5380       .                          __.
                             BY LIQUID CHROMATOGRAPHY
                                    METHOD 640
         Scope and Application
         1.1  This method covers the determination of mercaptobenzothiazole.
              The following parameter can be determined by this method:
              Parameter                            CAS No.
              Mercaptobenzothiazole               149-30-4
         1.2  This is a high performance liquid chromatographic (HPLC)
              method applicable to the determination of the compound  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 is listed in Table 1.  The MDL for a specific
                                            U.S. Environmental Protection Agency
                                            Region V, library
                                            230 South Dearborn Street
                                            Chic?*o, Illinois  60604

U.S. Environmental Protection Agency

         wastewater may differ from those listed, depending upon the nature
         of Interferences in 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
         necessary, in order to apply appropriate cleanup procedures.
    1.5  This method is restricted to use by or under the supervision of
         analysts experienced in the use of liquid chromatoqraoh.y and in the
         interpretation of liquid chromatograms.  Each analyst must demonstrate
         the ability to generate acceptable results with  this method using
         the procedure described in Section 8.2.
    1.6  When this method is 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, is solvent ex-
         tracted with methylene chloride using a separatory funnel.  The
         methylene chloride extract is dried and concentrated to 1.0 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.
    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 in solvents,
         reagents, glassware, and other sample processing apparatus that
         lead to discrete artifacts or elevated baseline 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 it.  Follow by washing
                with hot water and detergent and thorough rinsing with tap
                and reagent water.  Drain dry, and heat in 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 2.
4.  Safety
    4.1  The toxicity or carcinogenicity of each reagent used in 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
         is responsible for maintaining a current awareness file of OSHA
         regulations regarding the safe handling of the chemicals specified
         in this method.  A reference file of material data handling sheets
         should also be made available to all  personnel involved in the
         chemical analysis.  Additional references to laboratory safety are
         available and have been identified3-5 for the information of the
5.  Apparatus and Materials
    5.1  Sampling equipment, for discrete or composite sampling.

     5.1.1  Grab sample bottle - Amber boroslllcate or flint glass,
            1-Hter or 1-quart volume, fitted with  screw caps lined  with
            Teflon.  Foil may be substituted for Teflon if the sample is
            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
            silicone 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 is 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

     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
            1s 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  Snyder column, Kuderna-Danish - two-ball micro (Kontes
            K-569001-0219 or equivalent).
     5.2.8  Vials - Amber glass, 10 to  15 ml capacity with Teflon lined
     5.2.9  Erlenmeyer flask -  250-mL
     5.2.10 Graduated cylinder  - 1000-mL
     5.2.11 Volumetric flask -  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 in 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, analytical  columns,  detectors,
     and strip-chart recorder.   A data  system  is recommended  for
     measuring peak areas.

         5.6.1   Column 1  - Spherisorb-ODS,  5  micron,  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 - Lichrosorb RP-2, 5 micron,  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, acetonitrile,  ethyl  ether, and
         acetone distilled-in-glass quality or  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, 100-200 mesh,  dried for 12  hours
         at 150eC.
    6.5  IN. Sodium hydroxide.  Dissolve 4.0 grams of sodium hydroxide
         in 100 ml of distilled water.
    6.6  IN^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  ug/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 1n
                 distilled-1n-glass quality methanol and dilute to volume  in
                 a 10-mL  volumetric flask.  Larger volumes can be used at the
                 convenience of the analyst.   If compound  purity 1s 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 if comparison with  check standards  indicates  a
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 methanol.  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 injection 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
     1s not affected by method or matrix Interferences.  Due to these
     limitations, no Internal standard applicable to all samples can be
     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 methanol.   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 wL 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
                 AS  * Response for the compound to be measured.
                 AjS - Response for the internal standard.
                 Cfs " Concentration of the internal standard in yg/L.
                 GS  * 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, AS/A-JS
            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
8.  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 Is 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 1s established as described
                1n 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
                Is required to repeat the procedure 1n 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 1s described 1n 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 In 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.
            Uastewater background corrections must be made before R and
            s calculations are performed.
     8.2.4  Using the appropriate data from Table 2, 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 charts6 that are
            useful in observing trends in 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 aliquots 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
8.4  The laboratory 1s required to collect in 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 1s 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 1t 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 safeguard against
         laboratory contamination.
    8.6  It 1s 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 sodium hydroxide or
         sulfuric acid 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.  Allow
         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 emulsion 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 methyl ene 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-Danish (K-D) concentrator by attaching a 25-mL
     concentrator tube to a 500-ml evaporative flask.  Other concentra-
     tion devices or techniques may be used in place of the K-D if the
     requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
     10 cm 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 methyl ene 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 1s
     partially Immersed in the hot water, and the entire lower rounded
     surface of the flask 1s bathed with hot vapor.  Adjust the vertical
     position of the apparatus and the water temperature as required to
     complete the concentration in 15 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.
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 methyl ene
     chloride.  Add 1 or 2 clean boiling chips and attach a two-ball
     micro-Snyder column to the concentrator tube.  Prewet the

           micro-Snyder column with methylene chloride and concentrate the
           solvent extract as before.   When an apparent volume  of 0.5 ml is
           reached, or the solution stops boiling, remove the K-D apparatus
           and allow it to drain and cool for 10 minutes.
      10.8 Remove the micro-Snyder column and adjust the volume of the extract
           to 1.0 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 Section 10.9.  If the sample requires cleanup,  proceed to
           Section 11.
     10.9  Add 1 or 2 clean boiling chips to the concentrator tube along
           with 10-ml of methanol.  Attach a two-ball micro-Snyder column and
           prewet the micro-Snyder column with about 1 ml of methanol.  Concentrate
           the solvent extract as before to an apparent volume of 2 ml
           and allow it to drain and cool for 10 minutes.  Transfer the solvent
           extract to a 5 ml volumetric flask and dilute to the mark with methanol.
           Proceed with the liquid chromatographic analysis in  Section 12.
     10.10 Determine the original sample volume by refilling  the sample bottle
           to the mark and transferring the water to a 1000-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 not less than 85%.
11.2  The following silica gel  column cleanup procedure has  been demon-
      strated to be applicable to mercaptobenzothiazole.
      11.2.1 Add 10 g of silica gel  to 100 ml of ethyl  ether and 600 uL of
             reagent water in a 250-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
             methylene chloride.  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 thesolvent  to elute  from-the
             column until  the silica gel  is almost exposed to the air.
             Elute the column with 50 ml  of methylene chloride.   Discard
             this fraction.
      11.2.3 Elute the column with 50 ml  of 6%  acetone  in methylene  chloride
             and collect eluate in a K-D  apparatus.   Concentrate this
             fraction to 1 nt as described in Sections  10.6  and  10.7.
             Exchange solvent with methanol  as  described  in  Section  10.9
             and proceed with liquid chromatographic  analysis.

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 and 2.  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 is 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.   Record the
           resulting peak  sizes  in area or 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 In the Interpretation of
    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 1s required.
13. Calculations
    13.1 Determine the concentration of Individual  compounds in the sample.
         13.1.1 If the external standard calibration procedure is used,
                calculate the amount of material  injected from the peak
                response using the calibration curve or calibration factor
                in Section 7.2.2.  The concentration in the sample can be
                calculated as follows:
                     Concentration, yg/L
                   A  • Amount of material  injected, in nanograms.
                   V-j  « Volume of extract Injected in uL.
                   Vt  " Volume of total  extract in uL.
                   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:
                     Concentration, ug/L

                   As  * Response for the compound to be measured.
                   AJS « Response for the Internal standard.
                   Is  » Amount of Internal  standard added to each
                         extract 1n yg.
                   V0  - Volume of water extracted, 1n liters.
    13.2 Report results 1n mlcrograms 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  in Section 8.3,
         data for the affected compounds must be labeled as suspect.
14. Method Performance
    14.1 The method detection limit (HDL) is defined as the minimum concen-
         tration of a substance that can be  measured and reported with 99%
         confidence that the value is above  zero.11  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 5 to 1000 ug/L.
    14.3 In a single laboratory, Battelle Columbus Laboratories, using
         spiked wastewater samples, the average recoveries presented  in
         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

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 In 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.


                          Retention Time (Min)       MDL
	Parameter	Column 1     Column 2     (ug/L)

Mercaptobenzothiazole        8.4          9.5        1.7
Column 1 conditions:  Spherisorb-ODS, 5 micron, 250 x 4.6 mm;
1 mL/min flow; 50/50 acetonitrile/water.

Column 2 conditions:  Lichrosorb RP-2, 5 micron, 250 x 4.6 mm;
1 mL/min flow; 10/90 acetonitrile/water.

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