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APPLICATION NOTE 73745 Determination of sodium, potassium, and calcium in rice and wheat flours using ion chromatography Authors: Beibei Huang and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA Keywords: Dionex IonPac CS16 column, suppressed conductivity detection, RFIC system Goal To develop an ion chromatography method for the determination of sodium, potassium, and calcium in rice and wheat flours Introduction For the past 20 years, ion chromatography (IC) with suppressed conductivity detection has proven to be a robust and reliable technique for the determination of alkali and alkaline earth metals. The National Institute of Standards and Technology (NIST) established a Health Assessment Measurements Quality Assurance Program (HAMQAP) to support the measurement needs of the food, dietary supplement, and clinical communities.1 Participants measure concentrations of nutritional and toxic elements, fat- and water-soluble vitamins, fatty acids, active and/ or marker compounds, and contaminants in samples distributed by NIST. Rice and wheat flours were provided by NIST to measure nutritional elements including calcium, potassium, and sodium. Here, we employed IC to make those measurements. The method uses a Thermo ScientificTM DionexTM IonPacTM CS16 column, an electrolytically generated methanesulfonic acid (MSA) eluent, and suppressed conductivity detection on a Reagent-FreeTM Ion Chromatography (RFICTM) system. The Dionex IonPac CS16 column is a high-capacity cation exchange column that has medium hydrophobicity and is solvent compatible with 100% aqueous eluents, 100% acetonitrile, or 20% tetrahydrofuran without loss of performance.2 The high capacity of 3000 eq/column is achieved by using a particle diameter of 5.5 m and high density of grafted carboxylic acid cation exchange groups. The flour samples were extracted under acidic conditions and then separated on a Dionex IonPac CS16 column set. The cations were measured by suppressed conductivity detection. The contents of the cations in different flour samples vary greatly. Therefore, it is important to have a high capacity column to determine small amounts of one cation in the presence of large amounts of other cations, including the hydronium ions from the extraction solution. In our study, linearity, limits of detection and quantification, accuracy, and precision are demonstrated. Experimental Equipment · Thermo ScientificTM DionexTM ICS-6000 HPIC system* including: Thermo ScientificTM DionexTM ICS-6000 DP Pump module Thermo ScientificTM DionexTM ICS-6000 EG Eluent Generator module with high-pressure degasser module Thermo ScientificTM DionexTM ICS-6000 DC Detector/ Chromatography module CD Conductivity Detector Reagents and standards · Deionized (DI) water, Type I reagent grade, 18 M·cm resistivity or better · Thermo ScientificTM DionexTM Combined Six Cation Standard-II, 50 mL (P/N 046070) · Sodium chloride, (Crystalline/Certified ACS), Fisher ChemicalTM (Fisher Scientific P/N S271-500) · Potassium chloride, (Crystalline/Certified ACS), Fisher Chemical (Fisher Scientific P/N P217-500) · Calcium chloride dihydrate, (Certified ACS), Fisher Chemical (Fisher Scientific P/N C79-500) Tablet control · Thermo Scientific Dionex AS-AP Autosampler (P/N 074921) that includes sample syringe, 250 µL (P/N 074306) and buffer line, 1.2 mL (P/N 074989) Samples · One bottle of rice flour · One bottle of wheat flour The samples were provided by NIST. *This method can be run on any Thermo ScientificTM DionexTM Reagent-Free Ion Chromatograph (RFICTM) system, and an SP pump module can be used rather than the DP pump module. Consumables · Thermo ScientificTM DionexTM EGC 500 MSA Eluent Generator Cartridge (P/N 075779) · Thermo ScientificTM DionexTM CR-CTC 600 Continuously Regenerated Cation Trap Column (P/N 088663) · Thermo ScientificTM DionexTM CDRS 600 Cation Dynamically Regenerated Suppressor (2 mm, P/N 088670) · Thermo ScientificTM DionexTM IC PEEK ViperTM Fitting Kit for Dionex ICS-6000 2 mm systems with CD (Analytical) (P/N 302965) · Thermo ScientificTM NalgeneTM Syringe Filters, PES, 0.2 m (Fisher Scientific P/N 725-2520) · Air-TiteTM All-Plastic Norm-JectTM Syringes, 5 mL, Sterile (Fisher Scientific P/N 14-817-28) Software Thermo ScientificTM ChromeleonTM Chromatography Data System software version 7.2 IC conditions Columns Dionex IonPac CG16 Guard, 3 × 50 mm (P/N 079931) Dionex IonPac CS16 Analytical, 3 × 250 mm (P/N 059596) Eluent source Dionex EGC 500 MSA Eluent Generator Cartridge with CR-CTC 600 trap column Eluent 30 mM MSA Flow rate 0.36 mL/min Column temperature 40 ºC Detector temperature 20 ºC Injection volume 25 µL, (Full Loop) Detection Suppressed conductivity, Dionex CDRS 600 Suppressor (2 mm), recycle mode, Use the recommended voltage at constant voltage mode or 32 mA in constant current mode. System backpressure ~2600 psi (with backpressure coils) (100 psi = 0.6894 MPa) Background conductance ~0.2 µS/cm Run time 25 min Preparation of solutions and reagents Stock solution To prepare 1000 mg/L stock solutions of each cation of interest, accurately weigh the amounts of reagent-grade salts in Table 1, transfer to a 100 mL plastic volumetric flask, and fill to the mark with DI water. Mix thoroughly and store at 4 °C. Stock standards are stable for at least three months. Our experiences and official ISO standards indicate that all solutions used, including the standard solutions, should be acidified. This prevents false negative bias due to the formation of calcium and magnesium carbonates. 2 Table 1. Amounts of compounds used to prepare 100 mL of 1000 mg/L stock solutions Cation Sodium Potassium Calcium Compound Sodium chloride Potassium chloride Calcium chloride Mass (mg) 254.2 190.7 366.8 Working standard solutions calibration Prepare the calibration standard solutions by diluting the 1000 mg/L stock standard with DI water. The calibration concentrations were 0.02, 0.05, 0.2, 0.4, 2, 4, 10, and 20 mg/L for sodium; 0.05, 0.125, 0.5, 1, 5, 10, 25, and 50 mg/L for potassium; and 0.05, 0.125, 0.5, 1, 5, 10, 25, and 50 mg/L for calcium. For example, to prepare the 20 mg/L sodium, 50 mg/L potassium, and 50 mg/L calcium mixed standard, add 2 mL 1000 mg/L sodium, 5 mL potassium, and 5 mL calcium into a 100 mL plastic volumetric flask and bring to volume with DI water. Sample preparation Weigh 1 g of the flour samples and extract them in 100 mL 3% (w/w) acetic acid solution. Shake the mixture for at least 5 min at room temperature. Centrifuge sample solutions at 5000 rpm for 30 min; then pass through a 0.22 µm Nalgene PES syringe filter. Prepare three samples from one bottle of each flour provided. Recovery study The samples were spiked with appropriate known amounts of the analytes, then subjected to the sample preparation procedure. Use the overall mean of the unspiked samples for the recovery calculation. Results and discussion Separation and detection The separation of six common cations was performed on a Dionex IonPac CS16 column set with 30 mM MSA at a flow rate of 0.36 mL/min and a column temperature of 40 °C because the selectivity of the column for maximizing peak efficiencies is optimized at that temperature. Figure 1 shows the chromatogram of six common cations on a Dionex IonPac CS16 column set. Sodium, potassium, and calcium were well resolved and separated from other cations that could be in the sample. Column: Dionex IonPac CG16 Guard, 3 × 50 mm Dionex IonPac CS16 Analytical, 3 × 250 mm Eluent: 30 mM MSA Eluent source: Dionex EGC 500 MSA cartridge with CR-CTC 600 Temperature: 40 °C Flow rate: 0.36 mL/min Inj. volume: 25 µL Detection: Dionex CDRS 600 suppressor, 2 mm, recycle mode, 32 mA Peaks: 1 Conc. (mg/L) 1. Lithium 0.05 2. Sodium 0.20 3. Ammonium 0.25 4. Potassium 0.50 5. Magnesium 0.25 6. Calcium 0.50 3 12 4 5 6 µS/cm 0 -0 0 5 10 15 20 25 Minutes Figure 1. Separation of six common cations on a Dionex IonPac CS16 column set Calibration, limit of detection, and limit of quantitation Calibration curves with eight concentration levels were constructed from 0.02 mg/L to 20 mg/L for sodium, from 0.05 mg/L to 50 mg/L for potassium, and from 0.05 mg/L to 50 mg/L for calcium (Figure 2). Each of the standards was injected in triplicate. The results yielded a linear relationship of peak area to concentration with a coefficient of determination (r2) of 1.0 for each (Table 2). A signal-to-noise ratio 3:1 is generally considered acceptable for estimating the limit of detection (LOD), and a signal-to-noise ratio 10:1 for limit of quantification (LOQ).3 To determine the LOD and LOQ, the baseline noise is determined by measuring the peak-to-peak noise in a representative 1 min segment of the baseline where no peaks elute but close to the peak of interest. The LOD and LOQ were then calculated from the average peak height of five injections of 0.08 µg/L sodium, 0.2 µg/L potassium, and 0.2 µg/L calcium. The results of the calibration, LOD, and LOQ are summarized in Table 2. 3 Table 2. Method calibration, LOD, and LOQ Compound Sodium Range (mg/L) 0.0220 Potassium 0.0550 Calcium 0.0550 aLOD = 3 × S/N bLOQ = 10 × S/N Coefficient of determination (r2) 1.0 1.0 1.0 LODa (µg/L) 0.087 0.264 0.139 LOQb (µg/L) 0.290 0.880 0.462 Sample analysis To accurately determine sodium, potassium, and calcium, we assessed the performance of acetic acid concentration on the extraction of these cations from rice and wheat flours. Figures 3 and 4 show the overlaid chromatograms of rice and wheat flours treated with 0, 0.1, 0.5, 1, and 3% acetic acid solution. When the samples were extracted with acidified solution, the recoveries were improved compared to extraction with water. The addition of acetic acid was found to have more influence on rice flour than wheat flour. The lower percentages of acetic acid were effective too. To make the protocol of sample preparation universal and reproducible, the 3% acetic acid solution was standardized to extract rice and wheat flours. Area (µS*min) 40 Acetic acid 0% 0.1% 0.5% 1% 3% Peaks: 3 1. Sodium 2. Ammonium 3. Potassium 4 4. Magnesium 5. Calcium µS/cm Concentration (mg/L) Area (µS*min) 12 5 0 -5 0 5 10 15 20 25 Minutes Figure 3. Overlaid chromatograms of rice flour extracted with 0, 0.1, 0.5, 1, and 3% acetic acid solution Concentration (mg/L) 18 Acetic acid 3 0% 0.1% 0.5% 1% 3% Peaks: 1. Sodium 2. Ammonium 3. Potassium 4. Magnesium 5. Calcium 4 µS/cm Area (µS*min) 2 5 1 0 -2 0 5 10 15 20 25 Minutes Figure 4. Overlaid chromatograms of wheat flour extracted with 0, 0.1, 0.5, 1, and 3% acetic acid solution Concentration (mg/L) Figure 2. Calibration curves of sodium (0.0220 mg/L), potassium (0.0550 mg/L), and calcium (0.0550 mg/L) 4 The high capacity of the Dionex IonPac CS16 column allows loading of samples containing 3% acetic acid without affecting resolution of the target analytes. Six samples, three from each flour, were analyzed for sodium, potassium, and calcium contents. Figure 5 shows chromatograms of the three rice flour replicate samples and three wheat flour replicate samples. µS/cm 90 3 Rice flour 3 1 2 Rice flour 2 Rice flour 1 Wheat flour 3 Wheat flour 2 0 Wheat flour 1 Peaks: 1. Sodium 2. Ammonium 4 3. Potassium 4. Magnesium 5. Calcium 5 -10 0 5 10 15 20 25 Minutes Figure 5. Chromatograms of three rice flour and three wheat flour samples The concentrations (mg/L) of sodium, potassium, and calcium were calculated using their calibration curves. The contents (mg/kg) of sodium, potassium, and calcium in rice and wheat flours were calculated as below: Content (mg/kg) = Calculated concentration (mg/L) × 0.1 L 0.001 kg As shown in Table 3, rice flour contains an average of 15.8 mg/kg sodium, 2700 mg/kg potassium, and 113 mg/kg calcium, while wheat flour contains an average of 9.18 mg/kg sodium, 1220 mg/kg potassium, and 199 mg/kg calcium. Precision Method precision was evaluated by triplicate injections of the samples prepared and run on three separate days. The calculation of the relative standard deviation (RSD) was performed using all nine injections. The retention time RSDs were 0.1% and the peak area RSDs ranged from 0.14 to 0.60% (Table 4). If using manually prepared mobile phases, the precisions--especially for retention times-- likely will not be as low as when using electrolytically generated eluent. Table 3. The contents of sodium, potassium, and calcium in rice and wheat flours Rice flour 1 Rice flour 2 Rice flour 3 Average RSD (n=3) Sodium (mg/kg) 15.9 16.0 15.6 15.8 1.33 Potassium (mg/kg) 2740 2730 2640 2700 1.91 Calcium (mg/kg) 113 113 112 113 0.704 Wheat flour 1 8.97 1220 197 Wheat flour 2 9.19 1200 196 Wheat flour 3 9.38 1240 203 Average 9.18 1220 199 RSD (n=3) 2.23 1.77 1.63 Table 4. Retention time and peak area precisions (n=9) Sodium Potassium Calcium Rice flour Retention Peak area time RSD RSD 0.07 0.48 0.05 0.48 0.10 0.59 Wheat flour Retention Peak area time RSD RSD 0.05 0.60 0.05 0.14 0.08 0.34 Accuracy Method accuracy was validated by determining the recovery of sodium, potassium, and calcium in spiked rice and wheat flours in triplicate. The recoveries in rice flour were checked with 0.2 mg/L sodium, 2 mg/L potassium, and 0.5 mg/L calcium, while they were checked in wheat flour with 0.1 mg/L sodium, 2 mg/L potassium, and 2 mg/L calcium. Table 5 shows the recovery ranged from 79 to 110%, indicating that this method can be applied to the determination of sodium, potassium, and calcium in rice and wheat flours. Table 5. Method accuracy (n=3) Sodium Rice flour Spike level Recovery (mg/L) (%) 0.2 93 Potassium 2 79 Calcium 0.5 92 Wheat flour Spike level Recovery (mg/L) (%) 0.1 83 2 110 2 99 5 Conclusion This study describes an IC method for the determination of sodium, potassium, and calcium in rice and wheat flours. The method uses a Dionex IonPac CS16 column combined with suppressed conductivity detection on an RFIC system. The sample preparation was optimized for the extraction of sodium, potassium, and calcium in rice and wheat flours. The method showed broad linearity, excellent sensitivity, good accuracy, and high precision. References 1. U.S. National Institute of Standards and Technology (NIST). Health Assessment Measurements Quality Assurance Program. [Online] https://www.nist.gov/programsprojects/health-assessment-measurements-quality-assurance-program (accessed April 20, 2020). 2. Thermo Fisher Scientific. Product Manual for Dionex IonPac CS16 and CG16 columns. Doc No 031747 Revision 05, 2010. [Online] http://assets.thermofisher.com/ TFS-Assets/CMD/manuals/man-031747-ionpac-cs16-columns-man031747-en.pdf (accessed August 17, 2020). 3. ICH Guideline Q2B, Validation of Analytical Procedures, Methodology (CPMP/ICH/281/95), Geneva, Switzerland, November 1996. Find out more at thermofisher.com/IC © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Air-Tite and Norm-Ject are trademarks of Air-Tite Products Co., Inc. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all locations. Please consult your local sales representative for details. 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