Method 282.3 - The Determination
of Tributyltin Chloride in Marine
and Fresh Waters by Liquid-Solid
(LSE) and Gas Chromatography with
Electron-Capture Detection(GC/ECD)
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Method 282.3 The Determination of Tributyl tin Chloride in Marine and Fresh
Waters by Liquid-Solid Extraction (LSE) and Gas Chromatography
with Electron-Capture Detection (GC/ECD).
Version 1.0
October, 1989
Otis Evans
Betty Jacobs
Arnold Cohen
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring Systems
Laboratory - Cincinnati, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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INDEX
Section Number Subject
1 Scope and Application
2 Summary of Method
3 Definitions
4 Interferences
5 Safety
6 Apparatus and Equipment
7 Reagents and Consumable Materials
8 Sample Collection, Preservation and Handling
9 Calibration and Standardization
10 Quality Control
11 Procedure
12 Calculations
13 Method Performance (Precision and Accuracy)
14 References
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TABLES
1. Liquid-Solid Extraction and Gas Chromatography Experimental Conditions
2. Temperature Program
3. Accuracy and Precision Data for Eighteen Determinations of the Method
Analyte at 0.025 nq/L (100 ml) with Liquid-Solid Extraction and GC/ECD.
Using a C-18 Silica-Based Column (100 mg.)
4. Accuracy and Precision Data for Thirteen Determinations of the Method
Analyte at 0.1 ng/L (100 mL) with Liquid-Solid Extraction.
Using a C-18 Polymer-Based Column (Polystyrene) (100 mg.)
5. Accuracy and Precision Data for Eight Determinations of the Method
Analyte Using a 25 mm C-18 Teflon Enmeshed (Filter) Disk.
APPENDICES
1. Liquid-Solid Extraction Procedure
FIGURES
1. GC/ECD Chromatograms from Commercial C-18 Bonded Porous Silica
Columns(a),(b) Extracts from Two Pre-Conditioned Columns, 100 mg, 1 mL
(different manufacturers).
2. Schematic Diagram of Sample Filtration Apparatus
3. Photographs of LSE Column Processor
(a) Vacuum Manifold Displaying the Arrangement for Column (Cartridge)
and Disk Extractions
(b) Display of the Component Parts for Disk Extractions Using Glass
Filter Holder
4. Photograph of Stainless Steel Filter Holder for 25 mm Extraction (Filter)
Disks
IV
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5. GC/ECD Chromatograms of LSE Solutions from C-18 Disks.
(a) Extract from Pre-conditioned Disk
(b) 'Extract from Laboratory Reagent Blank
(c) Extract from Sea Water Containing 0.05 Aig/L Tributyltin Chloride
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1. Scope and Application
1.1 This method describes liquid-solid extraction (LSE) procedures
(1,2) for a particular dissolved organotin species in marine and
fresh waters followed by gas chromatography with electron-capture
detection (GC/ECD) (1,3-5).
Chemical Abstract Services
Analvte Registry Number (CASRN1
Tributyltin Chloride 1461-22-9
1.2. The analytical range has been verified to be linear from 1 ng/L
to 100 jxg/L of tributyltin chloride as Sn.
1.3. Quantitative measurements can be obtained by generating an
'external' calibration curve, or preferably by
preconcentrating/extracting the aqueous standards in the same
manner as the samples.
1.4. This method has been evaluated in a single laboratory and a method
detection limit (MDL) has been determined. Observed detection
limits will vary with sample types depending on the nature of the
interferences in the sample matrix, the particular extraction
device and the specific instrumentation used. For this work the
MDL was determined to be 6.7 parts-per-trillion of analyte as
tributyltin.
1.5. This method should be used by analysts experienced in LSE, and the
use of GC and in the interpretation of gas chromatograms.
2. Summary of Method
2.1. Samples containing 1-2% methanol (100 mL - 250 mL) adjusted to pH
4.5 are passed through one milliliter, 100 milligram octadecyl LSE
columns or Teflon enmeshed extraction disks at a rate of 5 mL/min.
The extraction devices are air dried and subsequently placed in a
desiccator for a least one hour, to ensure that all traces of
water have been removed from the adsorbent. The analyte is
desorbed with acidified ethyl acetate (HC1) into a calibrated GC
glass sample vial. The eluent is then adjusted to a final volume
of 0.5 mL with ethyl acetate (HC1). The sample extract is
refrigerated overnight (4°C) in order to allow solution
equilibration. The tin analyte is determined by capillary column
GC using electron capture detection (ECO).
3. Definitions
3.1. Field duplicate (FD1 and FD2)'--Two separate samples collected at
the same time and placed under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
1
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with sample collection, preservation and storage, as well as with
laboratory procedures.
3.2. Field reagent blank (FRB)--Reagent water placed in a sample
container In the laboratory and treated as a sample in all
respects, Including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the
FRB is to determine if method analytes or other interferences are
present in the field environment.
3.3. Laboratory duplicates (LD1 and LD2)--Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the
precision associated with laboratory procedures, but not with
sample collection, preservation, or storage procedures.
3.4. Laboratory Fortified Blank (LFB)--An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required detection limit.
3.5. Laboratory performance check solution (LPC)--A solution of method
analytes used to evaluate the performance of the GC instrument
system with respect to a defined set of method criteria.
3.6. Laboratory reagent blank (LRB)--An aliquot of reagent water that
is treated exactly as a sample. It is exposed to all the
glassware, liquid-solid extraction columns and disks, method
solvents, and reagents that are used with other samples. The
purpose of the LRB is to determine if method analytes or other
interferences are present in the laboratory environment, the
reagents, or the apparatus.
3.7. Laboratory fortified sample matrix (LFM)--An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the
LFM corrected for background concentrations.
3.8. Primary dilution standard solution—A solution of a single analyte
or several analytes prepared in the laboratory from stock standard
solutions and diluted as needed to prepare calibration solutions
and fortified blanks.
3.9.
Stock standard solution—A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the
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laboratory with an assayed reference compound. Stock standard
solutions are used to prepare primary dilution standards.
3.10. Calibration standard (CAL)--A solution prepared from the primary
dilution standard solution and/or stock standard solution. The
CAL solutions are used to calibrate the instrument to response
with respect to analyte concentration.
3.11. Quality control sample (QCS)--A sample matrix containing method
analytes or a solution of method analytes in a water miscible
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is issued to check laboratory performance with
externally prepared test materials.
3.12. Speciation--The determination of specific individual physico-
chemical forms of an element.
3.13. Organometallic compounds--.Compounds in which the carbon atoms of
organic groups are bound to metal atoms.
3.14. Liquid Solid Extraction (LSE)--A sample preparation technique
based on the separation mechanisms of liquid chromatography (1C).
The solubility and functional group interactions of sample,
sorbent and solvent are optimized to effect extraction and/or
elution. Also, more commonly known as solid-phase extraction
(SPE).
4. Interferences
4.1. Interferences in this method may be caused by contaminants in
solvents, reagents, glassware, Teflon and polycarbonate bottles,
liquid-solid extraction columns and disks and other sample
processing apparatus that lead to artifacts or elevated baselines
in gas chromatograms. All reagents and apparatus must be
routinely demonstrated to be free from interferences under the
conditions of the analysis by running field and laboratory reagent
blanks, including extracts of preconditioned columns.
4.1.1. Glassware, Teflon and polycarbonate bottles must be
scrupulously cleaned. All glassware, Teflon and
polycarbonate bottles should be soaked in 50% nitric acid
and rinsed thoroughly with organic free deionized,
distilled water.
4.1.2. The glassware, Teflon and polycarbonate bottles used for
organometal solution preparation and storage should be
sealed and stored containing deionized, distilled water.
4.1.3. The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents
by distillation in all-glass systems may be required.
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4.1.3.1. The extracting solvent, ethyl acetate, may
contain impurities, e.g., preservatives, etc.
and/or water which may give rise to extraneous
peaks and exacerbate the thermal sensitivity of
TBT chloride by enhancing its degradation in the
Injection port liner. Solvent blanks should be
analyzed for each new bottle or solvent before
use. An interference free solvent is a solvent
containing no peaks yielding data at 0.5 times
the MDL and at the retention times of the
analytes of interest. Indirect daily checks on
the extracting solvent are obtained by
monitoring laboratory reagent blanks (3.6) and
periodically monitoring the solvent by obtaining
solvent blank chromatograms. Whenever an
interference is noted in the laboratory reagent
blank, either impurity peaks or a depressed
analytical signal, the analyst should analyze
another solvent blank. Generally, low level
interferences can be removed by solvent
redistillation. The solvent container should be
kept tightly closed to minimize exposure to
moisture. Additionally, to insure a "dry"
solvent the non-acidified ethyl acetate may be
passed through a sodium sulfate drying column,
preferably just prior to use.
4.1.4. Liquid solid extraction columns and disks may contain
interfering impurities (Figures la and Ib) which can be
extracted by the solvent. Additionally, sample water
retained in the column interstices can give rise to a
negative interference, i.e., the analyte signal decreases
in magnitude over time (Section 4.1.3.1.).
4.1.4.1 Ethyl acetate can extract compounds from the
polypropylene housing, polyethylene frit, and
the C-18 bonded porous silica of the liquid-
solid extraction cartridges. Phthalates,
quinones, alkanes, cresols, etc. have been
identified in column extracts (b). A
representative number of columns in a given
batch (lot) should be analyzed before use. An
interference free column is a column containing
no peaks yielding data at 0.5 times the MDL at
the retention time of interest. Variability in
background between column lots and within a
particular column necessitates careful checking
of column performance via analyses of column
extracts.
Water must be completely removed from the
liquid-solid extraction cartridges and disks.
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Water aids In the thermal decomposition of
trlbutyltin (TBT) resulting in decreased analyte
response. The extraction devices should be air-
dried and placed in a desiccator for an extended
period of time (Table 3).
4.1.5. Syringes, and splitless injection port liners must be
cleaned carefully, re-silanized (if appropriate), and/or
replaced as needed.
4.1.5.1. At the end of each day's analyses it is
recommended that a solvent blank be analyzed.
The solvent blank should effectively clean the
syringe and remove trace amounts of TBT
chloride. TBT chloride has been found to be
incompatible with some solvents, e.g.,
methyltert-butyl ether (MTBE). The syringes
tend to "freeze" and require frequent cleaning.
4.1.5.2. Splitless injection port liners have a finite
life and should be checked frequently. Aging of
the injection port liner is indicated by
diminished peak height and significant peak
broadening (7.8). It is recommended that the
liner be changed or re-silanized every three
days. However, useful liner life may depend on
a) frequency of analyses, b) concentration of
the organotin analyte, etc.
4.1.6. Interfering contamination (carry over) (see 9.3.1.) may
also occur when a sample containing low concentrations of
analytes is analyzed immediately following a sample
containing relatively high concentrations of analytes. A
preventive technique is between-sample rinsing of the
syringe. After analysis of a sample containing high
concentrations of analytes, one or more laboratory reagent
blanks should be analyzed.
4.1.7. Matrix interferences may be caused by contaminants that
are present in the sample. The extent of matrix
interference will vary considerably from source to source,
depending upon the sample type.
5. Safety
5.1. The toxicity or carcinogenicity of each reagent chemical used in
this method has not been precisely defined; each chemical should be
treated as a potential health hazard, and exposure to these
chemicals should be minimized. Each laboratory is responsible for
maintaining a current awareness file of OSHA regulations regarding
the safe handling of chemicals used in this method. A reference
file of material safety data should also be made available to all
personnel involved in the chemical analysis. Additional references
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(9-11) to laboratory safety should be identified and made available
for the information of the personnel using this method.
5.2. Tributyltin (TBT) chloride is an extremely toxic substance (12,
13). Pure standard material (liquid) and stock standard solutions
of this compound should be handled with suitable protection to
skin, eyes, etc.
5.2.1. In the event of eye exposure - flush with copious amounts
of water.
5.2.2. In the event of skin exposure - remove any contaminated
clothing and flood skin with large volumes of water.
5.2.3. In the event of accidental ingestion - seek medical
attention promptly.
5.3. TBT chloride should be kept away from heat, sparks or open flames.
It is a COMBUSTIBLE LIQUID. In contact with acid or acid fumes,
highly toxic chloride fumes can be emitted.
5.4. TBT chloride spills should be covered with dry sand or dry
vermiculite, mixed well and transferred to specially marked
containers.
5.5. Solution pH adjustments should be made in the hood.
5.6. Disposal of waste (solvents, analyte(s), etc.) from the system
should be according to local regulations.
Apparatus and Equipment (All specifications are suggested.)
6.1. Sample containers-- One liter polycarbonate, Teflon, or amber glass
bottles fitted with Teflon-lined or polycarbonate lined screw caps.
Bottles in which high purity solvents were received can be used as
sample containers. These bottles must be" thoroughly cleaned,
sealed and stored containing deionized, distilled water prior to
use.
6.2. Balances
6.2.1. Analytical, capable of accurately weighing to the nearest
0.1 mg.
6.2.2. General purpose laboratory, metric, suggest automatic
calibration, full-range taring, readability to 0.01 g.
6.3. pH meter—Laboratory, capable of measuring to at least 0.01 pH
units.
6.4. Filtration Apparatus.
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6.4.1. Macro filtration—To filter sample waters. Use 250 ml
glass reservoir (connects to 1L bottle or vacuum flask),
funnel base and stopper, clamp, SS holder, screen and
Teflon gaskets (Figure 2). Recommend using 47 mm filter
(Millipore Type HA, 0.45
6.4.2. Laboratory or aspirator vacuum system. Sufficient
capacity to maintain a slight vacuum of 13 cm (5 in) of
mercury in the vacuum flask.
6.5. Volumetric flasks, various sizes.
6.6. Beakers, various sizes.
6.7. Liquid-Solid Extraction Apparatus.
6.7.1. LSE column processor-vacuum manifold (stainless steel
basin), vacuum hose fitting, cover with luer fittings and
gasket), vacuum gauge controller, vacuum manifold luer
plugs (Figure 3a) or equivalent.
6.7.1.1. Glass microanalysis (filter) holder, 25 mm
(filter size), 2.1 cmz-filtration area,
graduated volume- 15 mL, removable stainless-
steel mesh support screen and PTFE gasket.
Modified to fit column processor (Figure 3b) or
used in an arrangement analogous to Figure 2.
6.7.1.2. 304 stainless steel syringe (pressure filter)
holder, 25 mm (Figure 4). Should accommodate a
syringe with luer fittings, also accepts LSE
column/cartridges for easy interfacing to
column processor vacuum manifold. (Female LUER-
LOK® - inlet and male luer slip outlet).
Should have a stainless-steel support screen,
PTFE gasket's and o-rings. Can also be used in
arrangement analogous to Figure 2 with a No. 18-
20 LUER-LOK® syringe needle, to accommodate
laboratory/aspirator vacuum system_or manual
sample loading via metal LUER- LOK^ tipped glass
syringe.
6.7.1.2.1. Syringe-glass with LUER-LOK® TIP,
50 mL.
6.7.2. Extraction Column Reservoirs, 75 mL.
6.7.3. Extraction Column Adaptors, 1,3,6 mL.
6.8. Digital Automatic Pipettes, variable volumes.
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6.8.1. Disposable pipette tips, sizes: 1-100 /il, 10-1000 M! -
6.9. Gas Chromatography System
6.9.1. The GC must be capable of temperature programming and
equipped with a linearized electron capture detector,
fused silica capillary column, and splitless injector. An
auto-sampler/injector is desirable. An on-column injector
system may be an alternative to splitless injection
because tributyltin compounds are thermally sensitive and
may decompose in the injection port liner. Additionally,
in the presence of residual water thermal decomposition is
enhanced. NOTE: Element selective/specific detection,
e.g., atomic absorption spectrometry, induction-coupled
plasma spectrometry, induction-coupled plasma/mass
spectrometry coupled with ion Chromatography (1C) or
liquid Chromatography (LC) may be acceptable alternatives
to electron-capture detection.
6.9.2. GC analytical column
6.9.2.1. Fused silica capillary column. Any capillary
column that provides adequate resolution,
capacity, accuracy, and precision can be used.
A 30 m X 0.32 mm i.d. column with a 0.25 pm
(bonded) film thickness is recommended. (J & W
DB-1 or equivalent).
6.9.3. GC syringes
6.9.3.1. Micro liter syringe(s) - 10 nl, Hamilton 701N
series or equivalent.
7. Reagents and Consumable Materials
7.1. Helium carrier gas and 5% methane in argon (make-up) gas, as
contaminant free as possible.
7.2. Ethyl Acetate (CAS-141-78-6)--Spectrophotometry or Gas
Chromatography grade. It may be necessary to redistill the solvent
if impurities are observed which co-elute (interfere) with the
analyte of interest.
7.2.1. Acidified ethyl acetate: 15 /iL of 20% HC1/50 ml solvent.
7.3. Methanol (CAS-67-56-l)--High purity solvent.
7.4. Acetic acid, Glacial (CAS 64-19-7)--Ultrex grade for pH adjustment.
7.5. Ammonium hydroxide (CAS-1336-21-6)--Ultrex grade, 20%, for pH
adjustment.
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7.6. Hydrochloric acid (CAS-7647-01-0)--Ultrex grade, for preparation of
acidified ethyl acetate: 15 Atl of 20% HC1/50 ml solvent.
7.7. Deionized, distilled water (CAS-7732-18-5)— Prepared by passing
distilled water through mixed bed cation and anion exchange resins.
This water was adjusted to pH 4.5 (7.4 and 7.5). In this method,
this term will be used interchangeably with reagent water. Water
in which an interference is not observed at the method detection
limit (MOL) of the compound of interest.
7.8. Stock standard solution (100 /ig/mL)— Tributyltin chloride as tin
(Sn). Tributyltin chloride, 95 + %, liquid, d. 1.20720 (CAS-1461-
22-9). An individual solution of analyte is prepared by dissolving
56.8 uL in 250 ml of methanol (7.3). The neat liquid organometal
is pipetted into a 250 ml acid cleaned/pre-aged volumetric flask
and diluted to volume. Transfer this solution to a 250 ml Teflon
bottle and refrigerate (4°C) in the dark. This solution can be
stored and used for at least six months.
7.9. Primary dilution standard solution (10 jug/mL)— The stock standard
solution is diluted further with methanol to prepare a 10 Mg/mL
solution. Pipet 1 mL into a 10 ml volumetric flask and dilute to
volume with methanol.
7.10. Secondary dilution standard solution (1 /xg/mL)—Pipet 100 ^L of
the primary dilution standard solution into a 10 ml volumetric
flask and dilute to volume with methanol. Note: Further dilutions
as needed should be made to prepare less concentrated standard
solutions. Minimize the generation of excess organotin waste by
using small volumes; i.e., <10 mL.
7.11. Calibration Solutions—A series of calibration solutions (working
standards) are prepared by pipetting the appropriate volume and
concentration of standard solution and diluting to 10 ml with
acidified ethyl acetate (7.6). Prepared external calibration
solutions range from 0 to 100 ppb of analyte. These solutions
should be refrigerated and stored in the dark until used. These
solutions should remain tightly closed to minimize evaporation.
HANDLE WITH CARE.
7.12. Extracted external standard solutions — Prepare the calibration
standards in water (7.7) to be taken through the liquid solid
extraction (LSE) procedure. Assume 100% extraction efficiency and
prepare calibration standards to cover the range from 1 ppb to 100
ppb of analyte. For example, a 100 mL sample solution containing
•0.1 ng/mL of analyte when extracted, eluted, and brought to a final
volume of 0.5 mL in acidified ethyl acetate should yield a signal
equivalent to a 20 ng/mL solution.
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NOTE: It is recommended that the calibration standards be taken
through the LSE procedure for quantitation of the analyte. This
corrects for losses in extraction and concentration.
7.13. Liquid-solid extraction cartridges (columns) and disks.
7.13.1. The columns are comprised of polypropylene sample
reservoirs, and polyethylene fritted disks. The columns
should not contain adipates, quinones, phenols,
phthalates, siloxanes, and cresols, etc.that can be
extracted from the plastic by the eluting solvent. The
columns are prepacked with approximately 100 mg of silica
gel bonded phase (C-18) material. The packing should have
a narrow size distribution and should not leach any
organic compounds into ethyl acetate. One hundred
milliliters of water should pass through the column in
about 20 minutes with the assistance of a slight vacuum.
7.13.2. Polymer-based extraction columns have the same
specifications as their silica based counterparts. The
same restrictions also apply for the leaching/extraction
of certain organic plasticizers. One hundred milliliters
of water should pass through the column in about 1\
hours. This, however, is dependent on the degree of
cross-linking.
7.13.3. Teflon enmeshed filter disks feature chemically bonded
silica particles enmeshed in an inert PTFE matrix to
create a mechanically stable sorbent disk. The 25 mm disk
should pass 100 ml of sample in approximately 20 minutes
with the assistance of a slight vacuum.
8. Sample Collection Preservation and Handling
8.1. Sample collection. Samples should be collected in pre-aged
polycarbonate or glass containers. All samples should be collected
in duplicate. The containers should not be pre-rinsed with sample
prior to collection.
8.1.1. When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has
stabilized. Adjust the flow to about 500 mL/min. and
collect duplicate samples from the flowing stream.
8.1.2. When sampling from an open body of water (fresh or sea
water), fill the sample container with water from a
representative area. Sampling equipment, including
automatic samplers, must be free of plastic tubing and
other components that may leach interferents into the
water. Automatic samples that composite samples over time
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should use refrigerated polycarbonate or glass sample
containers (14-16).
8.2. Sample preservation. All samples should be iced or refrigerated at
4°C from the time of collection until filtration. The samples
should be filtered as soon as possible (Figure 2} upon return to
the laboratory.
8.3. Holding time(s). Samples should be analyzed immediately. If
immediate sample analysis is not possible, the pH of the sample
should be adjusted to 4.5 (optimum pH for analyte extraction),
with subsequent refrigeration at 4°C. The maximum sample (aqueous)
holding time should be 2 days.
8.3.1. Alternate. It is recommended that sample filtration, pH
adjustment and extraction be performed upon immediate
return to the laboratory. Laboratory studies confirm the
extraction, storage and preservation of tributyltin on
column for at least one month. Note: The implications
are that LSE of TBT is amenable to field sampling,
extraction (preconcentration), storage and preservation.
8.4. Field Blanks
8.4.1. Processing of a field reagent blank (FRB) is recommended
along with each sample set, which is composed of the
samples collected from the same general sample site at
approximately the same time. At the laboratory, fill a
sample container with reagent water, seal and ship to the
sampling site along with the empty sample containers.
Return the FRB to the laboratory with filled sample
bottles.
NOTE: The prevention of contamination and losses are of
paramount importance in TBT special ion and
analysis. Potential sources of contamination in
the laboratory environment are: dust, reagent
impurities and sample contact with the laboratory
apparatus (resulting in contamination by leaching
or surface desorption). Depletion via adsorption
(14-16) should also be strongly considered.
9. Calibration and Standardization
9.1. Demonstration and documentation of acceptable initial calibration
is required before any samples are analyzed and is required
intermittently throughout sample analysis as indicated by results
of continuing calibration checks. After initial calibration is
successful, a continuing calibration check is required at the
beginning of each 8 hour period during which analyses are
performed. Additional periodic calibration checks are good
laboratory practice.
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9.2. Establish GC/ECD operating conditions equivalent to those indicated
in Tables 1 and 2. Calibrate the GC system using the external
standard techniques or use the preferred technique, i.e., carry the
aqueous standards through the LSE extraction procedure (Appendix
1), then calibrate with the "extracted" standards.
9.3. Prior to calibration, the GC system must be conditioned. Column
conditioning and injection port liner conditioning are a
prerequisite for stable and reproducible analytical measurements.
System conditioning should be done each day that analyses are to be
performed.
9.3.1. The conditioning solution should be at least 100 ppb
tributyltin (TBT) as tin. (Analysts may convert this
value to reflect the intrinsic entity TBT if desired).
Lower concentrations will prolong the length of time
needed for conditioning (require more injections).
Following conditioning, laboratory reagent blanks should
be run to determine whether carry over will be a problem
in subsequent sample analyses.
9.4. External Standard Calibration Procedure
9.4.1. An external standard is a known amount of a pure compound
that is analyzed with the same procedures and conditions
used to analyze samples containing that compound. From
measured detector responses to known amounts of the
external standard, a concentration of that sample compound
can be calculated from measured detector response to that
compound in a sample.
9.5. Standards Carried Through the LSE Procedure
9.5.1. Standards carried through the LSE procedure provide an
inherent correction for recoveries, because they are
preconcentrated in the same manner as the samples.
9.5.1.1. The standards should be prepared in analyte free
solutions that resemble the aqueous matrix of
the sample as closely as possible.
9.5.1.2. The volume of the standard should be identical
to the volume of the sample(s) to be
extracted/concentrated.
9.5.1.3. The recoveries of the standards should bracket
the expected concentration range of the samples.
NOTE: External standards may be used to check
the "extracted" standards' curve. (See 9.6).
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9.5.1.4. The standards' extract should be diluted to a
known, predetermined volume (Table 3).
9.5.1.5. From measured detector responses to known
amounts of the standards, the concentration of a
sample compound can be calculated from its
measured detector response in the sample.
9.6. At least three calibration standards are needed. One should
contain the analytes at a concentration near to, but greater than,
the method detection limit for the compound; the other two should
bracket the concentration range expected in the samples, or define
the working range of the detector. For example, if the MDL is
1.0 ng/L, and a sample is expected to contain approximately 5 ng/L.
standards should be prepared at concentration of 2.0 ng/L, 5.0
ng/L, and 10.0 ng/L.
9.7. Preparation of Calibration Standards
9.7.1. To prepare an external calibration standard, add an
appropriate volume of each secondary dilution standard to
acidified ethyl acetate in a 10 mL volumetric flask and
fill to the mark. Mix by inverting several times.
9.7.2. To prepare standards to be carried through the LSE
procedure, add an appropriate volume of each secondary
dilution standard to solutions that closely mimic the
sample matrix.
9.8. Inject 2 ML of each calibration standard and tabulate peak heights
or area response versus the concentration of the standard. The
results are to be used to prepare a calibration curve for each
analyte by plotting the peak height or area versus the
concentration.
9.9. The working calibration curve must be verified on each working day
by the measurement of one or more calibration standards (and
when/if the injection liner is changed between analyses). If the
response for the analyte varies from the response predicted by the
calibration curve (9.8.) by more than + 10%, the test must be
repeated using a fresh calibration standard. If the results still
do not agree, i.e., the response is off by more than ± 10%,
generate a new calibration curve for each analyte. (Assuming that
the injection liner has become "activated," the analyst should
change it before proceeding further). Usually the liner can be
used 3 to 4 days before its response begins to deteriorate.
9.10. Some possible remedial actions.
9.10.1. Check and adjust GC operating conditions.
13
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9.10.2 Clean and/or replace the splitless injection port liner;
silanize injection port liner for later use.
9.10.3. Flush the GC column with solvent according to
manufacturer's instructions.
9.10.4. Elevate the temperature of the oven and the detector to
"bake-off" any residual components.
9.10.5. Break off a short portion (1 meter) of the column from the
injector end; or replace GC column. This action will
cause a change in retention times.
9.10.6. Check the mechanical action of the GC syringe daily.
Clean or replace as necessary. On occasion decreasing
peak height may be caused by clogging of the syringe as
opposed to a deteriorating injection port liner.
10. Quality Control
10.1. Each laboratory using this method is required to operate a quality
control (QC) program. The minimum requirements of this program
consist of the following: an initial demonstration of laboratory
capability and regular analyses of laboratory reagent blanks
(including sol vent/eluent blanks), and laboratory QC samples.
More specifically, GC/ECD procedural blanks from commercial C-18
bonded silica, and C-18 polymer cartridges and C-18/C-8 Teflon
impregnated filter disks should be obtained when new lots of
cartridges and/or disks are used. NOTE: The same lot number
should be used throughout any study. The laboratory must maintain
records to document the quality of the data generated.
10.2. Initial demonstration of low system background and acceptable
particle size and packing. Before any samples are analyzed, or
upon receiving a new supply of cartridges or disks from a
supplier, it must be demonstrated that a laboratory reagent blank
(LRB) is reasonably free of contamination that would prevent the
accurate determination of the analyte of interest. The analyst
should obtain chromatograms of column/disk extracts prior to and
after the conditioning phase. From this information the analyst
can determine if further conditioning is warranted or if other
action should be taken. These experiments can be used to
demonstrate that the particle size/packing of the LSE cartridge
are acceptable, and if the flow rate is consistent through the LSE
column or disk. (Stable flow rates indicate uniform particle
distribution and homogenous packing). Cartridges and disks may be
placed in series to test for breakthrough.
10.2.1. Liquid solid extraction (LSE) cartridges can be a major
source of contamination (LSE disks appear to be a more
attractive alternative with fewer interferences,
consistent reproducibility from disk to disk, and from lot
14
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to lot, etc.)- Cartridges may contain phthalate esters,
silicon compounds, plastlclzers and other contaminants
that could Interfere with and prevent the accurate
determination of the method analyte (b). The compounds
may be leached from the cartridges into acidified ethyl
acetate and produce a background of varying magnitude. If
the background contamination prevents accurate and precise
analyses, the condition must be corrected before
proceeding with the analyses. Figure la and Ib show
unacceptable background contamination from poor quality
commercial LSE cartridges. By contrast, Figures 5a, 5b,
and 5c show the lower backgrounds obtainable from LSE
disks. (It may be necessary for the analyst to evaluate
LSE cartridges from several sources before an acceptable
supply is identified.)
10.2.2. Additional sources of background contamination are the
solvents, reagents, and glassware.
10.2.3. One hundred milliliters of water should pass through the
100 mg silica-based cartridge of 25 mm Teflon impregnated
filter disk in about 25 minutes @ » 10 psi. The
extraction time should not vary significantly among LSE
cartridges (columns) or disks.
10.3. Initial demonstration of laboratory accuracy and precision.
Analyze at least seven replicates of a laboratory fortified blank
solution (laboratory QC samples) containing the analyte (see
regulations and maximum contaminant levels for guidance on
approximately concentrations).
10.3.1. Prepare each replicate by adding an appropriate aliquot of
the primary/secondary dilution standard solution, or other
certified quality control sample, to reagent water.
Analyze each replicate according to the procedure
described in Table 3 and in Section 11.
10.3.2. For each replicate, calculate the measured concentration
of analyte, and the mean accuracy (as mean percentage of
true value) and precision (as relative standard deviation,
RSD) of the measurements.
10.3.3. For the analyte at 25 pg/L as tin, the mean accuracy,
expressed as a percentage of the true value should be 92-
108% and the RSD should be < 8%.
10.3.4. Analysts should develop and maintain a system of control
charts to plot the precision and accuracy of analyte
measurements over time. For the analyte, the mean
accuracy, expressed as a percentage of the true value
should be 75-125% and the RSD should be < 25%.
15
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This quality control criteria is applicable to LSE
cartridges (Sections 7.13.1 and 10.2.1).
10.3.5. It is recommended that the laboratory periodically
document and determine its detection limit capabilities
for the analyte of interest.
10.4. Laboratory Reagent Blanks (LRB). Before processing any samples,
the analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of reagents are
changed, new solvent bottle opened, or the injection port liner
replaced or silanized, a LRB must be analyzed. If within the
retention time window of the analyte of interest the LRB produces
a peak that would prevent the determination of the analyte,
determine the source of the contamination and eliminate the
interference before processing samples.
10.5. A single laboratory fortified blank, containing the tin analyte
must be analyzed with each set of samples, at a concentration as
specified in 10.3.
10.6. A field reagent blank should be analyzed with each set of field
samples. Data/information from these analyses will be used to
help define and determine contamination related to field sampling
and transportation activities.
10.7. Each quarter, replicate laboratory fortified blanks must be
analyzed to determine the precision of the laboratory
measurements. The data will be used in documenting data quality.
10.8. Each quarter, the laboratory must analyze a quality control sample
obtained from an external source. A quality control sample should
be analyzed each time a new set of standards are used. The entire
analytical procedure must be checked, if unacceptable accuracy
data is obtained.
10.9. The laboratory must analyze an unknown performance evaluation
sample (if available) at least once per year. Results for each
analyte must be within established acceptance limits.
10.10. At various points in this document, quality control measures are
incorporated to alert the analyst to potential problems.
11. Procedure
11.1. Gas Chromatography
11.1.1. Tables 1 and 2 present the recommended operating
conditions for splitless mode GC/ECD, i.e., conditioning,
calibration and sample analysis.
11.1.2. Gas Chromatographic conditioning.
16
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11.1.2.1. The analyst must condition the analytical column
and the injection liner before starting any
tributyltin analyses.
11.1.2.2. The recommended conditioning solution is a 100
ng/mL solution of the tributyltin as tin.
11.1.2.3. Make approximately four 2-^L injections of
tributyltin in acidified ethyl acetate.
11.1.2.4. Monitor the peak response—either area and/or
height. When these parameters stabilize
analyses can begin. NOTE: If the injection
liner is replaced, conditioning must be
performed again. Fewer injections are required
during this phase. However, the analyst should
verify this in his/her laboratory.
11.2. Liquid-Solid Extraction
11.2.1. The liquid-solid extraction procedure is presented in
Appendix 1.
11.2.2. Set up the liquid-solid extraction column processor as
shown in Figure 3. Three options are shown, depending
upon the type of extraction device chosen. NOTE: This
identical arrangement is not required, but it is
convenient for handling small volumes of solution (< 200
ml).
11.2.2.1. Sample water is added to the reservoir and
drains from it through the LSE column or disks
into blunt stainless steel syringe needles
(LUER-LOK HUB) permanently mounted in the
manifold cover. The sample passes into waste
collection containers placed in the stainless
steel basin (6.7.1.).
11.2.2.2. A slight vacuum of « 10 mm Hg is used during
the course of sample extraction. The sample
flow rate is about 5mL/min. The pressure and
flow rate are critical. Variations during
operations may result in poor precision.
Approximately 20 minutes is required to pass 100
ml of sample solution through the system. This
however, is extraction device dependent.
11.2.2.3. Depending on the volume of water extracted, the
vacuum may have to be released in order to dump
the waste from the collection containers. The
17
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vacuum control valve should be closed slowly
followed by gently lifting one of the vacuum
manifold luer plugs. Releasing the vacuum too
quickly will cause splashing Inside the basin
and may result in solution getting into the
vacuum gauge. The analyst should be aware of
the volume of solution that has been added to
the column processor to prevent solution
overflow inside the vacuum manifold basin.
11.2.2.4. During sample application the solution should
never drop below the top edge of the packing in
the LSE column. Likewise, the LSE disk should
be immersed in solution at all times.
11.2.3. Following sample application the extraction device is air-
dried on the vacuum manifold for approximately 20 minutes,
then placed in a desiccator.
11.2.4. The analyte is eluted with two 250 ML portions of
acidified ethyl acetate into a GC vial mounted inside the
vacuum manifold basin.
11.2.5. The final volume of the eluate is adjusted to 0.5 mL with
a few drops of acidified ethyl acetate.
11.2.6. The extract is refrigerated overnight (4°C)
11.3. GC-ECD
11.3.1. Inject a 2 ML aliquot with the GC-ECD system under the
conditions shown in Tables 1 and 2 and section 9.8.
12. Calculations
12.1. Calculate analyte concentrations (in the sample) by utilizing the
calibration curve generated from the relative responses of the
standard (analyte) solution.
(a) The calibration curve is generated from the analyte response
produced from the "external standard" curves and/or
"extracted standard" curve, i.e., analyte standards taken
through the LSE procedure.
12.2. Data should be rounded to one decimal place and reported in M9/L
or pg/L.
12.3. The data should show which calibration technique is used. The
enhancement (or recovery) factor should be reported.
18
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13. Precision and Accuracy
13.1. In a single laboratory the method detection limit (MDL) (17) was
determined for the tin analyte. Seven aliquots of the fortified
sample are measured and the results used to calculate the MDL at
the 99% confidence level. The MDL is calculated using the
formula:
MDL=t (n-l.l-alpha =» 0.99)(S)
where:
(n-l.l-alpha = 0.99)
Students t value for the 99%
confidence level with n-1 degrees
of freedom, where n = number of
replicates, and S = standard
deviation of replicate analyses.
The reported MDL can be lowered substantially by extracting a
larger volume of sample and/or concentrating to a lower
known/constant volume. NOTE: The analyst should compensate for
the increased sample volume or smaller extract volume by lowering
the relative concentration of the analyte in the reagent water.
Analyte detection at the regulatory level should be achievable.
The MDL was difficult to determine for several reasons:
(a) unstable non-reproducible backgrounds which may
be attributed to low quality commercial LSE cartridges
(Section (10.2). Please see figures la and Ib. (b)
TBT is thermally unstable and therefore quantitative
measurements are difficult. However, an MDL limit of
6.7 parts-per-trillion of the analyte as tributyltin
was determined. For the MDL determination, seven
replicate measurements were made on solutions fortified
with the tin analyte at 0.025
19
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14. References
1. Junk, G.A., and Richard, J.G., "Solid Phase Extraction, GC
Separation and EC Detection of Tributyltin Chloride," Chemosphere,
1987,16,61.
2. Matthias, C.L., Bellama, J.M., and Brinckman, F.E., "Determination
of Tributyltin in Estuarine Water Using Bonded C-18 Silica Solid
Phase Extraction, Hydride Derivatization and GC-FPD, "Proceedings.
Oceans 87 Conference, Marine Technology Society, TEEE Ocean
Engineering Society, Halifax, Nova Scotia, Canada (1987).
3. Junk, G.A., and Richard J.J., "Organics in Water: Solid Phase
Extraction on a Small Scale," Anal. Chem., 1988,60,451.
4. Junk, G.A. and Richard, J.J., "Solid Phase Extraction on a Small
Scale," Jo. of Res. of the National Bureau of Standards, 1988,93,
274.
5. Junk, G.A., and Richard, J.J., "Solid Phase Versus Solvent
Extraction of Pesticides from Water," Mikrochim, Acta [Wien], 1986,
1,387.
6. Junk, G.A., Avery, M.J., and Richard, J.J., "Interferences in Solid-
Phase Extraction Using C-18 Bonded Porous Silica Cartridges," Anal.
Chem., 1988,60,1347.
7. Rodriguez-Vazquez, J.A., "Gas-Chromatographic Determination of
Organomercury, (II) Compounds," Talanta, 1978,25,299.
8. Silvis, P.H., "Optimizing Injection into 0.53-mm i.d. Capillary
Columns," in GC Troubleshooting, LC'GC, 1989,7,562.
9. "Carcinogens-Working With Carcinogens", Department of Health,
Education and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
10. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910,
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January, 1976).
11. "Safety in Academic Chemistry Laboratories", American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979.
12. Saxena, A.K., "Organotin Compounds: Toxicology and Biomedical
Applications", Applied Organometallic Chemistry, 1987,1,139.
13. Muller, B., M.D., Renberg, L., Rippen, G. "Tributyltin in the
Environment—Sources, Fate and Determination an Assessment of
Present Status and Research Needs," Chemosphere, 1989,18,2015.
20
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14. Carter, R.J., Turoczy, N.J., and Bond, A.M., "Container Adsorption
of Tributyltin (TBT) Compounds: Implications for Environmental
Analysis," Environ. Sci. Technol., 1989,23,615.
15. Maguire, R.J., Carey, J.H., and Hale, E.J., "Degradation of the Tri-
n-butyltin Species in Water." J. Agric. Food. Chem., 1983,31,1060.
16. Valkirs, A.O., Seligman, P.P., Olson, G.J., Brinkman, F.E.,
Matthias, C.L. and Bellana, J.M., "Di-and Tributyltin species in
Marine and Estuarine Waters. Inter-laboratory Comparison of Two
Ultratrace Analytical Methods Employing Hydride Generation and
Atomic Absorption or Flame Photometric Detection," August, 1987,
112,17.
17. Glaser, J.A., Foerst, D.L., McKee, G.O., Quave, S.A., and Budde,
W.L., "Trace Analyses for Wastewaters," Environ. Sci. Technol, 1981,
15,1426.
21
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TABLE 1. LIQUID-SOLID EXTRACTION
AND GAS CHROMATOGRAPHY
EXPERIMENTAL CONDITIONS
22
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TIN SPECIATION
(TRIBUTYLTIN)
Liquid-Solid Extraction (LSE)
with Gas Chromatography -
Electron Capture Detection (GC/ECD
Liquid-Solid Extraction:
Sample Volume
Sample pH
Extraction Devices:
Columns/Disks
Eluting Solvent
100 mL (50, 200, 250 mL)
4.5
1 mL, 100 mg silica or polymer-based
(C-18); Teflon enmeshed extraction disks (C-18)
Acidified ethyl acetate (HCl)-15/xL
of 20% HC1/50 mL of solvent
Gas Chromatography:
Column
Detector
Retention Time
Injection Volume
Injector
Carrier Gas
Make-up Gas
Linear Velocity
Fused Silica Capillary (DB-1, 30 mx 0.32 mm i.d.,
0.25 tun film)
Electron Capture
=14.6 min (TBT)
2 /iL
Splitless
Helium (3.86 mL-min'1)
Argon-Methane (30 mL-min"1)
20.8 cm/sec 0 160°C (measured isothermally)
23
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TABLE 2. TEMPERATURE PROGRAM
Initial Value ° 80°C
Initial Time =» 1.00 Min
Level 1
Program Rate = 15.00°C/min.
Final Value = 160°C
Final Time = 10.00 Min
Level 2
Program Rate = 20.00°C/min
Final Value = 230°C
Final Time = 8.00 Min
Injector Temp = 200°C, Oven Temp. = 80°C to 230°C,
Detector Temp = 260°C, Splitless injection with 30s delay
24
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Table 3. Accuracy and Precision Data for Eighteen Determinations of the
Method Analyte at 0.025 ng/L (100 uL) with Liquid-Solid Extraction
and GC/ECO Using a C-18 Silica-Based Column 100 mg.
Volume of Sample
Conc'n after Extraction
(uQ/L)
Expected Mean
Observed
Std. Rel. . Mean
Dev. Std. Method
Dev. Accuracy
(ml)
(M9/L)
(%) (% True
Conc'n)
200
250
10
12.5
10.3
12.0
0.8
1.4
7.7
11.7
102.9
96.1
25
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Table 4. Accuracy and Precision Data for Thirteen Determinations of the
Method Analyte at 0.1 /ig/L (100 ml) with Liquid-Solid-Extraction.
Using a C-18 Polymer-Based Column (Polystyrene-100 mg).
Concentration After Extraction
(ua/L
Mean
Expected Observed
(M9/L) (Mg/L)
Std.
Dev.
(M9/L)
Rel.
Std.
Dev.
(*)
Mean
Method
Accuracy
(% True
Conc'n)
20
18.8
1.01
5.4
93.8
200 ml sample volume, standards taken through the LSE procedure.
26
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Table 5. Accuracy and Precision Data for Eight Determinations of the Method
Analyte Using a 25 mm C-18 Teflon Enmeshed Extraction (Filter) Disk.
True Conc'n Mean Std. Rel. Mean Method
(ng/L) Observed Dev. Std. Accuracy
Cone. Dev. (% True Conc'n)
(ng/L) (ng/L) (%)
50 52.7 3.79 7.2 105.
200 ml sample volume, standards taken through the LSE procedure.
27
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APPENDIX 1. LIQUID-SOLID EXTRACTION PROCEDURE
(a) Adjust a typical 100 mL sample volume to pH 4.5, containing 1-2% (V/V)
methanol. (Add 1-2 mL of methanol/100 mL of sample).
(b) Use 1 mL, 100 mg silica or polymer-based (C-18) column or Teflon enmeshed
extraction disk (C-18, C-8)
(c) Add 3 column volumes* (3 mL) of non-acidified ethyl acetate (do not allow
the column to become dry during additions of column conditioners and
before the sample is added). Note: After pre-conditioning the Teflon
enmeshed filter disk with ethyl acetate, maintain vacuum to pull air
through for 5 minutes (air-dry the disk). The disk is not allowed to go
dry with subsequent conditioning and sample application.
(d) Add 4 column volumes* (4 mL) of methanol, 2 column volumes (2 mL) of
deionized water, and 2 column volumes of pH 4.5 deionized water.
(e) Attach sample reservoir to coluinn. (If the LSE disk is used the
reservoir is attached prior to step C).
(f) Add sample solution and adjust the flow rate to approximately 5 mL/min.
(g) Following sample application, the LSE column and/or disk is air-dried
(room air) (@ 10 mm Hg) for at least 20 minutes.
(h) Place the LSE column and/or disk in a desiccator for at least one hour.
Note: Polymer-based LSE columns mustjremain in the desiccator overnight to
affect removal of all residual water."
(i) The analyte (TBT) is eluted with two 250 ML portions of ethyl acetate
(HC1) into calibrated GC screw cap glass sample vials. (Each portion of
ethyl acetate (HC1) remains in contact with the column for at least 30
seconds). Note: TBT is eluted (under vacuum) from polymer-based LSE
columns with three 250 /xL portions of ethyl acetate (HC1).
(j) The final volume of eluate (column extract) is adjusted to 0.5 mL with a
few drops of ethyl acetate (HC1).
(k) The sample vial is refrigerated overnight (4°C) to allow the extract
solution to equilibrate. NOTE: We obtained higher recoveries and
consistent reproducibility after refrigeration.
Column volumes are estimated/approximate. Solutions are dispensed
with a squeeze bottle.
It is essential that all residual water be removed from LSE columns
and disks prior to elution of TBT with acidified ethyl acetate. This
is an absolute necessity when using splitless injection.
28
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Figure 1. GC/ECD Chromatograms from Commercial C-18 Bonded
Porous Silica Columns:
(a),(b) Extracts from Two Pre-Conditioned Columns,
100 mg, 1 ml (Different Manufacturers)
Computer Imaging by Steven Wai trip and James Dryer
29
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LU
CO
O
CO
O
LU
IUI
14
28
TIME (min.)
30
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FIGURE 2. SCHEMATIC DIAGRAM OF SAMPLE FILTRATION APPARATUS
Stainless Steel
Screen and
Teflon Gasket
Rubber Stopper
To Vacuum
250 ml Reservoir
Clamp
1 Liter
Vacuum Flask
31
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Figure 3. Photographs of LSE Column Processor
(a) Vacuum Manifold Displaying the arrangement for
Column (Cartridge) and Disk Extractions
(b) Display of the Component Parts for Disk Extractions
Using Glass Filter Holder
Photos by James O'Oell
32
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(CO
cb)
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Figure 4. Photograph of Stainless Steel Filter Holder for 25 mm
Extraction (Filter) Disks
Photo by James O'Oell
34
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'hoto by James O'Dell
-------
Figure 5. GC/ECD Chromatograms of LSE Solutions from C-18 Disks
(a) Extract from Pre-conditioned Disk
(b) Extract from Laboratory Reagent Blank
(c) Extract from Seawater Containing 0.05 /ig/L
Tributyltin Chloride
Computer Imaging by Steven Wai trip and James Dryer
36
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CO
CO
o
14
TIME (min.)
28
37
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