How Liquid Chromatography Detects Sugars in Food

Carbohydrates are one of the body’s most important sources of energy and are widely found in various foods. Carbohydrates share similar chemical structures, and traditional detection methods are often unable to accurately measure sugar content. High-performance liquid chromatography (HPLC) is the method of choice for detecting five common carbohydrates in food: fructose, glucose, sucrose, maltose, and lactose. This article will explain how HPLC detects carbohydrates in food, describe key analytical methods, and explain why it remains the standard analytical tool in modern food laboratories to this day.

What Are the Challenges in Carbohydrate Analysis?

The analysis and detection of carbohydrate compounds present multiple technical challenges, which is why traditional chemical methods are often inadequate.

1. High Structural Similarity Makes Separation Difficult

Fructose, glucose, sucrose, maltose, and lactose—commonly found in food—are all carbohydrates with highly similar molecular structures. Fructose and glucose are both monosaccharides with nearly identical molecular weights. Sucrose, maltose, and lactose are all disaccharides, but they are composed of different monosaccharide units linked by different glycosidic bonds. This high structural similarity results in very similar retention behavior when analyzed in the same chromatographic system. Achieving simultaneous baseline separation of these five sugars places high demands on the selectivity and resolution of the chromatographic column.

2. Lack of Characteristic UV Absorption

The molecular structures of the vast majority of sugars do not contain chromophores, resulting in extremely weak or even no response in conventional UV-visible detectors. This means that, unlike the direct quantitative analysis of food components such as vitamins and preservatives using UV detectors, special detectors or derivatization methods must be employed.

3. Complex Matrix Composition

Actual food samples often contain multiple interfering substances, such as proteins, fats, organic acids, and pigments. If sample preparation is improper, these co-existing components may contaminate the chromatographic column or elute simultaneously with the target sugars, severely affecting the accuracy of both qualitative and quantitative analysis.

Advantages of Liquid Chromatography for the Analysis of Sugars

In response to the challenges mentioned above, high-performance liquid chromatography (HPLC) offers a systematic solution, making it the preferred technique for sugar analysis.

1. Strong Separation Capability

HPLC uses high pressure to drive the mobile phase through a high-resolution column, enabling the individual separation of sugars with highly similar structures. It can achieve simultaneous baseline separation of five sugars—fructose, glucose, sucrose, maltose, and lactose—within a dozen or so minutes.

2. Flexible Detector Configuration

For compounds such as sugars that do not absorb UV light, HPLC systems can be configured with a refractive index detector (RID) or an evaporative light-scattering detector (ELSD). The refractive index detector responds by measuring the difference in refractive index between the mobile phase and the sample solution, providing a stable response signal for all sugars and offering simple operation. The evaporative light-scattering detector (ELSD), which operates through atomization, evaporation, and light scattering detection, offers higher sensitivity and is compatible with gradient elution. Both detectors have their own advantages and can be flexibly selected based on specific requirements.

3. Reliable Results

Liquid chromatography not only enables the qualitative identification of individual sugar components based on retention time but also allows for accurate quantification using either the external standard or internal standard methods. The linear correlation coefficient of the standard curve typically reaches 0.999 or higher, ensuring the reliability of quantitative results.

4. Wide Range of Applicable Samples

From clear beverages and fruit juices to viscous honey and syrups, as well as grain products and dairy products, liquid chromatography, when combined with appropriate pretreatment steps (dilution, extraction, filtration, etc.), can cover virtually all common food matrices.

How to Detect Fructose, Glucose, Sucrose, Maltose, and Lactose in Food

The following is the complete procedure for determining these five sugars using liquid chromatography with a refractive index detector (RID).

1. Instrument Configuration

High-performance liquid chromatograph (HPLC) equipped with a binary high-pressure gradient pump or isocratic pump

Refractive index detector (RID)

Autosampler

Column oven

Chromatography workstation

2. Chromatographic Conditions

Under these conditions, the five sugars elute in the following order: fructose → glucose → sucrose → maltose → lactose, with all peaks eluting within 16 minutes.

3. Instruments, Reagents, and Consumables

Reagents:

Ultrapure water

Acetonitrile (chromatographic grade)

Reference standards: Fructose (McLean), glucose (Damas-beta), sucrose (General-Reagent), maltose (McLean), lactose (Aladdin)

Supporting Equipment:

Analytical balance

Solvent filtration apparatus

Ultrasonic cleaner

Laboratory Supplies:

Filter membranes: Aqueous-phase filter membranes, 0.45 μm

4. Preparation of Standard Solutions

Prepare a mixed standard stock solution (20.0 mg/mL): Weigh approximately 1 g each of dried fructose (dried at 90 °C for 2 h), glucose, sucrose, maltose, and lactose (all dried at 96 °C for 2 h), accurate to 0.001 g. Dissolve in water and transfer to a 50 mL volumetric flask. Add 2.5 mL of acetonitrile and dilute to the mark with water. Store sealed at 0–4 °C; valid for 3 months.

Series of Standard Working Solutions: Pipette 0.100, 1.00, 2.00, 3.00, and 5.00 mL of the stock solution into separate 10-mL volumetric flasks. Dilute with water to the mark to obtain a series of standard solutions with concentrations of 0.200, 2.00, 4.00, 6.00, and 10.0 mg/mL.

5. Sample Preparation

Sample preparation methods vary slightly depending on the type of food:

Honey and syrups: Weigh 1–2 g of the sample, dissolve it in water, and make up the volume to 100 mL. Mix thoroughly, let stand, then collect the supernatant. Filter through a 0.45 μm microporous filter membrane before injection.

Beverages: Take the sample directly, degas it using an ultrasonicator, filter it through a 0.45 μm microporous filter membrane, and then inject it. If the concentration is too high, dilute it appropriately.

Dairy products and grain products: These require steps such as protein precipitation, fat removal, and extraction.

6. Quantitative Method

Quantification was performed using the external standard method. A calibration curve was plotted with the concentrations of a series of standard working solutions on the x-axis and the corresponding chromatographic peak areas on the y-axis. The peak areas of each component in the sample solution were substituted into the calibration curve equation. The mass concentrations of each sugar in the sample were calculated and then converted to actual content in the sample based on the sample weight and dilution factor.

7. Method Performance Validation

Validation results show that the key performance indicators of this method are as follows:

Resolution: The resolution between adjacent chromatographic peaks of the five sugars is greater than 1.5, meeting the baseline separation requirements.

Linearity: All sugars exhibit good linearity in the concentration range of 0.200–10.0 mg/mL, with a correlation coefficient R > 0.999.

Precision: For the same sample injected seven times consecutively, the relative standard deviation (RSD) of retention time was < 0.3%, and the RSD of peak area was < 1.5%, demonstrating excellent reproducibility.

Sensitivity: The limits of detection for each sugar using the differential refractive index detector meet the requirements for routine food testing.

Precautions for Chromatography Column Use

Amino-bonded columns are the most commonly used type of chromatography column in carbohydrate analysis, but the following precautions should be observed during use:

The acetonitrile content in the mobile phase should generally be at least 60%; otherwise, the amino-bonded phase is prone to hydrolysis and leaching, which shortens the column’s lifespan;

When the column is not in use for an extended period, it should be properly stored according to the manufacturer’s recommended solvent system;

Be sure to filter samples through a 0.45 μm filter membrane before injection to prevent particulate matter from clogging the column.

Common Issues and Troubleshooting

Peak tailing or splitting: This may be caused by column contamination or a decrease in column efficiency. The column can be regenerated by flushing with an appropriate solvent, or the guard column can be replaced.

Decreased resolution: Check whether the mobile phase ratios are correct, or whether the column has reached the end of its service life.

Baseline drift: The refractive index detector is sensitive to temperature. Ensure that the column oven and detector temperatures are stable, and that the mobile phase is thoroughly degassed.

Conclusion

High-performance liquid chromatography (HPLC), with its exceptional separation capabilities, flexible detector configurations, and well-established quantitative systems, has become the mainstream technique for the detection of sugars in food. Whether in terms of methodological maturity or the comprehensiveness of standards and regulations, the HPLC-RID/ELSD method stands up to rigorous scrutiny. For food manufacturers, mastering this method helps ensure precise control over product formulations and quality; for testing institutions, it serves as a vital technical foundation for ensuring food safety and protecting consumer health. With the continuous advancement of chromatography technology, the sensitivity, speed, and level of automation in carbohydrate analysis are expected to improve further.