ALLOWAY METHOD OUTLINE Standard Laboratory Method: SM Parameter: Methylene Blue Method: Colorimetric Reporting Level: Reference: 0.05 mg/l Standard Methods for the Examination Of Water and Wastewater; Method 5540C-2000 Originator: Section Supervisor: QA Manager Date: Date: Date: Page 1 of 8
1.0 Scope and Application: METHYLENE BLUE ACTIVE SUBSTANCES 1.1 This method is applicable to the measurement of methylene blue active substances in drinking waters, surface waters, domestic and industrial wastes. It is not applicable to measurement of surfactant-type materials in saline waters. 1.2 It is not possible to differentiate between linear alkyl sulfonate (LAS) and alkyl benzene sulfonate (ABS) or other isomers of these types of compounds. However, LAS has essentially replaced ABS on the surfactant market so that measurable surfactant materials will probably be LAS type materials. 1.3 The method is applicable over the range of 0.050 to 1.000 mg/l. Higher concentrations may be determined by appropriate sample dilution. 2.0 Summary of Method: 2.1 The dye, methylene blue, in aqueous solution reacts with anionic-type surface active materials to form a blue colored salt. The salt is extractable with chloroform and intensity of color produced is proportional to the concentration of MBAS. 3.0 Definitions 3.1 Accuracy-The degree of agreement between an observed value and an accepted reference value. Accuracy includes a combination of random error (precision) and systematic error (bias) components which are due to sampling and analytical operations; a data quality indicator. 3.2 Aliquot - A portion of the field sample to be processed through the analytical steps 3.3 Analytical Batch Composed of prepared environmental samples (extracts, digestates, or concentrates) which are analyzed together as a group. An analytical batch can include prepared samples originating from various environmental matrices. 3.4 Calibration To determine, by measurement or comparison with a standard, the correct value of each scale reading on a meter, instrument, or other device. The levels of the applied calibration standard must bracket the range of planned or expected sample measurements. 3.5 Calibration Curve The graphical relationship between the known values, such as concentrations, of a series of calibration standards and their instrument response. 3.6 Continuing Calibration Check - A check standard analyzed periodically to evaluate whether the analytical process remains in control. These results are compared to the initial calibration. Page 2 of 8
3.7 Duplicate Analyses- The analyses or measurements of the variable of interest performed identically on two sub-samples of the same sample. The results from duplicate analyses are used to evaluate analytical or measurement precision but not the precision of sampling, preservation or storage internal to the laboratory. 3.8 Homogeneous Sample - A sample which has an even distribution of material. 3.9 Instrument Detection Limit - The minimum concentration of an analyte that can be measured with 99% confidence that the analyte concentration is greater than zero. Determine the IDL with seven replicates of standard solution at the instrument with no specific analytical preparation. 3.10 Method Detection Limit - The minimum concentration of an analyte that can be measured with 99% confidence that the analyte concentration is greater than zero. Determine the MDL with seven replicates of standard solution taken through the preparative and analytical procedures. 3. Initial Calibration - A series of standards analyzed to produce a calibration curve in order to develop a linear system. 3.12 Laboratory Control Sample A sample matrix, free from the analytes of interest, spiked with verified known amounts of analytes from a source independent of the calibration standards or a material containing known and verified amounts of analytes. It is generally used to establish intra-laboratory or analyst/technician specific precision and bias or to assess the performance of all or a portion of the measurement system. 3.13 Matrix- The component or substrate that contains the analyte of interest. 3.14 Matrix Spike A sample prepared by adding a known mass of target analyte to a specified amount of matrix sample for which an independent estimate of target analyte is available. Matrix spikes are used, for example, to determine the effect of the matrix on a method s recovery efficiency. 3.15 Quality Assurance An integrated system of activities involving planning, quality control, quality assessment, reporting and quality improvement to ensure that a product or service meets defined standards of quality with a stated level of confidence. 3.16 Quality Control The overall system of technical activities whose purpose is to measure and control the quality of a product or service so that it meets the needs of users. 3.17 Reagent Blank or Method Blank A sample consisting of reagent(s), without the target analyte or sample matrix, introduced into the analytical procedure at the appropriate point and carried through all subsequent steps to determine the contribution of the reagents and of the involved analytical steps. 3.18 Reporting Limit - The practical quantitation level for an analyte, greater than the MDL, accounting for dilutions and matrix specific concerns. Page 3 of 8
3.19 Sample Duplicate Two samples taken from and representative of the same population and carried through all steps of the sampling and analytical procedures in an identical manner. Duplicate samples are used to assess variance of the total method including sampling and analysis. 4.0 Interferences: 5.0 Safety 4.1 Materials other than man-made surface active agents which react with methylene blue are inorganic ions such as nitrates and chlorides. However, the occurrence of these materials at interference levels is relatively rare and with the exception of chlorides may generally be disregarded. 4.2 Chlorides at concentration of about 1000 mg/l show a positive interference but the degree of interference has not been quantified. For this reason the method is not applicable to brine samples. 4.3 Naturally occurring organic materials that react with methylene blue are relatively insignificant. 5.1 The toxicity or carcinogenicity of each chemical and reagent used in this method has not been precisely defined. However, each one must be treated as a potential health hazard, and exposure to these chemicals should be minimized. Each analyst is responsible for adherence to the procedures outlined in the Chemical Hygiene Plan. Safety Data Sheets (SDS) are kept in the laboratory. 5.2 Some method analytes have been tentatively classified as known or suspected human or mammalian carcinogens. Pure standard materials and stock standard solutions of these compounds should be handled with suitable protection to skin, eyes, etc. 5.3 Safety glasses and lab coats must be worn when handling samples and solvents. Safety glasses must be worn when handling glassware. 6.0 Equipment and Supplies 6.1 Spectrophotometer for use at 652 nm, with a light path of 1 cm or longer. 6.2 Calibrated pipettors. 7.0 Reagents and Calibration Standards (Reference Reagent Traceability for all preparations.) 7.1 Methylene blue reagent 7.2 Surfactant stock solution-concentrated ampule available from ERA or equivalent at 1000 mg/l 7.2.1 Prepare a working standard of 25 mg/l from the stock standard (LCSA) Page 4 of 8
7.2.2 From the 25 mg/l standard, make the following standards every twelve (12) months (or whenever QC fails to meet established limits): 1.00: 2.0 ml of the 25 mg/l std to 50 ml 0.75: 1.5 ml of the 25 mg/l std to 50 ml 0.50: 1.0 ml of the 25 mg/l std to 50 ml 0.25: 0.5 ml of the 25 mg/l std to 50 ml 0.20: 0.4 ml of the 25 mg/l std to 50 ml 0.10: 0.2 ml of the 25 mg/l std to 50 ml 0.05: 0.1 ml of the 25 mg/l std to 50 ml 7.3 Chloroform - located flammable cabinet in supply room. 7.4.1 CAUTION: Chloroform vapors are toxic. Take appropriate precautions against inhalation. 7.4 NaOH-10N 7.5 H 2 SO 4-30 % 7.6 Surfactant Wash Solution 8.0 Sample Collection, Preservation, and Handling 8.1 Sample can be collected in a plastic or glass container. 8.2 Samples should be preserved by refrigeration at 0.5 C - 6 C. 8.3 The holding time is 48 hours from time of collection. The analysts must document the time of analysis on the analytical data sheet to prove the holding time was met. 9.0 Calibration and Standardization 9.1 A calibration curve of at least 5 points is entered and stored in a linear program in Excel. The correlation coefficient must be 0.995 or better. 9.2 All samples and quality control are compared to this initial curve. 10.0 Quality Control: 10.1 An Initial Calibration Blank (ICB) and a second source Initial Calibration Verification Standard (ICV) must be analyzed prior to sample analysis. The ICB must read less than the reporting limit and the ICV must read +/- 10% of the true value for the analysis to continue. 10.2 A Reporting Limit Check Standard (RDLCHK) must be analyzed for every batch of 20 samples and recovery must be +/- 25% of the true value. If recovery falls outside this limit, a new curve must be prepared. Page 5 of 8
10.3 One sample of every ten must be analyzed in duplicate and should read within 20% RPD. If the duplicate results are greater than 5 times the reporting limit and greater than 20% RPD, the sample must be flagged with a comment on the report. 10.4 One sample of every ten must be spiked with a known amount of standard. The spike should read +/- 25% of the true value. If the spike falls outside the acceptance range the sample must be flagged with a comment on the report. 10.5 A Continuing Calibration Blank (CCB) and a second source Continuing Calibration Verification Standard (CCV) must be analyzed after every ten samples and at the end of each analytical run. The CCB must read less than the reporting limit and the CCV must read +/- 10% of the true value..0 ANALYTICAL PROCEDURES.1 Clean with chromic acid, tap, and distilled water. 4-125 ml Separatory funnels 4-125 ml Beakers * 1-400 ml Beaker * 2-25 ml Volumetric flasks * 3 - Spectrophotometer cells 1 - Glass funnel small enough to use in 25 ml Volumetric flask * These items should be completely dried in the oven..2 From LAS standard prepare the ICV and RDLCHK standards.3 Add 50 ml of sample and standards to appropriately labeled separatory funnels..4 Add 1 drop of phenolphthalein indicator..5 Add 10 N. NaOH dropwise until pink..6 Add 30% H 2 SO 4 dropwise until colorless..7 Add 10 ml of methylene blue reagent and 5 ml chloroform..8 Shake for 30 seconds..9 Withdraw chloroform into clean beaker..10 Extract sample with two more 5 ml portions of chloroform, shaking for 30 seconds. Page 6 of 8
. Discard contents of funnel (water layer) and rinse funnels with distilled water..12 Pour chloroform extracts back into funnel..13 Rinse beaker with 2 to 3 ml of chloroform and pour into separatory funnel..14 Add 25 ml of wash solution to funnel and shake vigorously for 30 seconds..15 After layers have separated and settling has taken place, drain chloroform into beaker..15 Filter chloroform through phase separation paper..16 Dilute filtrate up to 25 ml with chloroform..17 Transfer to 1/2 inch tubes..18 Read on spectrophotometer at 652 nm. 12.0 Calculations and Quantitative Analysis 12.1 Concentrated readout x dilution factor = mg/l MBAS. Sample absorbance is compared to the prepared calibration curve of known standards to Determine concentration of MBAS. 13.0 Data Assessment 13.1 The primary analyst bears the responsibility of producing accurate data. A documented review is performed that includes a check of the following parameters: Calibration verification, QC criteria, calculation checks, Data entry into the LIMS 13.2 The initial data review is documented on the worksheet or benchsheet and dated by the primary analyst. An individual trained in the analytical procedure must perform a secondary review. 13.3 Data qualifiers are used whenever deviations occur while analyzing the samples. The qualifiers are included on the Certificate of Analysis that is presented to the client. Qualifiers include: samples that were not preserved, failed LFM recovery, qualified due to LRB contamination, estimated or elevated reporting limit due to sample matrix interference, etc. 14.0 Report Generation 14.1 A Certificate of Analysis is generated after each analysis run is complete. The report includes the EPA Method Number, sample identification, date and time sampled, date received, date reported, quantitative result, analysis date, and analyst initials. Page 7 of 8
15.0 Waste Management and Pollution Prevention 15.1 Samples are disposed of according to the SOP on Sample Disposal. For those samples which are deemed to be hazardous materials, these must be lab packed and removed by a third party firm. 15.2 For information about pollution prevention that may be applicable to laboratory practices consult "Less is Better for Laboratory Chemical Management for Waste Reduction" available from the American Chemical Society Department of Government Relations and Science Policy. Address: 55 16th St. N.W. Washington, DC 20036. 16.0 Reference: 16.1 Standard Methods for the Examination of Water and Wastewater; Method 5540C-2000 17.0 Revision history: 17.1 Revision 9 issued to revise to Alloway formatting, to add reference to Reagent Traceability, added Reporting Limit Check QC requirement, and instructions on use of wash solution. 17.2 Revision 10 issued to update method number reference on cover page and in reference section, changed MSDS to SDS, modified section 8 for preservation temperature and requirement to document time of analysis. 17.3 Revision issued to update section 7.2.2 to remove the 2.00 and 1.50 standards and to change 6 months to 12 months in same section. Page 8 of 8