Sugar Columns in Series: Empowering Superior Separation Performance in Sugar Analysis
Introduction
Sugars are among the most familiar and essential components in food. Not only do they contribute significantly to taste, but they are also key indicators of a food’s nutritional value. Common sweeteners such as fructose, glucose, sucrose, and maltose are frequently added during food processing, while sugar alcohols like mannitol and xylitol serve important functions in pharmaceutical applications.
To ensure the safety of food and pharmaceutical products and to meet the growing analytical demands of modern industries, sugar analysis using chromatography has become a critical tool. In these analyses, the most widely used HPLC columns are amino (NH₂) columns with silica gel bases and sulfonated polymer-based columns—commonly referred to as “carbohydrate columns”, “saccharide columns” or “sugar columns”.
Welch Materials offer two types of polymer-based sugar columns: Xtimate® Sugar-H and Sugar-Ca, which are strong cation exchange columns specifically designed for the purpose of sugar analysis.
In this article, we share a practical insight that significantly improves separation by using two sugar columns in series. When a single sugar column does not achieve the desired resolution—even after adjusting column temperature and flow rate, this strategy greatly helps.
Application 1: Sialic Acid Fermentation Broth
Chromatographic Conditions
- Column: Xtimate® Sugar-H (7.8×300mm, 8μm) / Xtimate® Sugar-H (7.8×150mm, 8μm)
- Mobile Phase: 25mmol/L sulfuric acid (H₂SO₄) aqueous solution
- Column Temperature: 60℃
- Detector: Refractive index detector (RID)
- Detector Temperature: 40℃
- Flow Rate: 0.4mL/min
- Elution Time: 40 minutes
- Injection Volume: 20μL
Preparation of Standard Solutions
- Sialic Acid Standard Solution: Weigh 20mg of sialic acid into a 10mL volumetric flask, dissolve in water, and dilute to the mark, then mix well.
- MCNNAC Standard Solution: Weigh 20mg of the standard into a 10mL volumetric flask, dissolve in water, and dilute to the mark, then mix well.
- GLCNAC Standard Solution: Weigh 20mg of the standard into a 10mL volumetric flask, dissolve in water, and dilute to the mark, then mix well.
- Mixed Standard Solution: Combine and mix 1mL of each standard solution.
| No. | Compound | Ret. Time (min) | Peak Area | Area% | Plates | Tailing Factor | Resolution |
|---|---|---|---|---|---|---|---|
| 1 | Sialic acid | 10.06 | 4909956 | 20.56 | 5501 | 0.64 | - |
| 2 | M-NAC | 16.07 | 9096636 | 38.10 | 12167 | - | 10.7 |
| 3 | G-NAC | 16.73 | 9869595 | 41.34 | 11687 | - | 1.1 |
| Sum | 28876186 | 100.00 |
| No. | Compound | Ret. Time (min) | Peak Area | Area% | Plates | Tailing Factor | Resolution |
|---|---|---|---|---|---|---|---|
| 1 | Sialic acid | 14.46 | 5232986 | 21.91 | 5283 | 0.66 | - |
| 2 | M-NAC | 23.67 | 8797475 | 36.84 | 20858 | 1.16 | 13.3 |
| 3 | G-NAC | 24.65 | 9851514 | 41.25 | 20068 | 1.20 | 1.5 |
| Sum | 23881975 | 100.00 |
Results:
Using a single Xtimate Sugar-H (7.8×300 mm, 8 μm) column yielded insufficient resolution for the latter two analytes (resolution: 1.1), while using Sugar-H (7.8 × 300 mm) and Sugar-H (7.8 × 150 mm) in series significantly improved resolution to 1.5, meeting analytical requirements.
Pressure Consideration
A common concern with column setups in series is increased backpressure. In the aforementioned application, we have:
- Single column pressure: ~1.2 MPa
- Column in series pressure: ~2.5 MPa
This remains well within the pressure tolerance of most HPLC systems, which typically handle up to 20 or 40 MPa. Welch sugar columns themselves tolerate up to 14 MPa, ensuring safe operation even in series configurations.
When using other column specifications, users should however remain mindful of backpressure.
Application 2: Electrolyte Injection Solution
Chromatographic Conditions:
- Column: Xtimate® Sugar-Ca (7.8×300 mm, 8 μm)
- Mobile Phase: Pure water
- Column Temperature: 80℃
- Detector: Refractive index detector (RID)
- Detector Temperature: 40℃
- Flow Rate: 0.5 mL/min
- Elution Time: 40 minutes
- Injection Volume: 10μL
| No. | Compound | Ret. Time (min) | Peak Area | Area% | Plates | Tailing Factor | Resolution |
|---|---|---|---|---|---|---|---|
| 1 | Maltose | 13.32 | 6764 | 0.38 | 11520 | - | - |
| 2 | Maltotriose | 13.92 | 11793 | 0.66 | 11187 | - | 1.2 |
| 3 | Glucose | 15.36 | 1762003 | 98.50 | 12518 | 1.08 | 2.7 |
| 4 | Fructose | 17.35 | 8362 | 0.47 | 12518 | 1.08 | 3.8 |
| Sum | 1788922 | 100.00 |
| No. | Compound | Ret. Time (min) | Peak Area | Area% | Plates | Tailing Factor | Resolution |
|---|---|---|---|---|---|---|---|
| 1 | Maltose | 25.60 | 12779 | 0.36 | 25828 | 1.01 | - |
| 2 | Maltotriose | 26.86 | 21296 | 0.59 | 23441 | 1.09 | 1.9 |
| 3 | Glucose | 29.85 | 3538666 | 98.58 | 21968 | 1.08 | 4.0 |
| 4 | Fructose | 34.33 | 16829 | 0.47 | 27632 | 1.06 | 5.5 |
| Sum | 3589569 | 100.00 |
Results
Single-column analysis showed limited separation between maltose and maltotriose (resolution: 1.2). Using two Sugar-Ca (7.8 × 300 mm) columns in series improved resolution to 1.9, offering more reliable quantification.
Sugar Column Overview
Matrix: Sulfonated crosslinked polystyrene-divinylbenzene
Counterions: H⁺ and Ca²⁺
Column Type: Strong cation exchange
Key Features and Applications:
- H-type Sugar Columns: Acid- and heat-resistant; ideal for organic acid and sugar separations. Recommended in the Chinese Pharmacopoeia for ribavirin analysis.
- Ca-type Sugar Columns: Also heat-resistant; typically use pure water as the mobile phase. Suitable for analyzing mono- and oligosaccharides, polyols, and approved in the Chinese Pharmacopoeia for mannitol testing.