Spectrometric determination of the glucose level in jam

A number of reactions between sugars and other chemical reagents produce coloured products; the intensity of colour is related to the initial concentration of sugar. The absorbance of sample solutions can be measured and compared to the absorbance of standard solutions of known sugar concentrations. Only a limited number of colour-change reactions are known for polysaccharides, and most involve simple sugars, usually reducing sugars (see box).

Glucose determination was chosen primarily because students know what sugars are – the activity has widespread appeal. Furthermore, determining the sugar content of jams may have industrial applications in aspects such as quality control.

One method to determine the sugar concentration of jam involves hydrolysing many of the non-reducing sugars (in jam, principally sucrose) to glucose, using sulphuric acid (H₂SO₄), after which the sample is neutralised with sodium hydroxide (NaOH). When heated with 3,5-dinitrosalicylic acid (DNSA; also known as 2-hydroxy-3,5-dinitrobenzoic acid), reducing sugars (e.g. glucose and fructose) produce a red-brown product. For more details of this reaction, see Miller (1959).

The concentration of the coloured complex can be determined with the spectrometer using the blue LED (430 nm): the initial sugar concentration of the jam samples can then be read off a calibration curve created using known glucose concentrations.

Equipment and reagents

• Spektra spectrometer (or other spectrometer)
• Cuvettes or blisters
• Pipettes
• 100 ml volumetric flasks
• Conical flasks
• Test tubes
• Balance
• Water bath
• Funnel
• Filter paper
• Jam samples
• DNSA reagent (3,5-dinitrosalicil acid)
• Sulphuric acid (H₂SO₄) solution (approximately 2 mol/l)
• Sodium hydroxide (NaOH) solution (w=10%)
• Sodium potassium tartrate (NaK(CH₂OH)₂(COO)₂.4H₂O)
• Glucose powder (C₆H₁₂O₆)

Preparation of solutions

DNSA reagent: To prepare the DNSA reagent, dissolve 10 g DNSA in 200 ml NaOH solution (about 2 mol/l). Heat the solution and stir thoroughly. Dissolve 300 g sodium potassium tartrate in 500 ml distilled water to form a colour stabiliser. Combine the two solutions, stir well and top up to 1 l with distilled water.

Jam (sugars): Weigh 1-2 g jam into a conical flask and add 10 ml sulphuric acid. Heat in a boiling water bath for 20 min, stirring periodically until hydrolysis is complete. Leave the sample to cool and carefully add 12 ml sodium hydroxide. Stir and filter into a 100 ml volumetric flask, and top up to 100 ml with distilled water. Using a pipette, transfer 10 ml solution into another 100 ml volumetric flask and top up to 100 ml with distilled water to produce the test solution. Stir well.

Jam (reducing sugars): Weigh 3.0 g jam into a conical flask, add 50 ml distilled water, heat and stir for 10 min. Filter into a 100 ml volumetric flask, and top up to 100 ml with distilled water. Using a pipette, transfer 10 ml solution into another 100 ml volumetric flask and top up to 100 ml with distilled water to produce the test solution. Stir well.

Stock glucose solution (15 mg/ml):  Place 1.5 g glucose in a 100 ml volumetric flask and top up to 100 ml with distilled water. Stir.

Creating the calibration curve

1. Mark five volumetric flasks (100 ml) with the letters A–E. Into each labelled flask, pipette the volumes of standard glucose solution and distilled water specified in Table 1.

2. Label and fill six test tubes as specified in Table 2.

3. Heat the test tubes and their contents in boiling water for 5 min; the DNSA reagent will react with any sugar present, producing a red-brown product.


4. Cool the test tubes, add 6 ml distilled water to each and shake well.


5. Using the blue LED (430 nm) of the spectrometer, measure the transmittance of each solution.

The readings from the Spektra instrument are transmittances expressed in percentages and should be divided by 100 to obtain the transmittance values used in the subsequent calculations. Transmittance is related to absorbance as described by the equation: A = –log T. See the second and third columns in Table 3.

The image to the right shows the calibration solutions; even the blank (water and DNSA with no glucose) is an intense colour. It is therefore necessary to measure all the samples, including the blank, against distilled water. The glucose-specific absorbance of the samples is then calculated by subtracting the absorbance measurement of the blank from the absorbance measurement of the sample (see the fourth column of Table 3).

Figure 4: The calibration solutions Image courtesy of Irena Štrumbelj Drusany

6. Plot glucose concentration against glucose-specific absorbance, as shown in Figure 5.

Measuring the jam samples

The jam samples should be treated similarly to the glucose solutions used for the calibration curve.

1. For each jam to be tested, put 1 ml prepared jam sample (see ‘Preparation of solutions’) in a test tube and add 1 ml DNSA reagent and 2 ml distilled water.


2. Heat the test tubes and their contents in boiling water for 5 min; the DNSA reagent will react with any sugar present, producing a red-brown product.


3. Cool the test tubes, add 6 ml distilled water to each and shake well.


4. Using the blue LED (430 nm) of the spectrometer, measure the transmittance value (T%) of each jam sample. Divide by 100 to obtain T, convert T to A using the equation A = –log T, and use that to calculate the glucose-specific absorbance (A – A_blank).

Table 4 shows an example of the transmittance values obtained and the calculated glucose-specific absorbance of each sample.

5. Using your calibration curve, convert the absorbance measurements (A) into the concentrations of glucose (mg/ml) in your samples.

Using the examples from Table 4, the glucose concentrations read off the calibration curve give:

Sample 1: 0.8 mg/ml

Sample 2: 0.5 mg/ml

6. From the glucose concentrations, calculate the mass of glucose in a 1 g jam sample using the following equation:

Mass glucose (g per 1g sample) = mass concentration (mg/ml) x 10 x 100 ml

where

mass concentration is the value read off the calibration curve

10 is the dilution (see ‘Preparation of solutions’)

100 ml is the volume of 1g of jam sample.

In our example,

Sample 1: mass glucose (g per 1g sample) = 0.8 mg/ml x 10 x 100 ml = 0.8 g

Sample 2: mass glucose (g per 1g sample) = 0.5 mg/ml x 10 x 100 ml = 0.5 g.

The calculations above assume that the initial jam sample was 1 g. If, for example, the sample had weighed 2 g, the figures above would need to be divided by 2 to get the mass of glucose in g per 1 g sample.

Reducing and non-reducing sugars

In chemical terms, a reducing sugar is an aldose such as glucose, which has an aldehyde group that can be oxidised to a carboxylic acid. The more common chemical test to detect reducing sugars is to warm them with either Benedict’s or Fehling’s solutions, which contain copper(II) ions that are reduced to copper(I) oxide and can be observed as a brown-orange precipitate.

Non-reducing sugars, such as sucrose, may have a ketone rather than an aldehyde functional group, which cannot reduce copper(II) ions. When treated with Benedict’s or Fehling’s solutions, non-reducing sugars produce no coloured precipitate.

Review

Analytical chemistry relies extensively on spectrometric analysis, but professional instruments are expensive and thus not easily available for average schools in many countries. This article presents a spectrometer developed by one of the authors to make spectrometric analysis affordable to every secondary school. The project website details several school experiments in spectrometry, and the laboratory protocol for one experiment is included in the article.

I would recommend this article for introducing analytical chemistry in science lessons (not only chemistry, but also biology and environmental science), particularly for secondary schools that do not have a well-equipped laboratory. The proposed approach is simple and friendly enough to encourage teachers and students to try the suggested experiments and to explore new ones.

Teachers could use the article for a discussion of the methodology of spectrometry and the theory of spectrometric measurements. The experimental analysis provides the opportunity to analyse the obtained data mathematically, thus linking chemistry and statistics.

The article could also be used for a comprehension exercise. For example:

• Given the procedure for making the calibration curve from the stock standard solution, complete the following table:

Giulia Realdon, Italy

Referee's recommendations: Chemistry, Biology, Environmental science, Optics, Statistics