🍎 Sugar profile — HPLC
Determination of glucose, fructose, sucrose, and sorbitol in food by HPLC with refractive index detector (RID). Basic sugar composition analysis.
Overview
Sugar profile is a key parameter in food analysis, used for labeling control (total sugar content), adulteration detection (e.g., addition of glucose-fructose syrup to juice), product characterization (glucose/fructose ratio), and quality control.
The HPLC method with refractive index detection (RID) allows simultaneous separation and determination of mono- and disaccharides and sugar alcohols (sorbitol) on an amino (NH₂) or ion-exchange column (Ca²⁺/Pb²⁺). Sample preparation includes water extraction, deproteination (Carrez I + II), and filtration.
Method principle
Sugars extracted from the sample with water solution and purified with Carrez reagents (protein precipitation) are separated on an HPLC column with aminopropyl phase (NH₂) or ion-exchange column (Ca²⁺). Refractive index detection (RID) measures the difference in refractive index between eluent and sample. Identification based on retention times, quantitative determination using a calibration curve.
Applications
- Labeling control — total sugar content (EU Reg. 1169/2011)
- Analysis of fruit juices — adulteration detection
- Analysis of honey — sugar profile (authenticity)
- Quality control of confectionery and beverages
- Analysis of dietetic products (simple sugars)
- Quality control of alcoholic beverages (beer, wine)
Key parameters
| Parameter | Value |
|---|---|
| Sugars determined | Glucose, fructose, sucrose, maltose, lactose, sorbitol |
| Measurement range | 0.1–50 g/100 g |
| Limit of detection | 0.05 g/100 g |
| Precision (CV) | 1–3% |
| Analysis time | 15–25 min |
Standard
- Standard number
- PN-EN 12630:2001
- Title (PL)
- Soki owocowe i warzywne — Oznaczanie glukozy, fruktozy, sorbitolu i sacharozy — Metoda HPLC
- Title (EN)
- Fruit and vegetable juices — Determination of glucose, fructose, sorbitol and sucrose — HPLC method
Step-by-step procedure
1. Extraction
Weigh 5 g sample into 100 mL flask. Add 50 mL warm water (60°C). Mix 30 min. Cool.
2. Deproteination
Add 2 mL Carrez I, mix. Add 2 mL Carrez II, mix. Fill to 100 mL with water.
3. Filtration
Filter through paper filter, then through 0.22 µm into HPLC vial.
4. Calibration
Prepare mixed sugar standard: 0.1–5 g/100 mL of each sugar.
5. HPLC analysis
NH₂ column, mobile phase: ACN/H₂O 75:25, flow 1.0 mL/min, RID detection, column temp. 30°C.
6. Calculations
Read concentrations from calibration curve. Total sugars = glucose + fructose + sucrose. Convert to g/100 g.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with RID detector | Agilent 1260 + RID, Shimadzu RID-20A, Waters 2414 | 200 000–350 000 PLN |
| Aminopropyl column (NH₂) | Shodex Asahipak NH2P-50 4E, Waters Sugar-Pak | 2 000–5 000 PLN |
| Centrifuge | Eppendorf 5804R | 10 000–30 000 PLN |
| Volumetric flask 100 mL | Class A, calibrated | 20–50 PLN/pc |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| D-Glucose (standard) | 50-99-7 | Purity ≥99.5%, certified CRM |
| D-Fructose (standard) | 57-48-7 | Purity ≥99%, certified CRM |
| Sucrose (standard) | 57-50-1 | Purity ≥99.5%, certified CRM |
| Carrez I reagent | — | K₄[Fe(CN)₆] · 3H₂O, 150 g/L — for deproteination |
| Carrez II reagent | — | ZnSO₄ · 7H₂O, 300 g/L — for deproteination |
| Acetonitrile HPLC grade | 75-05-8 | Mobile phase: ACN/H₂O 75:25 (for NH₂ column) |
Health and safety (OHS)
- Acetonitrile — toxic, flammable. Work in fume hood.
- Carrez reagents — irritants, gloves and safety glasses
- HPLC waste — collect separately