Unknown honey samples - student

With this exercise guide you will learn to understand how the refractive index changes according to the water content of unknown honey.

Download Appendix 1.

PDF in Danish here. 



Honey is a naturally sweet substance produced by honey bees from the nectar of flowers. The main component of honey is sugar, primarily glucose and fructose. However, honey also contains a wide variety of other substances, including proteins, vitamins, minerals, phenolic compounds, and a small amount of water. In particular, the moisture content of honey is very important for its stability; low moisture content (<20%) protects the honey from antimicrobial activities and allows it to be preserved for longer periods. Unfortunately, the dilution of honey with syrup is a common scam in modern food production. Therefore, good methods for analyzing the amount of water present in honey are of great importance. One such method is the measurement of refractive index. NanoCuvette ™ One is a normal cuvette with a nanosensor installed on one of the sides. By inserting the cuvette with the sensor in the light direction, spectrophotometers can be used to determine the refractive index of a liquid sample. Thus, non-absorbent samples, such as water, can be quantified using standard instruments.


Learning objectives

In this experiment you will investigate unknown samples and identify possible fraud in the honey industry.



  • Spectrophotometer
  • NanoCuvette ™ One
  • Different honey (dark and light colors)
  • Demineralized water (DI water)
  • Vial
  • Pipettes 
  • Stirrer

Safety considerations

There are no special safety considerations. Use normal laboratory procedures.



In this experiment, you will measure five unknown samples: four pure honey and one water diluted honey. The aim of the experiment is based on refractive index measurements to determine which is the diluted sample:

  • Turn on the spectrophotometer. Select a NanoCuvette™One and write down the serial number in the specified table (Appendix 1).
  • Check the ambient temperature (T) and write it down in the table (See Appendix 1). Note: The refractive index is temperature dependent, therefore it is important to know the actual temperature at the moment of the experiment.
  • Open the NanoCuvette software and follow the instructions.
  • Measure the refractive index of the NanoCuvette™One in air. Note:
    To measure the refractive index, make sure that the sensor on the cuvette faces the light beam.
  •  Use a pipette, transfer approx. 3 mL of the unknown sample in NanoCuvette ™ and measure both absorbance and refractive index. Note the absorbance and the refractive index value in the table (See Appendix 1).
  • Discard the sample. Clean NanoCuvette ™ by heating soapy water. Pipette it into the cuvette and leave it with the heated soapy water for 5 min. Clean after with DI water.
  • Dry the cuvette thoroughly before starting a new measurement.

  • Measure the rest of the samples as described above.


Data analysis 

  • Can you identify the diluted sample by looking at the refractive index values ?
  • ​​Based on the experiment where the refractive index calibration curve was found using the dilution series (see the data analysis from the experiment) can you predict the concentration as a percentage of the unknown sample?
  • Use the following Wedmor equation: w(%) = -0.2681 - log(RI-1)/0.002243 to calculate the water content of the diluted sample. Explain in words how this can affect the product if it is sold in the market. The value is calculated as a percentage (%) .w is the water content and RI is the refractive index obtained.




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Hørmarken 2, DK-3520 Farum, Denmark
Tel: +45 36 99 27 46