Troubleshooting Chemistry Issues for Colorimetric …c.ymcdn.com/sites/ Chemistry Issues for Colorimetric Analysis 22nd Annual Good Laboratory Practice Technical Conference 2016 Sarah

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 …c.ymcdn.com/sites/ Chemistry Issues for Colorimetric Analysis 22nd Annual Good Laboratory Practice Technical Conference 2016 Sarah