Extraction and Speciation of Poorly Crystalline Fe Minerals from Soils and Sediments*

by

Joel E. Kostka, Florida State University

(As Used in Laboratory of Joseph W. Stucki, University of Illinois)

 

* Note: This method is also commonly used to measure the extent of structural Fe reduction in clay minerals and is referred to as the “Ferrozine Method.” The validity of using it for this purpose, however, has yet to be fully investigated. Some indications suggest that it may be less than robust for this purpose.

References:

Equipment needed:

-uv/ vis spectrophotometer

-shaker table

-centrifuge

 

Chemicals/ supplies needed:

-15 ml centrifuge tubes

-HCl

-Hepes

-Ferrozine

-Hydroxylamine hydrochloride

-Ferrous ammonium sulfate

 

Preparation of Reagents and Extractant:

HCl solution. Prepare a 0.5 M HCl solution by adding 41.75 ml of concentrated HCl (12 N) to a 1 liter volumetric flask and bringing the volume up to 1 liter with distilled water.  This solution can be stored at room temperature indefinitely.

 

Fe(II) buffer.  Add 0.2 grams of ferrozine and 11.91 grams of Hepes to a 1 liter volumetic flask and bring the volume up to 1 liter with distilled water.  Place solution into a larger vessel, add a magnetic stir bar, and adjust the pH to 7 using 10 M NaOH while stirring.  This solution can be stored at room temperature indefinitely.

 

Total Fe buffer.  Add 1 g of hydroxylamine hydrochloride to 100 ml of the above Fe(II) buffer.  This solution is unstable and MUST BE MADE UP DAILY prior to analysis.

 

HCl extraction.  Thaw frozen soil/ sediment while dispensing reagent solutions.   It is best to analyze each sample in duplicate for solid Fe and no more than 4 to 6 samples should be run at a time.  Dispense 10 ml of 0.5 M HCl into each of 15 ml centrifuge tubes.   Cap and label the tubes.  Weigh each tube and record the mass.  Make sure the caps are on tight and place tubes with the samples and a few spatulas into the exchange of the anaerobic chamber.  Mix the thawed sediment or soil samples well with a spatula in each tube inside the chamber.  Use the spatulas to add 0.1 to 0.3 grams of wet soil/ sediment into each 10 ml aliquot of HCl.  Again, make sure caps are on tight and remove the tubes from the chamber.  Check for and wipe away any soil/ sediment on the outside of each tube.  Weigh each tube again and record the mass. Vortex the sample tubes, making sure to break up any large clumps, then place the tubes onto the shaker table, secure and cover them with an opaque cloth, begin shaking at 200 rpm.  Record the time you started shaking.  Remove the tubes from the shaker 1 hour from the start. Centrifuge for 5 min at 5000 rpm before adding sample extract to buffer.

 

Fe analysis.  While HCl extraction is ongoing, set up plastic scintillation vials in parallel with Fe(II) and total Fe buffer.  Dispense 10 to 20 ml of buffer into each vial depending upon the amount of Fe expected in each sample.  Turn on the spectrophotometer, adjust wavelength to 562 nm, and allow to warm up for at least 15 to 20 min.  Zero the spec with distilled water.  The absorbance of Fe, especially Fe(II), can be time-dependent.  Therefore, be sure that you have organized your materials and can rapidly measure the absorbance on the spec.  Also, you should be consistent about the order in which samples are extracted and analyzed. 

 

Pipet 0.05 ml to 0.2 ml of acid extract per 10 ml of buffer.  Pipet no more than 0.2 ml of extract into 10 ml of buffer since the pH of the buffer will be affected at higher volumes. Reagent blanks should be prepared by adding HCl to each buffer solution in the same proportion as the extracts.  Mix buffer + extract (swirling by hand) immediately after pipetting.  Record the time you begin pipetting.  Let Fe(II) vials sit for 10 mins and place into cuvets during this period.  After 10 min., immediately read the absorbance at 562 nm for the Fe(II) vials.  Cap the vials containing total Fe buffer + extract, and place in a dark place for 4 hours.  Then pour the buffer + extract into cuvets and read the absorbance at 562 nm. 

Reagent blanks should give an absorbance of approximately 0.002 for Fe(II) and 0.004 to 0.008 for total Fe.  Blank absorbances higher than these mean that the reagents may be contaminated with Fe.

 

Standard Calibration Curve.  Prepare a 10 mM standard solution of ferrous ammonium sulfate in 0.5 M HCl.  Dilute standard 1:10 using 0.5 M HCl.  These standard solutions should be stable in acid when stored in the dark in a clean plastic container.  Pipet 0.025, 0.050, 0.10, and 0.20 ml of diluted standard into 10 ml of Fe(II) buffer and 10 ml of total Fe buffer.  Prepare parallel reagent blanks.  Analyze for Fe as described above.  Subtract blank absorbance from measured absorbance for each dilution of the standard solution.  Run a regression of absorbance vs. concentration of the standard.  Use slope of regression to calculate the concentration of Fe in the extracts.  The resulting regression coefficient should be above 0.99 if the analysis is performed properly.