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The Use of Ion Chromatography in Anions Quantification

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Determination and Quantification of Anions in Aqueous Samples by Ion Chromatography


Inorganic anions were determined and quantified in tap, stream, and ground water samples using ion chromatography. Standards of chloride, bromide, nitrate, and sulfate were prepared ranging from 10.27 to 0.10 mM, 0.43 to 0.08 mM, 0.356 to 0.008 mM, and 0.8 to 0.2 mM for those respective ions. These standards and a five-ion standard containing fluoride, chloride, nitrate, phosphate, and sulfate were used to determine the ions present in the samples by comparing retention times. Linear regression techniques were used to plot relationships between concentration and conductivity, which was used to quantify the ions present. For stream water, tap, and ground water samples sulfate concentrations were 248 ± 22 μM, 796 ± 70 μM, and 720 ± 63 μM; nitrate concentrations were 10.7 ± 0.5 μM, 23.3 ± 1.2 μM, and 373 ± 19 μM; and chloride concentrations were 727 ± 27 μM, 763 ± 29 μM, and 1820 ± 73 μM respectively in the samples. The ground water sample additionally had 129 ± 16 μM bromide. All concentrations were determined within a 99.7% confidence interval. All water samples tested positive for unquantifiable trace levels of fluoride. The R2 values for the standard curves were 0.9991 for chloride, 0.9917 for bromide, 0.9973 for nitrate, and 0.9980 for sulfate, showing strong correlation between concentration and conductivity.


Numerous different anions may present in aqueous samples, in varying concentrations and oxidation states. Some of these ions play important roles as micronutrients and fertilizers, while others are toxic to human and animal life. For instance metabolization of nitrates result in a nitrite intermediate 1, which then oxidizes iron in hemoglobin, preventing transportation of oxygen in humans when in high enough concentration in a process called methemoglobinemia 2. However, this same ion is an important nutrient and source of nitrogen for plants used in agriculture 3. Likewise, many other ions such as sulfate, chloride, phosphate, and fluoride appear in water samples 4.

Ion chromatography (Figure 1) is a useful method for the detection and quantification of both anions and cations in aqueous samples 5. When measuring for anions an eluent, in this case an aqueous solution of carbonate and hydrogen carbonate, is pumped through the column and these ions interact with the positively charged quaternary ammonium groups on the beads in the column’s packing material. The sample is loaded into an injector that holds μL portions of the analyte in the sample loop, which then injects the sample into the guard column. The guard column contains packing material identical to the separatory column and is used to remove debris before it can reach the separatory column. The sample then flows into the separatory column, as the analyte ions go through the column they displace the eluent ions, interacting with the positively charged functional groups, these different ions separate into different bands within the column due to factors such as charge of the anion, hydrated radii, and its ability to contribute of disrupt hydrogen bonding in the surrounding water. These different factors affect the amount of time the ion stays within the column, retention time, allowing halides like fluoride to pass much more quickly through the column than others such as sulfate. After the column a suppressor is used to reduce background noise from the eluent, by exchange of Na+ for H+ from the acidic regenerant across a membrane, this increases the sensitivity for the analyte anions. As the anions exit the column and then the suppressor they cause a change in the conductivity of the solution, this is measured by a pair of electrodes in the conductivity detector as the eluate passes through a flow cell. The change in conductivity is a proportional linear relationship to concentration (equation 1) where Λ is the molar conductance, κ is the electrolytic conductivity, and c is the concentration. The electrolytic conductivity (equation 2) is related to the distance, d, between the electrodes, the area, A, of the electrodes, and the conductance, G, between the electrodes. The conductivity signal is recorded by a computer and displayed as a chromatogram, displaying peak conductivity against retention time.

Λ= κ/c (1) κ= d/A G (2)

Figure 1. Diagram of an ion chromatograph

Concentration of a sample ion can then be determined by measuring the conductivity of a set of standard solutions by ion chromatography and plotting the integral of the conductivity over the time the analyte eluted against the sample concentration. Use of linear regression techniques then yields the relationship between the peak area in µS·min against the concentration, where dividing the measured integral area of the sample by the first derivative of the determined linear relationship gives the sample concentration.


A Dionex ICS – 1100 RFIC ion chromatograph with a flow rate of 1.2 mL/min was used to determine and quantify the concentration of anions present in tap water, ground water, and stream water samples. A five ion standard containing fluoride, chloride, phosphate, sulfate, and nitrate was used to find the retention times of these ions with the settings used on the instrument. The retention times from the standards were compared to those from the samples to determine which ions were present. Standard curves were made for chloride, sulfate, nitrate, and bromide to find the linear relationship between concentration and area of the chromatogram peaks. The standards ranged from 10.27 to 0.10 mM for chloride, 0.43 to 0.08 mM for bromide, 0.356 to 0.008 mM for nitrate, and 0.8 to 0.2 mM for sulfate. All standards were diluted with ultra-pure deionized water, MQ water, with a purity of 18.2 MΩ·cm at 25 °C, in 10 ml volumetric flasks. All glassware except that which was used in preparation of the chloride samples, was acid washed with 10% hydrochloric acid and rinsed with MQ water to remove contaminants. Some of the water samples were diluted 1 to 10 due to high concentrations of ions to prevent clipping of signal, trailing, or malformed peaks in the chromatogram. The concentration of the different ions in the water samples were calculated by dividing the peak area by the slope from the standard curve for the respective ions.


The stream water sample was determined to have contain 727 ± 27 μM chloride, 10.7 ± 0.5 μM nitrate, and 248 ± 22 μM sulfate. The tap water contained 763 ± 29 μM chloride, 23.3 ± 1.2 μM nitrate, and 796 ± 70 μM sulfate. The ground water had 1820 ± 73 μM chloride, 373 ± 19 μM nitrate, 720 ± 63 μM sulfate, and 129 ± 16 μM bromide. These concentrations were all determined within a 99.7% confidence interval. All water samples contained trace non-quantifiable amounts of fluoride. The standard curves (Figure 2) showed a strong linearity between concentration and peak area with R2 values of 0.9991 for chloride, 0.9917 for bromide, 0.9973 for nitrate, and 0.9980 for sulfate.

The ions present in the samples were determined by comparing retention times to standards. From the 5-ion standard the retention times of fluoride was 2.74 min, chloride was 3.77 min, nitrate was 6.16 min, phosphate was 8.25 min, and sulfate was 10.01 min. The retention time of bromide, 5.48 min, was determined by preparation of a separate standard. The peaks retention times in tap water were 2.72 min, 3.79 min, 6.28 min, and 10.10 min. The peak retention times in stream water were 2.72 min, 3.81 min, 6.29 min, and 10.14 min. The peak retention times in ground water were 2.72 min, 3.74 min, 5.48 min, 6.21 min, and 10.09 min. These retention times matched very well with those of the standards, giving a high level of confidence that the ions were properly determined.

The limit of linearity of the instrument was not determined for any of the ions, at high concentrations there was some trailing and poor resolution in the peaks for chloride. The limits of detection for fluoride, chloride, nitrate, phosphate, and sulfate were 0.0065, 0.0061, 0.0115, 0.0049, and 0.0083 respectively. The limits of quantification for those same anions were 0.0218, 0.0204, 0.0384, 0.0163, and 0.0278. No quantity of phosphates was detected in any of the water samples, nor were any other peaks indicating other ions present.

Figure 2. Standard curves for chloride, bromide, nitrate, and sulfate with linear regression formulae and R2 values.


A very strong linear relationship was present between concentration and peak area, giving a strong measure of confidence in determining concentrations of anions in the water samples. The stream and tap water samples showed very similar levels of chloride; however the stream water had roughly half the nitrate and a third of the sulfate concentration of the tap water. The discrepancy between the tap water and the stream water may be due in part to the tap water staying in the pipes longer, allowing a buildup of trace ions. Another possible reason for the low concentrations of different ions in the stream water may be due to the filtration used to remove biological contamination. Some amount of the anions may have been physically filtered, or may have been within the cells of algae or bacteria collected on the filter. The ground water showed the highest levels of all the ions quantified, with over twice the chloride of the stream water and nearly forty times the concentration of nitrate. These concentrations are expected in the ground water sample due to leaching of minerals into the water from the well it was collected from. Furthermore, the level of nitrates in this sample may be due to fertilizers used in the area it was collected from, due to heavy agriculture and farming there.

The bromide present in the ground water was unexpected and could not be determined by comparing the retention times of the ions in the 5-ion standard. A solution of potassium bromide was prepared and its retention time was found to be 5.48 minutes, matching exactly with the unidentified peak in the ground water in ion chromatography under identical settings and conditions, confirming the presence of bromide. Care needed to be taken to avoid contamination and false positives in the samples. During preparation of the standards 10% HCl was used to wash the volumetric flasks, followed by MQ water. This caused minor peaks corresponding to chloride to appear in the chromatograms, which would not interfere with the sulfate, nitrate, and bromide standards. All glassware used to test for chloride was washed only with MQ water to prevent inaccuracies during analysis, however due to insufficient cleaning some minor contamination consistent with acetate was visible in the chromatograms. Due to the large quantities of chloride in the ground water sample there was major deformation in that peak on the chromatogram, displaying two adjacent peaks and trailing. To gather more accurate results this sample was diluted to 1:10 to reduce interference from overloading of the column. Although fluoride was detected in the samples no standard was prepared to attempt to find the concentration, this is due to the very low peak areas in the chromatogram which would not have allowed accurate quantification. All ions in the samples showed clear separation in the chromatogram with sharp symmetrical peaks, making adjustment of the eluent concentration or flow rate unnecessary for good resolution.


Ion chromatography is an effective method for determining quantities of anions in water samples and allows for effective determination of ion species, for instance allowing proper quantification of nitrate vs. nitrite. An identical sample tested through flame AAS would only read the total concentration of nitrogen due to the ion being broken down to the atomic level. This technique offers a wide range of sensitivity, able to determine ions over a few orders of magnitude of concentration, including proper identification of trace ions at concentrations too low to quantify through other methods. However, this technique has several disadvantages, such as very long run times, where heavy ions with a large negative charge such as sulfate and selenate can take over 10 minutes to analyze. Likewise, changes in the eluent concentration, the temperature, and packing of the column can vary the readings in the chromatogram significantly, over 5% between trials over a couple days. Further, care must be taken to maintain the instrument, samples saturated to a high concentration can damage the column, and improperly filtered samples or bacterial growth can clog the instrument. The ground, stream, and tap water samples all yielded concentrations of chloride, sulfate, and nitrate with expected ranges. This technique was effective at determining the ions present in the samples with high confidence and finding the concentrations with a good confidence interval with good R2 values. Coupling of this technique with others such as mass spectrometry would be effective at confirming the ion species present, likewise the use of flame AAS could be used to confirm the presence of elements in molecular ions to distinguish between ions of similar mass, size, and charge.

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