The Analysis of Sugars using Ion Chromatography

Carbohydrates are one of the most important components in many foods and they may be present as isolated molecules or may be physically associated or chemically bound to other molecules. Sugars are simple carbohydrates and important for everyday life functions, the majority of the natural sugars contain 6 or 12 carbon atoms in the molecules.

Sugars are crystalline, soluble in water and generally have a sweet taste.

The sugar of commerce is the disaccharide sucrose or white sugar, as it is often known. Sucrose is found in sugar beet, sugar cane and certain fruits and it is possible to hydrolyse the carbohydrate to glucose and fructose with dilute acid.

Monosaccharides are water-soluble crystalline compounds.

They are aliphatic aldehydes or ketones containing one carbonyl group and one or more hydroxyl groups. The reactive centres of monosaccharides are the carbonyl and hydroxyl groups.

The majority of natural monosaccharides have five (pentose) or six (hexose) carbon atoms.

Some common hexoses in food are glucose, fructose and galactose whilst pentoses include arabinose and xylose.

Oligosaccharides are relatively low molecular weight polymers of monosaccharides covalently bound through glycosidic linkages.

Disaccharides consist of two monomers whilst trisaccharides consist of three monomers. Polysaccharides comprise the majority of carbohydrates found in nature and are high molecular weight polymers of monosaccharides. Since carbohydrates do not contain chromophoric species, detection using Ultra Violet spectrometry is only possible when combined with some form of post column derivitisation.

In reality the analysis of sugars by UV detection offers poor sensitivity and few applications.

Historically refractive index detection has been used with anion exchange chromatography but this technique is less sensitive (with detection limits greater than 100 mg l-1) and less versatile (no gradient elution system) than pulsed amperometric detection (PAD).

What is Ion Chromatography? Chromatography is a method for separating mixtures of substances using two phases, one of which is stationary and the other mobile moving in a particular direction.

Chromatography techniques are divided up according to the physical states of the two participating phases.

The term Ion Exchange Chromatography or Ion Chromatography (IC) is a subdivision of High Performance Liquid Chromatography (HPLC).

A general definition of ion chromatography can be applied as follows;" ion chromatography includes all rapid liquid chromatography separations of ions in columns coupled online with detection and quantification in a flow-through detector".

A stoichiometric chemical reaction occurs between ions in a solution and a solid substance carrying functional groups that can fix ions as a result of electrostatic forces.

For anion chromatography these are quaternary ammonium groups.

In theory ions with the same charge can be exchanged completely reversibly between the two phases.

The process of ion exchange leads to a condition of equilibrium, the side to which the equilibrium lies depends on the affinity of the participating ions to the functional groups of the stationary phases.

The reaction products formed on the working electrode during an amperometric determination can alter its surface properties by adsorption.

In Pulsed Amperometric Detection (PAD) it is possible to apply further potentials cyclically in addition to the detection potential in order to ensure a constant electrode surface.

In this way the electrode surface is renewed after each current measurement and remains in this activated condition.

The major use of PAD detection in anion chromatography has been for the determination of carbohydrates.

At pH's of 11 and above, the sugars become anionic and can be detected by anion chromatography.

Gold electrodes are favoured in PAD over platinum electrodes due to the fact that oxygen gives a cathodic response at a platinum electrode over the complete range of anodic detection.

Using the gold electrode the oxygen interference can be avoided by optimisation of the detection potential.

The Metrohm 817 Bioscan is a PC controlled measuring instrument for the sensitive analysis of ion chromatography using pulsed amperometric detection.

The unit can be used as a standalone system when combined with the Metrohm 812 Valve Unit and Metrohm 709 IC Pump or integrated into an existing standard IC system.

To describe the Bioscan only as an instrument purely for the determination of carbohydrates is doing it an injustice as it can also determine alcohols and sugar alcohols as well as oxidisable substances.

Metrohm has a developed a relatively new high capacity separation column, the Metrosep Carb 1, for the determination of carbohydrates using alkaline eluents and pulsed amperometric detection.

The ion exchange material is based upon the robust styrene-divinylbenzene co-polymer giving stable operating conditions and good separation between the mono and disaccharides as well as being suited to the analysis of sugar alcohols and oligosaccharides.

The compact housing of the Bioscan contains several IC components.

The detector is a flow through cell with a three-electrode arrangement for amperometric detection in DC mode, scan modes or pulse mode.

The latter is used for determining carbohydrates with three different working potentials being applied in a cyclic fashion.

The detector cell comprises a gold working electrode, a stainless steel auxiliary electrode and a solid phase reference electrode and is virtually maintenance free.

In DC mode a constant potential is applied to the gold working electrode.

The analytes are oxidised or reduced according to their electrochemical properties.

The scan mode allows current-potential curves to be recorded and is helpful for determining the optimal settings for pulsed amperometric operation.

The pulse mode utilises three different working potentials that are applied in cyclic fashion.

The electrode surface is then freed from any adherent reaction products after each measuring point and a new activated surface is provided for the next measurement.

This is an essential requirement for carbohydrate analysis.

The respective working potential and times are freely programmable within a wide range and are stored in the associated system file.

The Bioscan offers a number of different measuring ranges from 10 nA to 5 m A to embrace the fact that electrochemical detection has a wide linear range.

With the time program it is possible to freely change the measuring range during the chromatographic run, a useful feature when analysing samples with extreme variances in concentration.

The perfect insulation of the column compartment provides not only stable thermal conditions for the separating columns but also shields the system from electromagnetic interference.

The detection cell, pulsation absorber and preheating capillary are also housed here. It is a prerequisite of amperometric determinations that one has extremely thermal stable conditions.

The built in column compartment oven ensures that all the important components can be set to a temperature some 10deg C above ambient up to 60deg C with a stability of +/- 0.1deg C.

Apart from the electrodes, all the components that come into contact with the eluent and sample are metal free.

The Bioscan contains its own analogue to digital converter for the detector signal allowing complete control of the instrument from a PC using a RS 232 interface.

Operation and control is via a PC using IC Net 2.1 software that meets the necessary criteria placed upon modern integration software such as one or multi point calibration or calibration with internal or external standards.

Different algorithms can be selected for the calibration, there are numerous integration modes with selectable parameters and integration events.

With the peak editor one can optimise the chromatography, the scaling of the recorded chromatograph is automatically adjusted and sized accordingly. Several chromatographs can be superimposed upon one another and saved chromatographs can be retrospectively treated as desired. Amperometric detection takes place with a flowing current and therefore a chemical conversion of the analyte.

The course of the chemical reaction depends directly upon various physical parameters.

The temperature influences the oxidation and reduction reactions occurring at the working electrode.

This applies to both the conversions of the analyte and the interfering reactions that produce the background current indicating why a constant temperature is a necessary precursor to reproducible analysis.

The pH of the eluent has a direct influence on the electrochemical reactions that occur at the working electrode.

A change in the working pH causes a displacement of the voltammograms (current versus potential curves) potentially resulting in a reduced signal intensity and lower signal to noise ratio.

In order to ensure a stable baseline and reproducible measuring conditions are attained, care should be taken that the pH of the eluent is correct.

As electrochemical reactions at the electrode surfaces depend on the transport of the reacting substances to the electrode, a constant, pulsation free delivery of eluent must be ensured and it is recommended to use the supplied pulsation absorber.

The Bioscan allows the determination of carbohydrates, alcohols and sugar alcohols in the most diverse of matrices, for example soft drinks, fruit juices, dairy products, sweeteners, chocolate, sweets and chewing gum.

The detection limits using the Metrosep Carb 1 separation column are of the order 20 to 100 m g l-1 under standard operating conditions.

Ion chromatography as an analytical technique has seen an enormous surge in popularity, due partly to the simplicity of many of the methods as well as other factors such as market forces driving down the expenditure costs of the equipment and improved instrument power.

Being able to quantify quantitatively certain sugars is of great importance.

Many sugar alcohols are used in confectionery products because they give a sweet taste without the calories that sugars normally provide. The sugar alcohols sorbitol and mannitol are commonly substituted instead of sucrose but their use in foods is well regulated because they can exhibit laxative and diuretic properties.

The monosaccharide fructose is the sweetest sugar that one can find in nature, so it is naturally important to determine and control its presence, it occurs in sweet fruits and honey.

Similarly lactose is an important disaccharide, found in many dairy products such as cheese, butter, yoghurt and milk, it is significant to measure and regulate its content.

The low running costs of ion chromatography with Metrohm instruments are surprisingly low requiring only the acquisition of chemicals for the eluent as well as a clean, reliable source of deionised water.

A major advantage of ion chromatography is that often little sample preparation is required as long as the sample is in an ionic, homogenous form.

The determination of sugars proves to be a good matrix for analysis as most are soluble in water.

Only a very small amount of sample is required for the analysis and the quantified results obtained within a matter of minutes.

Ion chromatography is a clean technique as all the reagents are enclosed, its robustness and reliability are assured demonstrating precisely the reason why it is rapidly becoming the method of choice for many analysts. References: * Ion Chromatography, (2000) 3rd edition, J.S Fritz and D.T Gjerde, Wiley-VCH, ISBN 3-527-29914-9. * The Essential Guide to Analytical Chemistry, (1999) 2nd edition, G.Schwedt, John Wiley & Sons, ISBN 0-471-97412-9. The www.metrohm.com internet site was also used extensively as a reference and can be used to obtain further information.