Different honeys - student

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

Download Appendix 1

PDF in Danish here. 


Honey is a naturally sweet substance produced by honeybees 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 measure the water content of a variety of honeys using a spectrophotometer and NanoCuvette™ One.



  • 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.


Experimental Procedure

  • In this experiment, you will need to measure different honey samples to test the difference in water content. Different types of honey will be used. 
  • Turn on the spectrophotometer. Select a NanoCuvette ™ and write down the cuvette 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 room temperature at the time of the experiment. 
  • Open the NanoCuvette software and follow the instructions. 
  • Measure the refractive index of the NanoCuvette ™ in air. Note: To measure the refractive index, make sure that the sensor on the cuvette faces the light beam.

  • Using a pipette, transfer approx. 3 mL DI water in NanoCuvette ™ and measure both absorbance and refractive index. Note the absorbance and refractive index value 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 well before starting a new measurement.

  • Measure the rest of the samples as described above.


6. Data analysis

  • How does the refractive index of different types of honey change.
  • How does the absorbance of different types of honey change.
  • Use the following Wedmore equation [1] to calculate the water content of the samples. The value is calculated as a percentage (%).

    w(%) = -0.2681 - log(RI-1)/0.002243

    where w is the water content and RI is the refractive index obtained.

[1] Giulio Sesta, Lorenzo Lusco. Refractometric determination of water content in royal jelly. Apidologie, Springer Verlag, 2008, 39 (2), pp.225-232. <hal-00892298>


  • Does the calculated water content fit your expectations? Justify your answer. Prove (graphically) the linear relationship between the refractive index and the water content. Explain this relationship in words.
  • Is the water content color dependent? Justify your answer.
  • Is the refractive index color dependent? Justify your answer.
  • Based on the above questions, describe how NanoCuvette ™ One can be used by industry to detect honey scams. 
  • Explain how this experiment can be used to control the quality of honey.