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step 4a: Once the sugars are extracted the amount of sugars that were present in the food is determined.

Reactions with specific enzymes.

Specific enzymes can react with specific sugars. During this reaction the compound produced can be directly or indirectly measured by its light absorbance. These methods are highly specific. Kits are available for most of these sugars. An example is the glucose oxidation method. In this method glucose is oxidized by the enzyme glucose oxidase into peroxide. Peroxide reacts with a dye in the presence of the enzyme peroxidase, giving a coloured product that is measured with a spectrophotometer or colorimeter. The coloured product is proportional to the glucose present.

Other examples of these methods involve biochemical enzymatic reactions followed by a reduction of coenzymes (NAD and NADP). As an example it is shown how the enzyme hexokinase is used for the analysis of a glucose and fructose mixture. First glucose and fructose are phosphorylated by the enzyme hexokinase.

Second, Glucose – 6 – phosphate is oxidized which lead to the formation of NADH. The amount of NADH formed during

this reaction is measured by light absorbance (by 340 nm) and is a measure for the amount of glucose.

Next, fructose–6–phosphate is converted to glucose-6-phosphate (using an isomerase enzyme). Finally, the glucose-6- phosphate formed during this reaction is also oxidized. Again NADH is measured. The increase in NADH is a measure of the amount of fructose that was present in the sample.

Reducing properties of the aldehyde (O=CH) or keto group of the sugars.

Free monosaccharides can reduce alkaline solutions of metal oxides. Many procedures are for example based on the measurement of copper oxide formed in the reaction of monosaccharides with copper salts (Fehling’s reagent). For many sugars appropriate calibration tables are available so the amount of copper oxide which is formed can be linked to the amount of sugar that was available in the sample. These methods were traditionally applied, but have been mostly replaced by enzymatic or HPLC methods.

| HPLC/GLC When a mixture of sugars is present, HPLC or GLC can be used for separation of the sugars. With these separation methods it is possible to achieve complete separation of the mono- and disaccharides present in most foods. In addition, they have been widely used in the analysis of oligosaccharides.
| HPLC (high performance liquid chromatography) is a technique that can be used to separate sugars. All forms of chromatography separation techniques involve a stationary phase, often hold in some type of tube called “column” and a mobile phase, here being a liquid flowing through the column. How fast a particular compound of the sample (e.g. a sugar) displaces through the column depends on the interaction of the compound with both the stationary and mobile phase. Different components displace with a different speed. This depends on the (chemical) structure of the component. The picture shows the HPLC apparatus. Move with your mouse over the different components for an explanation. Play the animation to learn more about HPLC (High performance Liquid chromatography).

Play the animation to learn more about GLC (Gas Liquid chromatography)

1. Sample

2. Inject-O-Matic

3. Injector oven

4. Carrier gas in

5. Column oven

6. Detector

7. Recorder

GLC is a separation technique that can be used to separate the sugars in a sample. All forms of chromatography separation techniques involve a stationary phase, often hold in some type of tube called “column” and a mobile phase, a gas (such as helium or hydrogen) flowing through the column. In GLC, the components in the sample are first volatilized at a high temperature and then passed on a column which is held in an oven. The stationary phase is a high boiling point liquid coated on support material. How fast a particular compound of the sample flows through the column depends on the affinity of the compound for the stationary phase. Different components flow with a different speed. This depends on the structure of the component.

Density (mass per unit volume)

When the food sample contains high concentrations of one single sugar, it is possible to measure the density (or specific gravity*) of the solution and to determine the sugar concentration. These methods are frequently used in the measurements of strength of sucrose syrups.

A hydrometer is used for measuring the specific gravity of liquids. It is usually made of glass and consists of a cylindrical stem and a weighted bulb to make it float upright. The liquid to be tested is poured into a tall jar, and the hydrometer is lowered into the liquid until it floats freely. The level at which the surface of the liquid touches the stem of the hydrometer is noted.

The density of a solution can also be measured in degrees Brix. This is a measurement of the dissolved sugar-to-water mass ratio of a liquid. A 25 °Bx solution is 25% (w/w), with 25 grams of sugar per 100 grams of solution. Brix is primarily used in fruit juice, wine making and the sugar industry. A similar “Plato” scale is used in beer-making.

*Ratio of the density of a liquid to the density of water.

Refractive index

The refractive index of a medium is a measure indicating how much the speed of light (or other waves such as sound waves) is reduced inside the medium.

In most cases the refractive index is linearly (or nearly linearly) related to the percentage of dissolved solids in a solution. By comparing the value of the refractive index of a solution with a standard curve the concentration of solute can be determined with good accuracy. The refractive index is also used in detection systems for sugars in HPLC. These methods are only suitable for simple solutions of a few sugars.

Optical rotation

Optical rotation is the rotation of linearly polarised light as it moves through certain materials. A polarimeter is used to measure the optical rotation. The optical activity of the sugars provides a method for measurement of concentration of sugars in solution. Methods based on polarimetric measurements have been widely used in the measurement of sucrose in the presence of other sugars. These methods are only suitable for simple solutions of a few sugars.

A large range of methods can be used, based on chemical, physical and enzymatic characteristics of the sugars. The choice of the most appropriate sugar measurement method depends on the complexity of the mixture of sugars and on the complexity of the food matrix. The most common methods are enzymatic methods and HPLC methods. Methods based on the physical characteristics of sugars are only applicable for pure solutions of one sugar, or simple mixtures of sugars.