Food for human consumption and animal feedstuffs contain a variety of mono-, di-, oligo-, and polysaccharides with different functions. In this article, specific liquid chromatographic (LC) systems (column, mobile phase, and detector) that are used to determine different carbohydrates in food and feed matrices are described. Cation-exchange columns with different cation counter ions (Na + , Ag + , Ca ++ , and Pb ++ ) in combination with a refractive index detector have been widely used for the analysis of mono-, di-, and oligosaccharides for many years. Currently high performance anion exchange chromatography with pulsed amperometric detection (HPAEC–PAD) is often applied in these analyses. Hydrophilic interaction liquid chromatography (HILIC) with mostly aqueous organic mobile phases combined with mass spectrometric detection is a very powerful tool for both the qualitative and quantitative analyses of complex carbohydrates.
Both qualitative and quantitative characterization of the different carbohydrates present in food and feed are very important to ensure the correct labelling of the product concerning the digestible carbohydrates content (part of the energetic value), dietary fibre, and prebiotic content.In addition, the carbohydrate characterization (both qualitative and quantitative) of raw materials for manufacturing the different (non starch polysaccharide) food ingredients is essential for a good food manufacturing process.
Liquid Chromatography of Carbohydrates
Detectors: Liquid chromatography (LC) is a very well-established technique for the separation and determination of carbohydrates. The bottleneck in the LC analysis of carbohydrates is the detection system. Because carbohydrates do not show a significant radiation absorption in the normal UV and visible range, UV–vis detectors are not appropriate in carbohydrate analyses unless they are derivatized with UV-adsorbing substituents. The most applied LC detectors, particularly in routine analysis, are the refractive index (RI) detector in combination with isocratic separations, the pulsed amperometric detector (PAD), and the evaporative light scattering detector (ELSD), which can both be applied with gradient elution LC systems. In addition, mass spectrometric (MS) techniques are becoming more commonly applied for carbohydrate characterizations in combination with LC.
Liquid Chromatographic Systems: Various LC systems have been described in the literature. The choice of the chromatographic system depends upon the required level of structural details (for example, total monosaccharides or separation between the monosaccharides [glucose, galactose, mannose, and fructose]), type of glycosidic bonding (α, β, or 1à4, 1à1, or 1à6), concentration level of the carbohydrate, and, of course, the sample matrix. In this mini review we will discuss three chromatographic systems: Cation-exchange chromatography; high performance anion-exchange chromatography; and hydrophobic interaction chromatography. These LC techniques are very important for the analysis of carbohydrates in food and feed.
Cation-Exchange Chromatography with RI Detection: For a long time LC separations of carbohydrates have been performed on strong cation exchange resins in the Ca++ form at elevated temperatures, applying an aqueous solution of 50 ppm calcium ethylenediaminetetraacetic acid (Ca-EDTA) as the mobile phase and RI detection (4). For all carbohydrates the refractive index increment (dn/dc) has the same value, which means that the sensitivity of the RI detector is the same for all carbohydrates irrespective of DP value or monosaccharide composition. This facilitates the quantitation of unknown carbohydrate peaks in the chromatograms. A severe drawback of RI detectors is that they can only be applied in combination with isocratic separations.
At room temperature the mutarotation of many reducing sugars is low. As a result, the separation of a mixture of carbohydrates at room temperature on a cation-exchange column will result in a needlessly complicated chromatogram with double peaks or peaks with shoulders because of the (incomplete) separation of the α- and β-anomers of the respective sugars. Increasing the temperature increases the mutarotation by a factor of approximately 2.5 for every 10 °C rise in temperature. As a result of fast mutarotation at elevated temperatures, the α- and β-anomers of the respective carbohydrates elute together in one relatively sharp peak (5). Mutarotation is also strongly catalyzed by high pH values in alkaline solutions. This alkaline catalytic effect on the mutarotation is applied in high-performance anion-exchange chromatography, which will be discussed later.
The separation mechanism is twofold: Firstly, it is based on a complex formation between the hydroxyl groups of the carbohydrates and the immobilized Ca++ ions on the resin (ligand exchange mechanism); and secondly, it is based on size-exclusion effects.
The complex formation between the hydroxyl groups in the carbohydrates and the immobilized counter ions in the cation-exchange resin depends on the conformation of the hydroxyl groups (equatorial or axial) and the carbohydrate itself (chair or boat conformation), and on the specific cation counter ions. H+, Na+, K+, Ag+, Ca++, or Pb++ are used as counter ions. Na+ and K+ form no complexes with the hydroxyl groups in the carbohydrates. Applying a column in the Na+ or K+ form instead of a column in the Ca++ form results in a loss of selectivity for the different monosaccharides and polyols. Almost all monosaccharides and polyols co-elute with each other. Columns in the Na+ or K+ form are mostly applied for the separation of (malto) oligosaccharides. For the separation of different monosaccharides, cation-exchange columns in the Pb++ form are frequently applied. As a result of the differences in structural conformation of the hydroxyl groups in the various monosaccharides, the respective monosaccharides form complexes with the immobilized counter ions in the cation exchange resin with different stability constants, resulting in different retention times of the sugars eluting from the chromatographic column.
Cation-exchange chromatography in combination with RI detection is still a good technique for analyzing simple mixtures of mono-, di-, and tri-saccharides in relatively clean sample matrices such as fruit juices and beverages. Various cation exchange columns with different specifications (for example, particle size, degree of cross-linking, and counter ions) can be commercially obtained from different manufacturers.