Jun 21, 2025

Sulfide Measurement Protocol V.1

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvrq1oolmk/v1
  • 1Method developed for Coon et al., in prep;
  • 2Massachusetts Institute of Technology
  • Bosak Lab
Bosak Lab Protocols
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvrq1oolmk/v1
Protocol Citation: Gage R. Coon, Bosak Lab Protocols 2025. Sulfide Measurement Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrq1oolmk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2025
Last Modified: June 21, 2025
Protocol  Integer ID: 220658
Keywords: sulfide, cline assay, sulfide measurement protocol photometric determination of sulfide, sulfide measurement protocol photometric determination, hydrogen sulfide, h2s concentration, sulfide level, water sample, form of hydrogen sulfide, proportional to the h2s concentration, absorbance of the methylene blue color, yielding methylene blue, h2, methylene blue, methylene blue color, absorbance, more concentrated sample
Abstract
Photometric determination of sulfide (S2-) in the form of hydrogen sulfide (H2S) in water samples: Based on diamine and H2S yielding methylene blue when in the presence of Fe3+. Absorbance of the methylene blue color at 670 nm is proportional to the H2S concentration. This protocol is optimized for sulfide levels of roughly 0 to 10 mM. You can use more concentrated samples as long as absorbance is below 1 so it still follows Beer’s Law. Samples can be stored in the fridge and just add diamine directly later. If unsure about dilutions, save two samples or extra volume.
Materials
All water should be deoxygenated by flushing with N2 for 15+ minutes. Store diamine reagent in a dark place at 4°C if possible.

- Diamine reagent: Add 3.75 g N,N-dimethyl-p-phenylenediamine sulfate and 5.625 g ferric chloride to a beaker of 250 mL 50/50 HCl/deoxygenated water, store in a dark container.
- 0.05M zinc acetate
- 1 mM working sodium sulfide standard: Dilute concentrated standard from fridge to 0.05 M zinc acetate for a final dilution. Do this quickly and keep the concentrated standard anoxic!
Procedure
Standard Curve
Create the following standards in Eppendorf tubes in the following format: Standard concentration: volume of 0.05M zinc acetate, volume of 1 mM sodium sulfide standard.
0 mM (blank): 1.00 mL, 0 µL
0.2 mM: 0.98 mL, 20 µL
0.5 mM: 0.95 mL, 50 µL
0.8 mM: 0.92 mL, 80 µL
1 mM: 0.90 mL, 100 µL
Prepare 1 mL samples diluted 100x in 0.05M zinc acetate (10 µL sample + 990 µL 0.05M zinc acetate).
Add 33 µL of the diamine reagent to each Eppendorf tube.
Vortex and store in the dark for 20+ minutes.
Measure absorbance at 670 nm.
Protocol references
@article{CLINE1969,
title = {SPECTROPHOTOMETRIC DETERMINATION OF HYDROGEN SULFIDE IN NATURAL WATERS1},
volume = {14},
ISSN = {1939-5590},
url = {http://dx.doi.org/10.4319/lo.1969.14.3.0454},
DOI = {10.4319/lo.1969.14.3.0454},
number = {3},
journal = {Limnology and Oceanography},
publisher = {Wiley},
author = {CLINE, JOEL D.},
year = {1969},
month = may,
pages = {454–458}
}