Troubleshooting Chemistry Issues for Colorimetric Analysis 22nd Annual Good Laboratory Practice Technical Conference 2016 Sarah Leibenguth Technical Support Chemist Topics for Discussion Colorimetric Methods • Ortho-Phosphate and Total Phosphorous • Silica • Ammonia and TKN • Nitrate and Nitrite Topics for Discussion • Method Principle • Digestion Protocol • Reagents • Interferences • pH and Matrix Matching • Troubleshooting Examples Ortho-Phosphate and Total Phosphorous Ortho-Phosphate Total Phosphorous Methods • Ortho-Phosphate • EPA 365.1, version 2 (1993) • Standard Methods 4500-P F (18th,19th,20th) • Total Phosphorus • EPA 365.1, version 2 (1993) • Standard Methods 4500-P B, F (18th,19th,20th) • Total Kjeldahl Phosphorus • EPA 365.4 Ortho-Phosphate Total Phosphorous Method Principle • TP and oP Colorimetric Reaction • Ammonium Molybdate • Orthophosphate + Molybdate → Phosphomolybdic Acid • Antimony Potassium Tartrate • Catalyst for Phosphomolybdic Acid • Ascorbic Acid • Phosphomolybdic Acid + Ascorbic Acid → Blue Phosphomolybdic Complex Ortho-Phosphate Total Phosphorous Total Phosphorous Digestion • Polyphosphates and Organic Phosphorus are Converted to Orthophosphate • Kjeldahl Digestions • Block Digestor • Persulfate Digestions • Hot Plate or Block Digestor • Sample Reconstitution • Persulfate Digestions • Autoclave Ortho-Phosphate Total Phosphorous Reagents • Ammonium Molybdate • Remake Monthly • Refrigerated and Stored in Plastic • Prone to Precipitate and Turbidity • Dry Chemical White in Appearance • Sulfuric Acid • Stored Indefinitely at Room Temperature • Antimony Potassium Tartrate • Remake Monthly for Best Results • Refrigerated and Stored in Glass Ortho-Phosphate Total Phosphorous Reagents • Ascorbic Acid Reagent • Stable 1 Week • Stored in the Refrigerator • Discard if Yellow • Prepared Color Reagent • Add Reagents in Specific Order • Sulfuric Acid • Antimony Potassium Tartrate • Ammonium Molybdate • Turbidity • 1 – 3 Week Stability • Store in Refrigerator in Plastic Bottle • 4 Hour Stability if Ascorbic Acid Added Ortho-Phosphate Total Phosphorous Interferences • Iron, Copper, and Silica • High Silica Concentrations May Cause Positive Interference • High Iron Concentrations May Cause Precipitation of Phosphorus • Salt Concentrations • Salt Error Up to 20% Salt Samples was Less Than 1% • Turbidity • Filtration • Arsenate • Positive Interference Ortho-Phosphate Total Phosphorous pH and Matrix Matching • Riley Ratio • Acidic Environment Needed to Form Phosphomolybdic Acid • The [ H+] : [ Mo ] Ratio for Stable and Rapid Results • Sample pH Changes Riley Ratio • Method Adjustments for Acidified or Digested Samples • Matrix Matching • Sample Preservation • Digested Blanks and Standards Ortho-Phosphate Total Phosphorous Troubleshooting Example 1 Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example • Do I Have High Background Absorbance? • Do I Have Failing Low Level Controls? • HCl Glassware Rinse • Have I Prepared New Reagents? • Have I Purchased New Dry Chemicals? Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example Ortho-Phosphate Total Phosphorous Troubleshooting Example • Do I have a passing correlation coefficient? • What is the age of my prepared reagent? • Does my reagent include surfactant? • How old is my dry chemical? Silica Silica Method Principle • Molybdosilicate Method • High Range and Low Range Samples • Ammonium Molybdate – Phosphomolybdic and Silicomolybdic Acid • Oxalic Acid Addition • Yellow Color Formation • Heteropoly Blue Method • Low Range • Reduction to Increase Sensitivity • Amino Naphthol Sulfonic Acid • Ascorbic Acid • Blue Color Formation Silica Reagents • Ammonium Molybdate • Stir to Prepare with Gentle Warming • pH Between 7 and 8 • Filter if Turbid • Remake if Blue in Color • Stable 2 Weeks • Oxalic Acid • Eliminates Phosphate Interferences • Increase Concentration if High Phosphate Levels • Store at Room Temperature • Stable 2 – 3 Days Silica Reagents • Hydrochloric Acid • Reducing Reagent • Filter Prior to Analysis • Discard if Dark or Insoluble • Store in Refrigerator • Limit Exposure to Light • Ascorbic Acid • Stable 1 week • Discard if Yellow • Store in Refrigerator Silica Interferences • Color or Turbidity • Tannin and Phosphate Interferences • Decreased with Oxalic Acid • Iron and Sulfide • Minimize Contact with Glass • Reagent Storage and Preparation Silica pH and Matrix Matching • Reagent Preparation • Reaction Occurs in Acidic Environment • Sample Preservation • Silica Precipitates in Acidic Solution Silica Troubleshooting Example Silica Troubleshooting Example Silica Troubleshooting Example • How old are my prepared reagents? • Have I prepared any new reagents? • What is the age of my surfactant? • What is my manifold cleaning procedure? • 1 N HCl for 15 minutes • 1 N NaOH for 15 minutes • 10% Bleach for 15 minutes • DI Water rinse Ammonia and TKN Ammonia and TKN Method Principle • Ammonia and TKN Colorimetric Reaction • Salicylate or Phenate Solution • Hypochlorite • Ammonia + Hypochlorite → Monochloramine • Salicylate • Monochloramine + Salicylate → Indosalicylate • Phenate • Monochloramine + Phenate → Indophenol • Nitroferricyanide • Catalyst Digestion Protocol Total Kjeldahl Nitrogen Digestion • TKN is Measured as Ammonia by Colorimetric Analysis • TKN Digestion Converts Organic Nitrogen to Ammonia • Sulfuric Acid • Potassium Sulfate • Heat to 380°C • Catalyst • Troubleshooting Digestion Protocol • Incomplete Digestions • Sample Acidity Level Digestion Protocol Temperature • Potassium Sulfate Increases the Boiling Point to 380°C • Insures all Compounds Decomposed • Decreases Time Required for Digestion • Evaporation Step in Protocol • Reduces Sample Volume Prior to Temperature Increase • Decreases Potential for Splatter • Final Volume • Example: Step to 160°C and Hold for 30 min Step to 190°C and Hold for 30 min Approximately 5 mL Final Volume Digestion Protocol Draft Shield Digestion Block Controller Digestion Protocol Temperature • Temperature Stability Across the Block • Flat Plate Heating and Graphite Rod Heating • Final Sample Volume and Acidity Level Digestion Protocol Acid to Salt Content • Samples with High Salt Content • Brines and Inorganic Salts • Loss of Nitrogen Above 400°C • Approximately 1 mL Sulfuric Acid per 1 g Salt Recommended • Salt Crystallization • Adjust Flow Rate of Exhaust System • Adjust Acid Concentration Digestion Protocol Reconstitution • Cooling • Acid Volume Remaining in Digestion Tube • DI Water Addition • Cool Digestion Tubes • Reconstitute • Vortex Mixer • Dispense Accurately • Bottle Top Dispenser or Auto-Pipette • Samples and Standards Same Final Volume • Volumetric Digestion Tubes Digestion Protocol Catalysts • Mercury • Documentation • Reproducibility • Colorless Digest • Preparing Digestion Reagent • Toxic • Waste Disposal Considerations • Copper • Substitute for Mercury Catalyst • Blue/Green Final Digest • Less Toxic Alternative Digestion Protocol Interferences • High Salt Content • Acid to Salt Ratio • Boiling Point • Nitrogen Loss • High Nitrate and Nitrite • Excess of 10 mg/L • Organic Matter • Consumption of Acid • Digestion Tube Considerations Colorimetric Detection Distillation Requirements • Distillation • EPA Requirements • 40 CFR 136.3 • Sample pH • Colorimetric Detection by Automated • Strong Buffer • Sample pH • Conversation from Ammonium to Ammonia • Buffer Aids in Colorimetric Reaction Colorimetric Detection Reagents • Sodium Hypochlorite • Purchased Reagents • Expiration Dates • Bleach Stable 1 Month if Opened • Solution is Unstable • Chlorine Evaporates if Exposed to Air • Reduced Free Chlorine Concentration • Store in Refrigerator • Sodium Hypochlorite Volatizes in Heat • DCI Stable 1 Day in Solution • Dry Chemical Storage Colorimetric Detection Reagents • Buffer Solution • Store at Room Temperature • Extended Shelf Life • Some Recipes Include EDTA or Sodium Potassium Tartrate • pH Adjustments • Sodium Potassium Tartrate • Ammonia Contamination • Alkaline Boil for 1 Hour • Adjust pH between 7 and 8 • Purchase from Vendor • Stable 6 Months in Solution Colorimetric Detection Reagents • Salicylate • Refrigerate and Store in Amber Bottle • Stable 1 Month • Filtration • Precipitates in Acidic Conditions • Nitroferricyanide • Stable 1 Month • Discard if Blue in Color • Phenate • Stable 1 Month • Discard when Dark Brown • Prepare Day Prior to Analysis Ammonia and TKN Interferences • Calcium and Magnesium • Precipitation • Addition of EDTA or Sodium Potassium Tartrate • Turbidity or Color • Filtration Ammonia and TKN pH and Matrix Matching • Testing Reaction pH • Phenate pH 12 to 12.4 • Salicylate pH 12.6 to 13.1 • Preserved Samples • Adjustments in Buffer Solution • Matrix Matching • Sample Preservation • Digested Blanks Ammonia and TKN Troubleshooting Example Ammonia and TKN Troubleshooting Example Ammonia and TKN Troubleshooting Example Ammonia and TKN Troubleshooting Example • Discrete Analyzer • Pipette Reagents into Beaker • Must Be Same Ratio and Order as Analyzer • SFA • Collect Liquid from Waste Line in Beaker • Low Reagent pH • Age of Hypochlorite Source • Addition of NaOH Ammonia and TKN Troubleshooting Example Ammonia and TKN Troubleshooting Example