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

• 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

• 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

• 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

• 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

• Have I tested the pH of my reaction?

• How are my samples acidified?

• What is my hypochlorite source? • What is the age? • Are there additives? Ammonia and TKN

Troubleshooting Example Ammonia and TKN

Troubleshooting Example Griess Reaction Nitrate to Nitrite Reduction

x Griess Reaction Nitrate and Nitrite

Method Principle • Nitrite in the Sample + Nitrite Now Present After Reduction

• Nitrite + Sulfanilamide = Diazonium Salt

• Addition of NEDD = Red AZO Dye Nitrate and Nitrite

Reagent Stability • Sulfanilamide-NEDD • Store in Refrigerator in a Dark Bottle • One Month Stability • Filter Before Use to Eliminate Color

• Ammonium Chloride Buffer • Adjust pH to 8.5 • Store at Room Temperature Cadmium Reduction Cadmium Reduction

Approvals • EPA (NPDES & NPDWR) • EPA 353.2 Rev. 2.0 (1993) • ASTM D3867-04 (A) − • SM 4500-NO3 F-200 • USGS I-2545-90 Applications • Saline • Surface • Drinking • Domestic • Industrial Cadmium Reduction

Hardware Requirements • Cadmium Column • Prepare or Purchase • Cadmium Coil • Purchase • Switching Valve • Necessary for Online Reduction on Discrete Analyzer User Requirements • Column Preparation • Column or Coil Conditioning • Activation with Copper Sulfate • Conditioned with High Standard Cadmium Reduction

Interferences

• Oil and Grease

• Suspended Matter • Filtration Prior to Analysis

• Iron, Copper, and other Metals • EDTA in Approved Method

• Residual Chlorine • Sodium Thiosulfate Cadmium Reduction

Things to Consider

• Hazard

• Cost

• Storage

• Stability Cadmium Reduction

Chemicals • Cadmium • Toxicity • Mesh Size of Cadmium Granules • Coated Cadmium Coil • Cost Effective • HCl • Store at Room Temperature • Handle with Care • Copper Sulfate • Ammonium Chloride • EDTA – Stable as Dry Chemical and Prepared Reagent Cadmium Reduction

Summary Pros Cons • Approval • Cadmium Safety • Wide Application • Cadmium Column Preparation • Few Interferences • Additional Hardware • Chemical Stability • Documentation Hydrazine Reduction Hydrazine Reduction

Approvals • EPA (NPDES) − • SM 4500-NO3 H-2000

Applications • Surface • Domestic • Industrial Hydrazine Reduction

Hardware Requirements

• Heating

User Requirements

• Hydrazine Optimization Hydrazine Reduction

Interferences

• Sulfide • 10% Nitrate and Nitrite Concentration Variations

• Turbidity • Filter Prior to Analysis

• Saline Samples Hydrazine Reduction

Chemical Requirements • Hydrazine Sulfate • Toxicity • Liquid Waste Product • Cost Effective • Copper Sulfate • Sodium Hydroxide • Handle with Care Things to Consider • Hazard • Cost • Storage • Stability Hydrazine Reduction

Summary Pros Cons • Approval • Not Applicable to Saline • Minimum Hardware Waters Requirements • Hydrazine Safety • Hydrazine Concentration Optimization Vanadium(III) Chloride Reduction Vanadium(III) Reduction

Approvals

• EPA (NPDES & NPDWR) • Easy (1-Reagent) Nitrate Method, Revision November 12, 2011

Applications • Drinking • Surface • Domestic • Industrial Vanadium(III) Reduction

Hardware Requirements • Heating • Capable of Heating to 80° C • Lower Temperature Heating Lengthens Reduction Period Interferences • Residual Chlorine • Sodium Thiosulfate • Turbidity • Filter Prior to Analysis • Sulfate, Phosphate • Concentrations Above 100 ppm May Reduce Reduction Efficiency Vanadium(III) Reduction

Chemicals • Vanadium(III) Chloride • Toxicity • Easily Oxidized • Liquid Waste Product • Moisture and Light Sensitive • HCl Things to Consider • Hazard • Cost • Storage • Stability Vanadium(III) Reduction

Summary Pros Cons • Approval • Not Applicable to Saline Waters • Few Interferences • Vanadium(III) Chloride Safety • Extended Reduction Time Enzymatic Reduction Enzymatic Reduction

Approvals • ATP Accepted • USGS I-2547-11 and USGS I-2548-11 • The Nitrate Elimination Company, Inc. Method N07-0003 • Method Update Rule – Proposed • Submitted for Approval Applications • Saline • Drinking • Surface • Domestic • Industrial Enzymatic Reduction

Hardware Requirements

• Heating • Reagent Cooler

User Requirements

• Manual Addition of Nitrate Reductase Enzymatic Reduction

Interferences • Turbidity • Filter Prior to Analysis • Metal Ions • EDTA in Approved Method • Sulfate, Chloride, Bromide • High Concentrations May Reduce Nitrate Recovery • NADH • Interference in Griess Reaction Enzymatic Reduction

Chemicals

• EDTA • Stable as Dry Chemical and Prepared Reagent • Potassium Phosphate • Skin and Eye Irritant • Potassium Hydroxide • Respiratory Irritant • Nitrate Reductase and NADH • Non-Hazardous • Prepared Stable for 8 Hours • Prepared Reagent Volume of 20 mL Enzymatic Reduction

Summary Pros Cons • Chemical Safety • Approval • Wide Application • Chemical Cost and Stability • Minimal Hardware • Extended Reduction Time Requirements Nitrate and Nitrite

Troubleshooting Example Nitrate and Nitrite

Troubleshooting Example Questions?