5378
           DETERMINATION  OF MBTS  AND  TCMTB  IN  MUNICIPAL AND  INDUSTRIAL
                       WASTEWATERS  BY LIQUID CHROMATOGRAPHY
                                   METHOD  637

     1.   Scope  and  Application
         1.1  This  method covers  the  determination of MBTS and TCMTB
             pesticides.   The  following parameters can be determined by  this method.
             Parameter     ,                       CAS No.
             MBTS                                 120-78-5
             TCMTB                               21564-17-0
         1.2  This  is  a liquid  chromatographic  (LC) method applicable to  the deter-
             mination of the compounds listed  above in municipal and industrial -
             discharges  as provided  under  40  CFR 136.1.  Any modification of
             this  method beyond  those expressly permitted,  shall be considered a
             major modification  subject to application and  approval of alternate
             test  procedures under 40 CFR  136.4 and 136.5.
         1.3  The method  detection  limit (MDL,  defined in Section 14) for each
             parameter 1s listed 1n  Table  1-   The MOL for a specific wastewater
             wastewater  may differ from those listed, depending upon the nature
             of  interferences  1n the sample matrix.
         1.4  The sample  extraction and concentration steps  in this method are
             essentially the same  as in certain other 600 series methods.  Thus, a
             single sample may be  extracted to measure the  compounds included in
             the scope of the  methods.  When  cleanup is required, the  concen-
             tration  levels must be  high enough to permit selecting aliquots, as

                                            U.S.  Environmental Protection Agency
                                            Region V, Library
                                            230 South Dearborn Street
                                            Chicago,  Illinois   60604

-------
D,S. Environmental  Protection Agency

-------
         necessary,  1n  order to  apply appropriate cleanup procedures.
    1.5  This  method 1s restricted  to use by or under the supervision of
         analysts  experienced in the use of liquid chromatography and in the
         interpretation of liquid chromatograms.  Each~a&*Xyat  must demonstrate
         the ability to generate acceptable results with this method using
         the procedure  described in Section 8.2
    1.6  When this method 1s used to analyze unfamiliar samples for any or
         all of the compounds above, compound  identifications should be
         supported by at least one  additional  qualitative technique.  This
         method describes analytical conditions for a second liquid
         chromatographic column  that can be used to confirm measurements
         made with the  primary column.
2.  Summary of Method
    2.1  A  measured volume  of sample, approximately 1 liter, 1s solvent ex-
         tracted  with  methylene chloride using a  separatory funnel.   The
         methylene  chloride extract is dried and  concentrated to 5.0 ml.
         Liquid chromatographic  conditions  are described which  permit the
         separation and measurement of the  compounds  in  the extract  by high
         performance liquid chromatography  with ultraviolet detection.1
    2.2  This method provides a  silica  gel  column  cleanup  procedure  to  aid
         in the elimination of interferences which may be  encountered.
3.   Interferences
    3.1  Method Interferences may be caused by contaminants 1n  solvents,
         reagents, glassware, and other sample processing  apparatus that
         lead to discrete artifacts or elevated baselines  in liquid

-------
     chromatograms.   All  reagents  and  apparatus  must be  routinely
     demonstrated to be free from  Interferences  under  the  conditions of
     the  analysis by running laboratory  reagent  blanks as  described In
     Section  8.5.
     3.1.1  Glassware must  be scrupulously cleaned.2   Clean all
            glassware as soon as possible after  use by thoroughly
            rinsing  with the last  solvent used 1n  1t.  Follow by washing
            with hot water  and detergent and  thorough  rinsing with tap
            and  reagent water.  Drain  dry, and heat 1n an  oven or muffle
            furnace  at 400°C for 15  to 30 min.   Do not heat volumetric
            ware.  Some thermally  stable materials such  as PCBs may not be
            eliminated by this treatment.  Thorough rinsing with-acetone
            and  pesticide quality  hexane may  be  substituted for the
            heating.  After drying and cooling,  seal and store glassware
            In a clean environment to  prevent any  accumulation of dust
            or other contaminants.  Store inverted or  capped  with
            aluminum foil.
     3.1.2  The  use of high  purity reagents  and solvents  helps to mini-
            mize  Interference problems.  Purification of  solvents by
            distillation in  all-glass systems  may be required.
3.2  Matrix  Interferences may be caused by contaminants that are  coex-
     tracted from the sample.  The extent of matrix interferences will
     vary considerably from  source  to source,  depending upon the  nature
     and diversity of the industrial  complex  or municipality sampled.
     The cleanup  procedure in Section 11  can be used to overcome  many of
     these interferences, but unique  samples may  require  additional
     cleanup approaches to achieve  the MDL listed in Table  1.

-------
4.  Safety
    4.1  The toxidty or carcfnogenlclty of each reagent used 1n this method
         has not been precisely defined; however, each chemical  compound
         should be treated as a potential  health hazard.  From this
         viewpoint, exposure to these chemicals must be reduced  to the
         lowest possible level by whatever means available.   The laboratory
         1s responsible for maintaining a current awareness  file of  OSHA
         regulations regarding the safe handling of the chemicals specified
         1n this method.  A reference file of material  data  handling sheets
         should also be made available to all  personnel  Involved 1n  the
         chemical analysis.  Additional references to laboratory safety  are
         available and have been identified3^ for the  Information of the
         analyst.
5.  Apparatus and Materials
    5.1  Sampling equipment, for discrete or composite sampling.
         5.1.1  Grab sample bottle - Amber borosllicate or flint glass,
                1-liter or 1-quart volume, fitted with screw caps lined  with
                Teflon.  Foil may be substituted for Teflon  if the sample  is
                       f
                not corrosive.  If amber bottles are not available,  protect
                samples from light.  The container and cap liner must be
                washed, rinsed with acetone or methylene chloride, and dried
                before use to minimize contamination.
         5.1.2  Automatic sampler (optional) - Must Incorporate  glass sample
                containers for the collection of a minimum of 250 ml.
                Sample containers must be kept refrigerated  at 4°C and
                protected from light during compositing.  If the sampler

-------
            uses a peristaltic pump,  a  minimum  length of compressible
            slUcone rubber tubing may  be used.  Before use,  however,
            the compressible tubing should be thoroughly rinsed with
            methanol, followed by repeated rinsings  with distilled water
            to minimize the potential-for contamination of the sample.
            An Integrating flow meter 1s required to collect  flow
            proportional  composites.
5.2  Glassware (All specifications are  suggested.  Catalog numbers are
     Included for Illustration only.)
     5.2.1  Separatory funnel - 2000-mL, with Teflon stopcock.
     5.2.2  Drying column - Chromatographic column 400 mm long x 10 mm
            ID with'coarse frit.
    '5.2.3  Chromatographic column -  400 mm long  x 19 mm ID with 250 ml
            reservoir at the top and  Teflon stopcock (Kontes  K-420290 or
            equivalent).
     5.2.4  Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
            K-570050-1025 or equivalent).   Calibration must be checked
            at the volumes employed In  the  test.   A  ground glass stopper
            Is  used to prevent evaporation  of extracts.
     5.2.5  Evaporative flask, Kuderna-Danish - 500-mL (Kontes
            K-570001-0500 or equivalent).   Attach to concentrator tube
            with springs.
     5.2.6  Snyder column, Kuderna-Danish - three-ball  macro  (Kontes
            K-503000-0121 or equivalent).

-------
         5.2.7  Vials - Amber glass, 10 to 15 mL capacity with Teflon  lined
                screw-cap.
         5.2.8  Erlenmeyer flask - 250-mL
         5.2.9  Graduated cylinder - 1000-mL
         5.2.10 Volumetric flasks - 5 ml, 10 ml
    5.3  Boiling chips - approximately 10/40 mesh carborundum.  Heat at
         400°C for 4 hours or Soxhlet extract with methylene chloride.
    5.4  Water bath - Heated, capable of temperature control (+2°C).  The
         bath should be used 1n a hood.
    5.5  Balance - Analytical, capable of accurately weighing to the nearest
         0.0001 g.
    5.6  Liquid chromatograph - Analytical system complete with liquid
         chromatograph suitable for on-column injection and all required
         accessories including syringes, analyical columns, detector,    ~
         and strip-chart recorder.  A data system is recommended for
         measuring peak areas.
         5.6.1 Column 1 - 5 micron Dupont Zorbax-CN, 250 mm long x 4.6 mm ID
               or equivalent.  This column was used to develop the
               method performance statements in Section 14.   Alternate
               columns may be used in accordance with the provisions
               described in Section 12.1.
         5.6.2 Column 2-5 micron Dupont Zorbax Silica, 250 mm long x 4.6 mm
               ID or equivalent.
6.  Reagents
    6.1  Reagent water - Reagent water is defined as a water in which
         an interferent is not observed at the method detection limit
         of each parameter of interest.

-------
 6.2  Methylene chloride,  methanol,  ethyl  ether,  and  hexane
      distilled-in-glass  quality of  equivalent.   Ethyl  ether must
      be free  of peroxides as  indicated  by EM Quant Test Strips (available
      from Scientific Products Co.,  Catalog  No. P1126-8 and other
      suppliers).  Procedures  recommended  for removal of peroxides are
      provided with the test strips.
 6.3  Sodium sulfate (ACS) Granular, anhydrous; heated  in a muffle
      furnace  at 400°C overnight.
6.4  Silica gel, Davison  Grade 923,  100-120  mesh, dried for
     12 hours at 150°C.
6.5  W Sodium hydroxide.  Dissolve  4.0 grams  of NaOH (ACS)  in
     100 ml of distilled water.
6.6  ™ Sulfuric acid.  Slowly add 2.8 ml of cone.  H2S04  (94%) to
     about 50 ml of distilled water.  Dilute to 100 ml  with  distilled
     water
6.7  Sodium phosphate, monobasic, ACS grade.
6.8  Sodium phosphate, dibasic, ACS  grade.
6.9  Stock standard solutions  (1.00  yg/uL) - Stock  standard  solutions
     can  be prepared  from pure standard materials or purchased as
     certified  solutions.
     6.9.1 Prepare  stock  standard solutions by accurately weighing about
           0.0100 grams of pure material.  Dissolve the material  in
           distilled-in-glass quality methylene chloride and dilute
           to volume  in a 10-mL volumetric flask.  Larger volumes can
           be used  at the convenience of the analyst.  If compound purity
           Is certified at 96% or greater, the weight can be used without
           correction to  calculate the concentration of the stock standard.
           Commercially prepared stock standards can be used at any
           concentration  if they are certified by the manufacturer or by
           an independent source.

-------
         6.9.2  Transfer the stock standard solutions Into Teflon-sealed
                screw-cap bottles.  Store at 4°C and protect from light.
                Frequently check standard solutions for signs of degradation
                or evaporation, especially just prior to preparing
                calibration standards from them.
         6.9.3  Stock standard solutions must be replaced after six months
                or sooner 1f comparison with check standards indicates  a
                problem.
                        •
7.  Calibration
    7.1   Establish liquid chromatographic operating parameters equivalent to
         those indicated in Table 1.   The liquid chromatographic system
         can be calibrated using the  external  standard technique (Section 7.2)
         or the internal standard technique (Section 7.3).                    _
    7.2  External standard calibration procedure:
         7.2.1  For each compound of  interest, prepare calibration standards
                at a minimum of three concentration levels  by adding volumes
                of one or more stock  standards to a volumetric flask and
                diluting to volume with methylene chloride.  One of the external
                standards should be at a concentration near, but above, the
                method detection limit.   The other concentrations should
                correspond to the range  of concentrations expected in the
                sample concentrates or should define the working range  of
                the detector.
         7.2.2  Using injections of 5 to 20 yL of each calibration standard,
                tabulate peak height  or  area responses against the mass
                Injected.  The results can be used to prepare a calibration
                curve for each compound.   Alternatively, the ratio of  the
                response to the mass  injected, defined as the calibration

-------
            factor (CF), can be calculated- for each  compound  at each
            standard concentration.   If the relative standard deviation
            of the calibration  factor is less than 10% over the working
            range, the average calibration factor can be used in  place
            of a calibration curve.
     7.2.3  The working calibration  curve or calibration factor must  be
            verified on each working shift by the measurement of  one  or
            more calibration standards.  If the  response for  any
            compound varies from the predicted response by more than
            ±10%, the test must be repeated using a  fresh calibration
            standard.  Alternatively, a new calibration curve or
            calibration factor must  be prepared  for  that compound.
7.3  Internal  standard calibration procedure.  To use this approach,  the
     analyst must select one or more internal  standards similar in  .
     analytical behavior to the compounds of interest.  The analyst must  -
     further demonstrate that the measurement  of the internal standard
     Is not affected by method or matrix interferences.  Due  to these
     limitations, no internal standard applicable to all  samples  can  be
     suggested.
     7.3.1  Prepare calibration standards at a minimum of three
            concentration levels for each compound of interest by adding
            volumes of one or more stock standards to a volumetric
            flask.  To each calibration standard, add a known constant
            amount of one or more Internal  standards, and dilute  to
            volume with methylene chloride.  One  of  the standards should be at
            a concentration near, but above, the  method detection limit.
            The other concentrations should correspond to the range of
            concentrations expected  in the sample concentrates or should
            define the working  range of the detector.

-------
     7.3.2  Using Injections of 5 to 20 vL of each calibration standard,
            tabulate the peak height or area responses against the
            concentration for each compound and internal standard.
            Calculate response factors (RF) for each compound as
            follows:
                          RF « (AsC1s)/(A1sCs)
            where:
                 AS  * Response for the compound to be measured.
                 AJS » Response for the internal standard.
                 Cjs * Concentration of the internal  standard in ug/L.
                 Cj  • Concentration of the compound to be measured in
            If the RF value over the working range is constant, less
            than 10% relative standard deviation, the RF can be assumed
            to be Invariant and the average RF can be used for
            calculations.  Alternatively, the results can be used  to
            plot a calibration curve of response ratios, Aj/A^
            against RF.
     7.3.3  The working calibration curve or RF must be verified on each
            working shift by the measurement of one or more calibration
            standards.  If the response for any compound varies from the
            predicted response by more than +10%, the test must be
            repeated using a fresh calibration standard.  Alternatively,
            a new calibration curve must be prepared for that compound.
7.4  Before using any cleanup procedure, the analyst must process  a
     series of calibration standards through the procedure to validate

-------
    • elution patterns and the absence of interferences from the
     reagents.
Quality Control
8.1  Each laboratory using this method 1s required to operate a formal
     quality control program.  The minimum requirements of this program
     consist of an Initial demonstration of laboratory capability and
     the analysis of spiked samples as a continuing check on
     performance.  The laboratory 1s required to maintain performance
     records to define the quality of data that is generated.
     8.1.1  Before performing any analyses, the analyst must demonstrate
            the ability to generate acceptable accuracy and precision
            with this method.  This ability is established as described
            in Section 8.2.
     8.1.2  In recognition of the rapid advances occurring in chromato-
            graphy, the analyst 1s permitted certain options to improve
            the separations or lower the cost of measurements.  Each
            time such modifications to the method are made, the analyst
            1s required to repeat the procedure in Section 8.2.
     8.1.3  The laboratory must spike and analyze a minimum of 10% of
            all samples to monitor continuing laboratory performance.
            This procedure is described in Section 8.4.
8.2  To establish the ability to generate acceptable accuracy and
     precision, the analyst must perform the following operations.
     8.2.1  Select a representative spike concentration for each
            compound to be measured.  Using stock standards, prepare a

-------
            quality control check sample concentrate 1n methanol 1000
            times more concentrated than the selected concentrations.
     8.2.2  Using a pi pet, add 1.00 ml of the check sample concentrate
            to each of a minimum of four 1000-ml aliquots of reagent
            water.  A representative wastewater may be used in place of
            the reagent water, but one or more additional aliquots must
            be analyzed to determine background levels, and the spike
            level must exceed twice the background level  for the test to
            be valid.  Analyze the aliquots according to the method
            beginning in Section 10.
     8*2.3  Calculate the average percent recovery (R), and the standard
            deviation of the percent recovery (s), for the results.
            wastewater background corrections must be made before R and
            s calculations are performed.
     8.2.4  Using the appropriate data from Table 3, determine the
            recovery and single operator precision expected for the
            method, and compare these results to the values measured in
            Section 8.2.3.  If the data are not comparable, the analyst
            must review potential problem areas and repeat the test.
8.3  The analyst must calculate method performance criteria and define
     the performance of the laboratory for each spike concentration and
     parameter being measured.
     8.3.1  Calculate upper and lower control limits for method
            performance as follows:
                     Upper Control Limit (UCL) » R + 3 s
                     Lower Control Limit (LCL) » R - 3 s

-------
            where R and s are calculated as in Section 8.2.3.  The UCL
            and LCL can be used to construct control charts$ that are
            useful 1n observing trends 1n performance.
     8.3.2  The laboratory must develop and maintain separate accuracy
            statements of laboratory performance for wastewater samples.
            An accuracy statement for the method 1s defined as R_+ s.
            The accuracy statement should be developed by the analysis
            of four allquots of wastewater as described in Section
            8.2.2, followed by the calculation of R and s.  Alternately,
            the analyst may use four wastewater data points gathered
            through the requirement for continuing quality control in
            Section. 8.4.  The accuracy statements should be updated
            regularly. *>
8.4  The laboratory 1s required to collect 1n duplicate a portion of
     their samples to monitor spike recoveries.  The frequency of spiked
     sample analysis must be at least 10% of all samples or one sample
     per month, whichever is greater.  One aliquot of the sample must be
     spiked and analyzed as described in Section 8.2.  If the recovery
     for a particular compound does not fall within the control limits
     for method performance, the results reported for that compound in
     all samples processed as part of the same set must be qualified as
     described in Section 13.3.  The laboratory should monitor the
     frequency of data so qualified to ensure that it remains at or
     below 5%.
8.5  Before processing any samples, the analyst should demonstrate
     though the analysis of a 1-liter aliquot of reagent water that all

-------
         glassware and reagents Interferences are under control.  Each time
         a set of samples 1s extracted or there Is a change in reagents, a
         laboratory reagent blank should be processed as a safequard against
         laboratory contamination.
    8.6  It Is recommended that the laboratory adopt additional  quality
         assurance practices for use with this method.  The specific
         practices that are most productive depend upon the needs of the
         laboratory and the nature of the samples.  Field duplicates may be
         analyzed to monitor the precision of the sampling technique.  When
         doubt exists over the Identification of a peak on the chromatogram,
         confirmatory techniques such as gas chromatography with a
         dissimilar column, specific element detector, or mass spectrometer
         must be used.  Whenever possible, the laboratory should perform
         analysis of standard reference materials and participate in
         relevant performance evaluation studies.
9.  Samples Collection, Preservation, and Handling
    9.1  Grab samples must be collected in glass containers.   Conventional
         sampling practices7 should be followed; however, the  bottle must
         not be prerinsed with sample before collection.  Composite  samples
         should be collected in refrigerated glass containers  in accordance
         with the requirements of the program.  Automatic sampling equipment
         must be as free as possible of plastic and other potential  sources
         of contamination.
                                                                           *
    9.2  The samples must be iced or refrigerated at 4°C from  the time  of
         collection until extraction.

-------
    9.3  Adjust the pH of the sample to 6 to 8 with sodiun hydroxide or
         sulfurlc add Immediately after sampling.
10. Sample Extraction
    10.1 Mark the water meniscus on the side of the sample bottle  for later
         determination of sample volume.  Pour the entire sample into a
         2-liter separatory funnel.  Check the pH of the  sample  with wide
         range pH paper and adjust to 6 to 8 with 1  N sodium  hydroxide
         or 1 N sulfuric acid.   Dissolve 5 grams of monobasic sodium phosphate
         and 5 grams of dibasic sodium phosphate in the sample.
    10.2 Add 60 ml of methylene chloride to the sample bottle, seal,  and
         shake 30 seconds to rinse the inner walls.   Transfer the  solvent to
         the separatory funnel  and extract the sample by  shaking the.funnel
         for 2 min with periodic venting to release  excess  pressure.   Allovr •
         the organic layer to separate from the water phase for  a  minimum of
         10 min.  If the emulsion interface between  layers  is more than one
         third the volume of the solvent layer, the  analyst must employ
         mechanical  techniques  to complete the phase separation.   The
         optimum technique depends upon the sample,  but may include
         stirring, filtration of the emul'sion through  glass wool,  centrifu-
         gation, or other physical methods.  Collect the  methylene chloride
         extract in  a 250-mL Erlenmeyer flask.
    10.3 Add a second 60-mL volume of methylene chloride  to the  sample
         bottle and  repeat the  extraction procedure  a  second  time, combining
         the extracts in the Erlenmeyer flask.  Perform a third  extraction
         in the same manner.

-------
10.4 Assemble a Kuderna-Danlsh (K-D) concentrator by attaching a 10-mL
     concentrator tube to.a 500-mL evaporative flask.  Other concentra-
     tion devices or techniques may be used in place of the K-D 1f the
     requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
     10 on of anhydrous sodium sulfate, and collect the extract in the
     K-D concentrator.  Rinse the Erlenmeyer flask and column with 20 to
     30 ml of methylene chloride to complete the quantitative transfer.
     Once the flask rinse has passed through the drying column, rinse
     the column with 30 to 40 ml of methylene chloride.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
     a three-ball Snyder column.  Prewet the Snyder column by adding
     about 1 ml methylene chloride to the top.  Place the K-D apparatus
     on a hot water bath, 60 to 65°C, so that the concentrator tube is
     partially immersed in the hot water, and the entire lower rounded
     surface of the flask is bathed with hot vapor.  Adjust the vertical
     position of the apparatus and the water temperature as required to
     complete the concentration in IS to 20 min.  At the proper rate of
     distillation, the balls of the column will actively chatter but the
     chambers will not flood with condensed solvent.  When the apparent
     volume of liquid reaches 1 ml, remove the K-D apparatus and allow
     it to drain and cool for at least 10 min.  If the sample
     extract requires no cleanup proceed with Section 10.7.  If
     the sample extract requires cleanup, proceed to Section 11.
10.7 Remove the macro-Snyder column and rinse the flask and its lower
     joint into the concentrator tube with 1 to 2 ml of methylene
     chloride.  Adjust the volume of the extract to 5.0 ml

-------
         with methylene chloride.   Stopper the concentrator  tube and
         store refrigerated if further processing will  not be  performed
         immediately.   If the extract is to be stored  longer than two
         days, transfer the extract to a screw capped  vial with a Teflon-
         lined cap.   If the sample extract requires  no further cleanup,
         proceed with the liquid chromatographic analysis in Section 12,
         If the sample requires cleanup, proceed to  Section  11.
    10.8 Determine the original sample volume by refilling the sample
         bottle to the mark and transferring the water to a  IQQO-mL
         graduated cylinder.  Record the sample volume to the  nearest
         5 ml.
11. Cleanup and Separation
    11.1 Cleanup procedures may not be necessary for a relatively clean
         sample matrix.  The cleanup procedure recommended in  this method
         has been used for the analysis of various clean waters and
         industrial  effluents.  If particular circumstances  demand the
         use of an alternative cleanup procedure, the  analyst  must
         determine the elution profile and demonstrate that  the recovery
         of each compound of interest is no less than  the recovery values
         reported in Table 2.
    11.2 The following silica gel  column cleanup procedure has been
         demonstrated to be applicable to the pesticides listed in
         Table 1.
         11.2.1 Add 10 g of silica gel to 100 ml of  ethyl ether and
                600 yL or reagent  water "in a250-mL Erlenmeyer  flask.
                Shake vigorously for 15 minutes.  Transfer the slurry to a

-------
                chromatographic column (silica gel may be retained with a plug
                of glass wool).  Allow the solvent to elute from the
                column until the silica gel is almost exposed to the air.
                Wash the column with 100 ml of 50% hexane in methylene
                chloride as before and discard.  Use a column flow of 2 to
                2.5 mL/min throughout the wash and elution profiles.
         11.2-2 Quantitatively add the sample extract from Section 10.8 to
                the head of the column.  Allow the solvent to elute from
                the column until the silica gel is almost exposed to the
                air.  Elute the column with 50 ml of 50% hexane in
                methylene chloride.   Discard this fraction.
         11.2.3 Elute the column with 50 ml of methylene chloride
                (Fraction 1) and collect eluate in a K-D apparatus.  Repeat
                process with 50 ml of 6% ethyl ether in methylene  chloride
                (Fraction 2).  The TCMTB elutes in Fraction 1 and  the MBTS
                elutes in Fraction 2.  Concentrate each fraction to 5.0 ml
                as described in Sections 10.6 and 10.7.  Proceed with
                liquid chromatographic analysis.
         11.2.4 The above-mentioned fractions can be combined before
                concentration at the discretion of the analyst.
12.  Liquid Chromatography
     12.1 Table 1  summarizes the recommended operating conditions for the
          liquid chromatograph.  Included in this table are estimated
          retention times and method detection limits that can be achieved
          by this  method.  Examples  of the separations achieved by Column 1
                                      ?••*

-------
     and Column 2 are shown in Figures 1, 2 and 3.  Other columns,
     chromatographic conditions, or detectors may be used if the
     requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described
     in Section 7.
12.3 If the Internal standard approach 1s being used, the analyst must not
     add the internal standard to the sample extracts until  immediately
     before Injection Into the Instrument.  Mix thoroughly.
12.4 Inject 5 to 20 yL of the sample extract by completely filling
     the sample value loop.  Record the resulting peak sizes in
     areas of peak height units.
12.5 The width of the retention time window used to make identifications
     should be based upon measurements of actual retention time
     variations of standards over the course of a day.   Three times  the
     standard deviation of a retention time for a compound can be used
     to calculate a suggested window size; however, the experience of
     the analyst should weigh heavily 1n the interpretation  of
     chromatograms.
12.6 If the response for the peak exceeds the working range  of the
     system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response Is prevented by the
     presence of Interferences, further cleanup is required.

-------
13. Calculations
    13.1 Determine the concentration of Individual  compounds in the sample.
         13.1.1 If the external standard calibration procedure 1s used,
                calculate the amount of material  Injected from the peak
                response using the calibration curve or calibration factor
                1n Section 7.2.2.  The concentration In the sample can be
                calculated as follows:
                                            (A)(Vt)
                     Concentration, yg/L »	—

                where:
                   A  * Amount of material  Injected, in nanograms.
                   V-f * Volume of extract Injected in uL.
                   Vt » Volume of total  extract in  VL.
                   Vs * Volume of water extracted In ml.
         13.1.2 If the Internal standard calibration procedure was used,
                calculate the concentration in the sample using the response
                factor (RF) determined in Section 7.3.2 as follows:
                                              (AS)(is)
                     Concentration, pg/L »	—	
                                            (Ais)(RF)(V0)
                where:
                   As  » Response for the compound  to be measured.
                   Ajs » Response for the internal  standard.
                   Is  * Amount of Internal  standard added to  each
                         extract in pg.
                   V0  * Volume of water extracted, in liters.

-------
    13.2 Report results  1n  nrfcrograms  per  liter without correction for
         recovery data.   When  duplicate and spiked samples are analyzed,
         report all  data  obtained with the sample results.
    13.3 For  samples  processed as part of a set where the laboratory spiked
         sample recovery  falls outside of the control limits 1n Section 8.3,
         data for the affected compounds must be labeled as suspect.
14. Method Performance
    14.1 The  method  detection  limit (MDL)  Is defined as the minimum concen-
         tration of  a substance  that can be measured and  reported with 99%
         confidence  that  the value  1s  above zero.    The  MDL concentrations
         listed In Table 1  were  obtained using  reagent water.1  Similar
         results were achieved using representative wastewaters.
    14.2 This method has  been  tested for linearity of recovery from spiked
         reagent water and  has been demonstrated to be applicable over the
         concentration  range from  10 x MOL to 1000 x MDL.
    14.3 In a single laboratory, Battelle  Columbus Laboratories, using
         spiked wastewater  samples, the average recoveries presented 1n
         Table 2 were obtained after silica gel cleanup.   Seven replicates of
         each of two different wastewaters were spiked and analyzed.  The
         standard deviation of the  percent recovery Is also included In
         Table 2.1

-------
                                 REFERENCES
1.   "Development of Methods for Pesticides 1n Wastewaters," Report for EPA
     Contract 68-03-2956 (In preparation).

2.   ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
     Preparation of Sample Containers and for Preservation," American
     Society for Testing and Materials, Philadelphia, PA, p. 679,  1980.

3.   "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, August, 1977.

4.   "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
     Occupational Safety and Health Administration, OSHA 2206 (Revised,
     January 1976).

5.   "Safety 1n Academic Chemistry Laboratories,"  American Chemical Society
     Publication, Committee on Chemical Safety, 3rd Edition, 1979.

6.   "Handbook for Analytical Quality Control  in Water and Wastewater
     Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
     Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
     45268, March 1979.

7.   ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
     Sampling Water," American Society for Testing and Materials,
     Philadelphia, PA, p. 76, 1980.

8.  Glaser, J. A. et al, "Trace Analysis for Wastewaters,"  Environmental
    Science and Technology. 15,  1426 (1981).

-------
TABLE 1.  CHROMATOGRAPHIC CONDITIONS AND ESTIMATED DETECTION LIMITS
Parameter
MBTS
TCMTB
Retention
Column 1
6.6
9.3
Time (Min.)
Column 2
6.3
7.9
MDL
(wg/L)
0.5
1.0
    Column 1 conditions: Dupont Zorbax-CN, 5 micron, 250 x 4.6 mm;
    1 mL/min flow; 15/85 methylene chloride/hexane.

    Column 2 conditions: Dupont Zorbax silica, 5 micron, 250 x 4.6 mm;
    1 mL/min flow; 90/9.5/0.5 hexane/methylene chloride/methane!.

-------
SB
o
1-4
en
M
u
u
a
i
u
CJ
ofi
o
s
u

u
2

cn
w
          o«

          s2  S


          3  0)
         •gj
          (O ">
            0
          c  &.
             ,
         CO  H
             
                                           •o
                                            01
                                                §
               0)   01

                    0)
               Ut   O)
               e   
               •r-       O
               •O  ^-   O)
               C   (O   -M
               o   CL  a>
               U  t-   T3
                                           5   ^
                                                     a>

-------
r o
     •

   in
•  in


   CO
r  o

   cJ

h  *-
 •  o
   in
I
r

r
 • o
     *

   co

         i
o




03
                OO
                u_
                o
LU



Z


O



u.

o
                o
                cc
                  I
                 o

-------
  o
    *

  LfS
  10
    •

  co
   o
    •

   CM
  o

  CP>
o
o
         E      u_
   If)
  o
•  in
                   i

                  _i
                  a.
                  CVJ
                  C9
   o
    •

   co

-------
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago,  Illinois  60604

-------
   o
    •

   in

   r—•






   in
     •

   CO
<•  o
     •

   CM
   in
                cxi
[>'    i
         o
   o   -
                 CO
                 O


                 C9
                 O
                 o
   in
o

5

   in
                 ro


                 LU

                 C£.


                 cs
                 ^^

                 u.
   o
     •

   CO
   IT)

-------