Liquid Chromatographic Separation of Sugars

Significance of the Topic

Sugar profiling is critical in food and beverage quality control, nutritional labeling, and authenticity assessment. Accurate separation and quantification of common mono- and disaccharides support regulatory compliance and product development, ensuring consistency in formulation and consumer safety.

Objectives and Study Overview

This study aimed to develop and validate a robust liquid chromatographic method for simultaneous analysis of eight sugars (arabinose, fructose, glucose, galactose, maltose, lactose, sucrose, and xylose). Particular emphasis was placed on resolving the glucose/galactose epimer pair, which poses a common analytical challenge due to their identical mass and similar structures.

Methodology and Instrumentation

A gradient HPLC method was established using water and acetonitrile as mobile phases on an Agilent ZORBAX Original 70Å Carbohydrate Analysis column (4.6 × 150 mm, 5 µm) at 25 °C. Separation was achieved on an Agilent 1290 Infinity III LC System coupled with a 1290 Infinity III Evaporative Light Scattering Detector (ELSD). Key ELSD settings included an evaporator temperature of 60 °C, nebulizer temperature of 90 °C, gas flow of 1.50 SLM, and a data rate of 10 Hz. Calibration standards ranged from 0.1 to 10 mg/mL and were prepared in water.

Main Results and Discussion

The method achieved baseline separation of all eight sugars within a 13-minute run time. The glucose/galactose epimers were distinctly resolved, demonstrating the column’s selectivity. Limits of quantification (LOQ) ranged from 0.03 to 0.11 mg/mL, with calibration curves exhibiting correlation coefficients (R²) above 0.998 using a log/log model. Repeatability testing (n=6) yielded retention time RSDs below 0.2% and peak area RSDs under 2%, confirming method precision.

Benefits and Practical Applications of the Method

• Gradient-compatible ELSD allows faster analysis compared to refractive index detectors.
• High sensitivity and low LOQs support trace-level sugar detection.
• Reliable separation of structurally similar sugars enhances nutritional labeling accuracy.

Future Trends and Potential Applications

Advancements in column technology and detector sensitivity may further reduce analysis times and detection limits. Integration with high-throughput automation and coupling with mass spectrometry could expand carbohydrate profiling to complex matrices, such as fermented beverages and plant extracts. Data-driven approaches may also enable rapid method optimization and real-time quality control.

Conclusion

The described HPLC-ELSD method provides a fast, sensitive, and reproducible approach for quantitative analysis of eight common sugars in food and beverage samples. Its ability to resolve critical epimer pairs and robust validation parameters support its adoption in routine quality and authenticity testing.

References

1. Debebe A.; Temesgen S.; Redi-Abshiro M.; Chandravanshi B. S.; Ele E. Improvement in Analytical Methods for Determination of Sugars in Fermented Alcoholic Beverages. Journal of Analytical Methods in Chemistry 2018, 2018, 1–10.