A Comparison Between Amino (NH2) Columns and Sugar Columns in Carbohydrate Analysis
Carbohydrates, as a vital source of energy and metabolic intermediates in living organisms, have always been a focus of attention. With continuous technological advancements in recent years, carbohydrate research has seen unprecedented development opportunities, with significant leaps in both depth and scope.
Carbohydrates are organic compounds composed of carbon, hydrogen, and oxygen. They are commonly found in nature in the form of polyhydroxy aldehydes, ketones, and their derivatives. Based on their composition and structure, carbohydrates can be classified into monosaccharides, oligosaccharides, and polysaccharides:
- Monosaccharides: The simplest form of carbohydrates, such as glucose and fructose.
- Oligosaccharides: Small molecules composed of 2-10 monosaccharide units condensed through dehydration, such as maltose and sucrose.
- Polysaccharides: Large molecules formed by the dehydration condensation of many monosaccharide units, such as starch and cellulose.
The Expanding Scope of Carbohydrate Research
In the 21st century, carbohydrate research has extended into multiple fields, including life sciences, material sciences, and energy sciences. As life sciences have developed, the interaction between carbohydrates and biomacromolecules has become a research hotspot.
For instance, carbohydrates play an essential role in the synthesis and modification of biological macromolecules like proteins and nucleic acids. Additionally, carbohydrates are involved in cellular recognition, signal transduction, and immune responses.
Main Categories of Carbohydrate Analysis
Carbohydrate analysis generally falls into two categories:
- The separation of monosaccharides, disaccharides, and oligosaccharides.
- The analysis of polysaccharides' molecular weight and distribution.
For carbohydrate analysis, amino (NH2) columns are typically used in liquid chromatography. However, with the advancement of chromatography technology, the variety of columns available for carbohydrate analysis has expanded.
Sulfonated columns based on styrene-divinylbenzene copolymer have become mainstream for carbohydrate testing. These polymeric sulfonated columns are further divided into hydrogen-type (such as SUGAR-H), calcium-type (such as SUGAR-Ca), sodium-type, etc.
With so many options, how should one choose the right column for carbohydrate analysis?
Pros and Cons of NH2 Columns and Sugar Columns
Detector Requirements: When detecting carbohydrate molecules, differential refractive index detectors are typically used. These detectors require a certain sample concentration to ensure detection sensitivity. Therefore, the sample needs to have a relatively high concentration.
Mobile Phase Differences: The mobile phase for sugar columns is pure water or an aqueous sulfuric acid solution. Carbohydrates generally dissolve well in these solvents. However, when separating with amino (NH2) columns, the mobile phase is usually a 70:30 acetonitrile-water mixture. When the sample contains multiple carbohydrates or has a high concentration, using 70:30 acetonitrile-water to dilute the sample can cause precipitation due to solubility differences, negatively impacting separation and detection accuracy.
It is also not recommended to use pure water for dilution instead, as solvent effects may lead to abnormal peak shapes. Additionally, from an economic and environmental perspective, sugar columns are more cost-effective and eco-friendly, as they avoid the use of organic solvents like acetonitrile.
Instrument Setup: Sugar columns require temperatures above 70°C, and some HPLC column ovens may not reach these temperatures. Therefore, sugar columns may incur higher equipment costs compared to NH2 columns.
Column Longevity: The amino-bonded phase can be unstable under acidic conditions, and silica-based columns can degrade under basic conditions. Reducing sugars like glucose, fructose, maltose, and lactose may react with amino groups, causing the amino phase to detach or denature, significantly shortening the lifespan of amino columns (NH2 columns).
Sugar columns, on the other hand, are based on styrene-divinylbenzene copolymers, which have lower cross-linking and thus, lower mechanical strength compared to silica-based columns. During use, flow rates must be kept stable between 0.2-0.3 mL/min before the column reaches the set temperature. Excessive stress can damage the column bed, and flow rate changes should be made in 0.1 mL/min increments to avoid rendering the column unusable.
Separation Mechanism Differences: Sugar columns primarily use a coordination exchange mechanism. This mechanism leverages the interaction between hydroxyl groups on carbohydrate molecules and metal ions on the column phase (i.e., coordination exchange energy) to achieve separation. Carbohydrates with varying numbers of hydroxyl groups and different spatial structures have different coordination exchange energies, allowing for effective separation.
The strength of the coordination bond also varies with the type of metal ion. Currently available columns include Ag+, Li+, Na+, Zn2+, Ca2+, Ba2+, and Pb2+, with increasing binding strength in that order. Amino (NH2) columns, in contrast, use aminopropyl groups as their main functional group and rely on hydrophilic interactions to achieve separation.
Therefore, amino (NH2) columns are advantageous for simultaneously separating carbohydrates of different polymerization degrees, such as monosaccharides, disaccharides, and trisaccharides. Sugar columns are better suited for separating monosaccharides, organic acids, and sugar alcohols. However, when the polymerization degree exceeds four (4), the separation efficiency of sugar columns tends to decrease.
Observations on Separation Efficiency
Figure 1 illustrates that amino (NH2) columns have difficulty separating monosaccharides from monosaccharides or disaccharides from disaccharides, but they perform well in separating monosaccharides from disaccharides.
Figures 2 and 3 show differences in separation efficiency between calcium-type sugar columns and amino (NH2) columns for sugars and sugar alcohols. NH2 columns struggle to separate sugar alcohols from sugars, while on calcium columns, sugar alcohols elute later than sugars (e.g., maltose and maltitol are poorly separated on NH2 columns but fully resolved on calcium columns).
Therefore, sugar columns are recommended when separating sugars from sugar alcohols. Moreover, research suggests that as the degree of polymerization increases, size exclusion effects cause retention to decrease, leading to reduced resolution on calcium columns.
In Figure 4, Sugar-H and Sugar-Ca columns exhibit different selectivities when separating sugars and sugar alcohols. In most cases, Sugar-H is used to separate various organic acids.
However, the sulfonic groups in Sugar-H can catalyze the decomposition of certain sugars (such as sucrose and raffinose). The decomposition rate increases with temperature, so it is recommended to use Sugar-H columns at temperatures below 40°C.
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