Determination of nitrite and nitrate in sugar using ion chromatography

Importance of the Topic

Determining nitrite and nitrate residues in sugar and sugar by-products is critical for food safety and regulatory compliance. Excessive levels of these anions can pose carcinogenic risks and contribute to food poisoning. Regulatory bodies such as the U.S. EPA and the EU have established strict maximum levels for nitrite and nitrate in drinking water and animal feed, motivating the need for reliable analytical methods in the sugar industry.

Objectives and Study Overview

This application note describes the development and validation of an ion chromatography (IC) method for quantifying nitrite and nitrate in sugar samples. The goal was to establish a sensitive, accurate, and precise method using a Thermo Scientific RFIC system equipped with a Dionex IonPac AS11-HC-4µm column, suitable for regulatory monitoring of sugar, molasses, and beet pulp.

Used Instrumentation

• Thermo Scientific Dionex ICS-6000 HPIC System with RFIC-EG eluent generator and suppressed conductivity detector (or equivalent ICS-5000+ or Integrion HPIC systems)
• Dionex AS-AP Autosampler with 250 µL syringe and temperature control
• Dionex Chromeleon Chromatography Data System (CDS) software
• Dionex IonPac AS11-HC-4µm Analytical Column (2×250 mm) and AG11-HC-4µm Guard Column (2×50 mm)
• Dionex EGC 500 KOH Cartridge and CR-ATC 600 Anion Trap Column
• Dionex ADRS 600 Suppressor in recycle mode

Methodology and Instrumentation

Sample Preparation:

• Dissolve 1 g sugar in 49 mL deionized water (50× dilution), shake, filter through 0.2 µm PES filter
• Add DI water to filtered extract for 100× final dilution
• Prepare spiked samples by adding known nitrite/nitrate standards before final dilution

Chromatographic Conditions:

• Column temperature: 30 °C; sample tray: 4 °C
• Eluent: potassium hydroxide gradient (5 mM for 0–5 min; 5–20 mM for 5–30 min; 65 mM for 30–36 min; return to 5 mM for 36–40 min)
• Flow rate: 0.37 mL/min; injection volumes: 2.5 µL or 25 µL
• Detection: suppressed conductivity (2 mm suppressor, 60 mA, recycle mode)

Calibration and Detection Limits:

• Mixed standards prepared daily at six levels: nitrite 0.01–25 mg/L; nitrate 0.02–50 mg/L
• Linearity (2.5 µL injection): r2 = 0.9999 (nitrite), 1.0000 (nitrate)
• Method detection limits (2.5 µL): 0.002 mg/L (nitrite), 0.006 mg/L (nitrate)

Main Results and Discussion

The optimized gradient achieved baseline separation of nitrite and nitrate from common interfering anions and organic acids within 20 minutes. Comparative injections (2.5 µL vs. 25 µL) showed sharper peaks and a wider linear range with the smaller volume. Precision studies (n=3/day over 5 days) yielded intraday RSDs ≤1.0% and interday RSDs ≤1.6%. Recovery experiments at two spiked levels demonstrated accuracy: nitrite 89–96% and nitrate 97–101%. Application to three commercial sugar samples (white cane, brown cane, beet syrup) revealed no nitrite/nitrate in cane sugars, while beet syrup contained 50 mg/kg nitrite and 544 mg/kg nitrate, exceeding the EU nitrite limit of 15 mg/kg.

Benefits and Practical Applications

• High sensitivity and selectivity for nitrite and nitrate in complex sugar matrices
• Automated eluent generation reduces manual preparation and variability
• Sufficient throughput for routine regulatory monitoring and quality control
• Adaptable to different HPIC platforms with equivalent performance

Future Trends and Applications

Ongoing advances in column chemistries and suppressor technologies will further improve resolution and detection limits. Integration with mass spectrometric detectors could extend the method to trace-level organic contaminants. Additionally, miniaturized IC systems and on-line sample preparation may enable real-time monitoring in sugar processing facilities.

Conclusion

This study presents a robust, validated RFIC method for the simultaneous determination of nitrite and nitrate in sugar products. The method exhibits excellent linearity, precision, and accuracy, with detection limits well below regulatory thresholds. It is directly applicable to routine compliance testing in the sugar industry.

References

1. US EPA National Primary Drinking Water Regulations for nitrite and nitrate.
2. USEPA Method 300.1: Determination of Inorganic Anions in Water by IC.
3. EU Directive 2010/63/EU: Undesirable substances in animal feed.
4. Mikoś et al., Int. Sugar J., 2015, 117, 790–797.
5. Thermo Fisher Common Anions Analysis by EPA 300.0 & 300.1.
6. Thermo Scientific Application Note 143: Organic Acids in Fruit Juices, 2016.
7. Antczak-Chrobot et al., Food Chem., 2018, 240, 648–654.