🍎 Preservatives (sorbates, benzoates) — HPLC
In short
Determination of sorbic acid and benzoic acid (preservatives E200, E210) in food by HPLC-UV. The test is performed according to PN-EN 12856:2001; Measurement range: 5–5000 mg/kg. The procedure comprises 5 steps (about 1 h 5 min in total); it is used for: Control of preservative use in food (EU Reg. 1333/2008), Analysis of soft drinks and juices, Analysis of fruit products (jams, marmalades).
At a glance
- Standard: PN-EN 12856:2001
- Category: Food Chemistry
- Procedure steps: 5
- Total time of stages: 1 h 5 min
- Substances determined: Sorbic acid (E200), benzoic acid (E210)
- Measurement range: 5–5000 mg/kg
- Limit of detection: 2 mg/kg
Overview
Sorbic acid (E200) and benzoic acid (E210) and their salts are the most commonly used preservatives in food. Their permitted concentrations are regulated by EU Regulation 1333/2008: sorbic acid 150–2000 mg/kg (depending on product category), benzoic acid 150–2000 mg/kg.
The HPLC-UV method involves extraction of preservatives from food sample, purification, separation on a C18 column, and UV detection: sorbic acid 255 nm, benzoic acid 228 nm. The method is simple, fast, and selective.
Method principle
The sample is homogenized with water, ultrasonically extracted, and deproteinated with Carrez reagents. After filtration (0.22 µm), the solution is analyzed by HPLC on a C18 column with buffered mobile phase (ammonium acetate pH 4.2 + methanol). UV detection at 228 nm (benzoic acid) and 255 nm (sorbic acid). Quantitative determination using a calibration curve.
Applications
- Control of preservative use in food (EU Reg. 1333/2008)
- Analysis of soft drinks and juices
- Analysis of fruit products (jams, marmalades)
- Quality control of dairy products (cheeses, yogurts)
- Analysis of sauces, dressings, and marinades
- Detection of undeclared preservatives
Key parameters
| Parameter | Value |
|---|---|
| Substances determined | Sorbic acid (E200), benzoic acid (E210) |
| Measurement range | 5–5000 mg/kg |
| Limit of detection | 2 mg/kg |
| Wavelength | 228 nm (benzoic), 255 nm (sorbic) |
| Precision (CV) | 2–5% |
Standard
- Standard number
- PN-EN 12856:2001
- Title (PL)
- Artykuły żywnościowe — Oznaczanie kwasu sorbowego i benzoesowego metodą HPLC
- Title (EN)
- Foodstuffs — Determination of acesulfame-K, aspartame and saccharin — HPLC method
Step-by-step procedure
-
Extraction
Weigh 5 g sample, add 40 mL water, ultrasonic 15 min. Deproteinate with Carrez I+II. Fill to 50 mL.
-
Filtration
Filter through 0.22 µm PVDF into vial.
-
Calibration
Standards: 5, 10, 50, 100, 500 mg/L mixture of sorbic and benzoic acid.
-
HPLC analysis
C18, 20 mM ammonium acetate pH 4.2 / MeOH gradient, 1 mL/min, UV 228+255 nm.
-
Calculations
Concentration from calibration curve, conversion to mg/kg product.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with UV/DAD detector | Agilent 1260, Shimadzu Nexera, Waters Alliance | 200 000–400 000 PLN |
| C18 column | Agilent Zorbax Eclipse Plus C18, 150×4.6 mm, 5 µm | 1 500–3 500 PLN |
| Ultrasonic bath | Bandelin Sonorex RK 510 | 3 000–8 000 PLN |
| Syringe filters 0.22 µm | PVDF or nylon, ø 13 mm | 1–3 PLN/pc |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Sorbic acid (standard) | 110-44-1 | Purity ≥99%, CRM |
| Benzoic acid (standard) | 65-85-0 | Purity ≥99.5%, CRM |
| Ammonium acetate | 631-61-8 | Mobile phase: 20 mmol/L, pH 4.2 |
| Methanol HPLC grade | 67-56-1 | Mobile phase component (gradient 10–60%) |
Health and safety (OHS)
- Methanol — toxic, flammable. Fume hood mandatory.
- Sorbic acid — irritant, nitrile gloves
- HPLC waste — organic waste container
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN 12856:2001 — “Foodstuffs — Determination of acesulfame-K, aspartame and saccharin — HPLC method”.
How does this method work?
The sample is homogenized with water, ultrasonically extracted, and deproteinated with Carrez reagents. After filtration (0.
What is the measuring range and accuracy?
Substances determined: Sorbic acid (E200), benzoic acid (E210); Measurement range: 5–5000 mg/kg; Limit of detection: 2 mg/kg; Wavelength: 228 nm (benzoic), 255 nm (sorbic).
How long does the test take?
The times given for the individual stages add up to approximately 1 h 5 min. The procedure comprises 5 steps.
What equipment is required?
HPLC system with UV/DAD detector, C18 column, Ultrasonic bath, Syringe filters 0.22 µm. Indicative cost of the core instrument (HPLC system with UV/DAD detector): 200 000–400 000 PLN.
Where is this test used?
Control of preservative use in food (EU Reg. 1333/2008); Analysis of soft drinks and juices; Analysis of fruit products (jams, marmalades); Quality control of dairy products (cheeses, yogurts); Analysis of sauces, dressings, and marinades; Detection of undeclared preservatives.
What safety precautions apply?
Methanol — toxic, flammable. Fume hood mandatory.; Sorbic acid — irritant, nitrile gloves; HPLC waste — organic waste container.
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 12856.