Troubleshooting Chemistry Issues for Colorimetric … · • Nitrate and Nitrite ... • Catalyst for Phosphomolybdic Acid ... • Sodium Thiosulfate Interferences. Cadmium Reduction

Troubleshooting Chemistry

Issues for Colorimetric Analysis

22nd Annual Good Laboratory Practice Technical Conference 2016

Sarah Leibenguth

Technical Support Chemist

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

Topics for Discussion

Ortho-Phosphate and 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


• 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


• 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


Ortho-Phosphate

Total Phosphorous


Ortho-Phosphate

Total Phosphorous


Ortho-Phosphate

Total Phosphorous


• 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


Ortho-Phosphate

Total Phosphorous


Ortho-Phosphate

Total Phosphorous


Ortho-Phosphate

Total Phosphorous


• 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?

Ortho-Phosphate

Total Phosphorous

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


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


• Silica Precipitates in Acidic Solution

Silica


Silica


Silica


• 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

Silica

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

Ammonia and TKN

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

Digestion Protocol

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


• Sodium Hypochlorite Volatizes in Heat

• DCI Stable 1 Day in Solution

• Dry Chemical Storage


Reagents• Buffer Solution


• 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


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


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


• Digested Blanks

Ammonia and TKN


Ammonia and TKN


Ammonia and TKN


Ammonia and TKN


• 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


Ammonia and TKN


• 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


Ammonia and TKN


Ammonia and TKN


Ammonia and TKN


Ammonia and TKN

Griess Reaction

Nitrate to Nitrite Reduction

x

Griess Reaction

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


Nitrate and Nitrite

Cadmium Reduction

Cadmium Reduction

• 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

Approvals

Cadmium Reduction

• 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

Hardware Requirements

Cadmium Reduction

• Oil and Grease

• Suspended Matter• Filtration Prior to Analysis

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

• Residual Chlorine• Sodium Thiosulfate

Interferences

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

Pros

• Approval

• Wide Application

• Few Interferences

• Chemical Stability

• Documentation

Summary

Cons

• Cadmium Safety

• Cadmium Column Preparation

• Additional Hardware

Hydrazine Reduction

Hydrazine Reduction

• EPA (NPDES)• SM 4500-NO3

− H-2000

Applications

• Surface

• Domestic

• Industrial

Approvals

Hydrazine Reduction

• Heating

User Requirements

• Hydrazine Optimization


Hydrazine Reduction

• Sulfide• 10% Nitrate and Nitrite Concentration Variations

• Turbidity• Filter Prior to Analysis

• Saline Samples

Interferences

Hydrazine Reduction

• Hydrazine Sulfate• Toxicity

• Liquid Waste Product

• Cost Effective

• Copper Sulfate

• Sodium Hydroxide• Handle with Care

Chemical Requirements

Things to Consider

• Hazard

• Cost

• Storage

• Stability

Hydrazine Reduction

Pros

• Approval

• Minimum Hardware

Requirements

Summary

Cons

• Not Applicable to Saline

Waters

• Hydrazine Safety

• Hydrazine Concentration

Optimization

Vanadium(III) Chloride

Reduction

Vanadium(III) Reduction

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

Applications

• Drinking

• Surface

• Domestic

• Industrial

Approvals


• Heating• Capable of Heating to 80° C

• Lower Temperature Heating Lengthens Reduction Period

Interferences

• Residual Chlorine• Sodium Thiosulfate


• Sulfate, Phosphate• Concentrations Above 100 ppm May Reduce Reduction Efficiency



• Vanadium(III) Chloride• Toxicity

• Easily Oxidized

• Liquid Waste Product

• Moisture and Light Sensitive

• HCl

Chemicals

Things to Consider

• Hazard

• Cost

• Storage

• Stability


Pros

• Approval

• Few Interferences

Summary

Cons

• Not Applicable to Saline Waters

• Vanadium(III) Chloride Safety

• Extended Reduction Time

Enzymatic Reduction

Enzymatic Reduction

• 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

Approvals

Enzymatic Reduction

• Heating

• Reagent Cooler

User Requirements

• Manual Addition of Nitrate Reductase


Enzymatic Reduction

Interferences


• Metal Ions• EDTA in Approved Method

• Sulfate, Chloride, Bromide• High Concentrations May Reduce Nitrate Recovery

• NADH• Interference in Griess Reaction

Enzymatic Reduction

• 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

Chemicals

Enzymatic Reduction

Pros

• Chemical Safety

• Wide Application

• Minimal Hardware

Requirements

Summary

Cons

• Approval

• Chemical Cost and Stability

• Extended Reduction Time


Nitrate and Nitrite


Nitrate and Nitrite


Nitrate and Nitrite

Questions?

Documents

Troubleshooting Chemistry Issues for Colorimetric … · • Nitrate and Nitrite ... • Catalyst for Phosphomolybdic Acid ... • Sodium Thiosulfate Interferences. Cadmium Reduction