APPARATUS
Eppendorfpipettes (1-5 cm³; 100 µl)
Volumetric flask
Cylinder (30 cm³)
Shaker for vertical rotation
Centrifuge
Centrifugetubes (50 cm³)
Photometer

REAGENTS:
HCl (1M)
NaOH (1N)
0,05 M HEPES (11.915 g/l)
1 g Ferrozine (= Ferrospectral)
1 g Hydroxyl ammonium chloride
Fe-II standard: Merck-Titrisol;  (NH₄)₂ Fe(SO₄)₂ ;  (0.1 mol/l)
Fe-standard: Merck-Titrisol; 1.000 g Fe/l ( FeCl₃ in 15 % HCl)

EXTRACTION:
Add 1-2 g wet sediment to 30 ml HCl (1M) and let it shake for 24 hours in the shaker.
(For carbonate-rich sediments more HCl has to be added until the pH of the suspension is smaller than 1.)
Then the samples are centrifuged for 15 min at the highest possible speed of rotation. The liquid phase is filtrated (0.2 µm), filled into bottles and used for the iron-determination.
The sediment remains in the centrifuge tube and is stored for the later determination of the dry mass.

PROCEDURE:
Solution 1: 11.915 g of HEPES and 1 g of Ferrozine are made up to 1000 cm³ with pure water.
From this solution you take 5 cm³, add 100 µl sample, shake well and wait for exactly 20 min. (Final pH-value  >5). Measure absorbance at 562 nm. (Determination of Fe-II.)

Solution 2: Take 100 cm³ of solution 1 and add 1 g of hydroxyl ammonium chloride (NH₄OH*HCl).
Take 5 cm³ of solution 2 and add 100 µl sample (final pH-value > 4). After exactly 20 min. measure the absorbance at 562 nm. (Determination of Fe-II and Fe-III.)

CALIBRATION:
Work standard: Dilute the stock-standard with pure water to obtain standards with different concentrations. Measure the standards in the same way as the samples and form a calibration curve (5-6 calibration points).
Calibration area: 0.05 mM- 1 mM

INTERFERENCES:
The pH values have to be kept within the limits defined above to avoid interferences. Fe (III) can be reduced to Fe (II) by humic and fulvic acids during the reaction with HEPES/ Ferrozine. The Fe (II) concentration can be monitored over time to estimate the extent of this reaction.