🍎 Sugar profile — HPLC
In short
Determination of glucose, fructose, sucrose, and sorbitol in food by HPLC with refractive index detector (RID). The test is performed according to PN-EN 12630:2001; Measurement range: 0.1–50 g/100 g. The procedure comprises 6 steps (about 1 h 45 min in total); it is used for: Labeling control — total sugar content (EU Reg. 1169/2011), Analysis of fruit juices — adulteration detection, Analysis of honey — sugar profile (authenticity).
At a glance
- Standard: PN-EN 12630:2001
- Category: Food Chemistry
- Procedure steps: 6
- Total time of stages: 1 h 45 min
- Sugars determined: Glucose, fructose, sucrose, maltose, lactose, sorbitol
- Measurement range: 0.1–50 g/100 g
- Limit of detection: 0.05 g/100 g
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
-
Extraction
Weigh 5 g sample into 100 mL flask. Add 50 mL warm water (60°C). Mix 30 min. Cool.
-
Deproteination
Add 2 mL Carrez I, mix. Add 2 mL Carrez II, mix. Fill to 100 mL with water.
-
Filtration
Filter through paper filter, then through 0.22 µm into HPLC vial.
-
Calibration
Prepare mixed sugar standard: 0.1–5 g/100 mL of each sugar.
-
HPLC analysis
NH₂ column, mobile phase: ACN/H₂O 75:25, flow 1.0 mL/min, RID detection, column temp. 30°C.
-
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
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN 12630:2001 — “Fruit and vegetable juices — Determination of glucose, fructose, sorbitol and sucrose — HPLC method”.
How does this method work?
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.
What is the measuring range and accuracy?
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%.
How long does the test take?
The times given for the individual stages add up to approximately 1 h 45 min. The procedure comprises 6 steps.
What equipment is required?
HPLC system with RID detector, Aminopropyl column (NH₂), Centrifuge, Volumetric flask 100 mL. Indicative cost of the core instrument (HPLC system with RID detector): 200 000–350 000 PLN.
Where is this test used?
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).
What safety precautions apply?
Acetonitrile — toxic, flammable. Work in fume hood.; Carrez reagents — irritants, gloves and safety glasses; HPLC waste — collect separately.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN 12630.