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\begin{document}

\title{On-line chloride removal from ion chromatography for trace-level
analyses of phosphite and other anions by coupled IC-ICPMS}



\author[1]{Abu S. Baidya}%
\author[1]{Eva E. Stueeken}%
\affil[1]{University of St Andrews}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Rationale:} Ion chromatography combined with inductively-coupled
plasma mass spectrometry is an ideal tool for measuring low
concentrations of anionic species such as phosphite; however, the high
concentration of chloride and other anions in natural solutions may
negatively impact chromatographic separation and data quality.
\textbf{Method:} We developed an on-line mechanism of removing chloride
from the sample within the ion chromatograph, using an additional valve
and a separation column that transfers chloride to waste while phosphite
and most other anions are retained. We installed this system in a
coupled IC-ICPMS system (ICS6000 and Element 2 in medium resolution
mode) and determined linearity and detection limits. In addition, we
measured phosphorous species by NMR for comparison as an alternative
method for phosphite determination. \textbf{Results:} Chloride was fully
removed from the samples while phosphite was retained and could be
analysed by IC-ICPMS. Concentrations could be measured down to 0.003
\selectlanguage{greek}µ\selectlanguage{english}mol/L and possibly less with good linearity over the explored range (up
to 1.615 \selectlanguage{greek}µ\selectlanguage{english}mol/L; r \textsuperscript{2} = 0.999). In contrast, the
detection limit by NMR was 6.46 \selectlanguage{greek}µ\selectlanguage{english}mol/L. \textbf{Conclusions:} The
on-line removal mechanism works well for simplifying sample matrices. It
removes the need for costly pre-analytical sample treatment with OnGuard
columns. We confirm that IC-ICPMS is the most powerful technique for
quantifying phosphite in natural solutions. The new Cl-removal method
may also be applicable to analyses of other anions.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Ion chromatrography is a common tool for quantifying the concentration
of different species of the same element in natural and experimental
solutions. Examples include, but are not limited to, nitrate and
nitrite, bromide and bromate, chloride and perchlorate, as well as an
array of organic compounds. In recent years, with the discovery of
reduced phosphorus species in natural
settings\textsuperscript{{[}1-3{]}}, phosphate, phosphite and
hypophosphite have moved into the focus of anion
chromatography\textsuperscript{{[}2, 4-6{]}}. Phosphite has even been
detected in ancient sedimentary rocks and holds potential as an
important substrate for prebiotic chemistry and early
life\textsuperscript{{[}7, 8{]}}. Reconstructing its biogeochemical
history over geologic time and its distribution in modern environments
therefore promises to yield new insights into the evolution of Earth's
biosphere. Phosphite is thermodynamically unstable in water (i.e., it
forms in the stability field of H\textsubscript{2}), but it is
kinetically stable, because oxidation to phosphate is slow and primarily
catalysed by micro-organisms today\textsuperscript{{[}7-9{]}}. Phosphite
can be produced biologically\textsuperscript{{[}10{]}} or abiotically
under dry, hot conditions\textsuperscript{{[}8, 11{]}}, by
lightning\textsuperscript{{[}12-14{]}}, or from the dissolution of
meteoritic FeP-minerals\textsuperscript{{[}15{]}}. However, as these
pathways are relatively rare, phosphite concentrations can be low in
natural fluids (e.g., 0.15-3 \selectlanguage{greek}µ\selectlanguage{english}mol/L in waters from
Florida\textsuperscript{{[}1{]}} and up to 0.45 \selectlanguage{greek}µ\selectlanguage{english}mol/L in a eutrophic
lake in China\textsuperscript{{[}3{]}}, but less than 0.06 \selectlanguage{greek}µ\selectlanguage{english}mol/L in
some geothermal waters\textsuperscript{{[}2{]}} and often undetectable).
Concentrations at the lower end of this range pose analytical
challenges, because the phosphite anion peak is typically dwarfed by
those of other anions such as chloride, sulfate, and phosphate in
typical chromatographic setups. Saturation of the column and/or detector
by those other more abundant anions may impact element separation and
detection of phosphite\textsuperscript{{[}6{]}}.

To overcome this issue, Han et al.\textsuperscript{{[}6{]}} as well as
Ivey \& Foster\textsuperscript{{[}4{]}} implemented the use of OnGuard
cartridges that simplify the sample matrix, particularly by the removal
of chloride -- the most common anionic species in most natural systems.
This allowed injection of larger sample volumes (500-800 \selectlanguage{greek}µ\selectlanguage{english}L loop size)
to overcome the detection limit. By coupling the ion chromatograph (IC)
with an inductively-coupled plasma mass spectrometer (ICP-MS), Ivey \&
Foster\textsuperscript{{[}4{]}} were able to achieve detection limits
around 0.002 \selectlanguage{greek}µ\selectlanguage{english}mol/L for phosphite. However, the authors also noted that
OnGuard cartridges can impact the phosphate and phosphite content of the
samples, introducing additional uncertainty into the analytical yield.
Furthermore, OnGuard cartridges add extra costs and labor to the sample
preparation protocol. Alternatively, phosphite can be analysed by UV-VIS
spectrophotometry\textsuperscript{{[}16{]}}; however, this may suffer
from interferences with other ions, and the reported detection limit of
0.36 \selectlanguage{greek}µ\selectlanguage{english}mol/L\textsuperscript{{[}16{]}} is not as good as with the coupled
IC-ICPMS system. Lastly, phosphite measurements can be made by nuclear
magnetic resonance (NMR)\textsuperscript{{[}1{]}}, but this technique is
generally not optimized for trace quantities. A detection limit has to
our knowledge not yet been published, possibly because it is dependent
on numerous instrument parameters (discussed below).

Here we present a new approach of removing chloride from the sample
matrix on-line within the ion chromatograph by splitting the sample
stream after passage of the chloride fraction through an additional
clean-up column. By coupling this method with an Element 2 ICP-MS in
medium-resolution mode, this allows us to achieve detection limits
better than 0.003 \selectlanguage{greek}µ\selectlanguage{english}mol/kg with a small sample volume (37.5 \selectlanguage{greek}µ\selectlanguage{english}L loop size
on Valve 1, Fig. 1a). We further present data collected by NMR with a
detection limit of ca. 6.46 \selectlanguage{greek}µ\selectlanguage{english}mol/L, which highlights the value of the
IC-ICPMS setup for natural samples.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1. Reagents and equipment}

All sample preparation and IC-ICPMS analyses were carried out in the St
Andrews Isotope Geochemistry laboratory (StAIG). NMR analyses were
carried out in the School of Chemistry at the University of St Andrews.
Solutions containing chloride, nitrite, nitrate, sulfate, phosphite,
phosphate and in some cases hypophosphite and pyrophosphate were
prepared in LDPE bottles from pure reagents (NaCl, Sigma-Aldrich p/n
1.06404.0500; KNO\textsubscript{2}, Fisher Scientific p/n 11328016;
NaNO\textsubscript{3}, Fisher Scientific p/n 10696842;
MgSO\textsubscript{4}, Fisher Scientific p/n 11377658;
Na\textsubscript{2}HPO\textsubscript{2}, Fisher Scientific p/n
222791000; Na\textsubscript{2}HPO\textsubscript{3}.5H\textsubscript{2}O,
Fisher Scientific p/n 11994281; Na\textsubscript{2}HPO\textsubscript{4},
Acros Organics p/n 204855000;
K\textsubscript{4}P\textsubscript{2}O\textsubscript{7}, Fisher
Scientific p/n 10378860) dissolved in 18.2
M\selectlanguage{greek}Ω\selectlanguage{english}[?]\selectlanguage{english}cm\textsuperscript{-1}deionized water, which was generated with a
Smart2Pure system. During the analysis, a 1 mmol/L NaOH solution was
used, which was prepared each day by dilution of a concentrated stock
solution (500 g/L, carbonate-free NaOH, VWR p/n 87938.290). This stock
bottle was stored and handled with minimal agitation to avoid mixing
with atmospheric CO\textsubscript{2} gas that may lead to elevated
carbonate concentrations. An aliquot of 0.16 ml was pipetted into 2 L of
DI-water, and the pipette was dipped as deeply into the stock bottle as
possible to avoid carbonate-enriched solution from the upper layer
closest to the lid. The bottle with the diluted 1 mmol/L NaOH solution
was only shaken up after purging with N\textsubscript{2} to remove air.
The headspace was then pressurized with N\textsubscript{2} to further
avoid ingrowth of atmospheric CO\textsubscript{2} during the analysis.
For the NMR analysis, 10-20 \% heavy water (D\textsubscript{2}O) was
used (Sigma-Aldrich p/n 151882-100G) to prepare a 0.6 ml sample
solution.

The ion chromatograph used in this study was a Dionex ICS-6000 from
Thermo Fisher, equipped with an AS-AP autosampler, a 37.5 \selectlanguage{greek}µ\selectlanguage{english}L sample
loop, a gradient pump, an eluent generator with an RFIC degasser, a
CR-ATC 600, an EGC 500 KOH cartridge, an AG17-C guard column, an AS17-C
analytical column, an ADRS 600 2mm suppressor, and a conductivity
detector (Thermo Scientific p/n 061830). It was run with a constant flow
rate of 0.5 ml/min. KOH was used as an eluent, and its concentration was
ramped from 1 mmol/L to 40 mmol/L over the course of each run. Analyses
with pyrophosphate were set to last for 55 minutes, and here the KOH was
ramped up between 5.5-23 minutes. After 45 minutes of total run time,
the KOH concentration was decreased back to 1 mmol/L over a duration of
5 minutes. Analyses of solutions without pyrophosphate were set to last
for 27 minutes, the KOH was ramped up between 5.5-23 minutes and ramped
back down between 23-26 minutes. Containers used for samples and
standards were soaked in hot 2M HCl overnight and rinsed several times
with deionized water prior to use.

The ICP-MS was an Element 2 from Thermo Fisher. It was equipped with a
Scott quartz spray chamber and a quartz nebulizer rated for a solution
flow rate of 1 ml/min. Argon gas flow rates were 16 L/min for the cool
gas flow, 0.8 L/min for the auxiliary flow, and 1 L/min for the sample
carrier flow. The RF power was set to 1250 W. Prior to the start of the
run, the ICP-MS was tuned with a multi-element solution at a
concentration of 1 ppb in 5\% HNO\textsubscript{3} (Thermo Scientific
p/n 1099601). The instrument was operated in medium resolution mode
(measured resolution was ca. 4200 \selectlanguage{greek}Δ\selectlanguage{english}m/m) to avoid HNO interferences with
phosphorus at m/z 31. Oxide \% measured determined from UO/O was ca.
3-5\%.

The NMR used in this study was a Bruker AVIII 500 MHz NMR instrument
equipped with nitrogen cooled broadband cryoprobe. It was operated in
proton-decoupled mode with 3000-7000 scans per analysis.

\textbf{2.2. Chloride removal set-up}

In the standard IC set-up, the sample is transferred from the
autosampler to a loop that is attached to a Valco-style (hereafter Valve
1). Initially, Valve 1 remains in load position until sample transfer is
complete. Then it switches to inject position, and KOH that comes from
the eluent generator pushes the sample from the sample loop to the guard
and separator column (Fig. 1a). For on-line chloride removal, the IC was
modified with the installation of an additional valve (hereafter Valve
2, supplied by Sunquest Scientific). This was placed between the sample
loop on Valve 1 and the guard column (Fig. 1c, d). The software
(Chromeleon) was modified such that Valve 2 remained in load position
for an additional 2.5 minutes after Valve 1 had switched from `load' to
`inject' (Fig. 1). During this time, a 1 mmol/L NaOH solution at a flow
rate of 1 ml/min was used to push the sample from the loop on Valve 1 to
the clean-up column installed on Valve 2. This additional NaOH solution
was supplied by an external pump (Dionex GP50 Gradient Pump).
Alternatively, an N\textsubscript{2}-pressurized reservoir may be used.
The clean-up column on Valve 2 was a AG11-HC 4x50mm column, which
separates anions contained in the solution similar to the guard and
analytical columns. Chloride eluted first and was allowed to pass into
the waste (Fig. 1c, d). Valve 2 was switched from `load' to `inject'
after chloride had passed but prior to the elution of other anions from
the clean-up column. The timing was calibrated manually at the start of
the installation. After 2.5 minutes, Valve 2 switched automatically to
`inject', and the KOH eluent pushed the sample out of the clean-up
column onto the guard column.

When the instrument was not in chloride-removal mode, Valve 2 was set to
stand-by (Fig. 1b). The clean-up column was flushed with NaOH and kept
moist with DI-water supplied from the external gradient pump. This
solution was sent to waste and did therefore not interfere with analyses
carried out in standard mode.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\emph{\textbf{Figure 1: Schematic of the change-over valves in the ion
chromatograph.} Valve 1 is the part of the standard setup; Valve 2 and
the associated small clean-up column were added for chloride removal.
(a) Valve 1 during normal operation without the chloride removal step.
The tube going to the column, suppressor, and conductivity detector can
optionally also extend further to transfer the sample into the ICP-MS.
(b) Valve 2 in stand-by mode when chloride removal is not needed. Here
the small column is constantly flushed with water to keep it hydrated.
(c) Valve 1 in chloride removal mode. NaOH is introduced by an
additional pump. (d) Valve 2 in chloride removal mode, receiving
solution from Valve 1, when Valve 1 is in the `inject' position. Valve 2
is in the `load' position while receiving sample from Valve 1 and
switches to `inject' after 2.5 minutes.}

\textbf{2.3. Coupling of IC to ICP-MS}

The IC was connected to the ICP-MS with black PEEK tubing. This was spun
between the outlet of the conductivity detector on the IC and the inlet
of the nebulizer on the ICP-MS. When the instruments were connected in
this way, the suppressor on the IC was regenerated with an external
water supply at a flow rate of ca. 0.5-1 ml/min, delivered from an
N\textsubscript{2}-pressurized container. (When the IC is not connected
to the ICP-MS, the suppressor is regenerated with the waste solution
from the detector). The ICP-MS method for this analysis was set up with
750 runs and 1 pass, equivalent to 3 minutes per analysis. In the
sequence file, the chromatographic output mode was selected for each
sample. While it is possible to run the ICP-MS in automatic mode where a
new analysis is externally triggered via a relay cable between the IC
and the ICP-MS, we decided against this option, because it would imply
that the slit plate used for medium resolution in the ICP-MS would be
corroded quickly over the course of several analyses that last up to 55
minutes each. Instead, we manually started the analysis on the ICP-MS
ca. 1 minute before the expected arrival of the phosphite peak at the
conductivity detector on the IC. The peak itself lasted for ca. 40
seconds. The ICP-MS method was allowed to run for 3 minutes, providing
sufficient data before and after the phosphite peak to determine
analytical background levels.

The final data were accessed via the Show program under Chromatogram
\textgreater{} From Info File \textgreater{} Display Chromatogram,
followed by Display \textgreater{} Data View and File \textgreater{}
Export (select ASCII format). This dataset could be opened in Excel or
Origin (Origin Lab) for further processing. Here, we used Origin for
smoothing the data (using the fast furrier transform filter with a
points of window value of 15), subtract background levels, and calculate
the area under the peak as a metric for signal intensity. We also
calculated the peak height, but the calibration curve was found to be
less scattered when peak area was used instead. The method was tested
with a series of standards containing 0.003 to 1.614 \selectlanguage{greek}µ\selectlanguage{english}mol/L phosphite
(corresponding to 0.1 to 50 ppb P).

\textbf{2.4. Nuclear Magnetic Resonance}

For the NMR, solutions were mixed with 10-20\% D\textsubscript{2}O to
make a total volume of 0.6 ml. The solutions were analyzed in
proton-decoupled mode with 3000-7000 scan. Typical run time for a 7000
scan was around 4 hours. The \textsuperscript{31}P chemical shifts are
referenced to phosphoric acid having a chemical shift of 0\selectlanguage{greek}δ. \selectlanguage{english}Standards
of known concentration (0.026 \selectlanguage{greek}µ\selectlanguage{english}mol/L (8 ppb) to 1614 \selectlanguage{greek}µ\selectlanguage{english}mol/L (50 ppm) P
were analysed to find the detection limit and building calibration
curves. After acquiring the data, phase correction, background
correction, peak identification, and area integral calculation were
executed in MestReNova software. Area integrals of the peaks were used
for building the calibration curve.

\textbf{3. RESULTS AND DISCUSSION}

\textbf{3.1. Removal of chloride in ion chromatograph}

The chromatograms of the IC in chloride-removal mode (Fig. 1c, d)
revealed complete removal of chloride while phosphite and other ions
contained in the solution were retained (Fig. 2b). The comparison to the
normal mode (Fig. 2a) shows that also nitrite was removed by this method
as it elutes close to chloride. However, nitrate, phosphite, sulfate and
phosphate were retained. Chloride removal was incomplete when the delay
on Valve 2 (i.e., the time before Valve 2 switches form `load' to
`inject' mode) was less than 2 minutes. It may be possible to also cut
off anions that elute later than phosphite (such as sulfate or
phosphate) by switching Valve 2 back to `load' as soon as phosphite has
been pushed from the clean-up column to the guard column. This was not
explored in this study.

The chromatograms with the Cl-removal method display a larger carbonate
peak than those generated in normal mode (Fig. 2) despite the use of
carbonate-free NaOH. We suspect that some carbonate was already
dissolved in the samples and standards, derived from atmospheric
CO\textsubscript{2}. Additional CO\textsubscript{2} may have dissolved
in the NaOH solution during preparation. Care was taken to minimize
ingrowth of atmospheric CO\textsubscript{2} (Section 2.1), but we could
not guarantee complete avoidance. The retention time of carbonate is
only slightly longer than that of phosphite, and therefore the carbonate
peak may reduce confidence in the detection and quantification of
phosphite with the conductivity detector on the IC. However, this
problem is resolved by IC-ICPMS coupling, as the ICP-MS measures
phosphite by mass and is therefore not impacted by the presence of
carbonate ions.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\emph{\textbf{Figure 2:} \textbf{IC traces with and without chloride
removal set-up.} A. Peaks of a 1 ppm solution (all species are at the
same concentration) in normal set-up. B. Peaks of the same solution in
Cl-removal set-up. The latter does not contain chloride or nitrite but
displays a slightly larger carbonate peak. The shift in retention time
for all ions in panel B compared to panel A is expected, because the
Cl-removal setup increases the path length for the solution as it
travels from the autosampler to the detector. Both panels show an
increase in the baseline over the course of the run which is due to the
ramp-up of the KOH concentration.}

\textbf{3.2. Calibration and detection limits of IC and IC-ICPMS}

We generated calibration curves with the IC in standard mode (without
chloride removal), and with the coupled IC-ICPMS setup with chloride
removal, using both the conductivity detector in the IC and the SEM
detector in the ICP-MS. In all cases, the data show good linearity over
the concentration ranges that were tested, with correlation coefficients
(r\textsuperscript{2}) better than 0.999 (Fig. 3, 4). As shown in Fig.
3, the performance of the conductivity detector in the IC is not
negatively impacted by the presence of the Cl-removal setup. Good
linearity was obtained with and without chloride removal, as illustrated
here with phosphite and nitrate. This observation demonstrates that the
Cl-removal may also be useful for analyses of ions such as nitrate that
cannot be analysed by ICP-MS. Furthermore, it gives confidence that the
clean-up column does not introduce random error performs as expected.

Regarding detection limits, the coupling of the IC to the ICP-MS
resulted in significant improvement, as expected. In standard mode
without chloride removal (Fig. 3a), we were able to confidently quantify
phosphite peaks down to ca. 0.32 \selectlanguage{greek}µ\selectlanguage{english}mol/L (10 ppb P) using the
conductivity detector alone. With the IC-ICPMS setup and the SEM
detector in the ICP-MS, a resolvable peak was obtained for as little as
0.003 \selectlanguage{greek}µ\selectlanguage{english}mol/L (0.1 ppb P, Fig. 4a). Slightly lower concentrations may
still be detectable. Our results are thus similar to the detection limit
of 0.002 \selectlanguage{greek}µ\selectlanguage{english}mol/L reported by Ivey \& Foster
(2005)\textsuperscript{{[}4{]}}, despite a significantly smaller sample
loop (37 \selectlanguage{greek}µ\selectlanguage{english}L instead of 800 \selectlanguage{greek}µ\selectlanguage{english}L). Reproducibility in our IC-ICPMS setup
was 13\% at 0.032 \selectlanguage{greek}µ\selectlanguage{english}mol/L.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\emph{\textbf{Figure 3: Calibration curves for phosphite and nitrate in
normal and Cl-removal set-ups with the IC conductivity detector.} A and
B: Calibration for phosphite and nitrate in normal set-up. C and D:
Calibration for phosphite and nitrate in Cl-removal set-up. Correlation
coefficients (r\textsuperscript{2}) values are \textgreater{} 0.999 for
all cases suggesting no disruption of other peaks by Cl removal.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\selectlanguage{greek}\emph{\textbf{Figure 4: IC-ICPMS traces and calibration curve for
phosphite}. A: Chromatogram of a blank and of phosphite-bearing
solutions with concentrations from 0.1 to 10 ppb (0.003 to 0.32 µmol/L).
Red lines are smoothened curves obtained by FFT filtering with a points
of window value of 15. The 0.003 µmol/L (0.1 ppb) solution has a
detectable phosphite peak as compared to the blank. B: Phosphite
calibration curve with the peak integral data.}\selectlanguage{english}

\textbf{3.3. Comparison of IC-ICPMS to alternative methods}

Previous workers also used NMR to quantify phosphite concentrations in
solutions\textsuperscript{{[}1{]}}. Our own measurements reveal a
detection limit around 6.46 \selectlanguage{greek}µ\selectlanguage{english}mol/L (200 ppb), which is substantially
higher than what is achievable by IC or IC-ICPMS. To first order, this
method therefore appears less suitable for phosphite-lean samples than
IC, IC-ICPMS and even UV-VIS spectrophotometry (Table 1). However, we
note that in NMR, the detection limit depends on the strength of the
magnetic field (i.e., resonance frequency), the nature of the probe, and
the number of scans (which in turn determine the length of the run per
sample). In our case, the strength of the magnetic field was 500 MHz.
The detection limit could be comparatively lower (\textless{} 200 ppb)
in an instrument with a stronger magnetic field (e.g., 700 mHz) and
higher (\textgreater{} 200 ppb), if the magnetic field is weaker (e.g.,
400 mHz). The NMR used in this study is equipped with a
liquid-nitrogen-cooled broadband cryoprobe. If an uncooled probe were
used, it is estimated that the detection limit could be roughly 2.5
times higher (nearly 500 ppb) if all other parameters remain the same.
Finally, we performed 7000 scans for phosphite analysis, which equated
to 4 hours of run time per sample, but it is possible to detect even
lower concentrations if the samples are analyzed with a higher number of
scans and accordingly longer runs.

A major advantage of NMR is its ability to detect a much wider range of
phosphorus species, including various
polyphosphates\textsuperscript{{[}17{]}}. These are relatively large
molecules that would likely be difficult to elute from the separator
column of an IC. And to our knowledge, no UV-VIS method has so far been
developed to measure polyphosphates other than pyrophosphate in
solution. Hence NMR, despite its limitations in the detection limit,
analytical time and installation costs (Table 1), is perhaps the best
method for measuring polyphosphate species. It is also unaffected by the
presence of chloride or other interferences.

UV-VIS spectrophotometry is probably the most cost-effective method for
phosphite\textsuperscript{{[}16{]}} as well as
phosphate\textsuperscript{{[}18{]}} measurements, and it too can be
conducted in the presence of high chloride
concentrations\textsuperscript{{[}16{]}}. However, other interferences
may persist, and its detection limit is significantly higher than with
the IC and ICP-MS, making it unsuitable for many environmental samples.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\selectlanguage{greek}\emph{\textbf{Figure 5: Selected \textsuperscript{31}P NMR patterns and
calibration curve for phosphite with NMR data.} A: NMR patterns for 4
different standards (8, 100, 250, and 500 ppb) containing 4 different
species (hypophosphite, phosphite, phosphate, pyrophosphate). 8 and 100
ppb standards (0.258 and 3.229 µM/L) are not detected in the present
analytical conditions. B: Calibration curve for phosphite with peak
integral data with a R\textsuperscript{2} value nearly 1.}\selectlanguage{english}

\emph{\textbf{Table 1: Method comparison.} The detection limit is given
in nanogram phosphorus per gram of solution.}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Method}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{Detection limit {[}µmol/L{]}}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Advantages}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Disadvantages}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{References}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
IC\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
0.002-0.32 (Dependent on size of the sample loop and column type;
detection limit may be impacted by the presence of carbonate eluting
close to phosphite)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Relatively cost-effective; separation from most interferences\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Low detection limit requires large sample volume, possibly causing
saturation by other ions; possibly interferences by ions with similar
retention times; can analyse multiple P species but not all\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
This study; {[}6{]}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
IC + ICP-MS\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
0.002-0.003 (Dependent on the size of the sample loop)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Low detection limits achievable with small sample sizes; separation of
interferents with similar retention times in the IC\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
High installation costs; can analyse multiple P species but not
all\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
This study; {[}4, 6{]}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
UV-VIS spectrophotometry\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.36\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Most cost effective; separation from many interferences; fast\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
High detection limit; some interferences may persist; can analyse
multiple P species but not all\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
{[}16{]}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
NMR\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
6.46 (Dependent on length of run or number of scans, strength of the
magnet, type of probe)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Can analyse greatest variety of P species; separation from
interferences\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
High installation costs; slow analysis; high detection limit\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{4. CONCLUSION}

Our results show that on-line removal of chloride with the clean-up
column and split valve (Valve 2 in Fig. 1d) in the ion chromatograph is
a viable method for simplifying the matrix of natural and experimental
solutions for phosphite analysis. For an element such as phosphite, this
tool can be combined with coupling of the IC to an ICP-MS to achieve
detection limits below 0.003 \selectlanguage{greek}µ\selectlanguage{english}mol/L (\textless{} 0.1 ppb P), in line
with previous studies\textsuperscript{{[}4, 6{]}} but without the need
for a large sample loop or pre-analytical sample treatment with OnGuard
cartridges.

Without the ICP-MS, the removal of chloride also simplifies analyses
with the conductivity detector of the IC alone, except for ions that
elute close to chloride (as those may be difficult to separate from
chloride) or those close to carbonate. The latter may be elevated by the
introduction of external NaOH. The carbonate problem may be mitigated if
a degasser is installed in-line with the NaOH supply, but we did not
explore that in this study. However, even without carbonate-removal from
the NaOH solution, we would expect that ions such as nitrate, sulfate or
phosphate, which typically have much shorter or much longer retention
times than carbonate with the AS17-C analytical column, would be easier
to quantify at low concentrations after on-line removal of chloride with
the setup described in this study. Our results show good linearity in
the conductivity detector both with and without the Cl-removal setup. In
addition, it may be possible to further modify the timing in the
software such that additional ions can be cut out from the sample. Our
study therefore presents a new approach for optimizing ion
chromatography and taking full advantage of the low detection limits of
ICP-MS.

We conclude that IC-ICPMS coupling with on-line chloride removal
provides perhaps the best way forward for phosphite analyses at low
concentrations, because detection limits are significantly better
compared to NMR and UV-VIS spectrophotometry. NMR holds the major
advantage that it can detect a more diverse range of phosphorus species,
including large polyphosphate ions, while UV-VIS spectrophotometry is
the most cost-effective method for phosphite analysis, but neither of
the two methods is able to achieve similar detection limits. The
IC-ICPMS approach may therefore be ideally suited for unlocking
phosphorus redox chemistry in the environment.

\textbf{ACKNOWLEDGEMENTS}

This work was financially supported by a NERC Frontiers grant to EES
(NE/V010824/1). We thank Lloyd Billingham from Sunquest Scientific for
support with the design and installation of the chloride-removal system
on the IC. Nathan Rochelle-Bates and Tomas Lebl are thanked for
technical support with the ICP-MS and NMR, respectively.

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