxylammonium chloride NH2OH * HCl (Merck, No. 104619)
  • Titriplex II C10H16N2O8 (edetinic acid, EDTA, Merck, No. 108417)
  • di-ammonium oxalate monohydrate C2H8N2O4 * H2O (Merck, No. 101192)
  • Oxalic acid dihydrate C2H2O4 * 2 H2O (Merck, No. 100495)
  • L(+)-ascorbic acid (vitamin C) (Merck, No. 100127)
  • Acetic acid C2H4O2 50% (diluted from glacial acetic acid, Merck No. 100063)
  • Ammonia solution NH3 25% (Merck 105432)
  • Nitric acid HNO3 65% subboiled (Merck: 100456, purified by subboiling distillation)
  • Hydrochloric acid HCl fuming 37% (Roth)
  • Ultrapure water ‘ThermoScientific 2GenPure’ UV-TOC)

Working instructions

Fraction I: Mobile heavy metals

  • Weighing: 2 g air-dried fine soil (<2 mm) to the nearest 0.01 g in centrifuge tubes (50 ml)
  • Extraction agent: 50 ml 1 M NH4NO3 (NH4NO3: 80.04 g/l, Roth, No. K299.1)
  • Shaking time: 24 h at 20°C and approx. 20 rpm (overhead shaker)
  • Obtaining the extraction solution: centrifuge for 15 min at 2500 rpm
  • Transfer the supernatant solution through a cellulose acetate filter into acid-rinsed bottles using a vacuum filtration apparatus
  • Stabilise the filtrates by adding 0.5 ml HNO3 (65%)

 

Fraktion II: Leicht nachlieferbare Schwermetalle

  • im Zentrifugenbecher verbliebene Probe mit 50 ml 1 M NH4OAc (pH 6,0) versetzen (CH3COONH4 (NH4OAc) : 77,08 g/l, Fa. Merck, Nr. 101116, eingestellt mit 50%iger Essigsäure auf pH 6,0)
  • Bei carbonathaltigen Bodenproben (> 5% CaCO3) zur Neutralisation der Carbonate (zur Gewährleistung der pH-Konstanz von pH 6,0) die entsprechende Menge verdünnter HCl p.a. zugeben
  • 24 h schütteln
  • 15 min bei 2500 U/min zentrifugieren
  • überstehende Lösung über Cellulose-Acetat-Filter per Vakuumfiltrationsapparatur in säuregespülte Flaschen überführen
  • Gewinnung der in der Bodenprobe verbliebenen Restlösung: Probe mit 25 ml 1 M NH4NO3 (Lösung Fraktion I) 10 min schütteln, zentrifugieren und filtrieren (die Filtrate werden vereinigt)
  • Filtrate durch Zugabe von 0,5 ml HNO3 (65%) stabilisieren

Fraction II: Easily replenishable heavy metals

  • Add 50 ml of 1 M NH4OAc (pH 6.0) to the sample remaining in the centrifuge beaker (CH3COONH4 (NH4OAc) : 77.08 g/l, Merck, No. 101116, adjusted to pH 6.0 with 50% acetic acid).
  • For soil samples containing carbonates (> 5% CaCO3), add the appropriate amount of diluted HCl p.a. to neutralise the carbonates (to ensure a constant pH of pH 6.0)
  • Shake for 24 h
  • Centrifuge for 15 min at 2500 rpm
  • Transfer the supernatant solution into acid-rinsed bottles via a cellulose acetate filter using a vacuum filtration apparatus
  • Recovery of the residual solution remaining in the soil sample: shake sample with 25 ml 1 M NH4NO3 (solution fraction I) for 10 min, centrifuge and filter (the filtrates are combined)
  • Stabilise the filtrates by adding 0.5 ml HNO3 (65%)

 

Fraktion IV: An organische Substanzen gebundene Schwermetalle

  • im Zentrifugenbecher verbliebene Probe mit 50 ml 0,025 M NH4-EDTA (pH 4,6) versetzen (Editinsäure: 7,31 g/l, Titriplex II, Fa. Merck, Nr. 108417, eingestellt mit verdünnter Ammoniak-Lösung auf pH 4,6)
  • 90 min schütteln
  • 15 min bei 2500 U/min zentrifugieren
    • überstehende Lösung über Cellulose-Acetat-Filter per Vakuum-Filtrationsanlage in säuregespülte Flaschen überführen
    • Gewinnung der in der Bodenprobe verbliebenen Restlösung: Probe wird mit 25 ml 1 M NH4OAc (pH 4,6) versetzt (NH4OAc : 77,08 g/l, Fa. Merck, Nr. 101116, eingestellt mit 50%iger Essigsäure (Fa. Merck, Nr. 63) auf pH 4,6), 10 min geschüttelt, zentrifugiert und filtriert (die Filtrate werden vereinigt)

Fraction V: Heavy metals bound to poorly crystalline Fe oxides

  • Add 50 ml of 0.2 M NH4 oxalate (pH 3.25) to the sample remaining in the centrifuge beaker (di-ammonium oxalate monohydrate: 28.422 g/l, Merck, No. 101192 and oxalic acid dihydrate: 25.214 g/l, Merck, No. 100495), adjusted to pH 3.25 with diluted ammonia solution
  • Shake for 4 h in the dark
  • Centrifuge for 15 min at 2500 rpm
    • Transfer the supernatant solution into acid-rinsed bottles via a cellulose acetate filter using a vacuum filtration system
    • Recovery of the residual solution remaining in the soil sample: add 25 ml of 0.2 M NH4 oxalate (pH 3.25) to the sample once (solution fraction V), shake for 10 min in the dark, centrifuge and filter (the filtrates are combined)

Fraction VI: Heavy metals bound to crystalline Fe oxides

  • Add 50 ml of 0.1 M ascorbic acid and 0.2 M NH4 oxalate (pH 3.25) to the sample remaining in the centrifuge beaker (0.1 M L(+)-ascorbic acid (vitamin C): 17.613 g/l, Merck, No. 100127 + 0.2 M NH4 oxalate buffer: 0.2 M di-ammonium oxalate monohydrate p.a.: 28.422 g/l; Merck, No. 101192 and 0.2 M oxalic acid dihydrate p.a.: 25.214 g/l; Merck, No. 100495), adjusted to pH 3.25 with diluted ammonia solution)
  • Extract for 30 min in a water bath at 96 +- 3°C
  • Centrifuge for 15 min at 2500 rpm
    • Transfer the supernatant solution via a cellulose acetate filter into acid-rinsed bottles using a vacuum filtration system
    • Recovery of the residual solution remaining in the soil sample: add 25 ml of 0.2 M NH4 oxalate (pH 3.25) (solution fraction V) to the sample once, shake for 10 min in the dark, centrifuge and filter (the filtrates are combined

More info

Extract from: Lewandowski, Jörg: Schadstoffe im Boden: eine Einführung in Analytik und Bewertung; Springer 1997, ISBN: 3-540-62643-3, S. 129-130

"In a sequential extraction, the weakest bound heavy metals are first dissolved with the weakest solvent. Then (sequentially) the next, slightly more tightly bound heavy metal fraction is extracted from the same soil sample with a stronger solvent.

Finally, the most strongly bound heavy metals are dissolved in several steps with the strongest solvent and the total content is determined. This means that an extraction agent ideally dissolves only one heavy metal fraction, as the more easily bound heavy metals have already been separated.

From an ecological point of view, it is not only the total content of heavy metals in the soil that is of interest, but above all their ecological availability. Depending on the form of binding, the heavy metals are either immediately available, can be mobilised in the short term, can be released in the long term or are immobile. The mobilisation of heavy metals is determined by chemical-physical soil parameters.

Sequential extraction 'attempts to capture heavy metals separately according to their binding forms' by increasing the strength of the extraction agents. This should provide information on the ecological availability of the heavy metals. (Federer 1993, p. 21)".

Contact

Ulrike Buhler