NBS
EFft
              U S Department
              of Commerce
             National
             Bureau of
             Standards
             Office of Environmental
             Measurements
             Washington. DC 20234
United States
Environmental Protection
Agency
Office of Environmental Engineering and
Technology
Washington DC 20460
EPA-600 7-79-211
November 1979
            Research and Development
             Assessing the
             Environmental
             Impact of Fossil-
             Fuel Based Energy
             Sources

             Interagency
             Energy/Environment
             R&D Program
             Report

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                RESEARCH  REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.  Environmental Health Effects Research
      2.  Environmental Protection Technology
      3.  Ecological Research
      4.  Environmental Monitoring
      5.  Socioeconomic  Environmental Studies
      6.  Scientific and Technical Assessment Reports (STAR)
      7.  Interagency  Energy-Environment Research and Development
      8.  "Special" Reports
      9.  Miscellaneous Reports

 This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
 RESEARCH AND DEVELOPMENT series. Reports in this series result from the
 effort funded under the 17-agency Federal  Energy/Environment Research and
 Development Program. These studies relate to EPA's mission to protect the public
 health and welfare from adverse  effects of pollutants associated with energy sys-
 tems. The goal of the  Program is to assure  the rapid development of domestic
 energy supplies in an  environmentally-compatible manner by providing the nec-
 essary environmental  data and control technology. Investigations include analy-
 ses of the transport of energy-related pollutants and their health and ecological
 effects;  assessments  of,  and development of, control  technologies for energy
 systems; and integrated assessments of a wide range of energy-related environ-
 mental issues.
  This document is available to the public through the National Technical Informa-
  tion Service, Springfield, Virginia 22161.

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        ASSESSING THE ENVIRONMENTAL IMPACT OF
          FOSSIL-FUEL BASED ENERGY SOURCES:
         MEASUREMENT OF ORGANOMETAL SPECIES
        IN BIOLOGICAL AND WATER SAMPLES USING
LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION
                         by
      William A.  MacCrehan and Richard A.  Durst
           Center for Analytical Chemistry
            National Bureau of Standards
                Washington, DC  20234
      Interagency Agreement No.  EPA-IAG-D5-E684
          EPA Project Officer:   J.  Stemmle
           Environmental Protection Agency
                Washington,  DC 20460
              This study was conducted
               as part of the Federal
           Interagency Energy/Environment
          Research and Development Program
                    Prepared for
      OFFICE OF ENERGY,  MINERALS,  AND INDUSTRY
         OFFICE OF RESEARCH AND DEVELOPMENT
        U.  S.  ENVIRONMENTAL PROTECTION AGENCY
                WASHINGTON, DC  20460

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                              DISCLAIMER
     This report has been prepared and reviewed by the Center for Analytical
Chemistry and the Office of Environmental Measurements, National  Bureau
of Standards, and reviewed by the U.  S.  Environmental  Protection  Agency,
and approved for publication.  Approval  does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency.  In order to adequately describe materials and experimental
procedures, it was occasionally necessary to identify commercial  products
by manufacturer's name or label.  In no instance does such identification
imply endorsement by the National Bureau of Standards or the U. S.
Environmental Protection Agency nor does it imply that the particular
products or equipment is necessarily the best available for that purpose.
                                  ii

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                          FOREWORD
The role of the National Bureau of Standards (NBS) in the Interagency
Energy/Environment R&D program, coordinated by the Office of Research
and Development, U. S. Environmental Protection Agency, is to provide
those services necessary to assure data quality in measurements being
made by a wide variety of Federal, state, local, and private industry
participants in the entire program.  The work at NBS is under the
direction of the Office of Environmental Measurements and is conducted
in the Center for Analytical Chemistry, the Center for Radiation Research
and the Center for Thermodynamics and Molecular Science.  NBS activities
are in the Characterization, Measurement, and Monitoring Program category
and addresss data quality assurance needs in the areas of air and water
measurement methods, standards, and instrumentation.  NBS outputs in
support of this program consist of the development and description of
new or improved methods of measurement, studies of the feasibility of
production of Standard Reference Materials for the calibration of both
field and laboratory instruments, and the development of data on the
physical and chemical properties of materials of environmental importance
in energy production.  This report is one of the Interagency Energy/
Environment Research and Development Series reports prepared to provide
detailed information on an NBS measurement method or standard development.
C. C. Gravatt, Chief
Office of Environmental Measurements
National Bureau of Standards
                                   iii

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                                   ABSTRACT

     A new measurement approach for the determination of trace organometals
in energy related environmental samples is described.  The method is
based on liquid chromatographic separation with electrochemical detection.
A detailed description of the development of the electrochemical detection
system, optimized for reducible analytes, is given.  The conditions for
the separation of methyl-, ethyl-, and phenylmercury in under 16 minutes
by charge-neutralization reverse-phase chromatography are developed.
Also, a separation of a number of organoleads, including trimethyl- and
triethyllead, are described.  The potential interferences in this type
of organometal determination are investigated.  Sample preparation
methods and improvement in the detector  selectivity  are described that
overcome these interferences.  A significant improvement in the selectivity
of the detection system has been investigated using  a differential  pulse
waveform.

      Sample preparation methods for  the  determination of methylmercury
in biological  tissue are  examined  and  measurements are made on  two
research materials:   lyophilized  tuna  and shark meat.  A  column preconcentration
procedure  for  methyl- and ethylmercury in natural  water samples  is
developed.

      This  report was submitted in partial fulfillment  of  Contract  EPA-
IGA-D5—E684  by the National Bureau of  Standards  under  the sponsorship of
 the  U.S.  Environmental Protection Agency.  This  report  covers work
performed during the period of May 1977 to June  1978.
Keywords:   Organometal  speciation,  electrochemical detection  in  liquid
chromatography,  methylmercury,  trimethyllead,  column preconcentration,
tissue  preparation.
                                    iv

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                               CONTENTS

Forward	 .11 i
Abstract	  , . . .iv
List of Illustrations 	vi
List of Tables	vii
     1 .   Introduction ..............................................  1
     2 .   Summary and Conclusions ....................................  2
     3 .   Organometal Chemistry ......................................  3
     4 .   Analytical Measurement of Organometals .....................  5
     5 .   Experimental Procedures ....................................  9

          Development of LCEC .......................................  9
          Electroactivity of Organometals ........................... 12
          LCEC System for Reductions ................................ ] 4
          Application of LCEC to Real Samples .................... , . . 44

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                         LIST OF ILLUSTRATIONS

Number                                                                     Page
  1    Thin layer electrochemical flow cell	18
  2    Amperometric detection in hydrodynamic voltammetry 	 22
  3    Background residual current as a function of applied potential .  .  . £4
  4    Optimization of detector response for (CH~)-Pb   	25
  5    Limit of detection for (CH3)~Pb  in the amperotnetrie mode of
          detection	26
  6    Calibration curve for CH~Hg  in the amperometric mode of detection . 28
  7    Elimination of the metal ion interference as organomercury
          measurement by improvements in the chromatography 	 29
  8    Differential pulse detection in hydrodynamic voltammetry 	 32
  9    Relationship between signal-to-noise ratio and pulse height
          in differential pulse detection 	 33
 10    Differential pulse current response as a function of applied
          potential	34
 11    Limit of detection for CH-Hg  in the differential pulse mode
          of detection	35
 12    Elimination of metal ion interference by increasing the
          selectivity of the detector	37
 13    Calibration curve for CH-Hg  in differential pulse mode of
          detection	38
 14    Separation of organomercury cations by charge-neutralization
          reverse-phase chromatography  	 41
 15    Separation of selected organometals  	 42
 16    Separation of organolead cations 	 43
 17    Block diagram of organomercury preconcentration  .... 	 46
 18    Determination of me thy liner cury in shark paste	c^
                                   vi

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Table 1.


Table 2.

Table 3.

Table 4.


Table 5.

Table 6.

Table 7.
                                 LIST OF TABLES
Major detection approaches for organometal analysis by gas
chromatography 	
Electrochemical detection modes for liquid chromatography	10

Electrode materials used in LCEC	• 11

Classes of compounds that would be readily amenable to electro-
chemical detection	"12

Reduction potentials for some organometal cations	"J3

Comparison of electrode materials for reductive potential range	15
Comparison of several mercaptans as complexing ligands
for the reverse-phase chromatography of methylmercury. .
                                                                              •39
Table 8.  Result of the methylmercury determination in the two fish samples... 51
                                       vii

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                                1.   INTRODUCTION
     Mounting concern over the presence and role of toxic substances in
the environment and living systems resulting from energy generation has
created the need for selective and sensitive measurement techniques.
Methodology now exists for the analysis of heavy metals (an important class of
pollutants) in a variety of matrices, and considerable effort is being devoted
to the collection of data on the levels of these elements in biota and water
samples.  However, some toxic elements can be transformed, by biological and
chemical processes, into organometals.  Organometallic species have different
properties from their inorganic counterparts.  Thus, in order to fully understand
the role of these toxic materials, it is necessary to measure the exact chemical
form of the element in the sample.  This research project describes the development
of a new analytical technique for the speciation of organometals from fossil-
fuel based energy sources.

     The measurement technique is based on a separation of several organometal
species by liquid chromatography with detection by reductive voltammetry.
Primarily, the work described here covers the optimization of the choice of
working electrode materials, solvent purification, and applied waveforms.
Separations of several organotnercury and organolead species are also described.

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                      2.  SUMMARY AND CONCLUSION
     This report describes the development of a new hybrid detection approach
for the measurement of organometal cations, liquid chromatography combined
with reductive electrochemical detection (referred to as LCEC).   Considerations
unique to reductive LCEC are discussed in detail including the choice of
working electrode material (gold amalgamated with mercury was selected),
purification of the solvent (deoxygenation and cathodic electrolysis),
and system construction (dampening of flow pulsations and the need for an
inert atmosphere).  The waveform applied to the detector cell affects the
sensitivity and selectivity.  Differential pulse detection has been found to
provide the best selectivity while still providing detection limits in the
low ppb (yg/g) range for several organometals.

     The separation of organomercury cations was accomplished by reverse-
phase chromatography with the addition of a strong complexing agent.
Organolead cations form only weak complexes, but can be separated directly
on a CT 0 column.
      lo
     The reduction potentials of many organotin, lead and mercury cations, in
a chromatographically compatable solvent, are reported.

     Methods have been developed for water sample preconcentration and
biological tissue preparation for the measurement of organomercury species
by LCEC.  Methylmercury has been determined in two reference materials.

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                          3.  ORGANOMETAL CHEMISTRY
     Organometals are becoming recognized as an important class  of
compounds in many phases of chemistry.   Their unique properties  make
them important in industrial, agricultural,  biochemical,  and environmental
systems.  The widespread influence of organometals has created the  need
for selective and sensitive analytical measurement techniques, which  has
provided the impetus for this research project.
Inorganic Chemistry of Organometals
     Organometallic compounds are those with direct carbon-to-metal (or
metalloid) bonds.  These many be pi interactions between the metal and
alkenes, alkynes, or arenes; or the bonds may be sigma as the alkyl
organometals.  Only the sigma bond organometals will be considered here
as these are the species most investigated for their biological interactions
(1).
     There are a number of metal and metalloid elements that form waterstable
organometallic species including Hg, Pt,  Au, Tl, Pb,  Sn, Pd, As, Sb, Co, Cr,
Te, and Se.  However, from an environmental standpoint the compounds of
Hg, Pb, Sn, As, and Se are the most important.


     The chemical behavior of organometallic compounds is dependent on the
central metal element and on the degree of coordination and character of
the hydrocarbon groups.  Completely alkylated metals  are usually volatile
liquids at room temperature.  The partially, organically coordinated species
of Pb, Hg, Sn, and Tl can form dissociated cations in aqueous solution (2).
This contrasts the similar compounds of As, Sb, and Se that do not form
free cations when dissolved in water.  Rather, the organometal-anion bond
shows mostly covalent character.

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     Organometals are generally organophilic  in behavior.   This is especially
true as the organic coordination increases and as the R group length is
increased.   The formation of strong neutral complexes of the cationic
organometals also enhances the organophilicity.

     It is possible to have interreaction between an organometal and a
metal ion with the transfer of one or more organic groups.  For example
trimethyltin cation will methylate mercury (II) ion (3):

             (CH3) 3Sn+ + Kg** + (CH3) 2Sn"H" -I- CH3Hg+

This type  of reaction is termed "transalkylation".

     Most  organometal species  are  subject  to hydrolysis in  strong acid  (4) but
are stable to  dilute acid  and  alkaline hydrolysis in  some cases  (5).   Some of
the organometals are subject  to photolysis with intense U.V.  light  (3).

     It  is important to  take  these characteristics  into account  when
developing an  analytical procedure for organometal  analysis.

Environmental  Importance

      Organometallic compounds have been  known to have toxic effects on
 living organisms since the 1800's (1).   The organometal compounds of arsenic
 and mercury were found to have biocidal  effects and were  therefore used in
 the treatment of disease.  But only recently, it has been discovered that a
 number of heavy metal (and also metalloid) elements could be transformed in
 living systems into organometallic compounds.  Early studies on these
 conversions have shown that certain bacteria and fungi can methylate inorganic
 metal ions (1).  Later it was found that  this  "biological methylation" can
 also occur in the presence of suitable coenzymes, namely vitamin 812 and S-
 adenosylmethionine (6).

      These "biomethylation" reactions are important  from two major  standpoints,
 First,  the methyl products are generally  much  more toxic than their inorganic
 counterparts  (1).  .For  example, CHaHg  is often quoted as  being  100 times
 more  toxic than Hg  .   The enhanced toxicity may be  related  to  their  enhanced
 lipophilicity and  the associated  increase in  the residence time  in  the
 tissues.   The toxicity  is further complicated  by the tendency of organometals
 to be "bioaccumulated"  throughout the food  chain of  a  living ecosystem.
 For example,  in a  marine  environment, the lower forms  of  life such  as
 plankton  accumulate mercury  to over 1,000 fold that  found  in the surrounding
 seawater  (7).   Much of  the mercury is retained as  life progresses  up  the
 food  chain.   Since humans are often at  the  top of  the food chain,  the
 levels  of organometals  are very important.   For example,  the outbreak of
 "Minimata disease" in Japan  was a result of high methylmercury  levels in
  fish  (caught  in mercury polluted water).

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     The second major influence of the biomethylation reactions is the
change in the mobility of toxic elements in the environmental systems.
Often the transport of heavy metals is facilitated by the formation of
soluble and/or volatile species.  Totally coordinated species such as
and (CH3)3As, and even complexes of the cationic organometals are sufficiently
volatile for atmospheric transport far from sources of contamination.

     Another very important source of a wide variety of organometals in the
environment is waste from their industrial and agricultural use.  Organo-
arsenic, mercury and lead compounds are widely used as fungicides and pesticides
(1).  Organotins are used in the polymer industry as catalysts and in
antifouling boat paint.  Totally methylated species such as (CHs^Cd,
(CH3)2Hg, and (CHs^As are used in electronics manufacture to deposit small
amounts of pure metals.  And of course, tetraethyllead is an important
gasoline additive.

     In order to evaluate the environmental impact of organometals, both
naturally formed and synthetically produced, selective and very sensitive
analytical techniques are needed (6,8).  Methods must be developed that can
"speciate" the different forms a metal may take in a sample.

                     4.  ANALYTICAL MEASUREMENT  OF  ORGANOMETALS

     There are many approaches to the speciation of organometals.  In all
cases there is a selective separation (either chemical or chromatographic)
and subsequent selective detection.

     Perhaps the most rapid separation approach for the volatile organometals
(or their derivatives) is gas chromatography.  This analytical technique is
in an advanced stage of development,  with a wide variety of commercially-
available high-performance column materials.  This approach depends heavily
on the volatility of organometal species.  For totally coordinated species
such as (CH3)2Hg (B.P. 96 °C) and (CH3)1|Pb (B.P. 110 °C), and for volatile
derivatives of the cationic organometals such as CHsHgCl (B.P. 170 °C) (9),
there is no difficulty using this separation technique.  However, for many
other organometals, easily volatile and thermally stable derivatives are
not available, limiting the applicability.

     There are four major detection approaches for organometal analysis by
gas chromatography.  The following table summarizes them:

                                    TABLE 1
Technique
       Principle
Sensitivity
    Selectivity
electron
 capture
(Continued)
analyte absorbs $
particle from Ni63
source and ions
formed are collected
at an anode
100 pg (10)
 20 pg (11)
fairly selective
but also responds
to halide, nitro,
and sulfur
compounds

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                               TABLE  1  (Continued)
 Technique
Principle
Sensitivity
Selectivity
 mass  spec-    low pressure  analyte
  trometry     gas is bombarded with
              electrons,  resultant
              positive  ions are
              accelerated and sepa-
              rated by  a  magnetic
              field, ions collected
              at  a cathode;  mass-to-
              charge ratio  may be
              scanned or  a  single
              mass may  be monitored

 flameless     analyte is  atomized
  atomic       in  a continuously
  absorp-      heated electrothermal
  tion        graphite  furnace and
              absorption  of atom reso-
              nant line is  measured

 microwave     analytes  are  introduced
  cavity       into inert  gas plasma
  emission     generated by  microwave
              absorption, emission
              by  excited  atoms is
              monitored
                       500 pg (12,
                        13)
                       100 pg (14)
                        10 ng (15)
                       0.5 pg (16)
                        10 pg (17)
                very selective
                but scanned
                spectrum may be
                cluttered with
                interferent's
                ions
                extremely high
                element  selec-
                tivity but no
                differentiation
                of  species of
                the same element

                very high element
                selectivity
     The electron capture detector is presently the most widely used in
organometal analysis because of its simplicity and sensitivity.  However,
the selectivi'ty is relatively poor and therefore rather extensive sample
cleanup is required before analysis with this detection approach.  For
example, when analyzing solid homogenates for methylmercury, it is first
necessary to form the chloride in 1 M HC1 and then extract into toluene.
Then the toluene layer is back extracted into basic, aqueous cysteine
solution and subsequently extracted after reacidification with HC1 into
toluene (5).  This cleanup is necessary to remove interfering sample con-
stituents.  This laborious preparation procedure,enhances the possibility
of systematic error and lowers recovery of CI^Hg  to between 80 and 90
percent.  The selectivity of mass spectrometry and atomic absorption is
much higher, but the apparatus is expensive and complex.  Also the sensitivity
is relatively poor and often extensive sample preconcentration is needed prior
to analysis (12).

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     Another separation technique that only deserves a brief mention in organo-
metal speciation is thin layer chromatography.  After a separation the
"spots" are either identified colorimetrically (18)  or analyzed by another
technique such as A.A. (19).  This approach is adequate when the character
of the samples is well known (as in quality control) and the amounts of
organometals are fairly high (i.e., mg/g).

     In special cases, selective chemical reactions can be used to differentiate
between the organometallic and inorganic forms of an element.  For example,
in one such approach volatile C^HgCl is formed and distilled over (Hg   is
not volatile under these conditions).  The CHsHgCl is then reduced to Hg°
and measured by cold vapor atomic absorption  (20).  Selective extraction
has been used to differentiate organoleads (21).  Radiochemical assay can
be used with selective chemistry for,the mercury species as (surprisingly)
exchange of radioactively labeled Hg   is rapid with CHsHg  in the sample
(22).  However, the scope of this approach is limited to differentiation
between organic and inorganic forms of only one metallic element, and
cannot be used to investigate new, or unexpected species in a sample.

     Some work has been done recently using liquid chromatography as the
separation approach followed by selective detection.  This approach has a
fundamental advantage over gas chromatography, in that the analytes do not
have to be volatile.  Therefore, the requirement of forming volatile,
thermally stable derivatives does not apply.  The problem with liquid
chromatography is finding a suitable detection system for the job.  Present
work in analyzing organometals is limited to  the use of atomic absorption
detection except for some work using atomic fluorescence by Van Loon  (23).

     Perhaps the primary consideration in liquid chromatography with
atomic absorption detection (LCAA) is the interface of the LC solvent
output to the final formation of free atoms.  An easily interfaced atom
reservoir is the premix flame (24).  The flow rates used in HPLC  (0.5  to  2
mL/minute) are just about right for the burner uptake rate  (1-3 mL/minute).
However, the flame as an atom reservoir is not very sensitive, perhaps a
few ppb can be detected for good elements (without accounting for the
dilution in the LC).

     A somewhat more sensitive atom reservoir is the electrothermally
heated graphite furnace.  This device may be  used in a continuous mode if
the sample stream is split  (25).  However, in using this approach most of
the LC effluent is wasted,  increasing the detection limit.   In another
approach, a small (about 100 yL) sample can be taken of the  effluent periodi-
cally, for subsequent analysis (26).  In the  discontinuous mode of operation
an autosampler takes a sample of the effluent every 50 seconds, dries  it  in
the furnace, and subsequently atomizes the sample.  However, this sample
rate is too slow to maintain the fidelity of  a high efficiency separation
and thus resolution and detection limits suffer  greatly (26) unless the
chromatography is run very  slowly.

     Perhaps the most serious drawback of LCAA is the poor  limit of detection.
Although graphite furnace atomic absorption is quite sensitive for some
elements (such as Pb), it is relatively poor  for many elements that form

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interesting organometallic species such as As, Sb, Hg, Sn, Se, and Te.
It must be remembered that the HPLC separation generally dilutes a 20 yL
sample to 0.2 to 2 mL losing one to two orders of magnitude in detection
limits for any detection system.

     A second serious problem unique to organometal analysis by GFAA is
that the analytes are very volatile species and it is difficult to
atomize them without great losses by direct volatilization as neutral
compounds (which are not detected).  In the autosampler mode, the desolvation
and ashing temperatures are kept quite low and only the atomization temperature
is high (26).  However, under these conditions it is absolutely necessary to
use background correction, as nonspecific absorption of other sample con-
stituents is very likely without a pre-ashing cycle.  This is a disadvantage
as the use of continuum background correction generally lowers the sensitivity
somewhat.  This nonspecific absorption problem becomes quite severe when
solvents with greater than 0.1 M ionic content are used for the chromatography.
This severely limits the applicability of this detection approach in ion
exchange.

     Although the LCAA approach provides very high element specificity
(in the background corrected mode of operation) it does not discriminate
between different species of the same element if they coelute in the
chromatography.  Worse yet there is no way to gain further qualitative
information on a suspected analyte peak (besides the element content).
It is necessary to use a second detection approach to confirm the absolute
identity of the analyte species in a completely unknown sample.

     Clearly, a more sensitive and reliable detection technique is
needed for the measurement of organometals separated by liquid chromatography.
This report of investigation describes the development of such a detection
system.

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                         5.  EXPERIMENTAL PROCEDURES
DEVELOPMENT OF LCEC

Applicability

     A promising recent development in the separation and measurement of
complex samples is liquid chromatography with electrochemical detection
(LCEC).   In this approach an amperometric (or coulometric) flow cell
detector is used to monitor components separated by HPLC, the selectivity
is provided by the chromatography and from the use of the electrochemical
cell.  This analytical approach shows promise in the measurement of
trace organometals since many of these species show electroactivity (27,28).

Historical Development

     LCEC got its start in the early 1950's with some work done by Ketnula (29)
using D.'C. polarography coupled t'o ion exchange chromatography.  He
termed the new approach "chromato-polarography" a name that has given
way to LCEC.  Most of the applications of this technique before 1970
employed amperometry at a DME (30-52).  Only reducible species were detected.

     Perhaps the most significant development in LCEC was the work done
by Ralph Adams and Peter Kissinger (53).   In this new approach a solid
electrode, carbon paste was used for the working electrode material.  This
gave a potential range primarily suited for the oxidation of organic com-
pounds.   Since this development many papers have been published by Kissinger
and his associates on the application of LCEC to environmental and clinical
analysis.  In the short span of five years or so, LCEC has become an important
tool for the selective analysis of complex mixtures.

Basic Principles

     The electrochemical detector for liquid chromatography consists of a
low volume (typically 1 to 100 uL capacity) flow cell containing a working,
auxiliary, and reference electrode.  There are basically three methods of
providing working electrode contact with the flowing stream.  The solution
can be passed over a flat electrode giving a laminar flow (53) , or it can
impinge directly with radial flow (52), or it can flow through a tubular
(often with a packed bed of electrode material) electrode which is particu-
larly useful in coulometric detection (54-56).  In all cases, the solution
flow decreases the thickness of  the Nernst layer  through which the  analyte

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 species  must  diffuse,  increasing  the  faradaic  current  relative  to  quiescent
 conditions.   When amperometry  is  used,  the faradaic  current  is  proportional
 to the flow rate to  the 1/2  to 1/3  power  (57).

       The sensitivity  of the electrochemical detector  depends on the  ratio
 of the analyte  faradaic current to  the  background  noise  currents.   In
 constant potential amperometric detection  the  limiting background  noise
 arises from flow fluctuations  in  the  faradaic  current  (generated by electro-
 active impurities in the solvent  or the solvent's  decomposition).   Therefore,
 to minimize noise levels in  the amperometric mode, the fluctuations in
 solution flow must be  minimized by  pulse dampening of  the  chromatographic
 pumping  system  and by  good hydrodynamic design of  the  detector  cell to
 minimize turbulence.   Also the magnitude of the unwanted solvent faradaic
 current  should  be minimized, since  the  noise is directly proportional to
 this  quantity (58).  Coulometric  detectors are  less  flow sensitive since
 all of the analyte is  converted in  the  electrochemical process  and this is
 the quantity  measured.   However,  the  background faradaic current at the
 large  electrode  (needed  to achieve  100  percent  conversion  of the analyte)
 does have a flow dependence  and ultimately limits  the  sensitivity  (usually
 a  factor of 10  poorer  than amperometry  at  the  same potential).  The differen-
 tial pulse mode  has  very low flow dependence (58)  and  thus flow fluctuations
 would  not produce the  limiting noise  except in  situations where either the
 flow pulsations  or the background current  were  exceedingly large.   Rather,
 the sensitivity  limitation is  a result  of  capacitative charging currents
 generated by  the pulsed  potential.  The following  table compares these
 three  waveforms  used in  LCEC:
                                    TABLE 2
  Waveform
Sensitivity in
 LCEC (mol/L)
    Selectivity
Advantages/Limitations
amperometric
coulometric
differential
 pulse
   5 x 10
                         -9
   1 x 10
                         -7
   1 x 10
                         —8
fairly low, senses
all electroactive
species below
applied potential
fairly low,
as above
very high, senses
only electroactive
species with E%
within pulse  ~
height (5-100 mV)
 simple, inexpensive,
 and sensitive; how-
 ever, it is flow
 dependent and not
 very selective

 absolute calibration
 of the coulomb, low
 flow dependence;
 however, it has a
 complex electrode

 high selectivity,
 low flow dependence,
 but instrument
 required is more
 complex
                                        10

-------
     Another important aspect in the application of LCEC is the choice of
working electrode material, since this determines the useable potential range.
The following table lists some electrode materials used in LCEC work:

                                    TABLE 3
Material
 Potential Range
   in V. PH 4
Advantages/Limitations
Application
carbon
 paste

DME
glassy
 carbon
mercury
 coated
 platinum

WIGE3
GAME
+1.0  to  0.0 (53)
+0.1  to -1.5 (50)
+1.0  to -1.0 (59)
-0.1  to -1.1 (60)
+1.2  to -1.2 (61)
platinum     +0.90 to -0.5 (61)
-0.1  to -1.2 (28)
easily resurfaced
continuously
resurfaced, large
charging current

difficult to
resurface
resurfaceable
resurfaceable
formation of surface
oxides, difficult
to resurface

easily resurfaced
oxidations
only

reductions
only
oxidations
and
reductions

reductions
only
oxidations
and
reductions

oxidations
only
reductions
only
  WIGE:  Wax Impregnated Graphite Electrode.
  GAME:  Gold Amalgamated Mercury Electrode.
Applications

     LCEC can be applied to a wide variety of electroactive analytes.  Table 4
lists some of the classes of compounds that would be readily amenable to
electrochemical detection.
                                         11

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                        TABLE 4.  APPLICABILITY OF LCEC
                   Oxidations                       Reductions


             Hydroxylated Aromatics              Heavy Metal Ions

             Quinolines                          Nitro Compounds

             Catecholamines                      Diazo Compounds

             Amides                              Organometals

             Amines                              Oxidations at Hg

             Mercaptans                          Halides

                                                 Sulfide, Cyanide
Possible Interferences

     The great potential of LCEC for analyzing complex mixtures lies in the
selectivity achieved from the liquid chromatographic separation and from
the electrochemical detection.  However, LCEC can suffer interferences from
sample constituents.  Electroactive species that coelute with the analyte
can be the cause of a positive additive interference, thus, it is incumbent
on the sample preparation to eliminate this type of interference.  Although
nonelectroactive species give no response at the detector electrode, the
presence of excessive amounts of strongly adsorbed (non-analyte) species
that coelute with the analyte could passivate the electrode.  This will
decrease the detector response to the analyte causing a negative, multiplica-
tive interference.  Finally, substances such as complexing agents that bind
the analyte so that it changes its behavior in the chromatographic system
will mask the analyte signal.

     As in any analytical method, it is important to be aware of, and to
avoid potential interferences in the analysis scheme, right from sample
collection to the final measurement step.

ELECTROACTIVITY OF ORGANOMETALS

Cyclic Voltammetry

     In order to develop a LCEC method it is necessary to first investigate
the electroactivity of the analyte species.  One rapid and simple method of
obtaining qualitative information about the redox characteristics of analytes
                                         12

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is to use cyclic voltammetry.  To begin this work, cyclic voltammograms
were run on many potentially interesting organometals in a solvent thought
appropriate for the chromatography.  The potential range of -0.1 to -1.4 V
(versus the detector's Ag/AgCl 3 M Cl  reference electrode) was scanned for
a 10 ^ M solution of the organometal in a solution that was 50 percent
MeOH/water, 0.05 M in NHi^OAc, pH 5.5.  This electrolyte was chosen to aid
the solubility of the organometals (with MeOH), discourage the formation of
OH  complexes (pH 5.5), and provide only a weakly complexing anion (acetate).
The GAME was the working electrode and was prepared as described in the
section comparing electrode materials (vide infra).  The peak potentials E
for the first reduction wave are tabulated in Table 5.                    P
                                    TABLE 5

Compound
CH3Hg+
CH3CH2Hg+
C6H5Hg+
(POH-C6H4)Hg+
CH3Sn+++
^Uri^/ r\ ^n
\ L»rl« ) n 311
O J J
(CH3)2TeI2
(nBu)3Sn
E (in V) Compound
-0 3S (r~a "\ QV,"*"*"
^••JJ \LiIl^^«OU
_n it /T H \ cu"*"*"
^•-JJ vVj.-il^/^oD
3 D 5 3
-0.20 (C6H )4Sb+
-1.07 (CHq),.Pb+
_Q Q ~J / pTT pTJ \ D"k
-0.90 (C,H ) Pb+
-0.45 (CH3CH2)2Pb"H"
-0.1 (C^)^"^
f\ 0*5 (C U \ TM
— U .yj \u..rlc/AJ-l
E (in V)
P
-0.94
-0.77
-0.80
-0.89
-0.57
-0.77
-0.50
-0.78
-0.65
-0.44

     In the reduction of most of the organometals tested, the first reduction
occurs at potentials lower than -1.0 V and therefore would be readily
amenable to LCEC analysis.

Possible Reduction Mechanisms

     Much electrochemical research has gone into the elucidation of the
reduction/oxidation mechanisms of organometals  (27) .  In many cases the
                                         13

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 monocationic organometal species are reduced with one electron per molecule
 to a free radical:

      Et3Pb+ + e  ->  Et3Pb-  +  Et6?b2                                    (27)


      CH3Hg+ + e  + CH3Hg- -»- (CH^g)2 •*• (CH3)2Hg -f Hg°                    (27,62)


      (CH-KSn* + e  -*• (CH,),Sn« -> (CH,),Sn-                               (27)
         j J             J j         j D   ^


      Seldom are  the first reduction products the metals  themselves.   This
 is in contrast to  the reduction  of  many  free metal ions.   The formation of
 soluble  products as above is an  advantage in LCEC analysis,  particularly at
 high analyte concentrations where the reduction product  (if  insoluble)
 would coat the solid  electrode causing passivation.   Thus  the electrochemical
 detection approach  is elegantly  suited to the measurement  of organometals.

 LCEC SYSTEM FOR  REDUCTIONS

 Construction Considerations

      In  order  to develop an LCEC system  that would have  the  high  selectivity
 necessary for  the measurement of organometals in environmental  and  biological
 samples,  it was  necessary to improve on  the  past work done on LCEC  for
 reductions.  Early  work  on reducible species used the DME, with the separated
 solution impinging  on the electrode (40).  The continual growing  and
 falling  of the mercury electrode gives rise  to changing  capacitative and
 faradiac currents.  The  current  fluctuates with the  electrode size  so that
 the  chromatographic readout had  a rather  annoying oscillating baseline,
 unless heavily damped.   In addition,  the  sensitivity of DME  amperometry  is
 rather poor (10  Tnol/L).   The DME detectors  were inconvenient because the
 capillaries  used to deliver the  mercury drops are easily clogged  due to
 impurities deposited  at  the tip.  Later workers investigated  the  use of
 single mercury drops,  in one application  a hanging mercury drop electrode
 was  used  (51) and in  another a tube filled with mercury  served  as the
 working  electrode (63).   Also mercury  coated platinum was  used  (51,60)  as
 an electrode with better hydrodynamics than  the spherical mercury drops.

      In  order to select  the optimum electrode for reductive  LCEC  several
 considerations were taken into account:   the cathodic potential limit,
 electrode  surface homogeneity, ease of refinishing,  and reproducibility.
 As a  test  electroactive  organometal,  the methylmercury cation was chosen
 because its redox characteristics were well  investigated (64) and the first
 reduction  step was  found  to be somewhat reversible at the HMDE.   For each
 electrode material, a  qualitative judgment of the reversibility of  this
 reduction was made  since  this reflects the ease of the electrochemical
 process at  the electrode  surface  and  to some extent,  the reproducibility in
 analytical measurements.

     The electrode  materials were evaluated  by recording cyclic voltam-
mograms of a 5 x lO"1* mol/L solution of CH3Hg  in  a  1.0 x 10~2  mol/L NaC104
                                         14

-------
medium with the pH adjusted (unbuffered) to approximately 2.8,  5.0,  and
8.0.  The reduced data is given in the following table;
                                    TABLE 6


Material
Carbon
paste
Hg-coated
carbon
paste
Cathodic Limit
(PH 5)
-0.6 V

-1.0 V


CH Hg Residual
reduction Current
irreversible low

quasi- high
reversible


Advantages /Limitations
too limited a
cathodic potential
too high residual cur-
rent, irreproducible

gold wire -1.0 V
Hg-coated -1.1 V
gold wire
glassy -1.2 V
carbon
Hg-coated -1.0 V
glassy
carbon
quasi-
reversible
quasi-
reversible
poorly
defined
quasi-
reversible
high
low
low
low
residual current
high
low residual current ,
easily refinished
poor reduction cur-
rent for CH-Kg
irreproducible

a                     +
  Potential at which H  reduction just began.
     These materials were chosen by examination of an excellent text on
solid electrode voltammetry by Adams (65).   The materials selected had the
best cathodic potential ranges.  The idea of mercury coating was to extend
the potential necessary for the H  reduction, as a result of the hydrogen
reduction overvoltage on mercury.  Gold was selected as a metallic substrate,
over platinum, because of the higher overvoltage of gold amalgam (0.80 V)
relative to platinum amalgam (0.10 V) (66).

     The electrodes tested were prepared in the following manner.  The gold
electrode was prepared by press-fitting a 1.0 mm diameter, high-purity gold
wire into an undersized hole in a %" teflon rod, exposing about 4 mm of
length.  Electrical contact was made by drilling a duct to the wire and
filling with mercury, with a copper contact wire.  The mercury-coated gold
electrode was prepared in a similar manner as above except an amalgam layer
was prepared by dipping the electrode into a pool of mercury for about 15
minutes.  Excess mercury was then wiped off with a Kimwipe.  It was necessary
                                         15

-------
to hold the electrode at a potential of -1.5 V for about 5 minutes in
solution to reduce mercuric oxide impurities before the electrode could be
used.  The glassy carbon electrode consisted of a 6 mm glassy carbon rod
epoxied into a machined plexiglas holder with a mercury contact.  The face
of the electrode had been polished smooth in a glass shop.  The carbon
paste electrode was prepared from a 1/2" teflon rod that had a well contacted
by a threaded brass rod.  The well was 6 mm across and was packed with
commercial carbon paste (pasted with wax).  The brass rod was slotted so
that a screwdriver could drive it inward forcing some paste out of the
well.  This made it exceedingly easy to resurface the carbon paste electrode.
A small amount of paste was extruded, then the electrode was rubbed over
glossy computer cards to obtain a smooth surface.  The electrical contact
to the brass rod was made by mercury and a copper lead wire.  The mercury
coating on the carbon paste and glassy carbon electrodes had to be prepared
by electrolysis of a 10 3 M solution of Hg   with stirring for 3 minutes
at -2.0 V.  This gave a dull gray appearance to the electrode.  The electrode
was then rinsed and transferred to the CI^Hg  solution for use.

     The initial electrode comparison indicated that the mercury-coated
glassy carbon electrode (MCGCE) and the gold amalgamated with mercury
(GAME) should be further compared using some other test organometals as
well as CH3Hg .  Performance criteria that were considered were residual
currents^ t pH 2.9 and 8,0, as well as the cyclic voltammetry of CH3Hg ,
(CH3)3Sb  , and (CH3)3Sn  at pH 2.9, 5.5, and 8.0.   However, the final
decision to use the GAME was a more pragmatic consideration of the reproduc-
ibility of analytical measurements.  It was quite difficult to provide a
reproducible mercury coating on the glassy carbon, even when the surface
had been cleaned by mild mechanical polishing.  The thin mercury layer gave
variable electrochemical results.  The GAME, however, was simple to refinish
and gave reproducible results.  The extra thick, liquid mercury surface
seemed much less affected by repeated use, where the MCGCE response changed
rapidly with use (probably due to electrodeposited impurities on the thin
mercury surface).  Since the reproducibility and longevity are important
considerations in choosing an analytical working electrode in LCEC, the
GAME was selected for use.

     In order to use the GAME in LCEC, an electrode holder was constructed
from a plexiglass electrode blank.  A slightly oversized hole was drilled
and the gold wire (1 mm diameter) was epoxied into place.  The electrode
was then polished flat with wet, #600 emery paper and finally with gem
polish type A on a wet felt cloth.  This gave a shiny, hydrodynamically
flat surface.  The gold was then amalgamated by soaking for about 5 minutes
in mercury.  Excess mercury was wiped off with a Kimwipe but enough was
left to form a slightly convex mercury surface.  When put into use a very
cathodic potential (i.e.,  -1.0 V) is applied to reduce surface impurities.
When in continuous use for more than two hours an anodic potential (-0.2 V)
was applied for about 5 minutes to oxidize and/or desorb accumulated coatings.
After a period of about one day's use, the electrode must be repolished
and coated with mercury since excessive impurities accumulate.
                                         16

-------
     The electrode holder is separated from the cell top, which incorporates
the reference and auxiliary electrodes, by means of a 5 mm thick teflon
gasket.  This arrangement is shown in figure 1.  The basic design is that
of Peter Kissinger (available from Bioanalytical Systems) except that the
reference and auxiliary electrode holder has been modified so that they are
less than 1 cm from the working electrode.  This configuration was constructed
from a Teflon adapter (1/2" normal pipe thread to 1/4") with the reference
electrode (isolated by a porous vycor frit) and auxiliary electrode (1/16"
stainless steel tubing with end fitting) press fit in place.  This design
was superior to the commercial design for two reasons.  First, the solution
resistance between electrodes was lowered since the distance is now about 1
cm rather than 3 cm.   This lowers the effective time constant of the series
solution resistance and electrode capacitance, improving response in the
pulsed mode of operation.  Also, the dead volume of the entire cell is now
about 100 yL rather than about 1000 yL, making it possible to collect
fractions, retaining their chromatographic integrity, for subsequent "off-
line" analysis.  The dead volume to the working electrode remains at about 1 yL.

     The amperometric and differential pulse detection was accomplished
using a Princeton Applied Research Model 174 which provides a potential
range of ±3 V, choice of pulse heights (5 to 100 mV) and times (0.5 to 5
seconds), and current sensitivity as high as 20 nA/10 V.

     The system was designed to maintain ambient electrical noises at a
minimum since high amplification of very small signals is inherent in the
detection approach.  To do this, the chromatograph was mounted on an aluminum
base plate which was grounded.  Aluminum screen and rods were used to
fabricate a demountable faraday cage.  Power line spikes (frequent at NBS)
going into the voltammetric detector supplies were reduced by means of a
"glich-free" power supply constructed from an isolation transformer and
parallel, bipolar capacitors.

     Another important consideration unique to LCEC in a reductive mode, is
the necessity to remove oxygen from the system.  This is a requirement
since oxygen is easily reduced in two stages:

     at around pH 5:   02 + 2e + 2H+ -> H202    E,  = -0.1 V

                       H202 + 2e + 2H+ -> 2H20  E,  = -0.9 V


These processes will give a response at the detector  (when held at potentials
greater than -0.1 V) that will contribute  to the background current and
hence noise. To reduce the D£ to a minimum, the solvent reservoir must be
continuously purged with high purity inert gas such as Linde oxygen-free
nitrogen  (where 02 is < 0.5 ppm).

     When the chromatograph was constructed with functional detector cell
and purged solvent reservoir, a disconcertingly large background current
was still obtained.  The chromatographic  system must  have been allowing
oxygen to reenter the solvent b'efore it reached the detector.  This seemed
unlikely  at first because most of the  system was constructed  from stainless
                                         17

-------
                          ELECTROCHEMICAL DETECTOR  CELL
oo
         REFERENCE  ELECTRODEx,
         (Ag/AgCI (3 mol/L CD)
         POROUS VYCOR FRIT
         AUXILLARY ELECTRODE
         (316 STAINLESS STEEL)
 M" ALTEX TO !/2" NPT TEFLON
 ADAPTER
SOLVENT INLET
           TEFLON GASKET
                   WORKING ELECTRODE  (GAME)
                       Figure 1. Thin layer electrochemical flow cell.

-------
steel components which are non-permeable to oxygen.  However, when the pump
was isolated in a plastic garbage bag filled with nitrogen, the excessive
background current dropped.  The Kel-F fittings in the pump were apparently
quite permeable to oxygen.  Actually this is of no great surprise as perfluoro-
alkanes (of which Kel-F is a polymer) are such good solvents for oxygen
they have been considered as whole blood replacements.  At any rate, because
there were teflon-type fittings in the pump, sampling valve, some of the
connections, and in the cell spacer gasket, it was decided to enclose the
entire chromatographic system within a nitrogen-purged plexiglass box.  The
enclosure was made 40 x 40 x 100 cm with a large hinged door with a rubber
seal and eight threaded latches.  As many external controls as possible
were incorporated into the box to avoid the necessity of opening the door
for trivialities.  Provisions that were included are external solvent
reservoir filling and draining tubes, two reservoirs with a solvent switch,
pump on/off and flow rate controls, external sampling valve switch and
syringe, and low volume solvent output switch from the detector to waste
(for off-line analysis) or to recycle to the reservoir; all of which were
carefully sealed with silicone rubber.  The nitrogen flow to the box is
regulated by a Whitey valve and measured with a calibrated Matheson, ball-
type flowmeter.  The oxygen level in the box is continuously monitored by
means of a Beckman Oxygen meter with a Clark-type amperometric detector
used directly in the gas phase.  It is possible to maintain the 02 level at
less than 0.1 percent with a flow of 50 ml/minute of NBS prepurified nitrogen.
A small, variac controlled muffin-fan is used to help maintain steady state
equilibrium inside the box, especially when filling with N2.

     Other reducible species, especially trace metals, are important to
consider in constructing an LCEC system for reductions.  It is necessary to
maintain reducible species at a minimum to decrease detector background
current and noise, and to protect the electrode material from accumulation
of reduced (i.e., solid metal or amalgam) deposits.  To minimize impurities
the solvent is prepared from high-purity reagents  (such as Merck Suprapur
acetic acid and ammonium hydroxide) and solvents (such as Burdick and
Jackson "distilled in glass" methanol).  Once prepared the chromatographic
solvent was further purified by electrolysis at a mercury cathode (Environ-
mental Science Associates model 214 PM) held at about -1.3 V versus Ag/AgCl.
In fact,the electrolysis cell also served as the solvent reservoir in the
chromatographic system so that contamination during transfer of the solvent
could be eliminated.  This also allowed continuous system operation by
means of a solvent recycle valve, where normally wasted solvent could be
returned for electrolytic purification.  When methanolic (or especially
those with mercaptans) solvents are used, it is necessary to discard the
solvent after 3-4 weeks use as oxidation products in the electrolysis cell
(occurring at the platinum counter electrode) begin to accumulate and
degrade chromatographic performance.

     The chromatographic system is primarily constructed of type 316
stainless steel, used to withstand the high pressures  (500-2000 psi) required
for high performance columns.  At first there was  concern that the stainless
would corrode  (especially in solvents containing complexing agents) releasing
electroreducible metal ions that would cause excessive background current.
                                        19

-------
An examination of the literature (67) indicated that type 316 stainless is
about 17 percent Ni, 12 percent Cr, 2 percent Mn, and roughly 60 percent Fe+
Fortunately most of the expected ions of these major constituents (Fe 2, Cr 3,
Ni 2, Mn 2) are not reducible at potentials below about -1.1 V (68).   This
still gives a potential range suitable for most organometal analyses.  The
formation of metal ions from the system can be discouraged by using teflon-
lined stainless steel tubing where possible and to employ only type 316
stainless to avoid possible electrolytic corrosion.  The presence of these
metal ions coming from the stainless is possibly evidenced when complexing
solvents are used, by a depression of the electrolytic purification cell
potential when the solvent is recycled (indicating excessive reduction at about
-1.1 V) and by large initial oxidation currents observed when the working
electrode is put at more positive potentials (i.e., -0.4 V) after several days
at -1.0 V.  Thus, only very small amounts of complexing agents could be used
with the stainless steel system.  It would be interesting to see how much
cathodic potential range (ultimately limited by solvent decomposition) could be
gained by constructing an all teflon system.  Unfortunately, these systems are
limited to 500 psi maximum, putting some constraints on the efficiency of the
column packing materials (because larger particles must be used, usually
37-40 ym rather than 5-10 ym).

     Another, often important, consideration in high sensitivity—high
performance liquid chromatographic detection is pulse dampening of the
solvent delivery system.  This is particularly important in a flow sensitive
detection such as amperometric, where the noise of the detector (and
hence sensitivity) is linearly related to flow fluctuations (58).  For our
system we chose a relatively inexpensive chromatographic metering pump
(Altex model 110) that provides some pulse compensation in a novel way.  In
this pump the single piston is driven by an eccentric cam, where the pump
output stroke is very long, but the refill cycle is short (200 msec).  Thus
the flow is steady for ^90 percent of the cycle.  The remaining pulsations
were dampened by means of a nitrogen gas-filled hydraulic dampener, consisting
of a capped, 60 cm vertical length of 1/4" stainless tubing joined through a
tee between the pump and sampling valve.  The gas becomes more compressed
during the forward pumping stroke when the pressure is highest.  During the
pump refill phase decompression of the gas forces stored solvent out of the
dampener completing the cycle.  This hydraulic dampening provides virtually
pulse-free operation at pressures of 500-2000 psi.  The disadvantages are
the large holdup volume (^5 ml) of the dampener which precludes its use in
gradient elution (as the change in solvent composition is slowed), and the
need to periodically replace the gas in the tube that is lost due to dissolution
in the liquid under the pressures used.  About once weekly, the cap of the
dampener tube is removed and the liquid is emptied with suction using a
flexible piece of 1/16" teflon tube.  Then the tube is connected to a high
purity nitrogen supply and the dampener tube is purged and recapped.

     The sampling valve chosen was a Rheodyne fixed volume loop type with
choice of a 20 yL or 100 yL loop.  Throughout this work the 20 yL loop was
used as it gave the best efficiency and resolution.  The 100 yL loop was
tried in order to increase the concentration detection limit, however, the
larger loop caused too much loss in resolution particularly of early eluting
                                        20

-------
components.   It was necessary to deoxygenate the samples before analysis by
LCEC in the reductive mode.   On all columns 02 showed_some retention (k* = 2)
and gave a rather large response since it is about 10   M in saturated
aqueous solutions.   The samples are purged with solvent-presaturated,  high-
purity nitrogen, while connected to the sample loop intake.  Several sample
container materials were tried.  Teflon was found to release too much
"stored" oxygen.  Plexiglas was better in that respect but adsorbed the
organometals readily causing losses at low levels and cross contamination
of solutions.  Finally, 5-ml pyrex sample vials with polyethylene caps were
chosen.  Three 1/16" holes in the caps allowed for gas entry and exit, and
sample loop intake.  These sampling cells showed no significant adsorption
and were simple and inexpensive.  With this sampling system about three
replicates could be obtained from a one mL sample with good reproducibility.
One recent suggestion (69) worthy of future investigation is to turn around
the problem of high permeability of oxygen in teflon by employing a long
length (^50 cm) of 1/32" teflon tube as a prevalve sample loop.  The teflon
loop would be inserted in a good oxygen scavenger solution (such as Cr 2 or
V 2) that could even be warmed to speed up the gas transfer equilibria.
This way the sample would not need a separate purge cycle, but could be
sampled directly, and degassed simultaneously.  This principle could also
aid in deoxygenation of the solvent before it is pumped.

     As always in HPLC, filters are used to protect the system from clogging
and abrasive particles.  A 5-um stainless steel filter is used at the
solvent pick up point in the reservoir and a second 2-ym filter is used
between the pump and sampling valve.  The column is protected from the
accumulation of "real world" sample impurities by means of a 6 cm precolumn.
The protective column is packed with the same bonded phase material as the
analytical column, although larger particle (37-40 ym), pellicular packings
are used, as these are easily and efficiently hand packed.

     The columns used were all high porosity silica bonded-phase columns
of 25 to 30 cm length and 4.6 mm inside diameter.  The particle size was
generally 10 ym (for irregularly shaped particles), although a 5 urn column
was also used (with spherical particles).  Surface functionalities of -
CH2CH2-NH2 (y-NH2), -CH2(CH2)6CH3 (C8), -CH2(CH2)16CH3  (C18), and -C6H5(-0)
were evaluated for organometal separations.

Choice of Waveform

Amperometry—
     The simplest detector waveform used in LCEC is constant potential
amperometry.  This approach allows discrimination against all nonelectro-
active species as well as those active ones that have a lower redox potential
than that chosen for the detection.  For example, consider the use of an
amperometric detector used for reducible species depicted in figure 2.  By
controlling the constant potential applied we can discriminate between the
species whose reduction waves are depicted as A, B, and C.  When a potential
of E! is applied the detector will give a response for  species A only, B,
and C will not be "seen" as the potential is below their minimum decomposition
potential.  However, at E2 a response will be given for both A and B so
                                        21

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 CONSTANT-POTENTIAL
 AMPEROMETRIC
 DETECTION
                 c
               1
1
               E2   E,

      APPLIED POTENTIAL
                                 E(-)
Figure 2. Amperometric detection in hydrodynamic voltammetry.
                22

-------
that it will be incumbent on the chromatography to separate A and B completely
from each other.  Component C will not be detected at either Ej  or E2  and
will not interfere.  Thus, some selectivity is available in amperometric
(or constant potential coulometric which is similar)  detectors by selection
of the potential.  Some qualitative information is inherent in the amperometric
LCEC measurement.  The peak height will follow the amperometric  current/poten-
tial function for the analyte as a function of detector potential.  This
can give electrochemical information about an unknown component  (by repeated
chromatography at different potentials) or more importantly it can confirm
the presence of a known analyte in an unknown sample.

     Limitation on the potential range available to the electrode in LCEC is
imposed at the cathodic end by the reduction of the solvent or solvent impur-
ities and at the anodic extreme by the oxidation of the solvent, or as in the
case of the GAME, the oxidation of the electrode material.  In order to examine
the potential window accessible for the GAME, a point-by-point measurement of
the electrolyte residual current during amperometric LCEC operation was made
as a function of applied potential.  Figure 3 shows this current at two pH's
for a 60 percent methanol solution.  At the anodic limit of about -0.2 V the
oxidation of the mercury on the electrode begins.  The cathodic limit is about
-1.0 V for the highest sensitivity and is probably a result of the decomposition
of the solvent hydrogen ions (particularly noting the current increasing
effect of decreasing pH as expected for hydrogen evolution).  Some of the
background current, especially when small amounts of complexing agents such
as 2-mercaptoethanol are used, is caused by leaching of the metals from the
stainless steel.  Potentials greater than about -1.2 V cannot be used in
this system because the solvent faradaic background current becomes so
large that even small flow pulsations and turbulence at the electrode give
rise to excessive background noise, severely limiting the detection limit.

     In order to evaluate the absolute detection limit for  the amperometric
detector, a series of dilute standard solutions were prepared.  As a test
organometal, trimethyllead cation was chosen because the optimum potential
for its detection was near the normal cathodic potential limit of -1.0 V.
In order to optimize the detection limit the peak current and background
current were measured for a 3 x 10 5 M solution, as shown in  figure 4.  The
peak currents were 32.8, 47.1, and 49.4 nA for -0.95, -0.975, and -1.00 V
respectively.  The highest potential (-1.0 V) gave the largest signal but
the background current was much larger than at the lower potentials.  Since
the detector noise is proportional to the magnitude of the  background current
(53) the optimal potential for maximum sensitivity was -0.975 V since this
would offer the best compromise between signal and noise.   The dilute
solutions were then analyzed at -0.975 V and also two blanks were run.  The
results are shown  in figure 5.  Examination of this data indicates that the
detection_limit is about 0.1 ng absolute, which translates  to a concentration
of 2 x 10 8 mol/L  or 5 ppb, for a signal-to-noise ratio of  2.  This is a
fairly good detection limit as L.C. detectors go.

     The linearity of the amperometric detector was demonstrated by chromato-
graphing a series  of standard solutions in the 10~7 to 10~3 mol/L range
using methylmercury.  The signal-to-noise ratio was optimized as before
                                       23

-------
    26
    24
    22
<  20
E  18
    16
LU
    14
3  12
o
QC
CD
^
CJ
<
QQ
    10
     8
     6
     4
     2
     0
   -2
      -0.2
                                                                     (-1.2, 43)
                                                             H2, 26)
                      0.4          -0.6           -0.8
                        APPLIED POTENTIAL (vs.  Ag/AgCI)
-1.0
  Figure 3.  Background residual current as a function of applied potential.  Conditions:
            electrode - GAME; solvent - 60% MeOH, 0.04 mol/L NH^OAc; flow rate - 1.0 mL/min.

-------
                            -1.00V
    -0.95V
 T
10 nA
                  0.975V
4.0
           4.6 nA
          0
                                  0  ZERO  CURRENT
    Figure 4. Optimization of detector response for
                       25

-------
                              BLANK
                              BLANK
                              0.20 ng  (CH3)3Pb
                               0.34  ng  (CH3)3Pb
                               0.67ng   (CH3)3Pb
 i   i    i    I    I    I    i    i
 0   1
 2J 3   4   5   6   7

  t
Figure 5.
   0

   MINUTES

Limit of detection for (CHg)3Pb  in the amperometric mode
of detection. Conditions: as figure 15 except detector
potential -0.975 V.
                        26

-------
  with  trimethyllead  except that  -0.83  V  was  optimal  for CHsHg  .  The log of
  the peak height  is  plotted against  the  log  of  the concentration in figure 6.
  A very  linear plot  was  obtained over  4  orders  of magnitude.   The data point
  at  1  x  10 7 tnol/L was difficult to  measure  precisely as a result of the
  excessive noise  present on that particular  day caused by residual oxygen in
  the solvent.   Normally,  the noise is  much lower so  that the curve could be
  extended into the 10 6  mol/L range.

       The applicability  of the LCEC  approach for organometals  in "real"
  samples  is going to be  limited  somewhat by  the presence of interferences.
  In  LCEC  there are basically two systems vulnerable  to interference:  the
  chromatographic  separation  and  the  amperometric detection.  To evaluate the
  performance of LCEC for  the measurement of  the organomercury  species,
  several  possible sources  of interference were  investigated.

       The reversed phase  separation  of the organomercury compounds relies on
  the addition  of  a complexing agent, 2-mercaptoethanol, to the chromatographic
  solvent  to form  neutral  species.  Thus the presence of a stronger (or
 nonlabile) complexing agent in  the  sample would pose a chromatographic
  interference.  To evaluate  this  suspicion a number of complexing agents
 were added to a methylmercury solution in 10 fold excess and the peak
 height compared  to a complex-free standard.   The following potential  inter-
 ferents, expected to be present in real samples, were evaluated:   iodide
 and chloride  (to simulate salt water samples),  cyanide and sulfide (as in
 polluted water) ,  cysteine (for protein sulfhydryl groups  in tissue) ,  and
 fulvic acid (from fresh water,  decaying plant material).   The fulvic  acid
 was prepared by extracting commercially available humic  acid with  10  percent
 methanol/water.  Of  the  interferents tested  I  , CN , and  Cl   showed no
 interference at all.  This was  expected as  the  formation  constants  for the
 CHsHg   complex was  less  than that of 2-mercaptoethanol  (70^.   However.  S~
 forms  much stronger  complexes  (Kf =  1021-2)  than HOC_H2CH2S~  (Kf =  lO1^-1)
 and  it was found  to  completely  obliterate  the CHaHg   chromatography.
 Cysteine (K.  = 1015'7) showed  only a minor  (about  5  percent)  decrease  in
 peak height for a 10-fold excess. Thus, in  order  to have an interference-
 free analysis, it would  be necessary to  selectively  remove the sulfur
 species  from  the  sample.

     Another  type of interference that depends  on  the selectivity of the
 chromatography and the detection is  the  coelution  of the other reducible
 species with  the  analyte species.  In  particular,  the presence of trace
metal  ions in  real samples could pose  a problem.  To assess the magnitude
of the interference, a series of cations were added  to methylmercury solutions.
The metals that would be  expected to be present in^real^ampln and^hat
hav_e_^reduction potentials belov,one vol^are:  Hg   , Cu   , Cd  , Pb  , and
Fe   •  Of these metal ions Hg   and Cu   directly  interfere by eating | at
          times very close to that of CH3Hg  .  The  presence of Cd  , Pb
and Fe    would not interfere as they are chromatographically resolved from
CH3Hg .  Figure 7-A shows an interfering mixture at the same concentration
as methylraercury.  This interference could be eliminated by a prior separation,
improvement of chromatographic resolution, or increasing the ..selectivity of
the detector.  For example, figure 7-B shows the improvement of the chromato-
graphic separation by a change in the chemistry of the system.   A complexing
                                      27

-------
OO
               + 2.0
                +1.0
                        CALIBRATION CURVE FOR CH3Hg+
           O.
           O
0.0
              -1.0
^ 1
-7 -6
1
-5
1 J
-4 -3
                 Figure 6.  Calibration curve  for Cl^Hg  in the amperometric mode of detection.

-------
                                          CH3Hg*
                         B
                                   i    i    i   i
                                   2468
                            INJECT
                                    MINUTES
Figure 7.  Elimination of the metal ion interference as  organomercury
           measurement by improvements in the chromatography.
           Conditions:  as figure 14 except detector potential -0.83 V
           (amperometric mode),  sample amounts — 10~5 mol/L CH3Hg+,
           Pb*4",  Cu4"4", and Cd44".   Figure A without  addition of EDTA;
           Figure B with addition of 10 ** mol/L  EDTA to  sample.
                                 29

-------
 agent  EDTA has been added in excess to the same solution as figure 7-A.
 All  of the ions except the organomercury cations form stronger EDTA complexes
 than the  2-mercaptoethanol present in the chromatographic solvent.  The
 anionic EDTA complexes formed apparently have little retention in the reversed
 phase  system and elute near the solvent front, eliminating the interference.
 The  use of a prior separation and the use of the differential pulse waveform
 will be discussed as alternative approaches to the metal ion interference
 as well.

     Finally, there are possibilities of interferences directly attributable
 to the detection approach.  Adsorption of non-electroactive species on the
 surface of the electrode could cause passivation.  This effect was tested
 using  benzene as a nonelectroactive, adsorbable, organic compound.  An
 injection of a 0.1 percent V/V benzene in 50 percent methanol/water showed
 only a slight dip in the baseline after the solvent front, indicating the
 elution of benzene.  This effect was caused by momentary coverage of some
 of the electrode surface, decreasing the available area for background
 faradaic  reaction.  However, this was such a small response for such a
 large  amount of benzene (1000 ppm) that the potential for this particular
 interference in a real sample is probably not important.

     As previously mentioned, the only easily variable parameter in amperometry
 to improve the selectivity is the applied potential.  This can be used to
 eliminate interferences of coeluting species only if the interferents'
 reduction potential is greater than that of the analyte.  If the interferents
 have lower reduction potentials they may cause a positive, additive inter-
 ference in the analysis.  However, it is possible to further verify that
 the  "analyte" peak is truly a result of the analyte expected at that
 retention time, by repeatedly performing the analysis at different applied
 potentials.  This works because the S shaped current-potential response
 function  is unique, since it depends on the reversibility of the reaction,
 the  number of electrons transferred in the electrode reaction per mole of
 analyte,  and the decomposition potential.  Thus, by measuring the peak
 height  at several potentials for the sample and a standard solution of the
 analyte,  the identity of the sample peak can be confirmed even for samples
 near the  detection limit.  Of course, a second detection approach can be
 coupled to the electrochemical detector for added qualitative information.
 For  example, cyclic voltammetry could be used to scan the electrochemical
 potential range to look for other electroactive species and to confirm the
 presence  of the analyte (61).  Also such techniques as UV/visible or atomic
 absorption, or mass spectrometry could be used when appropriate as confirma-
 tion of analyte identity.

Differential Pulse Voltammetry—
     A very promising method of improving the selectivity of the electro-
 chemical  detector is to use a differential pulse mode rather than the
 simple amperometry.   The constant potential differential pulse detection
waveform  consists of 5 to 100 mV fixed pulses of 50 msec duration superimposed
 on a constant applied potential.   The pulse repetition rate is 0.5 to 5
 seconds and the current is sampled for 15 milliseconds before and at the
 end of each pulse.   The difference of these two currents is amplified and
provides  the output signal.
                                       30

-------
     The pulsed waveform makes the differential pulse detection much  less
flow sensitive (58),  since the diffusion layer established only extends
slightly into the solution when compared to the B.C.  waveform.   The lower
flow sensitivity puts less constraint on the LCEC system.   The  control of
pump pulsations is less critical and the magnitude of the  background  current
is less crucial.  The detection limit in most cases is now limited by
capacitative charging current caused by the potential pulses rather than
flow fluctuations (58) except when the background faradaic current is
large.  The background current fluctuations begin to limit the  sensitivity
in this system using the differential pulse detection at potentials of
about -1.2 V where residual currents become large.

     The most important virtue of differential pulse detection  is the added
selectivity of the approach.  Only electroactive species that have their
redox wave within the pulse height used will be sensed.  Figure 8 illustrates
the selective detection of an analyte species in the presence of another
coeluting, more easily reduced, interferent.  At the applied potential
(E   -   ,) and the pulse height used (AE) only the analyte showed a marked
change In its reduction current, where the interferent shows no change in
current before and at the end of the pulse.  Thus the differential readout
will give a signal for only the species with a changing current over the
pulse range, the analyte.  A much greater selectivity is obtained with the
differential pulse detection as it would be impossible to eliminate this
interference in constant potential amperometry, even by varying the potential.

     In order to experimentally evaluate the differential pulse waveform
the sensitivity, linearity of response, and selectivity were examined.

     The limit of detection was measured for methylmercury by analyzing a
series of dilute stock solutions.  Several parameters were optimized to
achieve the greatest sensitivity in differential pulse operation.  The
signal-to-noise ratio was measured for three pulse heights and  these are
plotted in figure 9.  The best sensitivity would be achieved with the
maximum pulse height 100 mV, however, the selectivity  is the least because
of the large pulse range.  For some applications the 10, 25, or 50 mV pulse
height could be used to enhance the selectivity with only a small penalty
in sensitivity.  The pulse cycle time was varied from  0.5 to 5  seconds.
Little difference in sensitivity was noted, but  the effective time constant
of the 2 and 5 second sampling rates was too long causing loss  of chromato—
graphic resolution.  Since maximum resolution  is important, the 0.5 second
pulse repetition rate was used.  Finally, the  potential that gave the
maximum signal was found by repeated injection of a dilute solution of
CHsHg , illustrated in figure 10.  Little difference in the background noise
was noted over this narrow potential range, so the maximum signal became the
only criterion for potential selection.  Figure  11 shows some solutions very
near the detection limit obtained under  the optimum conditions.  This indicates
a detection limit of about 1 x 10 8 mol/L which  is 2.5 ppb  (aqueous) or 0.04 ng
(for a 20 yL sample) for methylmercury.  The sensitivity of the differential
pulse waveform is thus very similar to that of amperometric detection in our
system.

-------
      DIFFERENTIAL
      PULSE
      DETECTION
                                       E(-)
               EAPPLIED

            APPLIED  POTENTIAL

Figure 8.  Differential pulse detection in hydrodynamic voltammetry.
                      32

-------
    100
C/J
     50
C£

CO
       Figure 9.
    25                               100


    PULSE  HEIGHT  (IN  mV)

Relationship between signal-to-noise ratio and
pulse height in differential pulse detection.
Conditions:  analyte - 5 x 10~7 mol/L CHsHg ,
pulse time - 0.5 sec, time constant - 0.0 sec.
                               33

-------
                  MeHg
 20
 10
      -0.60
-0.70
-0.80
      APPLIED  POTENTIAL (IN V)

Figure 10.  Differential pulse current response as a function
        of applied potential. Conditions: pulse height -
        5 raV, pulse time - 0.5 sec, time constant - 0.0 sec.
                   34

-------
     T
    2 nA
                                        REAGENT

                                        BLANK
INJECT
Figure 11.
^jM0J*^a j*"M"fc *•
**• » 1 •' "*' ""^^"^1 • >


/w^N-^-w^r-*



CH3Hg+ _8
* 1.0 x 10 b
^J v-.-j-t.-.y-v^i,, / / n n n
•*» ** ii»-»-— ^ . 4. yj u u
0.043 ng

A 2.0 x 10~8
^J\^*r~~ -^- 4.3 ppb
0.086 ng
1 1 II 1 1 1
mol/L


mol/L



234567
                 MINUTES
         Limit of detection for CH3Hg  in the differential pulse mode

         of detection.  Conditions:  detector potential - -0.75 V,

         pulse height - 100 mV (-) ,  time constant - 0.3 sec.
                             35

-------
     The selectivity of the differential pulse measurement approach was
tested by the addition of several interfering ions to a methylmercury
solution,^ Figure 12 shows a comparison of the results for a mixture of
Cu   , Cd  , Pb  , CH3CH2Hg , and CH3Hg  all at the 5 x 10 6 M level, first
in the amperometric, then in the differential pulse mode of operation.
Serious interference could be seen using the simple D.C. mode but the
differential pulse waveform shows high selectivity for Clr^Hg  and
     The selectivity of differential pulse detection is so high for an
analyte that I like to call it "species specific" detection.  Using this
approach samples can be analyzed with a minimum of sample "cleanup" before
analysis.  However, this approach limits the number of simultaneously
detectable species to only three or four, unless the detector potential is
programmed during the chromatographic run to be reoptimized for each analyte.
The potential switching approach may be feasible as the electrode settling
time (sort of a capacitative and faradaic time constant) is quite rapid in
this mode of operation.  In the case of very similarly behaved (electro-
chemically) species such as methyl-, ethyl-, and phenylmercury, it is
possible to choose a compromise potential (-0.70 V) that will allow simul-
taneous detection (as previously illustrated in figure 10) .

     The last test was the linearity of response in the differential pulse
mode.  Stock solutions of CI^Hg  were prepared by volumetric dilution and a
calibration curve was run.  The results in figure 13 show good linearity
over 3 orders of magnitude.

     The two waveforms are both quite comparable in their detection limits
and linearity of response.  They offer the analyst a choice between "species
specific" selectivity for the differential pulse approach and simultaneous
multicomponent "promiscuity" with the amperometric detection.  Thus the
optimum waveform will be dependent on the sample to be analyzed.

Development of Separation Conditions

Organomercury Cations —
     An important aspect of any liquid chromatographic analysis scheme is
the development of optimum conditions for the separation of the analyte
species.  There must be adequate resolution of all the analyte components
from each other and from potential interferents.  The correct separation
mechanism must be chosen based on the chemistry of the analytes and inter-
ferents.  Variation in the mobile phase's solvent strength can "fine tune"
the separation.

     When electrochemical detection is used, the type of separation mechanism
is limited to partition (reverse-phase) and ion exchange.  This is a result
of the requirement of a conducting, very polar solution, needed for the
detection system.  The solvent must contain a supporting electrolyte, using
at least a 0.05 M solution of an ionic salt as a conductor, to lower the
solution resistance and prevent migration currents.  The commonly used
solvents that are suitable for electrochemical detection are water, methanol,
acetonitrile, and dimethyl-sulf oxide.  As supporting electrolytes salts,
buffers, or complexing ions may be used, dependent on the chromatography.
                                        36

-------
                                  CH3Hg
                                      EtHg*
                              CH,Hg+
                                       EtHg
                          t   '   '
                          I    2   4
I    I
6   8
                       INJECT
                               MINUTES
Figure 12.   Elimination of metal ion interference by increasing
            the  selectivity of the detector.   Conditions:
            Figure A as Figure 7A, Figure B as Figure A except
            detector mode - differential pulse,  potential -
            -0.70 V, pulse height - 25 mV (-), pulse time -
            0.5  sec, time constant - 0.3 sec.
                             37

-------
oo
             QC
             OC
                -1.5 -
                      -7
                   -6            -5            -4
                          LOG CONCENTRATION
-3
Figure 13.  Calibration curve for
                                                  in differential pulse mode of detection.

-------
     Although the solvent choice in LCEC is somewhat limited,  this provides
very little handicap in the analysis of organometals, as partition and ion
exchange are the logical separation mechanisms for these moderately polar
and ionic analyte species.
     The organomercury species (CE^Eg , Ct^Ct^Hg ,  CgHsHg  and even Hg  )
were chosen as the first group for separation. Initially, methylmercury was
used as a test compound for various chromatographic approaches.
     Direct injection of CHsHg  in reverse-phase systems using a yNH2 and
CQ bonded phase columns yielded poor results.  It was reasoned that CH3Hg
was too ionic in the acetate medium (K  for a neutral acetate complex is
only lO3-6) to be efficiently separated in the reverse-phase system.  In
order to favor neutral CHsHg  species, a strong complexing agent was needed.
The strongest methylmercury complexes are formed by mercaptans and sulfide
(71, 72, 70).

     A comparison of several polar mercaptans as complexing ligands was
made using a reverse-phase CIQ column.  Table 7 summarizes the results.
                                    TABLE 7
Reagent added 1% V/V
                          Structure
k1
                                                N(plates/M)
Characteristics
    of peak
CH2-CH2
2-mercaptoethanol j |
SH OH
sharp , some
1.63 7600 tailing

2-mercaptophenol
2-mercaptobenzoic
  acid
                        COOH
                                        1.85
                                                   4400
                                                               retained
                                                               infinitely
                      sharp ,
                      tailing
4-mercaptoaniline
                                        2.96
           850
                                                               broad,
                                                               excessive
                                                               tailing
 (Continued)
                                        39

-------
                              TABLE 7  (Continued)
 Reagent added 1% V/V
                                     Characteristics
Structure      kj	N(plates/M)	of peak
CH2CH20
1-mercaptoethylether |
OU fU flj
on \^n^\^n,
1-mercaptoheptane |
broad ing,
3.33 160 excessive
} tailing
, broad with
4.51 1200 tailing
                        SH
CH7CH7-COOH
3-mercaptopropanoic
1.26 2960
acid SH
CH9-CH-CH9
1-mercaptoglycerol
j 1.76 2160
SH OH OH
sharp
but
excessive
tailing
sharp
some
with

tailing
     Based on this data 2-mercaptoethanol was chosen as the complexing ligand
The separation was further optimized by the selection of a high performance
column material and adjustment of the percentage of organic solvent (73).

     The optimum conditions for organomercury analysis were:  column —
Spherosorb ODS (C18) (5 urn), solvent - 40 percent methanol/water with 0.06
mol/L NHijOAc, pH 5.5, flow rate 1.0 mL/minute and 0.01 percent V/V 2-
mercaptoethanol.   The separation is illustrated in figure 14.

Multielement Separation—
     One particularly promising column material for the separation of
organometals is the bonded-phase micro-NH2.  Figure 15 shows a good separation
of several different organometals in a 40 percent methanol/water solvent.
This separation illustrates quite nicely the multicomponent capabilities of
the amperometric  mode of detection.

Organoleads—
     Some good results were obtained with the organolead species on the
Spherosorb GIB column with 80 percent methanol/water with a trace of mercapto-
ethanol as shown in figure 16,   Surprisingly the addition of strong complexing
agents to the samples had no effect on the retention of the organoleads
shown.  The following ligands were tested:  EDTA, diethyldithiocarbamate,
dithizone, halides, and cyanide.  This would seem to indicate that the
complexing agents do not compete with the mercaptoethanol ligand.   The
                                       40

-------
      CH3Hg+
INJECT
  Figure 14.
                  8    10   12    14    16
               MINUTES
Separation of organomercury cations by charge-
neutralization reverse-phase chromatography.
Conditions: column — Altex Spherosorb ODS 5 ym,
4.6 x 250 mm; solvent — 40% MeOH, 0.06 mol/L NHi+OAc
pH 5.5,  0.01% ME, flow rate - 1.0 mL/min; detection
mode — differential pulse; potential 	0.70 V; pulse
height - 25 mV (-); pulse time — 0.5 sec.
                             41

-------
                        (CH3)3Pb +
   (CH3CH2)3Pb+
   0     1
                                     10  nA
                                        CH3Hg+
    I	L    I     I     I     I     I     I      I     I
2)   3
7     8    9
                           MINUTES
Figure 15.  Separation of selected organometals.  Conditions:
          column — Altex yNH2 10 vim, 4.6 x 250 mm;  solvent —
          40% MeOH 0.06 mol/L NHi+OAc pH 5.5;  flow rate -
          1.0 mL/min; detector mode — amperometry;  potential
          -1.00 V, time constant — 1.0 sec.
                         42

-------
                                     Me3Pb"
INJECT  1
                         MINUTES
 Figure 16.  Separation of organolead cations.  Conditions:
           column — Altex Spherosorb ODS 5 pm, 4.6 x 250 mm;
           solvent - 80% MeOH, 0.04 mol/L NH^OAc pH 5.5 0.001%
           ME;  flow rate — 1.0 mL/min, detection mode — amperometry,
           detector potential 	0.95 V; sensitivity 50 na/V;
           sample concentration — 5 x 10 5 mol/L of each.

-------
 complex-insensitivity of this separation is a good feature, as ligands in
 the  sample would not interfere with the chromatography.

     Work on  chromatographic separations of trace metal ions and of the
 organotin species is underway.  Little success has been achieved in the separa-
 tion of  the organotins in reversed phase systems.  Perhaps more careful
 examination of the complexation chemistry of organotins will point the way to
 their  separation.

 APPLICATION OF LCEC TO REAL SAMPLES

 Special  Considerations in Sampling Organometals

     Quantitative removal of the analyte from a "real world" sample is
 always an important problem in any analytical measurement.  Organometal
 ions have some characteristics that make them particularly difficult to
 extract.  The compounds are not stable to strong acid (due to hydrolysis)
 or oxidizing  agents (73), which are commonly used in wet ashing procedures
 for  trace metals (74) in solid samples.  For organometals, only homogenization
 with an  extractant solution or alkaline hydrolysis of the matrix can be
 used (5).  It is not advisable to heat homogenization mixtures strongly as
 organometallic species for form neutral, volatile complexes and can be lost
 in the gas phase.

     Organometal species can also be lost from prepared homogenate (or
 sample)  solutions.  Some organometals are subject to photolysis (3), so
 that it  is important to store the solutions as prepared in the dark.  These
 analytes may  also be lost on container walls when in very dilute solution.
 With glass containers, unacidified solutions can lose trace metal ions by
 exchange for  surface hydrogen ions on the Si-O-H groups of the glass.  Perhaps
 cationic organometal ions could follow the same path.  Organometals are
 readily  lost  from aqueous solutions by adsorption on plastic containers (75,76)
 such as  polyethylene because of the hydrophobilicity of the organic portion
 of the analytes.  A thorough study of the storage of organometals is needed
 and will provide future work in this project.

 Natural Water Preconcentration

     In order to monitor the levels of toxic organometals in water samples,
very sensitive detection methodology is needed.  Even in heavily polluted
waterways the filtered sample is usually quite low in heavy metals (total)
 relative to the suspended particulate material and underlying sediment
 (77,7).  Although levels of some toxic metals such as mercury may be in the
 low parts-per-billion range in the water, living organisms such as algae,
 plankton, and ultimately fish can accumulate this small amount up to the
 parts per million range in their tissues.  Thus, it would be important to
measure even very low levels of organometals in water samples.

     In order to make analytical measurements in the low and sub parts-per-
billion  (ng/g) range,  it is necessary to preconcentrate the organometals
                                        44

-------
before analysis.  There are four major approaches to metal ion preconcentration
that could be considered for organometals.   Direct concentration by low
pressure evaporation can be used but losses on the container walls or by
volatilization, combined with the increase in the concentration of interfer-
ences, suggest this is not a good approach in this case.  Coprecipitation
can be used sometimes, where a metal ion is carried down with an added
precipitation reagent.  This often gives irreproducible results since it is
too dependant on nucleation conditions. Also it gives a sample with a large
excess of a potentially interfering precipitant.  Solvent extraction is a
very good method, where neutral complexes of the ion are extracted into an
immiscible organic phase.  This is particularly attractive for organometals
because of their organophilicity. For example, methylmercury may be precon-
centrated as its neutral chloride complex from a dilute HC1 solution into
toluene.  However, it is difficult to use solvent extraction for preconcentra-
tions greater than a factor of 10 unless the distribution coefficient of
the analyte is quite large  (>103) and the solubility of the organic solvent
is quite low.  Also it is quite difficult to completely equilibrate two
phases at volume ratios much greater than 10 to  1.  For higher preconcentration
ratios (100 to 1000) a column procedure is often very effective.  For
organic compounds or neutral metal ions, a nonpolar stationary phase can be
used  (78).  Ionic species may be retained on ion exchange materials.  The
analyte may be analysed directly on the column  (as with neutron activation
analysis or x-ray fluorescence) or by elution in a very small volume of
solvent with the proper stripping reagent.  Thus, column preconcentration
is the approach we have chosen for the organomercury species.

      The choice of column packing material is very  important  as it must
quantitatively  retain  the organomercury species  even though  large volumes
of sample elute through.  Cation exchange would  not be  a  good  choice  for
the monocationic organomercury ions as these have a low charge-to-size
ratio. However, neutral halide complexes can be  made and  retained on  a
nonpolar stationary phase in an approach chemically similar  to  the solvent
extraction approach.

      The basic  apparatus  for the column preconcentration  of  methyl-  and
ethylmercury is shown  diagrammatically in  figure 17.  A 100  mL  water  sample
is acidified with  0.10 mL of concentrated  nitric acid  (making it  1.6  x  10  2 M)
to help prevent adsorption  by  formation  of neutral  hydroxy  or other  basic
complexes.  The sample is  collected in an  acid  washed borosilicate  glass
bottle with a  Teflon  liner  in  the  cap.   The  sample  is  filtered  through
a 8 urn Millipore Teflon  filter  and  any solid material  in  the filter  is
washed with several small portions  of  1.6  x  10    M  HNOs and  the washings
combined with  the  sample.   One milliliter  of  2  II ammonium citrate  pH 3.1  is
added to  the sample bringing the final buffered pH  to  2.5.   The water
sample  is  then placed  in the  sample reservoir.   The second  reservoir,  shown
in the  figure,  holds  a solution  of  1 M halide  (I ,  and  Cl  have been used).
The  feed  rate  of sample  to  halide  solution is  10 to 1  going into  the pump.
This  ratio  is  controlled by the  inside diameter of  the  tubing used to
connect  the  two reservoirs  to  the  "Tee"  joint.   A single  piston metering
pump  (LDC minipump) provides  a flow rate  of  2.8 ml/minute to the  column.   A
short (6  mm  long,  3.2  mm inside  diameter)  column is nacked with either Rohm
and  Haas  XAD-2 (120-170  mesh)  or with  Waters  Associates Styragel  500A.
                                        45

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                        ORGANOMERCURY PRECONCENTRATION
LU
I-
<
ce
LU
o

6
u
LU
100 ML ACIDIFIED
WATER SAMPLE (pH 2,5)

HALIDE SOLUTION 1

r w i
I ^ETHANOL I

in
lu
	 1-1




PUMP

PUMP





4 CM PRECQLUMN
STYRAGEL 500 A

COLUMN

4,
TO WASTE
1
1 ML SAMPLE
                Figure 17.  Block diagram of organomercury preconcentration.

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Both materials are polystyrene providing a nonpolar surface for adsorption
of the neutral organomercury halide complexes.  The sample is pumped through
the column and then the solvent is switched to tnethanol at a flow rate of
about 0.1 mL/minute.  This elutes the neutral complexes in a very small
volume of 1.00 mL (the first 200 uL corresponding to V  is discarded first).
If the recovery was 100 percent this set up would give a preconcentration
factor of 100.  However, recoveries were not quantitative.  When I  is
used the recoveries are:  CHsHg  95 percent, CHsCI^Hg  70 percent (n=5) .
     Although the recovery to date is not quantitative, it is sufficiently
reproducible (about ± 5 percent) to be useful for water samples where the
precision of the result is not critical.  To evaluate the utility of the
approach and to study the potential interferences, several synthetic sample
mixtures were prepared.  Previous investigation showed that sulfide ion and
certain heavy metal iors would interfere with the amperometric LCEC analysis.
However, the conditions of the preconcentration are such that these inter-
ferences should be eliminated.  Metal ions should not be retained in the
concentration step unless they form strong neutral complexes in dilute,
acidic, halide solution. Although many metals do form halide complexes, the
anionic complexes are not protonated to neutrality except in high concentra-
tions of acid.  For example, the quantitative extraction of Fe 3 into ethyl
ether requires 6 M HC1  (79) :




Fe+3 + 4C1" + H+
To, test the metal ion interference a
Fe 3, and Pb and 10"6 M CHsHg was
->• HFeCl4

solution of 10
preconcentrated

b M CuH
using

-+
»
the



Cd"1^, Hg
iodide
procedure above.  No concentration of the interfering metal ions occurred
and the expected recovery of the organomercury species was obtained.

     The complexing agent interference was studied by addition of a 10 fold
excess (10  5 M) of sulfide and cyanide to the organomercury species.  These
anions should not interfere at pH 2.5 as they will be converted to their
neutral acids  (H2S and HCN) and be unable to complex the CHsHg  (70) .  No
interference was experimentally observed.

     At this point a real sample was attempted,  so a portion of NBS pond
water was collected, acidified, filtered, and analysed.  No organomercury
species were found to the 100 ppt  (parts per trillion) level.  An identical
sample was  spiked with the analytes, and the expected recovery was obtained
for CHaHg   and CHsCI^Hg  .  A particularly complex sample, genuine seawater
was also analysed.  A 100 mL sample of Danish Standard Seawater Pi+g (chlorinity
19,370 °/00) was spiked  and analysed.  The spiked amount was successfully
recovered,  but no* organomercury cations were found  in  the seawater.

     Certainly, this preconcentration approach shows promise, but further
work is needed to improve recovery and verify the reproducibility.  Also it
should be possible to extend the procedure to include  CgHsHg  and  (CHs^Hg
with little modification.  The general approach  should be useful for  other
organometals as well.
                                         47

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Methylmercury in Fish

     There is concern for the levels of mercury (and other heavy metals) in
aquatic organisms, especially those which are frequently part of the human
diet.  Most aquatic species are good bioaccumlators of metals, and although
the metal ion level may be in the ppb range in the surrounding water, the
organisms may have tissue levels in the ppm range.  The Environmental
Protection Agency toxic "action level" for mercury in fish is 0.5 ppm.

     In order to provide a standard material to aid analytical researchers
in comparing techniques for trace analysis in fish, the NBS has prepared a
research material of lyophilized albacore tuna fillet.  The material has
been carefully homogenized and packaged in a nitrogen atmosphere to prevent
decomposition.  Analysis results for total mercury indicates 0.95 ±0.1 ppm
in the freeze dried sample (80).  Our task was to evaluate the amount of
methylmercury in the material.  We also made measurements of the methyl-
mercury levels in a sample of lyophilized shark paste prepared by the
Department of Agriculture of the University of Tokyo (81).

     Removal of organometals from solid material such as tissue poses
several problems.  It is not possible to use oxidative, acidic digestion to
remove the organic material as the methylmercury will decompose.  Present
methodology uses either alkaline digestion or aqueous hotnogenization to
provide a workable liquid sample (5).

     We have investigated a new sampling approach for methylmercury in
tissue.  This organometal ion will be completely protein-bound in tissue by
cysteinyl groups (70).  The sampling approach was based on competitive
complexation with 2-mercaptoethanol.  The ME forms slightly stronger complexes
(Kf = 1016'1) than cysteine (Kf = 1015'7) (9), and therefore could compete
for the CHsHg  in the tissue.

     For the analysis, a fresh 2.00 g sample of the lyophilized tuna was
weighed into a 50 ml pyrex centrifuge tube and about 7 g of 50 percent
methanol containing 0.1 percent mercaptoethanol was added.  Two samples
were prepared as above and four more were spiked with known amounts of
methylmercury.  The samples were homogenized with a Brinkmann Polytron
sonic/mechanical homogenizer for about 5 minutes.  Then they were warmed
for 45 minutes to 60°C (boiling chloroform) to hasten the equilibrium.  The
tubes were then centrifuged at high speed for 10 minutes and the supernatant
liquid (^3 mL) was taken for LCEC analysis.

     The measurement of the extract was made in the "specj.es specific"
differential pulse mode of detection, optimized for CHsjjIg .  The selectivity
for methylmercury is quite good.  The identity of CHsHg  in the tuna was
further substantiated by analysis at an alternate potential where the
expected signal would be about 2/3 of maximum.  The sample showed the same
potential dependence as a standard.

     For the quantitative analysis the standard additions curve for the
differential pulse mode was drawn.  A reasonably linear result was obtained
with r = 0.9977.  From the graph methylmercury was found to be approximately

-------
0.97 ppm in the sample.  This result was very close to the 0.95 ± 0.1 ppm
value for total mercury (80) and would seem to indicate that all of the
mercury present is methylmercury .  No ethylmercury could be detected, and
inorganic Hg   would not be detected under these conditions.  However, the
uncertainty in the CHsHg  value is fairly large by this sampling method as
the absolute recovery of Ct^Hg  is only 0.35 ppm by direct comparison to
standards. This low recovery (about 35 percent) could reflect the equilibrium
competition between sample sulfhydryl groups and the mercaptoethanol or the
distribution of the neutral CI^HgME complex between the solution phase and
the oily solid phase.  If this is the source of the low recovery, the
interference would be multiplicative (at least for standard additions close
in concentration to the analyte) .  Thus the recovery should be 35 percent
for both sample analyte and standard added CHsHg  and at least an approximate
result can be obtained by this approach.  A much more reliable result could
be obtained if the recovery could be improved to greater than 80 percent by
the development of a stronger mercapto-complexing agent for
     Another approach we briefly investigated was selective oxidation of
the methylmercury-complexing, sulfhydryl groups of the tissue protein.  If
these groups could be oxidized (mercaptans are quite easily oxidized to
sulfates) the methylmercury would be set free.  Unfortunately, I have yet
to find a reagent that will oxidize sulfhydryl groups that won't oxidize
CHaHg .  Dilute H2C>2 and 1 2 both oxidize cysteine and methylmercury.  Perhaps
weaker agents such as Sn ** and Fe(CN)g 3 will do the job selectively and
rapidly.

     The standard procedures presently used for the measurement of methyl-
mercury in tissue involve alkaline hydrolysis to remove most of the solid
matrix.  The hydrolysis is followed by acidification with hydrochloric
acid.  The methylmercury cation can then be extracted from the aqueous
solution with toluene as the neutral chloride complex.  The procedure
employed in this work for the measurement of methylmercury follows the
recommendation of the Analytical Methods Committee of the Chemical Society
(5) , with some modification.  The sample and reagent amounts were reduced
by 4/5.  The aqueous back-extraction solution used was 0.01 mol/L disodium
thiosulfate (82) buffered to pH 5.5 with 0.05 mol/L ammonium acetate.  This
extraction solution was compatable with our chromatographic separation, and
analysis was performed directly on this aqueous extract, eliminating the
final toluene extraction.

     In all cases a standard additions procedure was used, with known
amounts of diluted CHsHg  solution added to the solid material before the
 hydrolysis  step.  Duplicates  of  each  sample  and  of  two  standard  additions
 were  run.   Figure 18  shows  duplicate  injections  of  two  portions  of  the  shark
 sample.  The  relative average deviation  is 0.75  percent.

      Table  8  shows  the result of the  methylmercury  determination in the two
 fish  samples.   The  values obtained  are in fairly good agreement  with the
 values  obtained by  other methods for  total mercury.  The  high proportion of
 methylmercury to  total mercury is consistent with the results of other
 workers (84,85,86).
                                        49

-------
    CH3Hg+
        I     I    I
        246

INJECT
        MINUTES
   Figure 18.  Determination of Methylmercury in Shark Paste.
                     50

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                                    TABLE 8
               Sample                CH-Hg  in ppm          Total Hg
Research material 50
Shark paste
0
8
.93
.4
± 0
± 0
.1
.1
0
7
.95
.4
± 0.
(83)
1

     The accuracy of these results are totally dependent on the purity of
the commercial C^HgOAc (Alfa Inorganics), stated as 99 percent.  However,
reliable standards are needed to improve the certainty of the values reported
in Table 8.  One approach to the preparation of a methylmercury standard
solution could be controlled-potential coulometry.  Any Hg   impurities can
be prereduced at low applied potential, then the potential can be stepped
to that necessary for the first methylmercury reduction.  The total current
flow is measured, and through coulombs law the number of moles of methyl-
mercury could be calculated.  Future work will examine this approach to the
preparation of a standard.

Conclusion

     The electrochemical detection approach to liquid chromatography promises
to provide a selective and sensitive method for the determination of organo-
metal cations in environmentally important samples with a mimimum of sample
preparation.
                                       51

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