Making the Invisible Visible – Analysis of Hydrogen Peroxide Concentrations in Seawater

Chemiluminescence of HOOH and AE

During the cruise I will be measuring the concentrations of hydrogen peroxide (H2O2), superoxide (O2), and antioxidants.   All of these species are colorless, odorless, and found at extremely low concentrations.  The challenge for analytical chemists is to make these invisible species visible.   A good example of a qualitative analytical measurement is the use of litmus paper to detect the acid/base properties of solutions (litmus paper is red in acid and blue in base).   In my measurements I need a more sensitive technique that can be automated to perform hundreds of measurements per day with as little human intervention as possible.   The technique that I have selected is chemilumniescence – a chemical reaction that produces light.   Recent advances in light detector technology makes it possible to measure individual photons produced by a chemical reaction.  By adding an acridinum ester reagent to seawater I can produce the blue chemiluminescence shown above.   The method is very sensitive because of sensitive light measurements and because Avogadro’s number is big, very big!

Consider adding ten drops of 3% (1 molar) hydrogen peroxide to a 6 lane swimming pool. This is a 2.5 billion fold dilution of H2O2 resulting in a concentration of 0.4 nM (4×10-10 M).   This concentration is close to the lower limit of H2O2 I can detect in seawater – an extremely low concentration.   However, in only 1 drop of the pool water we will still have 40 billion of molecules of hydrogen peroxide because Avogadros number (6.02×1023 molecules/mole) is so huge.  It is relatively easy to measure billions, millions, and even thousands of photons.  I “simply” need to convert the H2O2 into light to make the measurement.

Structure of AE. Click on image to see reaction with HOOH.

The chemiluminescent reagent used in this method is 10-methyl-9-(p-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate, abbreviated AE.  AE reacts selectively with H2O2 in seawater to produce blue light.    The reaction only occurs under basic conditions, so I add base to a mixture of seawater and AE to trigger the reaction.  The mixing is performed in a glass flow cell that helps automate the reaction and maximize photon collection by the detector.   The schematic below shows the tubing system that I use  to deliver the sample and reagents to the flow cell. A peristaltic pump pushes the solutions through the green valve on the way to the flow cell.  The valve can select for solutions in loop 3 or 5 automatically every few minutes.   When the valve is set to inject (loop 5) I measure the light coming from the AE-H2O2 reaction in the flow cell.   On the ship I replace the syringe shown in the diagram with a direct connection to the flowing seawater line coming from the sea chest on the ship.   This way I can get real-time measurements of H2O2 in the surface ocean.

Instrument for measuring hydrogen peroxide (tubes at the top are connected to the ships seawater system)

The photograph to the left shows the analytical system running on the Melville. Typical data from the instrument shows an increase in hydrogen peroxide near the surface and decreasing concentrations with depth.   This is what I would expect for a photochemical species, a compound formed by the interaction of sunlight with seawater. Interestingly, the maximum concentration of hydrogen peroxide is not at the surface, suggesting that another source of hydrogen peroxide may be present.   We know that some phytoplankton produce hydrogen peroxide.    Do my profiles support a biological source of HOOH?   If yes, why would phytoplankton produce HOOH at concentrations high enough to be detected in the surface ocean.  These are a few of the questions I hope to answer on the cruise.

Profile of HOOH in the surface ocean. Notice that oceanographers plot things backwards.

Check back for future blogs describing the other instruments on the ship and more oceanographic data.


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