Methods for Separating Sugars
Detection Methods (1)
In the previous page, we described methods for separating sugars. Here we will discuss methods for detecting sugars (mainly neutral sugars).
Types of Detection Methods
When choosing a detection method for a given substance, typically we would first check the structure of that substance. However, when it comes to the structure of sugar, the only notable functional structure is the hydroxide group, so how should they be detected? The major methods available for detecting sugars using HPLC are listed below.
- ■ UV-Vis Detection Directly (190 to 195 nm) or derivatized (pre-column and post-column)
■ Refractive Index
■ Fluorescence Detection; Derivatized (pre-column and post-column)
■ Electrochemical Detection; Cu electrode or Au electrode
■ Evaporative Light Scattering Detection
Each method is briefly described below:
UV absorption for sugars can only be detected near the 190 to 195 nm. Consequently, since direct detection of UV absorption is limited to this low wavelength range, it should be considered impractical unless only water is used as the mobile phase and the sample is relatively uncontaminated.
To enable UV-visible detection, in the past, a color reaction, such as the orcinol-sulfuric acid method, was used as a post-column derivatization method. However, this is no longer used, since it requires a corrosion-resistant instrument and is troublesome to handle. No pre-column methods are in practical use either.
Refractive Index Detection
Refractive index detection is a general-purpose detection method that can detect almost any sample components that have a different refractive index from the mobile phase. It is the standard detection method widely used for analyzing sugars. In the past, detection sensitivity was not very good, but instrument performance has improved in recent years and now can detect sugars in an aqueous mobile phase down to the nanomole level. However, even though it is a general-purpose detection method, selectivity of detection is poor.
Since it does not allow gradient elution methods, it is not useful for analyzing oligosaccharides using the partition method or for multi-analyte analysis using the borate complex anion exchange method.
Furthermore, it can cause the baseline to undulate during high-sensitivity analysis using the partition method. This is thought to be caused by slight variations in the mobile phase (acetonitrile/water mixture) equilibrium that occur near the surface of the stationary phase, due to slight temperature variations, which are detected by the detector as variations in refractive index.
Note also that when using the borate complex anion exchange method, ghost peaks from the boric acid (system peaks) can appear as sugar peaks.
Fluorescence detection offers far superior sensitivity and detection selectivity, but it requires that components emit fluorescence. Since sugars are not fluorescent, various derivatization methods are being studied.
Pyridylamino derivatization, which uses 2-aminopyridine as a reagent for pyridine amino derivatization, is one such pre-column method that is widely used for the structural analysis of sugar chains in glycoproteins and enables detection down to picomole levels. However, the pretreatment process for derivatization requires considerable time.
In contrast, post-column methods are preferred for routine analysis because they allow automating the derivatization reaction. Many post-column methods were developed in the 1980s, such as methods that use 2-cyanoacetamide and ethanolamine. During that same era, Shimadzu discovered that arginine, which is a basic amino acid, generates strongly-fluorescent derivatives in a thermal reaction with sugar in the presence of boric acid, and built an analytical system based on this principle. These systems are still being used by many customers today.
Electrochemical detection is also a highly-sensitive detection method. In the case of sugars, it includes methods using either copper or silver electrodes. In particular, combining an Au electrode with a pulse mode enables detecting sugars down to several picomoles.
However, in this method the reaction solution must be kept in a strong alkaline state. Depending on the mobile phase conditions, this requires using a separate pump to add concentrated sodium hydroxide to the column eluate. Furthermore, detection selectivity is not very high, which in some cases can cause problems in separating target components from contaminants.
Evaporative Light Scattering Detector
Evaporative light scattering detectors are general-purpose detectors that can detect any non-volatile substance by spraying the column eluate to remove the mobile phase by evaporation, then irradiating the solute with light and detecting the scattered light. Commercial ELSD systems have been available for about 20 years ago, but were not widely adopted, at first, due to issues with sensitivity and operability.
However, more recently, as models with improved performance have appeared (such as the ELSD-LTII, for example), they are now re-evaluated as general-purpose detectors. In terms of detecting sugars, they are useful for analyzing oligosaccharides using the partition method in particular, because they permit using gradient elution. However, due to their detection principle, note that these detectors do not allow using nonvolatile mobile phases (such as buffer solutions).
Therefore, as described above, there are many choices for analyzing sugars, not only in terms of separation methods, but also detection methods. Please select the optimal combination for your objectives.