Attachment 05 - NIH Triazoles.1714658486079.docx

Attachment 05: NIH Triazoles (HPLC/GC Method)

Summary High-performance liquid chromatography or high-pressure liquid chromatography (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry to identify, quantify or purify the individual components of the mixture. Generally, environmental samples containing triazoles require pre-concentration, such as solid phase extraction (SPE), before analysis.

Concurrent acetylation–dispersive liquid–liquid microextraction (DLLME) combined with gas chromatography–mass spectrometry (GC–MS) has been reported for analysis of triazoles. Generally, LC-MS is the preferred analytical technique over GC-MS. LC-MS can make determination-based characteristics such as whether the analyte is conjugated or unconjugated, whereas GC-MS is restricted to determinations based on volatility and molecular weight. Limited performance of gas chromatography methods for benzotriazole compounds has been overcome using ionic liquid coated columns, derivatization processes, such as methylation or acetylation, and the use of two-dimensional gas chromatography.

Sample Collection NIH shall collect samples, and the Contractor shall pick up the samples from NIH Bethesda, Building 11.

The Contractor’s personnel assigned to pick up the samples shall be trained in sampling procedures, and they shall adhere to the correct procedures for sample collection, sample preservation, sample handling, sample identification, and prompt transfer of the sample to the laboratory.

The Contractor shall provide NIH with sampling kits for collecting each sample.

In the event that the Contractor also collects the sample, they shall follow the following procedure:

Samples shall be collected in plastic or glass bottles. All bottles shall be thoroughly cleaned and rinsed with reagent water. Fill a prepared 1000 mL bottle as applicable from a free-flowing sample tap or other sources as applicable. The volume collected shall be sufficient to ensure a representative sample, allow for replicate analysis and laboratory fortified matrix analysis, if required, and minimize waste disposal.

Sample Preservation and Storage The method of preservation and the holding time for samples analyzed by this method are determined by the azoles of interest. In each sample, the anion that requires the most preservation treatment and the shortest holding time will determine the preservation treatment. It is recommended that all samples be cooled to 4°C and held for no longer than 28 days.

Method of Shipping and Duration The Contractor shall place the sample into a cooler with ice for immediate delivery or shipment to the laboratory. The sample shall be received with a chain of custody (COC). The COC shall have the name of the sample collector, date and time of sample collection, signature or initial of the sample collector, location of the sample and type of sample to be analyzed.

Lab Procedure for the Analysis The Contractor shall use the following procedure:

A. Start the instrument timer. 5-minute reaction time starts. Low results will occur if the UV photolysis is more than or less than 5 minutes. A yellow color will show if triazole is in the sample.

B. When the timer expires, turn off the lamp. Remove the lamp from the mixing bottle. Swirl the mixing bottle to mix thoroughly.

C. Use a 5-mL volumetric pipet, Class A and pipet filler and deionized water D. Prepare a 5.0 mg/L benzotriazole standard solution as follows:

a. Use a pipet to add 10.0 mL of a 500-mg/L benzotriazole standard solution into the volumetric flask.

b. Dilute to the marked level with deionized water. Mix well. Prepare this solution daily as needed.

E. Use the test procedure to measure the concentration of the prepared standard solution.

F. Compare the expected result to the actual result.

Calibration Procedure The Contractor shall use the following procedure for calibrating equipment:

Install the instrument cap on the DR 900 cell holder before “ZERO” or “READ” is pushed. Wear UV safety goggles while the UV lamp is on. Do not touch the UV lamp surface with bare fingers. Fingerprints can damage the glass. Rinse the lamp and wipe with a soft, clean tissue between tests. The sample temperature must be 20–25 °C (68–77 °F) for accurate results. Complete this procedure to make sure that the UV lamp operates correctly. If the result is significantly low, replace the lamp. The normal UV lamp life is approximately 5000 hours.

Analysis Method and Protocol Benzotriazole or tolyltriazole, used in many applications as corrosion inhibitors for copper and copper alloys, are determined by a proprietary catalytic ultraviolet (UV) photolysis procedure that takes less than 10 minutes to complete. The measurement wavelength is 425 nm for spectrophotometers or 420 nm for colorimeters High-performance liquid chromatography or high-pressure liquid chromatography (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry to identify, quantify or purify the individual components of the mixture. Generally, environmental samples containing triazoles require pre-concentration, such as solid phase extraction (SPE), before analysis.

Maintenance Plan for the Instrument Analytical system complete with chromatograph and all required accessories including syringes, analytical columns, compressed gasses and detectors shall be maintained according to the Good Laboratory Practice manual or manufacturer's guidelines. The maintenance protocol shall be available and be delivered to NIH upon request.

Interference Limited performance of gas chromatography methods for benzotriazole compounds has been overcome using ionic liquid coated columns, derivatization processes, such as methylation or acetylation, and the use of two-dimensional gas chromatography.

Adjust the sample pH to 4–6 with 1 N sulfuric acid, then start the test procedure. If the sample contains more than 4000 mg/L, dilute the sample.

References HACH method 8079