METHOD 300.0 

DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY 

John D. Pfaff 

Inorganic Chemistry Branch 

Chemistry Research Division 

Revision 2.1 

August 1993 

ENVIRONMENTAL MONITORING SYSTEMS LABORATORY 

OFFICE OF RESEARCH ANO DEVELOPMENT 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

CINCINNATI, OHIO 45268 

300.0-1 
METHOD 300.0 

DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY 

1.0 SCOPE AND APPLICATION 

1.1 This method covers the determ1nat1on of the following inorganic 

anions: 

PART fl. 

Bromide 

Chloride 

Fluoride 

Nitrate 

PART B. 

Bromate 

Chlorate 

Nitrite 

Ortho-Phosphate-P 

Sulfate 

Chlorite 

1.2 The m.1lrices applicable to each method are shown below: 

fl. Drinking water, surface water. mixed domestic and industrial 

wastewdters, groundwater, reagent waters. solids (after 

extraction 11.7), leachates (when no acetic acid is used). 

B. Drinking water and reagent waters 

1.3 The single laboratory Method Detect1on Limit (MOL defined in Sect. 

3.2) ror the above analytes is listed in Tables lA and lB. The MOL 

for a specific matrix may d1ffer from those l isted, depending upon 

the nature or the sample. 

1. 4 Me Lhod A is recommended for drinking and wastewaters. The 

mullilaboratory ranges tested for each anion are as follows: 

Analyte 

Brom1de 

Chlonde 

~ 1 uoride 

Nitrate-N 

Nitrite-N 

Ortho-Phosphate-P Sulfate 

mLI. 

0.63 - 21.0 

0.78- 26 .0 

0.26 - 8. 49 

0.42 - 14.0 

0.36 - 12.0 

0 . 69 - 23 .I 

2.85 - 95.0 

300.0-2 
1.5 Thi~ method is recommended for use only by or under the superv1s1on of analysts experienced In the use of 1on chromatography and in the interpretation of the rpsulting 1on chromatograms. 

1.6 When this method is used to analyze unfamiliar samples for any of the above anions, anion ldentiftcation should be supported by the 

use of a fortified sample matrix covertng the anions of Interest. The fortification pror.eclure is described in Sect. 11.6. 

1.7 Users of the method data should state the data-quality objectives prior to analysis. Users of the method mu~t demonstrate the ability to generate acceptable results w1th this melhod, using the procedures described in Sect. 9.0. 

2.1 A small volume of sample, typically 2 to 3 ml, is introduced into 

an ion chromatograph. The anions of interest are separated and measured, using a system comprised of a guard column, analytical column, suppressor device, and conductivity detector. 

2.2 lhe main differences between Parts A and 8 are the separator columns and guard columns. Sections 6.0 and 7.0 will elicit the differences. 

2.3 An extraction procedure must be performed to use this method for solids (See 11.7). 

2.4 Limited performance- based method modifications may be acceptable provided they are fully documented and meet or cxcPed requirements expressed in Sect. 9.0, Quality Contro l. 

3.0 DEFINITIONS 

3.1 CALIBRATION BLANK (CB) -- A volume of reagent watpr fortified with the same matrix as the calibration standards, but without the analytes, internal standards, or surrogate analytP~ . 

3.2 CALIBRATION STANDARD (CAL) -- A solution prepared from the prtmary dilution stdndard solution or stock standard solutions and the internal standards and surrogate analytes. The CAL solutions are used to calibrate the instrument response with respect to analyte concentration. 

3.3 FIELD DUPLICATES (FD) -- Two separate samples collected at the same time and place under identical circumstances and treated exactly the 

same throughout field and laboratory procedures. Analyses of field duplicates 1ndicate the precision assoc1ated with sample collection, preservation and storage, as well as with laboratory procedures. 

3.4 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) -- A ~olution of one or 

more method analytes, ~urrogates, internal standards, or other test 

300 .0-3 
substances used to evaluate the performance of the instrument system 

with respect to a defined set of criteria. 

3.5 LABORATORY FORTIFIED CLANK (LFB) --An aliquot of reagent water or 

other blank matrices to which known quantities of the method 

analytes are added in the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine whether the 

methodology is in control, and whether the laboratory is capable of 

making accurate and precise measurements. 

3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- An aliquot of an 

environmental sample to which known quantities of the method 

analytes are added in the laboratory. The LFM is analyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The background concent1·ations of the analytes in the sample matrix must. be 

determined in a separate aliquot and the measured values in the LFM 

corrected for background concentrations. 

3.7 LABORAlORY REAGENT BLANK (LRB) --An aliquot of reagent water or 

other blank matrices that are treated exactly as a sample including 

exposure to all glassware, equipment, solvents, reagents, internal 

standards, and surrogates that are used with other samples. The LRB 

is used to determine if method analytes or other interferences are 

present in the laboratory environment, the reagents, or the 

apparatus. 

3.8 LINEAR CALIBRATION RANGE (LCR) --The concentration range over which 

the instrument response is linear. 

3.9 MATERIAL SAFElY DATA SHEET (MSDS) -- Written information provided by vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and reactivity data including storage, spill, and 

handling precautions. 

3.IO METHOD DETECTION LIMIT (MDL) --The minimum concentration of an 

analyte that can be identified, measured and reported with 99% 

confidence that the analyte concentration is greater than zero. 

3.11 PERFORMANCE EVALUATION SAMPL[ (P[) --A solution of method analytes distributed by the Quality Assurance Research Division (QARO), 

Environmental Monitoring Systems Laboratory ([MSL-Cincinnati), U. S. 

Environmental Protection Agency, Cincinnati, Ohio, to multiple laboratories for analysis. A volume of the solution is added to a 

known volume of reagent water and analyzed with procedures used for 

samples. Results of analyses are used by QARO to determine 

statistically the accuracy and precision that can be expected when a 

method is performed by a competent analyst. Analyte true values are 

unknown to the analyst. 

3.12 QUALITY CONTROL SA~1PLE (QCS) -- A solution of method analytes of 

known concentrations that Is used to fortify an aliquot of LRB or 

300.0-4 
sample matrix. The QCS is obtalned from a source external to the laboratory and different from the source or calibration standards. It is used to check 1 aboratory performance with extern a 11 y pr~pared 

test materia 1s. 

3.13 STOCK STANDARD SOLUTION (SSS} --A concentr~ted solution containing one or more method analytes prepared in the laboratory using assayed reference materials or purchased from a reputable commercial source. 

4.0 INTERFERENCES 

4.1 Interferences can be caused by substances wilh retention times that 

are similar to and overlap those of the anion of interest. Large amounts of an anion can 1nterfere with the peak resolution of an adjacent anion. Sample dilutton and/ or fortification can be used to solve most interference problems associated with retention times. 

4.2 The water dip or negative peak that elutes ncar, and can Interfere with, the fluoride peak can usually be eliminated by the addition of the equivalent of 1 ml of concentrated eluent (7 .3 IOOX) to 100 ml of each standard and sample. 

4.3 Method Interferences may be caused by contdminants in the reagent water, reagents. glassware. and other ~ample processing apparatus that lead to discrete artifacts or elevated bas~line In 1on chromatograms. 

4.4 Samples that contain particles larger than 0.45 microns and reagent solutions that contain parti cles larger than 0.20 microns require filtration to prevent damage to Instrument columns and flow systems. 

4.5 Any anion that is not retained by the column or only slightly retained will elute in the area or fluoride and interfere. Known coelutlon Is caused by carbonate and other small organic anions. At concentrations of fluoride above 1.5 mg/L. this interference may not be significant, however. it is the responsibility of the user to gPnerate precision and accuracy information in each sample matrix. 

4.6 The acetate anion elutes early during the chromatographic run. The retention times of the antons also seem to differ when large amounts of acetate are present. Therefore, thh method is not recommended for leachates of solid samples when acetic dCid is used for pH adjustmP.nt.. 

4. 7 Th~ quantlt.atlon of unretained peaks should be avoided, such as low molecular weight organic actds (formate, acetdte, propionate etc .) which arc conductive and coelute wtth or neir fluoride and would bias the fluorldP quantitation 1n some drinking and most waste 

waters. 

4.8 Any residual chlorine dioxide present in the sample will result In the formation of andltional chlorite prior to analysis. If any 

300 .0-5 
5.0 SAFETY 

concentration of chlorine dioxide is suspected in the sample purge the sample with an inert gas (argon or nitrogen) for about five 

minutes or until no chlorine dioxide remains. 

5.1 The toxicity or carcinogenicity of each reagent used in this method 

have not been fully established. Each chemica 1 should be regarded 

as a potential health hazard and exposure should be as low as 

reasonably achievable. Cautions are included for known extremely hazardous materials or procedures. 

5.2 Each laboratory is responsible for maintaining a current awareness 

file of OSHA regulations regarding the safe handling of the 

chemicals specified in this method. A reference file of Material 

Safety Data Sheets (MSDS) should be made available to all personnel involved in the chemical analysis. The preparation of a formal 

safety plan is also advisable. 

5.3 The following chemicals have the potential to be highly toxic or 

hazardous, consult MSDS. 

5.3.1 Sulfuric acid (7.4) 

6.0 Equipment and Supplies 

6.1 Balance-- Analytical, capable of accurately weighing to the nearest 

0.0001 g. 

6.2 Ion chromatograph -- Analytical system complete with ion 'hromatograph and all required accessories including syringes, analytical columns, compressed gasses and detectors. 

6.2.1 Anion guard column: A protector of the separator column. If 

omitted from the system the retention times will be shorter. 

Usually packed with a substrate the same as that in the 

separator column. 

6.2.2 Anion separator column: This column produces the separation shown in Figures I and 2. 

6.2.2.1 Anion analytical column (Method A): The 

separation shown in Figure I was generated using a 

Olonex AS4A column (P/N 37041). An optional column may be used if comparable resolution of 

peaks is obtained, and the requirements of Sect. 

9.2 can be met. 

6.2.2.2 Anion analytical column (Method C). The 

separation shown in Figure 2 was generated using a 

Dionex AS9 column (P/N 42025). An optional column 

may be used if comparable resolution of peaks is 

300.0-6 
obtained and the requirements of Sect. 9.2 can be 

met. 

6.2.3 Anion suppressor device: The data presented in this method 

were generated using a Dlonex anion micro membrane 

suppressor (P/N 37106}. 

6.2.4 Detector -- Conductivity cell: approximately 1.25 ~l 

internal volume, {Dionex, or equivalent} capable of providing data as required in Sect. 9.2. 

6.3 The Dionex Al-450 Data Chromatography Software was used to generate all the data in the attached tables. Systems using a strlpchart recorder and integrator or other computer based data system may achieve approximately the same MOL's but the user should demonstrate this by the procedure outlined in Sect. 9.2. 

7.0 Reagents and Standards 

7.1 Sample bottles: Glass or polyethylene of sufficient volume to allow replicate analyses of anions of interest. 

7.2 Reagent water: Distilled or deionized water, free of the anions of interest. Water should contain particles no larger than 0.20 microns. 

7.3 Eluent solution (Method A and Method 8): Sodium bicarbonate (CASRN 144-55-8} 1.7 mM, sodium carbonate (CASRN 497-19-8) 1.8 mM. Dissolve 0.2856 9 sodium bicarbonate (NaHC03} and 0.3816 9 of sodium carbonate (lla2C03) In reagent water (7 .2) and dilute to 2 L. 

7.4 Regeneration solution (micro membrane suppressor): Sulfuric acid {CASRN-7664-93-9} 0.025N. Dilute 2.8 ml cone . sulfuric acid {H2S04} to 4 l with reagent water. 

7.5 Stock standard solutions, 1000 mg/L (1 mg/ml): Stock standard solutions may be purchased as certified solutions or prerared from ACS reagent grade matPrials (dried at 105•c for 30 min) as listed below. 

7 .5.1 

7.5.2 

7.5.3 

Bromide (Br') 1000 mgJL: Dissolve 1.2876 g sodium bromide (NaBr, CASRN 7647-15-6} in reagent water and dilute to J l. 

Bromate {Br03

') 1000 mg/L: Dissolve 1.1798y of sodium bromate {NaBr03 , CASRN 7789-38-0) 1n reagent water and dilute to I l. 

Chlorate {C103') 1000 mg/L: Dissolve 1.2753g of sodium chlorate (NaC!03 , CASRN 7775-09-9} in reagent water and dilute to I l. 

300.0-7 
7.5.4 

7.5.5 

7.5.6 

7.5.7 

7.5.8 

7.5 .9 

7. 5.10 

Chloride (CL') 1000 mg/L: Dissolve 1.6485 g sodium 

chloride (NaCl, CASRN 7647-14-5) in reagent water and 

dilute to 1 L. 

Chlorite (C10~") 1000 mg/L: Dissolve 1.3410g of sodium 

chlorite (NaC!02, CASRN 7758-19-2) in reagent water and 

dilute to 1 L. 

Fluoride (F") 1000 mgJL: Dissolve 2.2100g sodium fluoride 

(NaF, CASRN 7681-49-4) in reagent water and dilute to l L. 

Nitrate (NO" -N) 1000 mg/L: Dissolve 6.0679 g sodium 

nitrate (NaJo3 , CASRN 7631-99-4) in reagent water and 

dilute to 1 L. 

Nitrite (NO" -N) 1000 mg/L: Dissolve 4.9257 g sodium 

nitrite (Na~ • CASRN 7632-00-0) in reagent water and 

d i 1 ute to 1 L. 

Phosphate (PO" -P) 1000 mg/L: Dissolve 4.3937 g potassium phosphate (KH)'Ot, CASRN 7778-77-0) in reagent water 

and dilute to 1 . 

Sulfate (S04"} 1000 mg/L: Dissolve 

sulfate (K~S0 , CASRN 7778-80-5} in 

dilute to J L. 

1.8141 g potassium 

reagent water and 

NOTE: Stability of standards: Stock standards (7.5} are 

stable for at least 1 month when stored at 4"C. 

Except for the chlorite standard which is only stable 

for two weeks. Dilute working standards should be 

prepared weekly, except those that contain nitrite 

and phosphate should be prepared fresh daily. 

7.6 Ethylenediamine preservation solution: Dilute 10 ml of 

ethylenediamine (99%} (CASRN 107-15-3} to 200 ml with reagent 

water. Use 1 ml of this dilution to each 1 l of sample taken. 

8.0 sample Collecti on. Preservation and Storage 

8.1 Samples should be collected in plastic or glass bottles. All 

bottles must be thoroughly cleaned and rinsed with reagent water. 

Volume collected should be sufficient to insure a representative sample, allow for replicate analysis, if required, and minimize 

waste disposal. 

8.2 Sample preservation and holding times for the anions that can be 

determined by this method are as follows: 

Analyte 

Bromate 

Preservation 

None required 

300.0-8 

Holdjng [ime 

28 days 
Bromide 

Chlorate 

Chloride 

Chlorite 

n uoride 

Nitrate-N 

Combined 

(Nitrate/Nitrite} Nitrite-N 

0-Phosphate-P 

Sulfate 

tlone required tlone required tlont> requirt>d Cool to 4°C 

None requ1red Cool to 4'C 

cone. HzS04 

to a pll < 2 

Cool to 4'C 

Cool to 4'C 

Cool to 4'C 

28 days 28 days 28 days 1nrnedately 

28 days 48 hours 

28 days 

48 hours 

48 hours 

28 ddyS 

NOTE: If the determined value for the combined 

nitrate/nitrite exceeds 0.5 mg/L as N , a re~ample 

must be analyzed for the individual concentrations 

of nitrate and nitrite. 

8.3 The method of preservation and the holding time for samples analyzed by this method are determined by the anions of interest. 

In a given sample, the anion that requires the most preservation treatment and the shortest holding time will determine the preser- vation treatment. It is recommended that all ~amples be cooled to 

4"C and held for no longer than 28 days for Method A and analyzed immediately in Method B. 

NOTE: If the sample cannot be analyzed for chlorite wilhin s 10 

minutes, the sample may be preserved by adding I ml of the 

ethylenediamine (EDA} preservation solution (7.6) to 1 L 

of sample. This will preserve the concentration of the 

chlorite for up to 14 days. This addition of EOA has no 

effect on bromate or chlorate, so they can also be 

determined in a sample preserved with EOA. Residual 

chlorine dioxide should be removed from the sample (per 4.8) prior to the addition of EDA. 

9.0 QUALITY CONTROL 

9.1 Each laboratory using this method is required to operate a formal 

quality control (QC) program. The minimum requirements of this 

program consist of an initial demonstration of laboratory capability, and the periodic an,lysis of laboratory reagent blanks, forti fied blanks and other laboratory solutions as a continuing check on performance. The laboratory is required Lo maintain per- formance records that define the quality of the data that are 

generated. 

9.2 INITIAL DEMONSTRATION OF PERFORMANCE 

9.2.1 The initial demonstration of performance 1s used to 

characterize instrument performance (determ1nation of LCRs 

and analysis of QCS) and laboratory performance 

300.0-9 
(determination of HOLs) prior to performing analyses by this 

method. 

9.2.2 Linear Calibration Range (LCR) -- The LCR must be determined 

initially and verified every 6 months or whenever a 

significant change in instrument response Is observed or 

expected. The initial demonstration of linearity must use 

sufficient standards to insure that the resulting curve Is 

linear. The verification of linearity must use a minimum of 

a blank and three standards. If any verification data 

exceeds the inithl valuP.s by t 10%, linearity must be 

reestablished. If any portion of the range is shown to be 

nonlinear, sufficient standards must be used to clearly define the nonlinear portion. 

9.2.3 Quality Control Sample (QCS) --When beginning the use of 

this method, on a quarterly basis or as required to meet 

data-quality needs, verify the calibration standards and 

acceptable instrument performance with the preparation and 

analyses of a QCS. If the determined concentrations are not 

within t 10% of the stated values, performance of the 

determinative step of the method is unacceptable. The 

source of the problem must be identified and corrected 

before either proceeding with the initial determination of 

MDls or continuing with on-going analyses. 

9.2.4 Method Detection l. imlt (MDL) -- MDLs must be established for 

all analytes, using reagent water (blank) fortified at a 

concentration of two to three times the estimated instrument 

detection limit.<6> To determine MDL values, take seven 

replicate aliquots of the fortified reagent water and 

process through thP entire analytical method. Perform all 

calculations deflnPd In the method and report the 

concentration values 1n the appropriate unlts. Cilcuhte 

the MDL as follow~: 

MOL • (t) x (S) 

where, t - StudPnt's t value for a 99% confidence level 

and a standard deviation estimate with n-1 degrep~ of freedom [t • 3.14 for seven 

rei>l I cates]. 

S • standard deviation of the replicate analyses. 

MDLs should be determined every 6 months, when a new 

operalur begins work or whenever there is a significant chanye in lhc background or instrument response. 

9.3 ASSESSING LABORATORY PERFORMANCE 

300.0-10 
9.3.1 laboratory Reagent Blank (lRB) -- The laboratory must 

analyze at least one lRB with each batch of samples. Data 

produced are used to assess contamination from the 

laboratory environment. Values that exceed the HDl indicate 

laboratory or reagent contamination should be suspected and 

corrective actions must be taken before continuing the 

analysis. 

9.3.2 laboratory Fortified Blank (LFB) -- The laboratory must 

analyze at least one IFR with each batch of samples. Calculate accuracy as percent recovery (S~ct. 9.4.2). If 

the recovery of any analyte falls outside the required control limits of 90- 110%, that analyte Is judged out of 

control, and the source of the problem should be identified 

and resolved before continuing analyses. 

9.3.3 The laboratory must use LFB analyses data to assess 

laboratory performance against the required control limits 

of 90-110%. When sufficient internal performance data 

become available (usually a minimum of 20-30 analyses), optional control limits can be developed from the percent 

mean recovery (x) and the standard deviation (S) of the mean 

recovery. These data can be used to establish the upper and 

lower control limits as follows: 

UPPFR CONTROL LIMIT • X + 35 

LOWFR CONTROL LIHIT = X - 35 

The optional control limits must be equal to or better than 

the required control limits of 90-110%. After each five to 

ten new recovery measurements, new control limits can be 

calculated using only the most recent 20-30 data points. Also, the standard deviation (S) data should be used to 

establish an on going precision state~ent for the level of 

concentrations included In the LFB. These data must be kept 

on file and be available for review. 

9.3.4 Instrument Performance Check Solution (IPC) -- For all 

determinations the laboratory must analyze the !PC (a midrange check standard) and a calibration blank immediately following daily calibration, after every tenth sample (or 

more frequently, if required) and at the end of the sample 

run. Analysis of the !PC solution and calibration blank imm~diately following calibration must verify that the 

instrument is within± 10% of calibration. Subsequent andlyses of the IPC solution must verify the calibration is 

still within± 10%. If the calibration cannot be verified 

within the specified limits, reanalyze the !PC solution. If 

the second analysis of the JPC solution confirms calibrat1on 

to be outside the limits, sample analysis must be 

discontinued, the cause determined andjor in the case of 

drift, lhe instrument recalibratetl. All samples follow1ng 

300. ()-11 
the last acceptable IPC solution must be reanalyzed. The 

analysis dala of the calibration blank and !PC solution must 

be kept on file wilh the sample analyses data . 

9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY 

9.4.1 Laboratory Fortified Sample Matrix (LFM) -- The laboratory 

must add a known amou nt of analyte to a minimum of 10% of 

the routine samples. In each case the LFM aliquot must be a 

duplicate of the al iquot used for sampl e analysis. The 

analyte concentration must be high enough to be detected 

above the original sample and should not be less than f011 r 

times the MDL. The added analyte concentration should he 

the same as that used in the laboratory fortified blank. 

9.4.1.1 If the concentration of fortification is less than 

25% of the background concentration of the matrix 

the matrix recovery should not be calcul ated. 

9.4.2 Calcul ate the percent recovery for each analyte, corrected 

for concentrations measured in the unfortified sample, and 

compare these values to the designated LFM recovery range 90-110%. Percent recovery may be calculated using the 

following equation: 

R • c. - C 

....:...._.,....$- )( 100 

where, R • percent recovery. C • fortified sample concentration. 

c• • sample background concent rat ion. 

s • concentration equivalent of analyte added to 

sample. 

9.4.3 Unti l sufficient data becomes available (usually a m1n1mum 

of 20 to 30 analysis), assess laboratory performance against 

recovery limits for method A of 80 to 120% and 75 t o 125% 

for method B. When sufficient internal performance data 

becomes available develop control limits from percent mean 

recovery and the standard deviation of the mean recovery. 

9.4.4 If the recovery of any analyte falls outside the des1gnated LFM recovery range and t he l aboratory performance for t hat 

analyte is shown to be i n control (Sect. 9.3), the recovery 

problem encountered with the LFH is j udged to be either 

matrix or sol ution rel ated, not system related. 

9.4.5 Where reference materials are avai l abl e, they should be 

analyzed to provide additional performance data. The 

300.0-12 
analysis of reference samples is a valuable tool for 

demonstrating the ability to perform the method acceptably. 

9.4.6 In recognition of the rapid advances occurring in chromatog- raphy, the analyst is permitted certain options, such as the 

use of different columns andjor eluents, to improve the 

separations or lower the cost of measurements. Each time 

such modifications to the method are made, the analyst is 

required to repeat the procedure in Sect. 9.2. 

9.4.7 It is recommended that the laboratory adopt additional 

quality assurance practices for use with this method. The 

specific practices that are most productive depend upon the 

needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to monitor the precision of 

the sampling technique. When doubt exists over the 

identification of a peak in the chromatogram, confirmatory techniques such as sample dilution and fortification, must 

be used. Whenever possible, the laboratory should perform analysis of quality control check samples and participate in 

relevant performance evaluation sample studies. 

9.4.8 At least quarterly, replicates of LFBs should be analyzed to 

determine the precision of the laboratory measurements. Add 

these results to the on-going control charts to document 

data quality. 

9.4.9 When using Part B, the analyst should be aware of the pur1ty of the reagents used to prepare standards. Allowances must 

be made when the solid materials are less than 99% pure. 

10.0 Caljbratjon and Standardization 

10.1 Establish ion chromatographic operating parameters equivalent to 

those indicated in Tables lA or lB. 

10.2 For each analyte of interest, prepare calibration standards at a 

minimum of three concentration levels and a blank by adding accurately measured volumes of one or more stock standards (7.5) to 

a volumetric flask and diluting to volume with reagent water. If 

a sample analyte concentration exceeds the calibration range the 

sample may be diluted to fall within the range. If this is not 

possible then three new calibration concentrations must be chosen, 

two of which must bracket the concentration of the sample analyte of 

interest. Each attenuation range of the instrument used to analyze 

a sample must be calibrated individually. 

10.3 Using injections of 0.1 to 1.0 ml (determined by injection loop volume) of each calibration standard, tabulate peak height or area 

responses ~gainst the concentration. The results are used to 

prepare a calibration curve for each analyte. During this pro- c:edure, retention times must be recorded. 

300.0-13 
10.4 The calibration curve must be verified on each working day, or whenever the anion eluent is changed, and after every 20 samples. If the response or retention time for any analyte varies from the expected values by more than ± 101, the test must be repeated, using fresh calibration standards. If the results are still more than± 10%, a new calibration curve must be prepared for that analyte. 

10.5 Nonlinear response can result when the separator column capacity is exceeded (overloading). The response of the detector to the sample when diluted 1:1, and when not diluted, should be compared. If the calculated responses are the same, samples of this total anionic concentration need not be diluted. 

11. 0 Procedure 

11.1 Tables 1A and 18 summarize the recommended operating conditions for the ion chromatograph. Included in these tables are estimated retention times that can be achieved by this method. Other columns, chromatographic conditions, or detectors may be used If the requirements of Sect. 9.Z are met. 

11.2 Check system calibration daily and, if required, recallbrate as described in Sect. 10. 

11.3 load and inject a fixed amount of well mixed sample. Flush injection loop thoroughly, using each new sample. Use the same size loop for standards and samples. Record the resulting peak size in 

area or peak height units. An automated constant volume injection system may also be used. 

11.4 The width of the retention time window used to make identifications should be based upon measurement~ of actual retention time varia- tions of standards over the course of a day. Three times the standard deviation of a retention lime can be used to calculate a suggested window size for each analyle. However, the experience of the analyst should weigh heavily in the interpretation of chromatograms. 

11.5 If the response for the peak exceeds the working range of the 

system, dilute the sample with an dppropriatc amount of reagent water and reanalyze. 

11.6 If the resulting chromatogram fails to produce adequate resolution, or if identification of specific anions is questionable, fortify the sample with an appropriate amount or standard and rcanaly7e. 

NOTE: Retention time is inversely proportional to concentration. Nitrate and sulfate exh1b1t the greatest amount of change, although all anions are affected to some degree. In some 

cases this peak migration may produce poor resolution or Identification. 

300.0-14 
11.7 The following extraction should be used for solid materials. Add an amount of reagent water equal to ten times the weight of dry solid material taken as a sample. This slurry is mixed for ten minutes using a magnetic stirring device. Filter the resulting slurry before injecting using a 0.45 # membrane type filter. This can be the type that attaches directly to the end of the syringe. Care should be taken to show that good recovery and identification of peaks is obtained with the user's matrix through the use of fortified samples. 

11.8 It has been reported that lower detection limits for ~ornate 

(•7 pg/L) can be obtained using a borate based eluent<>. The use of this eluent or other eluents that improve method performance may be considered as a minor modification of the method and as such still are acceptable. 

11.9 Should more complete resolution be needed between peaks the eluent (7.3) can be diluted. This will spread out the run but will also cause the later eluting anions to be retained longer. The analyst must determine to what extent the eluent is diluted. This dilution should not be considered a deviation from the method. 

12.0 DATA ANALYSIS AND CALCULATIONS 

12.1 Prepare a calibrat1on curve for each analyte by plotting instrument response against standard concentration. Compute sample concentration by comparing sample response with the standard curve. Multiply answer by appropriate dilution factor. 

12.2 Report only those values that fall between the lowest and the highest calibration standards. Samples exceeding the highest standard should be diluted and reanalyzed. 

12.3 Report results in mg/L. 

12.4 Report N02

" as N 

No;· as H 

HP04" as P 

13.0 METHODS PERFORMANCE 

13.1 Tables lA and 2A give the single laboratory (EHSL-Cincinnati) MOL fur each anion included in the method under the conditions listed. 

13.2 Tables 2A and 28 give the single laboratory (EHSL-Cincinnati) standard deviation for each anion included in the method in a variety of waters for the listed conditions. 

13.3 Multiple laboratory accuracy and bias data (51

) and estimated single operator values (50 ) for reagent, drinking ana waste water using 

JOO.D-15 
method A arc given for each anion in Tables 3 through 9. Data from 

19 laboratories were used for this data. 

13.4 Some of the bias statements, for example chloride and sulfate, may be misleading due to spiking small increments of the anion into 

large naturally occurring com;entrations of the same anion. 

14.0 POLLUTION PREVENTION 

14.1 Pollution prevention encompasses any technique that reduces or 

eliminates the quantity or toxicity of waste at the point of 

generation. Numerous opportunities for pollution prevention exist 

in laboratory operation. The EPA has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. 

Whenever feasible, laboratory personnel should use pollution prevention techniques to address their waste generation. When 

wastes cannot be feasibly reduced at the source, the Agency recommends recycling as the next best option. 

14.2 Quantity of the chemicals purchased should be based on expected 

usage during its shelf life and disposal cost of unused material. 

Actual reagent preparation volumes should reflect anticipated usage and reagent stability. 

14.3 For information about pollution prevention that may be applicable to 

laboratories and research institutions, consult "less is Better: 

laboratory Chemical Management for Waste Reduction," available from 

the American Chemical Society's Department of Government 

Regulations and Science Policy, 1155 16th Street N.W., Washington 

D.C. 20036, (202) 872-4477. 

15.0 WASTE MANAGEMENT 

15.1 The Environmental Protection Agency requires that laboratory waste 

management practices be conducted consistent with all applicable rules and regulations. Excess reagents, samples and method 

process wastes should be characterized and disposed of in an 

acceptable manner. The Agency urges laboratories to protect the 

air, water, and land by minimizing and controlling all releases from 

hoods and bench operations, complying with the letter and spirit of 

any waste discharge permit and regulations, and hy complying with 

all solid and hazardous waste regulations, particularly the 

hazardous waste identification rules and land disposal restrictions. For further information on waste management consult 

lhe "Waste Management Manual for Laboratory Personnel," available 

from the American Chemical Society at the address listed in Sect. 

14.3. 

300.0-16 
16.0 REFERENCES 

1. "Determination of Inorganic Disinfection By-Products by Ion Chromatography", J. Pfaff, C. Brockhoff. J. Am. Water Works Assoc., Vol BZ, No. 4, pg 192. 

2. Standard Methods for the Examination nf Water and Wastewater, Method 4110B, "Anions by Ion Chromatography", 18th Ed1tion of Standard Methods (1992) . 

3. Dionex, System 4000 Operation and Maintenance Manual, Dionex Corp., Sunnyvale, California 94086, I988. 

4. Method Detection Limit (MDL) as described in "Trace Analyses for Wastewater,• J. Glaser, D. Foerst, G. McKee, S. Quave, W. Budde, lnvironmental Science and Technology, Vol. 15, Number 12, page 1426, December, 1981. 

5. American Society for Testing and Materials. Test Method for Anions in Water by Chemicolly-SuppressPd Jon Chromatography D4327-91. Annual Book of Standards, Vol 11.01 (1993). 

6. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B. 

7. Hautman, D.P. & Bolyard, M. Analysts of Oxyhalide Disinfection By- products and other Anions of Interest tn Drinking Wa ter by Ion Chromatography. Jour. of Chromatog., 602, (1992), 65-74. 

300.0-17 
17.0 TABLES. DIAGRAMS. FLOWCHARTS AND VALTDATION DATA 

TABLE lA. CHROMATOGRAPHIC CONDITIONS AND DETECTION LIMITS 

IN REAGENT WATER (PART A) 

ANALYTE 

Fluoride 

Chloride 

Ni trite-N 

Bromide 

Ni trate-N 

o-Phosphate-P 

Sulfate 

Standard Conditions: 

* RETENTION 

PEAK I TIHE(HIN) 

1 

2 

3 

4 

5 

6 

7 

1. 2 

1.7 

2.0 

2.9 

3.2 

5.4 

6.9 

MDL 

mg/L 

0.01 

0.02 

0.004 

0.01 

0.002 

0.003 

0.02 

Columns: as specified in 6.2.2.1 

Delector: as specified in 6.2.4 

Eluent: as specified in 7.3 

Pump Rate: 2.0 mLjmin. Sample Loop: SO ~L 

t1DL calculated from data system using a y-axis selection of 

1000 ns and with a stripchart recorder with an attenuator 

setting of I uMHO ful l scale. 

* See Figure I 

300.0-18 
TABLE lB. CHROMATOGRAPHIC CONDITIONS AND DETECTION LIMITS 

IN REAGENT WATER (PART B) 

AHALYTE 

Chlorite 

Bromate 

Chlorate 

Standard Conditions: 

* PEAK I 

1 

2 

4 

Column: as specified in 6.2.2.2 

Detector: as specified in 6.2.4 

Eluent: as specified in 7.3 

* See Figure 2 

RETENTION 

TTME(MIN) MDL 

mg/L 

2.8 

3.2 

7.1 

0.01 

0.02 

0.003 

Pu~p Rate: 1.0 ml/min. 

Sample Loop: 50 ~L 

Attentuation - 1 

y-axis - 500 ns 

300.0-19 
TABLE 2A. SINGLE-OPERATOR ACCURACY AND BIAS Of STANDARD ANIONS 

(METHOD A} 

KNOWN NUMBER MEAN STANDARD 

SAMPLE CONC. OF RECOVERY DEVIATION 

ANALYTE TYPE Cmg/Ll REPLICATES " (p!q/ll 

Bromide RW 5.0 7 99 0.08 

ow 5.0 7 lOS 0.10 

sw 5.0 7 9S 0.13 

ww 5.0 7 lOS 0.34 

GW 5.0 7 92 0.34 

so 2.0 7 82 0.06 

Chloride RW 20.0 7 96 0.35 

ow 20.0 7 108 1.19 

SW 10.0 7 86 0.33 

ww 20.0 7 101 5.2 

GW 20.0 7 114 1.3 

so 20.0 7 90 0.32 

Fluoride RW 2.0 7 91 0.05 

ow 1.0 7 92 0.06 

SW 1.0 7 73 0.05 

ww 1.0 7 87 0.07 

GW 0.4 7 95 0.07 

sn 5.0 7 101 0.35 

Nitrate- N RW 10.0 7 103 0. 21 

ow 10.0 7 104 0.27 

SW 10.0 7 93 0.17 

ww 10.0 7 101 0.82 

GW 10.0 7 97 0.47 

so 10.0 7 82 0.28 

Nitrite- N RW 10.0 7 97 0.14 

ow 10.0 7 121 0.25 

sw 5.0 7 92 0.14 

ww 5.0 7 91 0.50 

GW 10.0 7 96 0.3S 

so 2.0 7 98 0.08 

o Phosphate- P RW 10.0 7 99 0.17 

ow 10.0 7 99 0.26 

sw 10.0 7 98 0.22 

ww 10.0 7 106 0.85 

GW 10.0 7 95 0.33 

300.0-20 
Sulfate RW 

ow 

sw 

ww 

GW 

RW • Reagent Water 

OW - Drinking Water 

SW • Surface Water 

TABLE 2A (CONT ' O) 

20.0 

50.0 

40.0 

40.0 

40.0 

7 

7 

7 

7 

7 

99 

105 

95 

102 

112 

0.40 

3.35 

1.7 

6.4 

3.2 

WW ~ Mixed Domestic and Industrial Wastewater 

GW % Groundwater 

SO% USEPA QC Solid (shale} 

300. 0- 21 
TABLE 2B. SINGLE-OPERATOR ACCURACY AND BIAS OF BY-PRODUCT 

(PART B) 

NUMBER MEAN STANDARD 

SAMPLE SPIKE OF RECOVERY DEVIATION 

ANALYTE TYPE (mg/L) REPLICATES % (mgfL) 

Bromate RW 5.0 7 103 0.07 

1.0 7 98 0.04 

0.1 7 155 0.005 

0.05 7 122 0.01 

ow 5.0 7 95 0.04 

1.0 7 85 0.02 

0. I 7 98 0.005 

0.05 7 98 0.005 

Chlorate RW 5.0 7 101 0.06 

1.0 7 97 0.01 

0. I 7 100 0.01 

0.05 7 119 0.05 

OW 5.0 7 101 0.04 

1.0 7 115 0.01 

0.1 7 121 0.005 

0.05 7 110 0.01 

Chlorite RW 5.0 7 100 0.04 

1.0 7 98 0.01 

0.1 7 86 0.01 

0.05 7 94 0.01 

ow 5.0 7 96 0.03 

1.0 7 100 0.02 

0.1 7 76 0.00 

0.05 7 96 0.01 

RW = Reagent Water 

OW = Drinking Water 

300.0-22 
TABLE 3. 

WATER M'T ADDED 

1119/L 

Reagent 0.26 

0.34 

2.12 

2.55 

6.79 

8. 49 

Drinking 0. 26 

0.34 

2.12 

2.55 

6.79 

S.49 

Waste 0.26 

0.34 

2.12 

2.55 

6. 79 

8.49 

~LTIPLE LABORATORY (n•l9) DETER"INATION OF BIAS FOR FLUORIDE 

A"'T FOUND 

mg/L 

st so BIAS 

" 0.25 0.08 0.11 -3.8 0.29 0.11 -14.7 2.12 0.07 0.12 0.0 2.48 0.14 -2.7 6.76 0.20 0.19 -0. 4 8.46 0.30 -0. 4 

0.24 0.08 0.05 -7.7 0.34 0.11 0.0 2.09 0.18 0.06 -1.4 

2.55 0.16 0.0 6.84 0.54 0.25 +0.7 

8.37 0. 75 -1.4 

0.25 0.15 0.06 -3.8 

0.32 0.08 -5.9 

2.13 0.22 0.15 +0.5 2.48 0.16 -2.7 6.65 0.41 0.20 -2.1 8.27 0.36 -2.6 

300.0-23 
TABLE 4. ~ULTIP LE LABORATORY (n•l9) DETER~INATION OF BIAS FOR CHLORIDE 

WATER AI1'T ADDED Ari'T rOUND s, s. BIAS 

mg/L mg/L " 

Reagent 0. 78 0.79 0.17 0.29 +1.3 

1.04 1.12 0.46 +7.7 

6.50 6.31 0.27 0. 14 -2.9 

7.80 7.76 0.39 -0.5 

20.8 20.7 0.54 0.62 -0.5 

26 .0 25.9 0.58 -0.4 

Drinking 0.78 0.54 0.35 0.20 -30.8 

1.04 0.51 0. 38 -51.0 

6.50 5.24 1.35 1.48 -19.4 

7.80 6.02 1.90 -22.8 

20.8 20.0 2.26 1.14 -3.8 

26.0 24.0 2.65 -7.7 

Waste 0.78 0.43 0.32 0.39 -44.9 

1.04 0.65 0.48 -37.5 

6.50 4.59 1.82 0.83 -29. 4 

7.80 5.45 2.02 -30.1 

20.8 18.3 2. 41 I. 57 -II .8 

26.0 23.0 2.50 -II .5 

300.0-24 
TABLE 5. MULTIPLE LABORATORY (ncl9) 

DETERHINATJON OF BIAS FOR NITRITE - NITROGEN 

WATER AH 'T ADDED A" 'T FOUND s, s. BIAS mg/L mg/L " Reagent 0.36 0. 37 0.04 0.04 -t2.8 0.48 0.48 0.06 0.0 3.00 3.18 0.12 0.06 16 .0 3.60 3.83 0.12 +6. 4 

9.60 9.84 0.36 0.26 +2.5 12 .0 12.1 0.27 +0. 6 

Drinking 0.36 0.30 0.13 0.03 - 16.7 0.48 0. 40 0.14 - 16.7 3.00 3.02 0.23 0.12 +0.7 3.60 3.62 0.22 ~0.6 9.60 9.59 0.44 0.28 0.1 12 .o 11.6 0.59 3.1 

Waste 0.36 0.34 0.06 0.04 5.6 0. 48 0.46 0.07 4.2 3.00 3.10 0.13 0.10 ~6.0 

3.60 3.76 0.18 •4.4 9.60 9.74 0.49 0.26 +1.5 12.0 12.0 0.56 +0.3 

300.0-25 
TABLE 6. "ULTIPLE LABORATORY (n~l9) 

DETERMINATION OF BIAS FOR BROIIIDE 

WATER A"'T ADDED AII'T FOUND st s. 

mg/L mg/L 

Reagent 0.63 0.69 0.11 0.05 

0.84 0.85 0.12 

5.24 5.21 0.22 0.21 

6.29 6.17 0.35 

16.8 17.1 0. 70 0.36 

21.0 21.3 0.93 

Drinking 0.63 0.63 0.13 0.04 

0.84 0.81 0.13 

5.24 5.11 0.23 0. J 3 

6.29 6.18 0.30 

16.8 17.0 0.55 0.57 

21.0 20.9 0.65 

Waste 0.63 0.63 0.15 0.09 

0.84 0.85 0.15 

5.24 5.23 0.36 0.11 

6.29 6.27 0.46 

16.8 16.6 0.69 0.43 

21.0 21.1 0.63 

300.0-26 

BIAS 

" 

+9.5 

+1.2 

-0.6 

-1.9 

+1.6 

+1.5 

0.0 

-3.6 

-2.5 

-1.7 

+0.9 

-0.4 

0.0 

+1.2 

-0.2 

-0.3 

-1.0 

+0.3 
TABLE 7. ~LTJPLE LABORATORY (n•l9} 

DETER"JNATION OF BIAS FOR NITRATE - NITROGEN 

WATER A" 'T ADDEO APII 'T FOUND s, s. BIAS 

mg/l mg/L " 

Reagent 0.42 0.42 0.04 0.02 0.0 

0.56 0.56 0.06 0.0 

3.51 3.34 0.15 0.08 -4.8 

4.21 4.05 0.28 -3.8 

11.2 11.1 0.47 0.34 - 1.1 

14.0 14.4 0.61 +2.6 

Drinking 0.42 0.46 0.08 0.03 t 9.5 

0.56 0.58 0.09 +3.6 

3.51 3.45 0.27 0.10 - 1.7 

4.21 4.21 0.38 0.0 

11.2 ll. 5 0.50 0.48 +2.3 

14 .0 14.2 0. 70 t l.6 

Waste 0.42 0.36 0.07 0.06 - 14.6 

0.56 0.40 0.16 -28.6 

3. 51 3.19 0.31 0.07 -9.1 

4.21 3.84 0.28 -8.8 11.2 10.9 0.35 0.51 -3.0 14.0 14.1 0. 74 +0.4 

300.0-27 
TABLE 8. MULTIPLE LABORATORY (n~19 ) 

DETERMINATION OF BIAS FOR ORTHO-PHOSPHATE 

WATER AM'T ADDEO AM 'T FOUND s, s. BIAS 

mg/L mg/L ~ 

Reagent 0.69 0.69 0.06 0.06 0.0 

0.92 0.98 0.15 +6.5 

5. 77 5.72 0.36 0.18 -0.9 

6.92 6.78 0.42 -2.0 

18.4 18.8 1.04 0.63 +2 .1 

23.1 23.2 0.35 +0 .4 

Drinking 0.69 0. 70 0.17 0.17 +1.4 

0.92 0.96 0.20 +4 .3 

5. 77 5.43 0.52 0.40 -5.9 

6.92 6.29 0.72 -9.1 

18.4 18.0 0.68 0.59 -2.2 

23 .1 22.6 1.07 -2.0 

Waste 0.68 0.64 0.26 0.09 -7.2 

0.92 0.82 0.28 -10.9 

5.77 5.18 0.66 0.34 -10.2 

6.92 6.24 0. 74 -9.8 

18.4 17.6 2.08 1.27 -4 . 1 

23.1 22.4 0.87 -3.0 

300.0-28 
TABLE 9. MULTIPLE LABORATORY (nel9) 

DETERMINATION OF BIAS FOR SULFATE 

WATER AM'T ADDED AM'T FOUND s, mgfl mg/L 

Reagent 2.85 2.83 0.32 

3.80 3.83 0.92 

23.8 24.0 1.67 

28.5 28.5 1.56 

76.0 76.8 3.42 

95.0 95.7 3.59 

Drinking 2.85 1.12 0.37 

3.80 2.26 0.97 

23.8 21.8 1.26 

28.5 25.9 2.48 

76.0 74.5 4.63 

95.0 92.3 5.19 

Waste 2.85 1.89 0.37 

3.80 2.10 l. 25 

23.8 20.3 3.19 

28.5 24.5 3.24 

76.0 71.4 5.65 

95.0 90.3 6.80 

300.0-29 

so BIAS 

% 

0.52 -0.7 

+0.8 

0.68 +0.8 

-0.1 

2.33 +1.1 

+0.7 

0.41 -60.7 

-40 .3 

0.51 -8 .4 

-9 . I 

2.70 -2 .0 

-2.8 

0.24 -33.7 

-44.7 

0.58 -14.7 

-14.0 

3.39 -6 .] 

-5.0 
Method A 

Peak 

1 

2 

3 

2 4 

5 

6 

7 

7 

5 

1 3 

ll 4 \. 6 

I I I I I 

0 2 4 6 8 

Minutes 

Figure 1. Clv'omatogram Show1ng separallon using the AS4A eolurM 

I 

0 

I 

I 

2 

1 2 

3 

I 

4 

Minutes 

Method B 

Peak 

1 

2 

3 

4 

4 

I I 

6 8 

Figure 2. Chromatogram showing separation uSing lhe AS9 cotumn 

Ret. Time lon mg/L 

1.17 F- 2 

1.73 Cl" 20 

2.02 NO- 2 I 

2.95 Br 2 

3.20 NO -

l 10 

5.38 HPO >- • 2 

8.92 so>-• 60 

Ret. Time I on mg/L 

2.75 CIO2 0.1 

3.23 BrO -

) 0.1 

3.63 Cl" 0.1 

7.08 CIO-3 0.1