STANDARD OPERATING PROCEDURE FOR ANALYSIS OF BROMIDE USED

AS A NON-REACTIVE TRACER

1.0 SCOPE AND APPLICATION

1.0.1. This SOP describes the analytical procedures utilized by University of Florida for analysis of aqueous samples containing bromide, which is used as a non-reactive tracer, in both lab and field studies, to identify the flow behavior of displacing water. This SOP was written by H.K. Kim, D.P. Dai, and P.S.C. Rao at the University of Florida. This version (v.2) was updated on July 19, 1995.

1.0.2. The method involves high performance liquid chromatography (HPLC) techniques for estimation of the concentration of bromide in water samples; a ultraviolet detector (UV) is used to quantify the analyte concentrations in the sample.

1.0.3. The method has been found to provide reliable and reproducible quantitation of bromide for concentrations > 0.5 mg/mL; this value is then considered to be the method detection level (MDL). The standards calibration curve for FID response was found to be linear up to 30 mg/mL for bromide.

1.0.4. Samples selected for HPLC analysis may be chosen on the basis of preliminary screening to determine approximate concentration ranges, and select appropriate HPLC parameters (e.g., sample injection volumes; concentration range for standard curves, etc.) However, the strategies for sample screening themselves are outside the scope of this SOP.

1.0.5. Water samples from lab and field experiments are sub-sampled in to 2-mL RPLC autosampler vials for analysis.

2.0 PURPOSE

2.0.1. The purpose of this SOP is to ensure reliable and reproducible analytical results of bromide in water samples for laboratory-based HPLC analyses, and to permit traceability of possible causes of error in analytical results.

3.0 PROCEDURES

3.1 Sample Containers, Collection, Transportation and Storage

3.1.1. Sample Containers. Water samples are contained in 5-mL glass sample vials

(Cat # 06406-19F) purchased from Fisher Scientific Co. These vials are equipped with

Teflon-faced septa caps. The glass vials or the caps are not reused.

3.1.2. Sample Collection. Each sample vial is completely filled with aqueous samples, such that no headspace of air exists, and capped. The vials are not opened until the time of sub-sampling or analysis.

3.1.3. Transportation and Storage. For field studies, all the samples are stored in coolers containing "blue ice", and later sampled to refrigerators on site. Samples are packed in coolers and shipped via overnight air express (e.g., Fe~x). The samples will be stored in the cold storage room or refrigerator at 4 degrees Celsius, until they are ready for HPLC analysis. After sub-sampling, the samples are returned to cold storage. For lab studies, the samples are stored in a refrigerator if analysis is expected to take more than 12 hours.

3.2. Sub-Sampling and Dilution

3.2.1. Disposable, Pasture glass pipettes (Fisher Catalog # 13-678-20B) are used to transfer samples from 5-mL sample vials to the 2-mL HPLC autosampler vials. Sample may need to be diluted with HPLC grade water prior to HPLC analysis. The dilution necessary (usually 2x to 20x) is determined from preliminary screening analysis.

4.0 APPARATUS AND MATERIALS

4.0.1. Glassware. Disposable micro-pipettes (10, 100 mL; Fisher catalog # 21-175B;

21-175F) and Class A volumetric pipettes (1 or 2 mL) are required for sample dilution. Disposable Pasteur glass pipettes (Fisher Catalog # 12-678-20B) are required for sub-sampling. HPLC autosampler vials (2-mL) with Teflon-faced caps (Fisher Catalog # 03-375-16A) are required for HPLC analysis. Volumetric Class A pipettes (0.5, 1, 2, 5, 10 ml)are required for preparations of the calibration standards.

4.0.2. High Performance Liquid Chromatograph System. An analytical HPLC system is required, complete either with an integrator or a PC-based data acquisition/analysis software. Also required are other accessories, including analytical columns and guard column. The HPLC system used consisted of the following components: Eldex Model 9600 pump; Shimadzu Model SIL-9A Autoinjector; Waters Model 490 Multi-wavelength UV detector; and HP Model 3396A integrator.

•Column. Anion exchange column used was a Dionex lonpac AS4A-SC (4 250 mm; Dionex Catalog #043174).

•Eluent. Na₂CO₃/NaHCO₃ buffer solution was used as eluent. Reagent-grade Na₂CO₃ and NaHCO₃ were dissolved into HPLC-grade water where the concentrations were 0.9 mM and 0.85mM, respectively.

4.0.3. Reagents. Reagent water is defined as water in which an interferant is not observed at the method detection level (MDL) of the parameters of interest. Laboratory reagent water used is HPLC-grade or OPTIMA-grade water (Fisher Catalog # W5-4, W7-4).

4.0.4. Standard Solutions. Analytical Standards Solution are prepared from reagent grade potassium bromide in the laboratory'. Stock standard solutions with 1000 mg/mL was prepared in HPLC water kept in glass container with minimal headspace. These stock solution was stored at 4 degrees Celsius. Old stock solutions should be discarded, and a fresh batch prepared every month. Any time a comparison with the check standards indicates a problem, a new batch of standards must be prepared

4.0.5. Working Calibration Standards (Secondary Dilution Standards). Working calibration standards are prepared by diluting stock standard solutions in reagent water.

4.0.6. Calibration Check Standards. Calibration check standards are prepared by diluting stock standard solution in water.

5.0 CALIBRATION

5.0.1. Calibration Standards. Prepare ten calibration levels by adding 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 30.0,40.0 and 50.0 mL of the calibration standard solution into 100ML volumetric flask followed by filling with HPLC grade water to meniscus (unit mg/ML)

Level 1 2 3 4 5 6 7 8 9 10

Bromide 0.2 0.5 1.0 2.0 5.0 10.0 20.0 30.0 40.0 50.0

6.0 QUALITY CONTROL

6.0.1. The following quality control procedures will be followed:

•An acceptable method blank analysis must be performed once for each 12-hour time period.

•Standard calibration must be run every time restart the HPLC pump or recharge the eluent.

•Check standard and blank (reagent water) should be run every 10 to 12 samples.

7.0 INSTRUMENTAL PROCEDURES

High Performance Liquid Chromatography Conditions. Recommended operating conditions are as follows:

System temperature ambient

Eluent composition programming isocratic

Eluent flow rate 2.0 ml/min.

Sample injection volume l00 mL

Detection wavelength 205 nm

Typical retention time for bromide using these settings is about 3 mm. A copy of the representative chromatograms for several standards are shown in Figure 1.

8.0 SAMPLE PREPARATIONS

8.0.1. Sub-sampling. Samples are transferred from sample vials to HPLC vials and capped with open -op Teflon-lined septa caps.

8.0.2. Dilution. Prepare a sample dilution if responses for any peaks in the sample exceed the calibration ranges. The dilution is required to obtain an adequate detector response within the analytical range; screening analysis results may be used to determine if initial dilution is required.

9.0 SAMPLE ANALYSIS

9.0.1. Analysis. The samples are allowed to reach ambient temperature prior to HPLC analysis. The vials with sub-sampled (and diluted, if necessary) samples are loaded on the HPLC autosampler. After turning the UV detector lamp on, 10-20 minutes warm-up period is required to achieve a steady baseline. In order to avoid HPLC pump malfunction caused by air bubble entrapment in the pump head, it is recommended to degas the eluent before and/or during sample analysis. Because almost all HPLC parts are made of stainless steel, they are vulnerable to corrosion. Thus, it is also recommended to detach the analytical column right after sample analysis, and to wash the system with HPLC grade water.

9.0.2. Analyte Identification. The sample component must elute at the same relative retention time as the standard. The primary criterion is that the relative retention time of the sample component must be within +/- 0.1 relative retention time units of the standard.

9.0.3. Analyte Quantitation. When an analyte has been identified, concentration will be based on the peak area, which is converted to concentration using a linear standard calibration curve.

10.0 INTERFERENCES

10.0.1. Contamination by carry over can occur whenever high-level and low-level samples are analyzed sequentially. To reduce carry over, the injector syringe should rinsed with reagent water between analyses. Whenever a sample with unusually high concentration is encountered it should be followed by an analysis of reagent water to check for cross contamination.

11.0 SAFETY

11.0.1. The following safety procedures should be followed:

•High-pressure compressed-gas cylinders must be secured to a firm mounting point, whether they are located internally or externally.

•The effluent level in the waste reservoir should be checked on the regular basis otherwise the overflow of water solution could damage any electric parts of the system.

•The operator should not touch or manipulate inside of the HPLC autosampler while the autosampler needle is actuated for sample injection.

•Refer to the Materials Safety Data Sheets (MSDS) for additional information on environmental health and safety of the chemicals used.