Protocol Citation: Jian Gong 2020. Dissolved sulfide concentrations (H2S, HS-, S2-) colorimetric assay using a plate reader (96-well plate). protocols.io https://protocols.io/view/dissolved-sulfide-concentrations-h2s-hs-s2-colorim-bd7ji9kn
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: In development
We are still developing and optimizing this protocol
Created: March 25, 2020
Last Modified: July 13, 2020
Protocol Integer ID: 34763
Keywords: Total dissolved sulfide, colorimetric assay, plate reader,
Abstract
This protocol describes the adaption of a sulfide colorimetric assay originally described in Cline 1969, modified for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA), using standard 96-well plates for rapid measurements of < 400 μL of water samples. This assay measures the concentration of total sulfide species (H2S, HS-, S2-) in solution.
Samples for this assay should be filtered (0.2 µm syringe filter) and placed in 1.5 mL Eppendorf tubes. Care should be taken to avoid oxidation of the sample by oxygen in the atmosphere, by completing the first step of the assay quickly right after sampling, which stabilizes the sample against further oxidation.
Guidelines
Working principle
N,N-dimethyl-p-phenylenediamine (diamine) and ferric ion together form a stable colored complex with zinc sulfide at pH 0.35, with maximum absorbance at λ= 670 µm. This property is used to design the colorimetric assay described here, useful to measure the concentration of sulfide in a large range of natural waters. This method is free from the interference effect of salts up to 40 ‰ and sulfite up to 100 μM. Thiosulfate is known to inhibit the color-development of this assay; this effect is also time-dependent.
Table 1. Suggested reagent concentrations and dilution factors to be used for the determination of sulfide-sulfur (modified from Cline 1969). Bold text indicates the concentrations applied in this assay.
| Sulfide concen | Diamine concen | Ferric concen | Dilution factor | Path length | |
| (µM) | (g/100 ml) | (g/100 ml) | (ml:ml) | (cm) | |
| 1-3 | 0.1 | 0.15 | 1:1 | 10 | |
| 3-40 | 0.4 | 0.6 | 1:1 | 1 | |
| 40-250 | 1.6 | 2.4 | 2:25 | 1 | |
| 250-1000 | 4.0 | 6.0 | 1:50 | 1 |
Measurement range
Refer to Table 1 for the recommended preparation of diamine and ferric chloride reagent specific to the range of sulfide concentrations to be measured. Prepare standards and dilutions accordingly. Note that the spectrophotometer at the Bosak Lab uses a plate reader format, and the path length is roughly 4 mm when loading 200 μL wells, rather than the standard 10 mm on other spectrophotometers. Therefore, a dilution is applied to the sample (sample: reagent A = 4:10) to make the measurements consistent (Step 1 below). An accurate way to perform dilution is by measuring mass instead of volume. A pipette (which measures volume) has about 1-3% accuracy, whereas an analytical balance (0.1-1 mg accuracy) is vastly better. Do not switch pipettes during colorimetric assay procedures and pay careful attention.
Materials
Materials
Reagents
All reagents are prepared with nanopure water and stored in 4 °C fridge in the dark.
(A) Zinc acetate solution: 0.05 Molarity (M) of zinc acetate (Zn(CH3COO)2·2H2O, FW 219.51, 98%, Aldrich #383058) is prepared in nanopure water and used for sample stabilization, dilution, as well as the preparation of sulfide standards.
(B) Diamine reagent: Referring to Table 1, the reagent is prepared from 0.4 g N,N dimethyl-p-phenyldiamine sulfate salt ((CH3)2NC6H4NH2·H2SO4, FW 234.27, Aldrich #186384) and 0.6 g of anhydrous ferric chloride (FeCl3, FW 162.20, 97%, Aldrich #157740) dissolved in 100 mL of 6 M (N) hydrochloric acid (diluted from stock hydrochloric acid : HCl suprapur, 30%, 9.46 Molarity (M) , Aldrich #1.00318). This reagent is prepared for detecting sulfide at a concentration between 3-40 µM (see Table 1) and is a good starting point if the concentration of sulfide is unknown in a given sample. Store this reagent at 4 °C in the dark.
(C) Sulfide standard stock: 0.1 Molarity (M) of sodium sulfide nonahydrate (Na2S·9H2O, Aldrich #431648, 99.99%) is prepared in anaerobic nanopure water that is previously allowed to boil at 100 °C. The sulfide standard solution is kept in a hermetically sealed serum bottle. The headspace of the bottle was flushed with CO2: N2 (20:80 vol) atmosphere using standard anaerobic techniques to maintain anoxic conditions. The exact concentration of Na2S stock solution can be verified by precipitating an exact volume of Na2S with an excess volume of 0.3 M silver nitrate (AgNO3, Aldrich #209139, 99%).
Standards
Fresh standard solutions are prepared around the same time of sampling, by diluting reagent C with reagent A. This series of standard solutions should be prepared to cover the range of sulfide concentrations expected.
Safety warnings
Use safety goggles and nitrile gloves when performing the steps outlined in this assay. Dispose of left-over chemicals by evaporating off all liquids inside a chemical hood (96-well plate), or by collecting these liquids in designated chem-waste jars.
Before start
Samples for this assay should be filtered (0.2 µm syringe filter) and placed in 1.5 mL Eppendorf tubes. Care should be taken to avoid oxidation of the sample by oxygen in the atmosphere, by completing the first step of the assay quickly right after sampling, which stabilizes the sample against further oxidation.
Sample stabilization: Add 400 µL of liquid sample in 1 ml of reagent A upon sampling. This step makes zinc sulfide and stabilizes the sample from further oxidation (should be already done during sampling). Here 400 µL corresponds roughly to a detection range at 3 - 40 µM sulfide-sulfur. This amount can be further reduced (for example, adjusted to 200 µL) if higher sulfide concentrations are expected. This step dilutes the sample while also stabilizes the sample.
Colored-complex development: Transfer 900 µL of the stabilized mixture to a new tube and 15 µL of reagent B is added. Mix by pipetting up and down a few times. The reaction is timed for 20 minutes, in the dark.
Measurement: Measure absorbance at 670 nm. The system at the Bosak Lab is a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA). Measure by loading quadruplicate 200 μl mixtures on a 96-well plate.