During the early development of HPLC for sugar analysis, RI and, to a lesser extent, UV detectors were used almost exclusively. Steady improvement in detector design and the introduction of alternative modes of detection have resulted in a much wider range of options. These are now discussed.
1.6.2.1 Refractive Index Detectors
Refractive index detectors for HPLC analysis work on the principle of differ-ential refractometry and are typically of the deflection type. The flow cell is divided into a reference liquid compartment and a sample liquid compartment.
A light beam passing between these two compartments is deflected in propor-tion to the difference in refractive index between the reference and sample liquids. The deflection is measured by the displacement on a light-receiving element. Modern instruments are very much improved over instruments avail-able in the past, offering much lower noise and better baseline stability as well as greater sensitivity. This has been achieved by improved optical and electrical FIGURE 1.13 Analysis of apple juice sugars. Column, CarboPac PA1 (Dionex), 250 mm × 4 mm; mobile phase, 150-mM NaOH, 1.0 ml/min; pulsed amperometric detection (gold-electrode). 1, Sorbitol; 2, fructose; 3, glucose; 4, sucrose.
4 1
2 3
0 5 10
Minutes
systems and better temperature stability. Refractive index is temperature dependent, and a thermostated flow cell and liquid lines are necessary for good baseline stability. Because of the effect of small changes in temperature on the baseline, it is normally advisable also to thermostat the HPLC column using a column oven (typically set at 35°C).
The analysis of common sugars in foods normally does not require high sensitivity. Individual sugar concentrations down to 0.05% may be measured in the routine analysis of foods, and this is usually adequate. If greater sensi-tivity is required, alternative detectors must be used.
The refractive index detector is often referred to as a “universal” detector because of its nonselective response; however, this lack of specificity can be a disadvantage when other significant components of the food matrix interfere in the separation of the sugar profile. The limits of detection quoted above may then be more difficult to achieve.
Another disadvantage of the refractive index detector is its incompatibility with gradient operation. Such changes in mobile phase composition generally result in an unacceptably steep baseline change. Analysis of sugar mixtures requiring different elution conditions must therefore be conducted using more than one analytical run.
Despite these disadvantages, the refractive index detector remains a good, low-cost choice for the routine analysis of common food sugars.
1.6.2.2 Ultraviolet Detectors
Sugars may be detected by ultraviolet spectrophotometry either directly or as UV-absorbing derivatives. Direct detection is possible by monitoring at a wavelength below 200 nm; however, at this wavelength problems are likely to be encountered with solvent purity and interferences, so this is not a practical approach for routine use. Derivatization of sugars to form strong UV-absorbing chromophores allows the problem of interferences to be reduced and at the same time provides enhanced sensitivity over RI detection; however, artifacts formed by the derivatization process may give rise to interferences. A variety of derivatives have been used. Perbenzoates [48] and 4-nitrobenzoates [49]
have been prepared, but these give rise to several anomeric derivatives. A single peak for each mono- and disaccharide may be obtained using the benzoyloxime–perbenzoyl derivatives [50]. These can be determined at the nanogram level.
1.6.2.3 Pulsed-Amperometric Detectors
Carbohydrates can be oxidized at a gold or platinum electrode giving a high initial current [51]; however, as an oxide layer forms and the products of oxidation rapidly coat and poison the electrode surface, further oxidation is inhibited, causing the current to decay rapidly. By rapidly pulsing between
Mono- and Disaccharides: Analytical Aspects 31
high positive and negative potentials, the electrode surface is cleaned and reactivated (oxide layer reduced) between measurement of the carbohydrate oxidization current.
Pulsed-amperometric detection (PAD) provides highly specific detection of carbohydrates and, at the same time, high sensitivity. Limits of detection for glucose and sorbitol of 30 ppb have been reported [51]. Sample preparation is simplified when using this method of detection because nonsugar coextrac-tives do not interfere and clean-up procedures are generally not required.
Removal of some potential column contaminants may, however, be advisable (e.g., high protein levels, halides, sulfate).
A high pH (above 11) is necessary for the detector to function correctly.
This will generally be provided by the mobile phase when using anion-exchange chromatography; however, if the separation requires the use of a weakly alkali mobile phase, the necessary alkali strength at the detector may be provided by the postcolumn addition of, typically, 0.3-M NaOH at 0.5 ml/min. Postcolumn addition is also used when hydroxide gradients are required and gives a stable baseline, unaffected by the changing alkalinity of the mobile phase.
Because significant changes in pH will affect the detector response and cause baseline shifts it is usual practice in anion-exchange chromatography with gradient operation to maintain a fixed hydroxide concentration while changing the acetate concentration. In some applications requiring both a changing hydroxide and acetate gradient, a pH counter electrode may be used to replace the standard Ag/AgCl reference electrode.
1.6.2.4 Evaporative Light-Scattering Detectors
The evaporative light-scattering detector (ELSD) developed out of the need for a universal HPLC detector that, unlike RI detectors, could achieve high sensitivity and would be compatible with mobile phase gradient operation.
The principle of detection is simple. Column eluent is atomized in a heated air or nitrogen stream. Mobile phase solvents (including water) are vaporized, and any nonvolatile components are left as a fine mist of particles that then passes through a light beam (modern instruments use a laser light source).
Light scattered by the particles is detected by a photomultiplier positioned at an angle of 120° to the light beam. The intensity of the scattered light is proportional to the number of particles and the amount of material. The response is not linear over a wide concentration range, although a linear region of operation is usually possible. It may be necessary to produce a logarithmic calibration curve. The detector requires a high flow rate of air or nitrogen (the latter being advisable when using inflammable solvents) and a fume hood or extraction facility to handle the solvent vapors (essential if one is using acetonitrile).
Macrae and Dick [32] described an early use of the ELSD for sugar analysis. Using an aminopropyl column and a gradient of 20 to 30% water in acetonitrile, they separated sucrose, raffinose, and stachyose in a soya extract within 20 min. Morin-Allory and Herbreteau [52] used a light-scattering detec-tor to demonstrate the use of supercritical fluid chromatography to analyze a range of monosaccharides and polyols. Clement el al. [53] recently compared ELSD and RI detection for the analysis of a range of mono-and disaccharides and concluded that the ELSD offers better sensitivity and a more stable baseline than an RI detector and it makes gradient elution possible. Although it overcomes many of the disadvantages of the RI detector, the ELSD is not selective like the PAD, so coextractives can still interfere.
As a universal detector, the ELSD is very powerful. For example, it is the detector of choice for lipid analysis and, if other such applications are required in the food analysis laboratory, it might well be considered as an alternative to the RI detector for use in sugar analysis.