Carbohydrates refractive index detection

Femia, R. E. and Weinberger, R., Determination of reducing and non-reducing carbohydrates in food products by liquid chromatography with post-column catalytic hydrolysis and derivatization comparison with refractive index detection, /. Chromatogr., 402, 127, 1987. 

Polarimetry takes advantage of the optical activity of carbohydrates. The high selectivity of this procedure makes it especially suitable in the case of complex food extracts, where other components would interfere with ultraviolet or refractive index detection. However, a major disadvantage is its lower sensitivity. The use of immobilized enzymes (51) with detection by fluorescence or electrochemistry has also been applied in fermentation juices (52) and other particular cases. 

There are many substances which would appear to be good candidates for LC-EC from a thermodynamic point of view but which do not behave well due to kinetic limitations. Johnson and co-workers at Iowa State University used some fundamental ideas about electrocatalysis to revolutionize the determination of carbohydrates, nearly intractable substances which do not readily lend themselves to ultraviolet absorption (LC-UV), fluorescence (LC-F), or traditional DC amperometry (LC-EC) [2], At the time that this work began, the EC of carbohydrates was more or less relegated to refractive index detection (LC-RI) of microgram amounts. The importance of polysaccharides and glycoproteins, as well as traditional sugars, has focused a lot of attention on pulsed electrochemical detection (FED) methodology. The detection limits are not competitive with DC amperometry of more easily oxidized substances such as phenols and aromatic amines however, they are far superior to optical detection approaches. 

Figure 7.1. HPLC analysis of carbohydrates (simple sugars) in soft drink using resin column and refractive index detection. HPLC conditions column BioRad Aminex HPX-87C (300 x 7.8mmi.d.) injection volume 10 pL mobile phase water, flow rate 0.6mL/min at 85°C detection refractive index. Chromatogram courtesy of PerkinElmer, Inc.

Figure 7 Refractive index detection in liquid chromatography. (A) Dual-triangular section flow cell for detector based on Snell s law. (B) Separation of detection of carbohydrates with LC and refractive index detection. Sample 4ng each of (1) xylose, (2) fructose, (3) sucrose, (4) maltose hydrate, and (5) lactose. (Reproduced with permission from Munk M (1993) Refractive index detection. In Parriott D (ed.) A Practical Guide to HPLC Detection. San Diego Academic Press.)...

Figure 8.87 Separation of organic adds, carbohydrates, and alcohols in wine using refractive index detection. Separator column HPX-87 H column dimensions 300 mm x 7.7 mm i.d. column temperature 65 °C eluent 6S mmol/L H2SO4 flow rate 0.8 mlVmin detection refractive index injection volume 5 pU sample wine...

Dextrose Quantitation. The dextrose yield was determined by BioRad Aminex Carbohydrate HPX-87C HPLC at 85 C eluting with glass distilled water at a flow rate of 0.7 ml/minute. Detection was by refractive index. 

Hancock DO, Synovec RE. Microbore liquid-chromatography and refractive-index gradient detection of low-nanogram and low-ppm quantities of carbohydrates. Journal of Chromatography 464, 83-91, 1989. 

In the recent review on column LC by Majors et al. (21), a survey on the use of detector types was carried out in the same manner as that for the use of the various separation modes already mentioned. The results, shown in Table VI, were tabulated for the periods 1982-83 and 1980-81. The increased use of electrochemical and refractive index detectors is significant in these data. The authors speculated that the increased use of refractive index detectors resulted from the increased number of publications on the separation of carbohydrates. The increased use of electrochemical detection is probably a function of many different factors cell designs that are easier to use, expanding sales... 

Also known as the mass detector, this is an evaporative analyzer in which the mobile phase is removed by nebulization and evaporation prior to the determination of nonvolatile carbohydrates by light scattering (44). Unlike the refractive index detector, it allows gradient elution (eluent is removed before detection) and is more sensitive. The detection limit can go up to a few tens of nanograms injected. 

The refractive index detector was one of the first on-line HPLC detectors used. Detection is based on changes in the refractive index of the effluent when an analyte is present versus the solvent alone. This detector is commonly used when an analyte does not have a suitable UV chromophore. An example would be the detection of carbohydrates in drug preparations or acetylcholine in an ophthalmic solution [102,103]. Figure 5.4 is a chromatogram of propylene glycol, propylene carbonate, and resorcinol in anhydrous ointment detected by a refractive index detector [104]. 

The carbohydrate being eluted from a GPC column can be detected by a number of physical or chemical means (e.g., variation in refractive index or viscosity and colorimetric or fluorometric spectroscopic analysis). For the purpose of these experiments, the cellulose was tagged with a fluorescent label, dichlorotriazinylaminofluorescein (DTAF), which permits easy detection of very small quantities. The chromatographic system was set up to allow for convenient analysis of cellulose with a maximum resolution of the molecular weight distribution and a minimum of change to the sample. 

With the major constituents in foods the choice of LC detector is often the most important issue. Compounds such as vitamins, carbohydrates etc. may not have a strong ultraviolet (UV) chromophore. Therefore refractive index (RI) detection and, increasingly, electrochemical detection are often used. As discussed later, the choice of detector is even more important when determining the concentration of components in the foodstuff rather than the bulk constituent. 

For detection of carbohydrates in principle, ultraviolet (UV), laser-induced fluorescence, refractive index, electrochemical, amperometric, and mass spec-trometric detection can be used. Mass spectrometry, with its various ionization methods, has traditionally been one of the key techniques for the structural determination of proteins and carbohydrates. Fast-atom bombardment (FAB) and electrospray ionization (ESI) are the two on-line ionization methods used for carbohydrate analysis. The ESI principle has truly revolutionized the modern mass spectrometry of biological molecules, due to its high sensitivity and ability to record large-molecule entities within a relatively smaU-mass scale. 

Refractive index monitors are used in food analysis, for detecting carbohydrates, alcohols, and other substances with weak or no UV absorption. 

The first practical refractive index detector was described by TiseUus and Claesson [1] in 1942 and, despite its limited sensitivity and its use being restricted to separations that are isocratically developed, it is stiU probably the fifth most popular detector in use today. Its survival has depended on its response, as it can be used to detect any substance that has a refractive index that differs from that of the mobile phase. It follows that it has value for monitoring the separation of such substances as aliphatic alcohols, acids, carbohydrates, and the many substances of biological origin that do not have ultraviolet (UV) chromophores, do not fluoresce, and are nonionic. 

The basic mechanism of separation of carbohydrates is by ligand exchange chromatography but is quite similar to ion-exclusion chromatography described earlier in this chapter for weak organic and inorganic acids. The column contains fully sulfonated polystyrene polymer beads cross-linked with polydivinylbenzene. The polymers are fully hydrated and contain occluded water within the gel polymer matrix, just as in ion-exclusion polymer beads. Analytes partition between the occluded water within the bead matrix and the mobile phase. Water is most often used as the mobile phase and the detection method is most often refractive index. 

Sugars Common carbohydrates are monosaccharides (glucose, fructose), disaccharides (sucrose, maltose, lactose), trisaccharides (raffinose), and polysaccharides (starch). HPLC offers a direct, quantitative method for simple sugars, which requires a specialty cationic resin-based column and refractive index or evaporative light scattering detection.7,8 UV detection at low wavelengths (195 nm) can be used but is more prone to interferences.8... 

The refractive index (RI) detector is the only detector in LC that responds to almost every compound. It is very useful for detecting species that are not ionic, do not absorb in the UV and do not fluoresce, e.g. polymers and carbohydrates. The detection principle involves measuring the difference in the refractive index of the background eluent and that of the eluent with analyte. The refractive index of an analyte is a function of its concentration so any change in concentration is reflected as a change in the RI. The... 

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