Applications of in situ HPLC

The first in-situ HPLC was developed under my dissertation research through collaboration between Georgia Tech and Analytical Instrument Systems, Inc. for the purpose of in situ measurement of marine and sediment interstitial waters (Beckler, 2014; Beckler et al., 2014).  Because traditional anion chromatography requires complex instrumentation, it is not very suitable for deployment in situ.  Therefore, we developed an HPLC technique using UV detection that has been employed by the in situ chromatograph configured both "sipping" from a sediment incubation chamber on a benthic lander to measure sediment fluxes, and also from a porewater profiling probe that is affixed to a micromanipulator to collect discrete sediment porewater samples as a function of depth alongside Au/Hg voltammetric microelectrodes.

The in situ HPLC operated on the benchtop, sipping interstitial samples from the sediment core and measuring concentrations of major marine anions.

Figure 1a) An overlay of chromatograms obtained using the Analytical Instrument Systems ISEA-IV-LC when sipping pore waters from sediments using the sample sipping probe; b) A corresponding sediment profile from a station 9 km upstream (salinity 0.63) from the mouth of the Altamaha River demonstrate the AIS ISEA-IV-LC can resolve all discrete redox layers  (Aerobic respiration, denitrification, Mn and Fe reduction) and salinity features (Bromide) using a combination of in situ voltammetry and HPLC. 

Brevetoxin production is increased under low nutrient regimes and in the presence of competing organisms, and toxins may persist long after blooms have dissipated. On the other hand, toxins are not always present when cells are present (Abraham et al., 2006; Twiner et al., 2007).  Public health decisions such as closing a shellfishery are often made at a threshold cell concentration of 5,000 cell/L, even though brevetoxin may be below detection. A need exists to measure brevetoxin concentration routinely, rapidly, and autonomously, to provide economical data for management, and to enhance our understanding of the coupling between K. brevis blooms and toxin production. We have begun work at MML to modify the in situ High Performance Liquid Chromatograph (HPLC) for brevetoxin measurement. Mote is currently funded by the State of Florida to develop an analytical method for brevetoxin detection appropriate for use on a similar in situ system. Briefly, HPLC will be used to physically separate the molecules of interest from the bulk of seawater constituents, and UV absorbance measurements used to quantify the separated molecules.  Brevetoxins are normally measured using LC/MS, allowing for both the analytes retention time on the LC column and its m/z ratio to be used for identification (Hua et al., 1995).  Instead, the new rapid screening method will use HPLC retention time and UV/VIS spectral fingerprinting (similar to how the Optical Phytoplankton Discriminator can identify phytoplankton) to identify and quantify brevetoxins. The instrument will autonomously collect water samples, filter, preconcentrate, quantify, and transmit data periodically throughout the deployment. Deployment at a fixed location in tandem with an OPD is scheduled in less than two years, and continuous brevetoxin and K. brevis cell concentration reporting to SO-COOL (coolcloud.mote.org) will become an additional feature of our HAB observatory.