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Part 4000 INORGANIC NONMETALLIC CONSTITUENTS
4010 INTRODUCTION
The analytical methods included in this part make use of classical wet chemical techniques and their automated variations and such modern instrumental techniques as ion chromatography. Methods that measure various forms of chlorine, nitrogen, and phosphorus are presented. The procedures are intended for use in the assessment and control of receiving water quality, the treatment and supply of potable water, and the measurement of operation and process efficiency in wastewater treatment. The methods also are appropriate and applicable in evaluation of environmental water-quality concerns. The introduction to each procedure contains reference to special field sampling conditions, appropriate sample containers, proper procedures for sampling and storage, and the applicability of the method.
4020 QUALITY ASSURANCE/QUALITY CONTROL
4020 A. Introduction
Without quality control results there is no confidence in analytical results reported from tests. As described in Part 1000 and Section 3020, essential quality control measurements include: method calibration, standardization of reagents, assessment of individual capability to perform the analysis, performance of blind check samples, determination of the sensitivity of the test procedure (method detection level), and daily evaluation of bias, precision, and the presence of laboratory contamination or other analytical interference. Details of these procedures, expected ranges of results, and frequency of performance should be formalized in a written Quality Assurance Manual and Standard Operating Procedures.
For a number of the procedures contained in Part 4000, the traditional determination of bias using a known addition to either a sample or a blank, is not possible. Examples of these procedures include pH, dissolved oxygen, residual chlorine, and carbon dioxide. The inability to perform a reliable known addition does not relieve the analyst of the responsibility for evaluating test bias. Analysts are encouraged to purchase certified ready-made solutions of known levels of these constituents as a means of measuring bias. In any situation, evaluate precision through analysis of sample duplicates.
Participate in a regular program (at a minimum, annually, and preferably semi-annually) of proficiency testing (PT)/performance evaluation (PE) studies. The information and analytical confidence gained in the routine performance of the studies more than offset any costs associated
with these studies. An unacceptable result on a PT study sample is often the first indication that a test protocol is not being followed successfully. Investigate circumstances fully to find the cause. Within many jurisdictions, participation in PT studies is a required part of laboratory certification.
Many of the methods contained in Part 4000 include specific quality-control procedures. These are considered to be the minimum quality controls necessary to successful performance of the method. Additional quality control procedures can and should be used. Section 4020B describes a number of QC procedures that are applicable to many of the methods.
4020 B. Quality Control Practices
- Initial Quality Control See Section 3020B.1.
- Calibration
See Section 3020B.2. Most methods for inorganic nonmetals do not have wide dynamic ranges. Standards for initial calibration therefore should be spaced more closely than one order of magnitude under these circumstances. Verify calibration by analyzing a midpoint or lower calibration standard and blank as directed. Alternatively, verify calibration with two standards, one near the low end and one near the high end, if the blank is used to zero the instrument.
- Batch Quality Control See Section 3020B.3 a through d.
4110 DETERMINATION OF ANIONS BY ION CHROMATOGRAPHY*#(1)
4110 A. Introduction
Because of rapid changes in technology, this section is currently undergoing substantial revision.
Determination of the common anions such as bromide, chloride, fluoride, nitrate, nitrite, phosphate, and sulfate often is desirable to characterize a water and/or to assess the need for specific treatment. Although conventional colorimetric, electrometric, or titrimetric methods are available for determining individual anions, only ion chromatography provides a single instrumental technique that may be used for their rapid, sequential measurement. Ion chromatography eliminates the need to use hazardous reagents and it effectively distinguishes
among the halides (Br –^ , Cl –^ , and F–^ ) and the oxy-ions (SO 3 2–^ , SO 4 2–^ or NO 2 –^ , NO 3 –^ ).
This method is applicable, after filtration to remove particles larger than 0.2 μm, to surface,
before analyzing samples. F–^ can be determined accurately by ion chromatography using special techniques such as dilute eluent or gradient elution using an NaOH eluent or alternative columns.
- Apparatus
a. Ion chromatograph, including an injection valve, a sample loop, guard column, separator column, and fiber or membrane suppressors, a temperature-compensated small-volume conductivity cell and detector (6 μL or less), and a strip-chart recorder capable of full-scale response of 2 s or less. An electronic peak integrator is optional. Use an ion chromatograph capable of delivering 2 to 5 mL eluent/min at a pressure of 1400 to 6900 kPa.
b. Anion separator column, with styrene divinylbenzene-based low-capacity pellicular
anion-exchange resin capable of resolving Br –^ , Cl –^ , NO 3 –^ , NO 2 –^ , PO 4 3–^ , and SO 4 2–^ .*#(2)
c. Guard column, identical to separator column†#(3) to protect separator column from fouling by particulates or organics.
d. Fiber suppressor or membrane suppressor: ‡#(4) Cation-exchange membrane capable of continuously converting eluent and separated anions to their acid forms. Alternatively, use continuously regenerated suppression systems.
- Reagents
a. Deionized or distilled water free from interferences at the minimum detection limit of each constituent, filtered through a 0.2-μm membrane filter to avoid plugging columns, and having a conductance of < 0.1 μS/cm.
b. Eluent solution, sodium bicarbonate-sodium carbonate, 0.0017 M NaHCO 3 -0.0018 M
Na 2 CO 3 : Dissolve 0.5712 g NaHCO 3 and 0.7632 g Na 2 CO 3 in water and dilute to 4 L.
c. Regenerant solution, H 2 SO 4 , 0.025 N : Dilute 2.8 mL conc H 2 SO 4 to 4 L. d. Standard anion solutions, 1000 mg/L: Prepare a series of standard anion solutions by weighing the indicated amount of salt, dried to a constant weight at 105°C, to 1000 mL. Store in plastic bottles in a refrigerator; these solutions are stable for at least 1 month. Verify stability.
Anion§^ Salt
Amount g/L Cl–^ NaCl^ 1. Br –^ NaBr^ 1. NO 3 –^ NaNO^3 1.3707 (226 mg NO 3 –^ -N/L)
NO 2 –^ NaNO^2 1.4998i (304 mg NO 2 –^ -N/L)
PO 4 3–^ KH 2 PO^4 1.4330 (326 mg PO 4 3–^ -P/L
Anion§^ Salt
Amount g/L SO 4 2–^ K 2 SO^4 1.
§ (^) Expressed as compound. i Do not oven-dry, but dry to constant weight in a desiccator.
e. Combined working standard solution, high range: Combine 12 mL of standard anion
solutions, 1000 mg/L (¶ 3 d ) of NO 2 –^ , NO 3 –^ , HPO 4 2–^ , and Br –^ , 20 mL of Cl –^ , and 80 mL of
SO 4 2–^. Dilute to 1000 mL and store in a plastic bottle protected from light. Solution contains 12
mg/L each of NO 2 –^ , NO 3 –^ , HPO 4 2–^ , and Br –^ , 20 mg/L of Cl –^ , and 80 mg/L of SO 4 2–^. Prepare
fresh daily.
f. Combined working standard solution, low range: Dilute 25 mL of the high-range mixture (¶ 3 e ) to 100 mL and store in a plastic bottle protected from light. Solution contains 3 mg/L each
of NO 2 –^ , NO 3 –^ , HPO 4 2–^ , and Br –^ , 5 mg/L Cl –^ , and 20 mg/L of SO 4 2–^. Prepare fresh daily.
g. Alternative combined working standard solutions: Prepare appropriate combinations
according to anion concentration to be determined. If NO 2 –^ and PO 4 3–^ are not included, the
combined working standard is stable for 1 month. Dilute solutions containing NO 2 –^ and PO 4 3–
must be made daily.
- Procedure
a. System equilibration: Turn on ion chromatograph and adjust eluent flow rate to approximate the separation achieved in Figure 4110:1 (about 2 mL/min). Adjust detector to desired setting (usually 10 to 30 μS) and let system come to equilibrium (15 to 20 min). A stable base line indicates equilibrium conditions. Adjust detector offset to zero out eluent conductivity; with the fiber or membrane suppressor adjust the regeneration flow rate to maintain stability, usually 2.5 to 3 mL/min.
b. Calibration: Inject standards containing a single anion or a mixture and determine approximate retention times. Observed times vary with conditions but if standard eluent and
anion separator column are used, retention always is in the order F–^ , Cl –^ , NO 2 –^ , Br –^ , NO 3 –^ ,
HPO 4 2–^ , and SO 4 2–^. Inject at least three different concentrations (one near the minimum
reporting limit) for each anion to be measured and construct a calibration curve by plotting peak height or area against concentration on linear graph paper. Recalibrate whenever the detector
setting, eluent, or regenerant is changed. To minimize the effect of the ‘‘water dip’’##(5) on F– analysis, analyze standards that bracket the expected result or eliminate the water dip by diluting the sample with eluent or by adding concentrated eluent to the sample to give the same
ions by ion chromatography. Anal. Chem. 53: 1935. WEISS, J. 1986. Handbook of Ion Chromatography. E.L. Johnson, ed. Dionex Corp., Sunnyvale, Calif. PFAFF, J.D., C.A. BROCKHOFF & J.W. O’DELL. 1994. The Determination of Inorganic Anions in Water by Ion Chromatography. Method 300.0A, U.S. Environmental Protection Agency, Environmental Monitoring Systems Lab., Cincinnati, Ohio.
4110 C. Single-Column Ion Chromatography with Electronic Suppression of Eluent Conductivity and Conductimetric Detection
- General Discussion
a. Principle: A small portion of a filtered, homogeneous, aqueous sample or a sample containing no particles larger than 0.45 μm is injected into an ion chromatograph. The sample merges with the eluent stream and is pumped through the ion chromatographic system. Anions are separated on the basis of their affinity for the active sites of the column packing material. Conductivity detector readings (either peak area or peak height) are used to compute concentrations.
b. Interferences: Any two species that have similar retention times can be considered to interfere with each other. This method has potential coelution interference between short-chain acids and fluoride and chloride. Solid-phase extraction cartridges can be used to retain organic acids and pass inorganic anions. The interference-free solution then can be introduced into the ion chromatograph for separation.
This method is usable but not recommended for fluoride. Acetate, formate, and carbonate interfere in determining fluoride under the conditions listed in Table 4110:VIII. Filtering devices may be used to remove organic materials for fluoride measurements; simultaneously, use a lower eluent flow rate.
Chlorate and bromide coelute under the specified conditions. Determine whether other anions in the sample coelute with the anions of interest.
Additional interference occurs when anions of high concentrations overlap neighboring anionic species. Minimize this by sample dilution with reagent water.
Best separation is achieved with sample pH between 5 and 9. When samples are injected the eluent pH will seldom change unless the sample pH is very low. Raise sample pH by adding a small amount of a hydroxide salt to enable the eluent to control pH.
Because method sensitivity is high, avoid contamination by reagent water and equipment. Determine any background or interference due to the matrix when adding the QC sample into any matrix other than reagent water.
c. Minimum detectable concentration: The minimum detectable concentration of an anion is a function of sample volume and the signal-to-noise ratio of the detector-recorder combination. Generally, minimum detectable concentrations are about 0.1 mg/L for the anions with an
injection volume of 100 μL. Preconcentrators or using larger injection volumes can reduce detection limits to nanogram-per-liter levels for the common anions. However, coelution is a possible problem with large injection volumes. Determine method detection limit for each anion of interest.
d. Prefiltration: If particularly contaminated samples are run, prefilter before or during injection. If the guard column becomes contaminated, follow manufacturer’s suggestions for cleanup.
- Apparatus
a. Ion chromatograph , complete with all required accessories including syringes, analytical columns, gases, detector, and a data system. Required accessories are listed below.
b. Filter device , 0.45 μm, placed before separator column to protect it from fouling by particulates or organic constituents.*#(6)
c. Anion separator column , packed with low-capacity anion-exchange resin capable of resolving fluoride, chloride, nitrite, bromide, nitrate, orthophosphate, and sulfate.†#(7)
d. Conductivity detector , flow-through, with integral heat-exchange unit allowing automatic temperature control and with separate working and reference electrodes.
e. Pump, constant flow rate controlled, high-pressure liquid chromatographic type, to deliver 1.5 mL/min.
f. Data system, including one or more computer, integrator, or strip chart recorder compatible with detector output voltage.
g. Sample injector: Either an automatic sample processor or a manual injector. If manual injector is used, provide several glass syringes of > 200 μL capacity. The automatic device must be compatible and able to inject a minimum sample volume of 100 μL.
- Reagents
a. Reagent water: Distilled or deionized water of 18 megohm-cm resistivity containing no particles larger than 0.20 μm.
b. Borate/gluconate concentrate: Combine 16.00 g sodium gluconate, 18.00 g boric acid, 25.00 g sodium tetraborate decahydrate, and 125 mL glycerin in 600 mL reagent water. Mix and dilute to 1 L with reagent water.
c. Eluent solution , 0.0110 M borate, 0.0015 M gluconate, 12% (v/v) acetonitrile: Combine 20 mL borate/gluconate concentrate, 120 mL HPLC-grade acetonitrile, and 20 mL HPLC-grade n -butanol, and dilute to 1 L with reagent water. Use an in-line filter before the separator column to assure freedom from particulates. If the base line drifts, degas eluent with an inert gas such as helium or argon.
d. Stock standard solutions: See Section 4110B.3 e. e. Combined working standard solutions , high-range: See Section 4110B.3 e.
b. Generate accuracy and precision data with this method by using a reference standard of known concentration prepared independently of the laboratory making the analysis. Compare with data in Precision and Bias, below.
c. Analyze a quality control sample at least every 10 samples. Follow general guidelines from Section 4020.
- Precision and Bias Precision and bias data are given in Table 4110:IX.
- Reference
- GLASER, J., D. FOERST, G. MCKEE, S. QUAVE & W. BUDDE. 1981. Trace analyses for wastewater. Environ. Sci. Technol. 15:1426.
4120 SEGMENTED CONTINUOUS FLOW ANALYSIS*#(8)
4120 A. Introduction
- Background and Applications
Air-segmented flow analysis (SFA) is a method that automates a large number of wet chemical analyses. An SFA analyzer can be thought of as a ‘‘conveyor belt’’ system for wet chemical analysis, in which reagents are added in a ‘‘production-line’’ manner. Applications have been developed to duplicate manual procedures precisely. SFA was first applied to analysis of sodium and potassium in human serum, with a flame photometer as the detection device, by removing protein interferences with a selectively porous membrane (dialyzer).
The advantages of segmented flow, compared to the manual method, include reduced sample and reagent consumption, improved repeatability, and minimal operator contact with hazardous materials. A typical SFA system can analyze 30 to 120 samples/ h. Reproducibility is enhanced by the precise timing and repeatability of the system. Because of this, the chemical reactions do not need to go to 100% completion. Decreasing the number of manual sample/solution manipulations reduces labor costs, improves workplace safety, and improves analytical precision. Complex chemistries using dangerous chemicals can be carried out in sealed systems. Unstable reagents can be made up in situ. An SFA analyzer uses smaller volumes of reagents and samples than manual methods, producing less chemical waste needing disposal.
SFA is not limited to single-phase colorimetric determinations. Segmented-flow techniques often include analytical procedures such as mixing, dilution, distillation, digestion, dialysis, solvent extractions, and/or catalytic conversion. In-line distillation methods are used for the determinations of ammonia, fluoride, cyanide, phenols, and other volatile compounds. In-line digestion can be used for the determination of total phosphorous, total cyanide, and total nitrogen
(kjeldahl + NO 2 + NO 3 ). Dialysis membranes are used to eliminate interferences such as
proteins and color, and other types of membranes are available for various analytical needs. SFA also is well-suited for automated liquid/liquid extractions, such as in the determination of MBAS. Packed-bed ion exchange columns can be used to remove interferences and enhance sensitivity and selectivity of the detection.
Specific automated SFA methods are described in the sections for the analytes of interest.
- Bibliography BEGG, R.D. 1971. Dynamics of continuous segmented flow analysis. Anal. Chem. 43:854. THIERS, R.E., A.H. REED & K. DELANDER. 1971. Origin of the lag phase of continuous flow curves. Clin. Chem. 17:42. FURMAN, W.B. 1976. Continous Flow Analysis. Theory and Practice. Marcel Dekker, Inc., New York, N.Y. COAKLEY, W.A. 1978. Handbook of Automated Analysis. Marcel Dekker, Inc., New York, N.Y. SNYDER, L.R. 1980. Continuous flow analysis: present and future. Anal. Chem. Acta 114:3.
4120 B. Segmented Flow Analysis Method
- General Discussion
a. Principle: A rudimentary system (Figure 4120:1) contains four basic components: a sampling device, a liquid transport device such as a peristaltic pump, the analytical cartridge where the chemistry takes place, and the detector to quantify the analyte.
In a generalized system, samples are loaded onto an automatic sampler. The sampler arm moves the sample pickup needle between the sample cup and a wash reservoir containing a solution closely matching the sample matrix and free of the analyte. The wash solution is pumped continuously through the reservoir to eliminate cross-contamination. The sample is pumped to the analytical cartridge as a discrete portion separated from the wash by an air-bubble created during the sampler arm’s travel from wash reservoir to sample cup and back.
In the analytical cartridge, the system adds the sample to the reagent(s) and introduces proportionately identical air-bubbles to reagent or sample stream. Alternatively, another gas or immiscible fluid can be substituted for air. The analyzer then proportions the analyte sample into a number of analytical segments depending on sample time, wash time, and segmentation frequency. Relative flow and initial reagent concentration determine the amount and concentration of each reagent added. The micro-circulation pattern enhances mixing, as do mixing coils, which swirl the analytical system to utilize gravitational forces. Chemical reactions, solvent separation, catalytic reaction, dilution, distillation, heating, and/or special applications take place in their appropriate sections of the analytical cartridge as the segmented stream flows toward the detector.
A typical SFA detector is a spectrophotometer that measures the color development at a
back into shape immediately after compression. Also see manufacturer’s manual and specific methods.
b. Electrical equipment and connections: Make electrical connections with screw terminals or plug-and-socket connections. Use shielded electrical cables. Use conditioned power or a universal power supply if electrical current is subject to fluctuations. See manufacturer’s manual for additional information.
c. Automated analytical equipment: Dedicate a chemistry manifold and tubing to each specific chemistry. See specific methods and manufacturer’s manual for additional information.
d. Water baths: When necessary, use a thermostatically controlled heating/cooling bath to decrease analysis time and/or improve sensitivity. Several types of baths are available; the most common are coils heated or cooled by water or oil. Temperature-controlled laboratories reduce drift in temperature-sensitive chemistries if water baths are not used.
- Reagents Prepare reagents according to specific methods and manufacturer’s instructions. If required, filter or degas a reagent. Use reagent water (see Section 1080) if available; if not, use a grade of water that is free of the analyte and interfering substances. Run blanks to demonstrate purity of the water used to prepare reagents and wash SFA system. Minimize exposure of reagents to air, and refrigerate if necessary. If reagents are made in large quantities, preferably decant a volume sufficient for one analytical run into a smaller container. If using a wetting agent, add it to the reagent just before the start of the run. Reagents and wetting agents have a limited shelf-life. Old reagents or wetting agents can produce poor reproducibility and distorted peaks. Do not change reagent solutions or add reagent to any reagent reservoirs during analysis. Always start with a sufficient quantity to last through the analytical run.
- Procedure For specific operating instructions, consult manufacturer’s directions and methods for analytes of interest. At startup of a system, pump reagents and wash water through system until system has reached equilibrium (bubble pattern smooth and consistent) and base line is stable. Meanwhile, load samples and standards into sample cups or tubes and type corresponding tags into computer table. When ready, command computer to begin run. Most systems will run the highest standard to trigger the beginning of the run, followed by a blank to check return to base line, and then a set of standards covering the analytical range (sampling from lowest to highest concentration). Construct a curve plotting concentration against absorbance or detector reading and extrapolate results (many systems will do this automatically). Run a new curve daily immediately before use. Calculation and interpretation of results depend on individual chemistry and are analogous to the manual method. Insert blanks and standards periodically to check and correct for any drift of base line and/or sensitivity. Some systems will run a specific standard periodically as a ‘‘drift,’’ and automatically will adjust sample results. At end of a run, let system flush according to manufacturer’s recommendations.
- Quality Control See Section 4020 and individual methods for quality control methods and precision and bias data.
4130 INORGANIC NONMETALS BY FLOW INJECTION ANALYSIS*#(9)
4130 A. Introduction
- Principle Flow injection analysis (FIA) is an automated method of introducing a precisely measured portion of liquid sample into a continuously flowing carrier stream. The sample portion usually is injected into the carrier stream by either an injection valve with a fixed-volume sample loop or an injection valve in which a fixed time period determines injected sample volume. As the sample portion leaves the injection valve, it disperses into the carrier stream and forms an asymmetric Gaussian gradient in analyte concentration. This concentration gradient is detected continuously by either a color reaction or another analyte-specific detector through which the carrier and gradient flow.
When a color reaction is used as the detector, the color reaction reagents also flow continuously into the carrier stream. Each color reagent merges with the carrier stream and is added to the analyte gradient in the carrier in a proportion equal to the relative flow rates of the carrier stream and merging color reagent. The color reagent becomes part of the carrier after it is injected and has the effect of modifying or derivatizing the analyte in the gradient. Each subsequent color reagent has a similar effect, finally resulting in a color gradient proportional to the analyte gradient. When the color gradient passes through a flow cell placed in a flow-through absorbance detector, an absorbance peak is formed. The area of this peak is proportional to the analyte concentration in the injected sample. A series of calibration standards is injected to generate detector response data used to produce a calibration curve. It is important that the FIA flow rates, injected sample portion volume, temperature, and time the sample is flowing through the system (‘‘residence time’’) be the same for calibration standards and unknowns. Careful selection of flow rate, injected sample volume, frequency of sample injection, reagent flow rates, and residence time determines the precise dilution of the sample’s original analyte concentration into the useful concentration range of the color reaction. All of these parameters ultimately determine the sample throughput, dynamic range of the method, reaction time of the color reaction discrimination against slow interference reactions, signal-to-noise ratio, and method detection level (MDL).
- Applications
FIA enjoys the advantages of all continuous-flow methods: There is a constantly measured
matrix neutral organics, water, and cations do not interfere with anion analysis, and fluoride is well resolved from monovalent organic acids. Sample preparation typically is dilution with reagent water and removal of suspended solids by filtration. If necessary, hydrophobic sample components such as oil and grease can be removed with the use of HPLC solid-phase extraction cartridges without biasing anion concentrations.
4140 B. Capillary Ion Electrophoresis with Indirect UV Detection
- General Discussion
a. Principle : A buffered aqueous electrolyte solution containing a UV-absorbing anion salt (sodium chromate) and an electroosmotic flow modifier (OFM) is used to fill a 75-μm-ID silica capillary. An electric field is generated by applying 15 kV of applied voltage using a negative power supply; this defines the detector end of the capillary as the anode. Sample is introduced at the cathodic end of the capillary and anions are separated on the basis of their differences in mobility in the electric field as they migrate through the capillary. Cations migrate in the opposite direction and are not detected. Water and neutral organics are not attracted towards the anode; they migrate after the anions and thus do not interfere with anion analysis. Anions are detected as they displace charge-for-charge the UV-absorbing electrolyte anion (chromate), causing a net decrease in UV absorbance in the analyte anion zone compared to the background electrolyte. Detector polarity is reversed to provide positive mv response to the data system (Figure 4140:1). As in chromatography, the analytes are identified by their migration time and quantitated by using time-corrected peak area relative to standards. After the analytes of interest are detected, the capillary is purged with fresh electrolyte, eliminating the remainder of the sample matrix before the next analysis.
b. Interferences: Any anion that has a migration time similar to the analytes of interest can be considered an interference. This method has been designed to minimize potential interference typically found in environmental waters, groundwater, drinking water, and wastewater.
Formate is a common potential interference with fluoride; it is a common impurity in reagent water, has a migration time similar to that of fluoride, and is an indicator of loss of water purification system performance and TOC greater than 0.1 mg/L. The addition of 5 mg formate/L in the mixed working anion standard, and to sample where identification of fluoride is in question, aids in the correct identification of fluoride.
Generally, a high concentration of any one ion may interfere with resolution of analyte anions in close proximity. Dilution in reagent water usually is helpful. Modifications in the electrolyte formulation can overcome resolution problems but require individual validation for precision and bias. This method is capable of interference-free resolution of a 1:100 differential
of Br –^ to Cl –^ , and NO 2 –^ and NO 3 –^ to SO 4 2–^ , and 1:1000 differential of Cl –^ and SO 4 2–^.
Dissolved ferric iron in the mg/L range gives a low bias for PO 4. However, transition metals
do not precipitate with chromate because of the alkaline electrolyte pH.
c. Minimum detectable concentrations : The minimum detectable concentration for an anion is a function of sample size. Generally, for a 30-s sampling time, the minimum detectable concentrations are 0.1 mg/L (Figure 4140:2). According to the method for calculating MDL given in Section 1030, the calculated detection limits are below 0.1 mg/L. These detection limits can be compromised by analyte impurities in the electrolyte.
d. Limitations : Samples with high ionic strength may show a decrease in analyte migration time. This variable is addressed by using normalized migration time with respect to a reference peak, chloride, for identification, and using time-corrected area for quantitation. With electrophoresis, published data indicate that analyte peak area is a function of migration time. At high analyte anion concentrations, peak shape becomes asymmetrical; this phenomenon is typical and is different from that observed in ion chromatography.
- Apparatus
a. Capillary ion electrophoresis (CIE) system :*#(11) Various commercial instruments are available that integrate a negative high-voltage power supply, electrolyte reservoirs, covered sample carousel, hydrostatic sampling mechanism, capillary purge mechanism, self-aligning capillary holder, and UV detector capable of 254-nm detection in a single temperature-controlled compartment at 25°C. Optimal detection limits are attained with a fixed-wavelength UV detector with Hg lamp and 254-nm filter.
b. Capillary : 75-μm-ID × 375-μm-OD × 60-cm-long fused silica capillary with a portion of its outer coating removed to act as the UV detector window. Capillaries can be purchased
premade *^ or on a spool and prepared as needed.
c. Data system :*#(12) HPLC-based integrator or computer. Optimum performance is attained with a computer data system and electrophoresis-specific data processing including data acquisition at 20 points/s, migration times determined at midpoint of peak width, identification based on normalized migration times with respect to a reference peak, and time-corrected peak area.
- Reagents
a. Reagent water: See Section 1080. Ensure that water is analyte-free. The concentration of dissolved organic material will influence overall performance; preferably use reagent water with
<50 μg TOC/L.
b. Chromate electrolyte solution: Prepare as directed from individual reagents, or purchase electrolyte preformulated.
- Sodium chromate concentrate, 100 m M: In a 1-L volumetric flask dissolve 23.41 g sodium chromate tetrahydrate, Na 2 CrO 4 ⋅4H 2 O, in 500 mL water and dilute to 1 L with water.
Store in a capped glass or plastic container at ambient temperature; this reagent is stable for 1 year.
- Electroosmotic flow modifier concentrate, 100 m M: In a 100-mL volumetric flask
Anion Salt
Amount g/100mL Nitrate NaNO (^3) 0.1371 (1000 mg NO 3
−/L = 225.8 mg NO 3
− −N/L)
Phosphate Na 2 HPO 4 † (^) 0.1500 (1000 mg PO 4
3 −/L = 326.1 mg PO 4
3 − −P/L)
Sulfate Na 2 SO 4 † (^) 0.1480 (1000 mg SO 42 −/L = 676.3 mg SO 42 −^ −S/L)
- Do not oven-dry, but dry to constant weight in a desiccator over phosphorous pentoxide. † Potassium salts can be used, but with corresponding modification of salt amounts.
d. Mixed working anion standard solutions: Prepare at least three different working anion standard solutions that bracket the expected sample range, from 0.1 to 50 mg/L. Add 5 mg formate/ L to all standards. Use 0.1 mL standard anion solution/100 mL working anion solution (equal to 1 mg anion/L). (Above 50 mg/ L each anion, chloride, bromide, nitrite, sulfate, and nitrate are no longer baseline-resolved. Analytes that are not baseline-resolved may give a low bias. If the analytes are baseline-resolved, quantitation is linear to 100 mg/L.) Store in plastic containers in the refrigerator; prepare fresh standards weekly. Figure 4140:3 shows representative electropherograms of anion standards and Table 4140:I gives the composition of the standards.
e. Calibration verification sample: Use a certified performance evaluation standard, or equivalent, within the range of the mixed working anion standard solutions analyzed as an unknown. Refer to Section 4020.
f. Analyte known-addition sample: To each sample matrix add a known amount of analyte, and use to evaluate analyte recovery.
- Procedure
a. Capillary conditioning: Set up CIE system according to manufacturer’s instructions. Rinse capillary with 100 m M NaOH for 5 min. Place fresh degassed electrolyte into both reservoirs and purge capillary with electrolyte for 3 min to remove all previous solutions and air bubbles. Apply voltage of 15 kV and note the current; if the expected 14 ± 1 μA is observed, the CIE system is ready for use. Zero UV detector to 0.000 absorbance.
b. Analysis conditions: Program CE system to apply constant current of 14 μA for the run time. Use 30 s hydrostatic sampling time for all standard and sample introduction. Analysis time is 5 min.
c. Analyte migration time calibration: Determine migration time of each analyte daily using the midrange mixed working anion standard. Perform duplicate analysis to insure migration time stability. Use the midpoint of peak width, defined as midpoint between the start and stop integration marks, as the migration time for each analyte; this accounts for the observed non-symmetrical peak shapes. (Use of peak apex may result in analyte misidentification.) The
migration order is always Cl –^ , Br –^ , NO 2 –^ , SO 4 2–^ , NO 3 –^ , F–^ , and PO 4 3–^. Dissolved HCO 3 –^ is the
last peak in the standard (see Figure 4140:1). Set analyte migration time window as 2% of the
migration time determined above, except for Cl–^ , which is set at 10%. Chloride is always the first peak and is used as the reference peak for analyte qualitative identification; identify anions on the basis of normalized migration times with respect to the reference peak, or migration time ratio. (See Figure 4140:1 and Table 4140:II.)
d. Analyte response calibration: Analyze all three mixed working anion standards in duplicate. Plot time-corrected peak area for each analyte versus concentration using a linear regression through zero. (In capillary electrophoresis peak area is a function of analyte migration time, which may change during analyses. Time-corrected peak area is a well-documented CE normalization routine, i.e., peak area divided by migration time. ( NOTE: Do not use analyte peak
height. ) Calibration is accepted as linear if regression coefficient of variation, R^2 , is greater than 0.995. Linearity calibration curves for anions are shown in Figure 4140:4, Figure 4140:5 and Figure 4140:6.
e. Sample analysis: After initial calibration run samples in the following order: calibration verification sample, reagent blank, 10 unknown samples, calibration verification sample, reagent blank, etc. Filter samples containing high concentrations of suspended solids. If peaks are not baseline-resolved, dilute sample 1:5 with water and repeat analysis for unresolved analyte quantitation. Resolved analytes in the undiluted sample are considered correct quantitation. Electropherograms of typical samples are shown in Figure 4140:7, Figure 4140:8, and Figure 4140:9.
- Calculation Relate the time-corrected peak area for each sample analyte with the calibration curve to determine concentration of analyte. If the sample was diluted, multiply anion concentration by the dilution factor to obtain original sample concentration, as follows:
C = A × F
where:
C = analyte concentration in original sample, mg/L, A = analyte concentration from calibration curve, mg/L, and F = scale factor or dilution factor. (For a 1:5 sample dilution, F = 5.)
- Quality Control
a. Analytical performance check: Unless analyst has already demonstrated ability to generate data with acceptable precision and bias by this method, proceed as follows: Analyze seven replicates of a certified performance evaluation standard containing the analytes of interest. Calculate mean and standard deviation of these data. The mean must be within the performance evaluation standard’s 95% confidence interval. Calculate percent relative standard deviation (RSD) for these data as (SD × 100) / mean; % RSD should conform to acceptance limit
