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
-------
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.
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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.
-------
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.
-------
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.
-------
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).
-------
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
-------
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).
-------
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
-------
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.
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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.
-------
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
-------
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
-------
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
-------
REFERENCES
1. Thayer, J. S. J. Organometal. Chem., 76:265, 1974.
2. Tobias, R. S. Organometal. Chem. Rev., 1:93, 1966.
3. Jewett, K. L., F. E. Brinkman, and J. M. Bellama. Marine Chemistry in
the Coastel Environment. ACS Symp. Series #21, 1975. p. 305.
4. Baughman, G. L., J. A. Gordon, N. L. Wolfe, and R. G. Zepp. Chemistry
of Organomercurials in Aquatic Systems. EPA publication #660/3-73-012,
1973. p. 32.
5. Analytical Methods Committee, Analyst, 102:769, 1977.
6. Ridley, W. P., L. J. Dizikes, and J. M. Wood. Science, 197:329, 1977.
7. Brinckman, F. E., and W. P. Iverson. Marine Chemistry in the Coastal
Environment. ACS Symp. Series #18, 1975. p. 319.
8. Brinckman, F. E., G. E. Parris, W. R. Blair, K. L. Jewett, W. P. Iverson,
and J. M. Bellama, Environmental Health Perspectives, 19:11, 1977.
9. Handbook of Chemistry and Physics, 51st Edition, CRC Press, 1971.
10. Zimak, J., and E. Cwiertniewski. Roczn. panst. Zakl. Hig., 27:19, 1976.
11. Cappon, C. J., and J. C. Smith. Anal. Chem., 49:365, 1976.
12. Ohkoshi, S., T. Takahoshi, and S. Toshio. Japan Analyst, 22:593, 1975.
13. Knof, H., and G. Albers. Erdoel Kohle Erdas Petrochem. Brennst.-Chem.,
28:534, 1975.
14. Robinson, J. W., E. L. Kiesel, J. P. Goodbread, R. Bliss, and R. Marshall.
Anal. Chim. Acta, 92:321, 1977.
15. Parris, G. E., W. R. Blair, and F. E. Brinckman. Anal. Chem., 49:378,
1977.
16. Talmi, J. Anal. Chim. Acta, 74:107, 1975.
17. Reamer, D. C., T. C. O'Haver, and W. H. Zoller. Methods and Standards
for Environmental Measurement. NBS Special Publication #464, 1977.
p. 609.
-------
18. Vastogh, A. Z. Anal. Chem., 279:366, 1977.
19. Graffeo, A. P., C. J. Riggie, and B. L. Sgontv. Proceedings of 4th
Annual Symposium on Environmental Monitoring, paper 202.
20. Floyd, M., and S. E. Sommers. Anal. Lett., 8(8):525, 1975.
21. Hancock, S., and A. Slater. Analyst, 100(1191):422, 1975.
22. Stary, J., and J. Prosilova. J. Radiochem. Radioanal. Lett., 26(1):33,
1976.
23. Van Loon, J. C. Chemical Speciation Studies Using Atomic Absorption
and Fluorescence Detectors with Chromatography and Other Separational
Techniques. NBS Analytical Symposium, Feb. 14, 1978.
24. Botre, C., F. Cocace, and R. Cozzani. Anal. Lett., 9(9):825, 1976.
25. Epstein, M. private communication, 1978.
26. Brinckman, F. E., W. R. Blair, K. L. Jewett, and W. P. Iverson.
J. Chrom. Sci., 15:493, 1977.
27. Mairanovskii, S. G. Russian Chem. Rev., 45(4):298, 1976.
28. MacCrehan, W. A., R. A. Durst, and J. M. Bellama. Anal. Lett., 10(14):
1175, 1977.
29. Kemula, W. Roczn. Chem., 26:281, 1952.
30. Robertus, R. L., R. J. Cappell, and G. W. Bond. Anal. Chem., 30:1825,
1958.
31. Van Duyne. R. P., and D. A. Aikens. Anal. Chem., 40:254, 1968.
32. Buchanen, E. B., and J. R. Bacon. Anal. Chem., 39:615, 1967.
33. Blaedel, W. J., and J. W. Todd. Anal. Chem., 30:1821, 1958; also,
33:205, 1961.
34. Corfield, M. C., and A. Robson. Biochem. J., 84:146, 1962.
35. Koen, J. G., J. K. Huber, H. Poppe. and G. den Boef. J. Chrom. Sci.,
8:192, 1970.
36. Tustanowski, S. J. Chromatog., 31:266, 1967.
37. Stillman, R., and T. S. Ma. Microchim. Acta, 1973:491, 1973; also,
1974:641, 1974.
53
-------
38. Mohnke, M., R. Schmunk, and H. Schultze. Z. Anal. Chem., 219:137, 1966.
39. Sandi, E., Z. Anal. Chem., 167:241, 1959.
40. Mann, C. K. Anal. Chem., 45:1864, 1957.
41. Kemula, W., and K. Butkiewicz. Roczn. Chem., 39:73, 1965.
42. Kemula, W., D. Sybilska, and A. Kwiecinska. Roczn. Chem., 39:1101, 1965.
43. Kemula, W., B. Behr, K. Chlebicka, and D. Sybilska. Roczn. Chem.,
39:1315, 1965.
44. Kemula, W., D. Sybilska, and K. Chlebicka. Roczn. Chem., 39:1499, 1965.
45. Kemula, W., and D. Sybilska. Anal. Chim. Acta, 38:97, 1967.
46. Kemula, W., and J. Witwicki. Roczn. Chem., 29:1153, 1955.
47. Kemula, W., and Z. Stachurski. Roczn. Chem., 30:1285, 1956.
48. Brook, A. J. Chem. Ind., 48:1434, 1968.
49. Gierst, L., and W. Dubru. Bull. Soc. Chim. Beiges, 63:379, 1954.
50. Joynes, P. L., and R. J. Maggs. J. Chrom. Sci., 8:427, 1970.
51. Wasa, T., and S. Musha. Bull. Chem. Soc. Japan, 48(7):2176. 1975.
52. Fleet, B., and C. J. Little. J. Chrom. Sci., 12:747, 1974.
53. Kissinger, P. T., C. Refshange, R. Dreiling, and R. N. Adams. Anal.
Lett., 6:465, 1973.
54. Johnson, D. C., and J. Larochelle. Talanta, 20:959, 1973.
55. Johnson, D. C., and R. J. Davenport. Anal. Chem., 46:1971, 1974.
56. Taylor, L. R., and D. C. Johnson. Anal. Chem., 46:262, 1974.
57. Feher, Z., G. Nagy, K. Toth, and E. Pungor. Analyst, 99:699, 1974.
58. Swartzfager, D. G., Anal. Chem., 48:2189, 1976.
59. Lankelma, J., and H. Poppe. J. Chromatog., 125:375, 1976.
60. Buchta, R. C., and L. J. Papa. J. Chrom. Sci., 14:213. 1976.
61. MacDonald, A., and P. D. Duke. J. Chromatog., 83:333, 1973.
62. Durst, R. A., F. E. Brinckman, K. L. Jewett. and J. E. Doody. Methods
and Standards for Environmental Measurement. NBS SP 464, 1977. p. 473.
54
-------
63. Robenstein, D. L., and R. Doetre. Anal. Chem., 49:1036, 1977.
64. Heaton, R. C., and H. A. Laitenin. Anal. Chem., 46:547, 1974.
65. Adams, R. N. Electrochemistry at Solid Electrodes. Marcel Dekker Inc.,
1969.
66. Vydra, F., K. Stulik, and E. Julakova. Electrochemical Stripping Analysis.
John Wiley and Sons Inc., 1976. p. 141.
67. LaQue, F. L., and H. R. Copsur. Corrosion Resistance of Metals and
Alloys. ACS Monograph Series #158, Chapter 15.
68. Sawyer, D. T., and J. L. Roberts. Experimental Electrochemistry for
Chemists. John Wiley and Sons, Table 7-4, 1974.
69. Freeman, D. H. private communication (1977).
70. Zepp, R. C., G. L. Baughman, N. L. Wolfe, and D. M. Cline. Environ. Let.,
6:117, 1974.
71. Simpson, R. B. J. Am. Chem. Soc., 83:4711, 1961.
72. Schwarzenback, G., and M. Schellerberg. Helvetica Chim. Acta, 48:28,
1965.
73. MacCrehan, W. A. Ph.D. Thesis, University of Maryland, College Park, MD,
1978.
74. Maienthal, E. J., and D. C. Becker. A Survey of Current Literature on
Sampling, Sample Handling, and Long Term Storage for Environmental
Materials. NBS IN 927, 1976.
75. Youssef, M. K., A. Sina, A. A. Kassem, S. A. Ilrahin, and M. A. Attia.
Indiam J. Pharm., 35:155, 1973.
76. Brinckman, F. E. private communication 1977.
77. deGroot, A. J. Influence of Civil Engineering Projects on Water Quality
in Deltaic Regions. NBS Inorg. Sem. Series, Nov. 29, 1978.
78. Burnham, A. K., G. V. Calder, J. S. Fritz, G. A. Junk, H. J. Svec, and
R. Willis. Anal. Chem., 44:139, 1972.
79. Morrison, G. H., and H. Freiser. Solvent Extraction in Analytical
Chemistry. John Wiley and Sons, 1957. p. 62.
80. LaFleur, P. D., and W. P. Reed. Report of Investigation-Research
Material 50 - Albacore Tuna.
81. Dokiya, Y., M. Taguchi, S. Toda, and K. Fuwa. Anal. Chem., 50:533, 1978.
55
-------
82. Cappon, C. J., and J. C. Smith. Anal. Chem., 49:365, 1976.
83. Rains, T. C. Report of Analysis, NBS, 1978.
84. Westoo, G. Acta Chem. Scand., 20:213, 1966.
85. Westbb, G. Science, 181:567, 1973.
86. Bache, C. A., W. H. Gutenmank, and D. J. Lisk. Science, 172:951, 1971.
56
------- |