Determination of 5-(Hydroxymethyl)-2-furaldehyde in honey using thin layer chromatography


5-(Hydroxymethyl)-2-furaldehyde or Hydroxymethylfurfural (HMF) is an organic compound derived from dehydration of fructose, e.g. when honey is heated. To reduce the viscosity and ease filling processes honey is heated but not to more than 40°C. Higher temperatures can irreversibly damage enzymes and other components. In freshly centrifuged honey, HMF is not detectable, or only in minute amounts. The HMF content increases during storage at a rate of about 2-3 mg/kg per year, depending on the pH and storage temperature. With a storage temperature of 21°C, the HMF content can rise already up to 20 mg/kg in one year. The European Union calls for an HMF content of not more than 40 mg/kg for honey produced in Europe. National associations of beekeepers, such as in Belgium and Italy, are even stricter. The German Beekeepers Association (DIB) chose the label "Genuine German honey" for an HMF content of not more than 15 mg/kg. In this study, we have used high performance thin layer chromatography (HPTLC) to analyze and quantify HMF in honey with a minimum of sample preparation. The samples were only diluted with water and adequate aliquots spotted onto the TLC plate.

Densitogram of the developed plate at 290 nm

Figure 1: Densitogram of the developed plate at 290 nm. HMF detected at Rf of 0.82 = hRf of 82.

Results and Discussion

HMF was detected under UV light at a hRf value of 82. Quantitation was carried out using peak height measurement after scanning the plate at 290 nm with a TLC Scanner, see figure 1. HMF standards ranging between 1.0 to 25.0 mg/mL were used to construct an adequate calibration curve, see Figure 2. HMF concentrations between 2.7 to 18.0 mg/kg were determined in the different samples, see Table 1, and only one of the samples had a HMF value above the DIB limit.

Calibration curve of HMF standard (grey) and samples (red)

Figure 2: Calibration curve of HMF standard (grey) and samples (red).


HPTLC is a straightforward analytical technique for the analysis of honey samples. The viscous sample matrix with high sugar content only requires dilution with water before samples can be spotted onto a TLC plate by an automatic sample applicator, followed by quantitation with a TLC scanner after the plate is developed.


Table. 1: Determined HMF values:
Honey No 37 13.7 mg/kg
Honey No 38 18.0 mg/kg
Honey No B5 2.7 mg/kg


Chromatographic data

Track Compound Conc. [μg/mL] Application
volume [μL]
1, 8 HMF standard 1 1 6 82
2, 9 HMF standard 2 2.5 6 82
6, 13 HMF standard 3 10 6 82
7, 14 HMF standard 4 25 6 82
3, 10 Honey No. 37 250 6 82
4, 11 Honey No. 38 250 6 82
5, 12 Honey No. B5 250 6 82


Application data

Plate HPTLC Silica gel 60 F254 MS-grade
Sample preparation 1 g honey stirred in 10 mL Water, filtration
Sample application ATS 4 sample applicator (Camag), 6 mm bandwise
Application volume 6 μL
Mobile phase 1. Ethanol / Methanol 9/1 (v/v), 2. Ethyl acetate
Migration distance 2 cm / 5 cm
Migration time 1.8 min / 2.8 min
Documentation equipment -
Wavelength scan under UV-light at 290 nm with TLC scanner (Camag)
Staining none

Ordering information

Description Product No.
HPTLC Silica gel 60 F254 MSgrade, 20x10 cm 1.00934
Ethanol gradient grade for liquid chromatography LiChrosolv® 1.11727
Ethyl acetate gradient grade for liquid chromatography LiChrosolv® 1.00868
Methanol hypergrade for LCMS LiChrosolv® 1.06035
Millex®-FH filter, 0.45 μm hydrophobic PTFE, 25 mm, nonsterile SLFH025