Based on the separation mechanism, purification techniques can be divided into three categories: physical purification, chromatographic purification, and chemical precipitation. In recent years, a single purification method has been seldom used, and the combination of several separation methods and several devices has been employed to improve the purification results.
Fractional precipitation is suitable for polysaccharides with large differences in solubility and molecular weight [ 29 , 30 , 31 ]. In addition, the long chain quaternary ammonium salt chemical precipitation method and the metal complex method are also applied. The principles of precipitation are shown in Table 1. Column chromatography is an efficient purification method for the separation and the purification of natural components.
Based on the physicochemical properties of the target substance, the most suitable stationary phase and mobile phase are selected for achieving high yield of the target substance. According to the working principle of the stationary phase filler, column chromatography can be divided into cellulose column chromatography, ion exchange column chromatography, gel column chromatography, and affinity column chromatography. In recent years, dicthylaminoethyl DEAE -cellulose anion exchange column chromatography and gel column chromatography have been used in tandem to purify polysaccharides [ 32 , 33 , 34 , 35 ].
The separation of polysaccharides by anion-exchange column chromatography is generally used as the primary stage for the purification of crude polysaccharides [ 36 ] and is based on the principles of adsorption and partition chromatography. For the ion exchange resin, chromatographic separation is achieved by reversible exchange, electron—dipole interaction, or adsorption among surface charged groups of the stationary phase, ions of the sample, and ions of the mobile phase. Acidic polysaccharides can be adsorbed on the exchanger at pH 6, whereas neutral polysaccharides cannot. Moreover, the different acidic polysaccharides can be eluted by using a buffer with the same pH but different ionic strength.
In addition, if the column used is alkaline, the neutral polysaccharides can also be adsorbed, where the adsorption capacity depends on the number of acidic groups in the molecule. Different polysaccharides are separated by gel column chromatography GPC based on the molecular sieve action of the gel porous network structure in three dimensions , which depends on the speed of motion of the polysaccharides with different molecular sizes and shapes in the chromatography column.
Before purification, a gel with small voids can be used to remove impurities such as small molecules and inorganic salts.
Gels for example, dextran gel, polyacrylamide gel, and agarose gel are commonly used as the stationary phase, and deionized water or dilute salt solution are used as the eluent. Different gels are appropriate for polysaccharides of different molecular masses. Therefore, the specific gel column should be selected according to the relative molecular mass of the target polysaccharide [ 39 ]. In most cases, anion-exchange chromatography is used in the first step, followed by gel column chromatography, as shown in Figure 4 [ 40 , 41 , 42 ].
This combined method is simple and mild but may be potentially effective for the separation of viscous polysaccharides that tend to undergo adhesion. Depending on the size, the shape, and the charge characteristics of the polysaccharides, the migration speed of the polysaccharide under the action of an electric field varies, and preparation zone electrophoresis can be used to separate various polysaccharides. In addition, homogenous polysaccharides can be separated by ultracentrifugation.
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A comparison of the main separation and purification methods mentioned in this section is presented in Table 1. Generally, it is difficult to obtain pure polysaccharides by one method; thus, the combination of multiple methods is needed to achieve efficient separation of polysaccharides. The scope and the order of application of each method should also be considered in this process.
Unfortunately, there has been no major breakthrough in the purification methods and materials in recent years. The discovery of new methods and materials is required for elucidating the structural characteristics of polysaccharide molecules. Therefore, an in-depth study on the structure of existing polysaccharides can promote the innovation of polysaccharide purification. At present, most researchers pay more attention to the primary structures rather than the advanced structures of polysaccharides.
Therefore, a feasible technology for analyzing the structure of polysaccharides is urgently needed.
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Homogeneity of a polysaccharide suggests that the components of the purified polysaccharide are homogeneous or pure. In order to lay a good foundation for subsequent structural identification, determination of the purity of polysaccharides is a necessary process to ensure the uniqueness of the polysaccharides. Chromatography is currently the most common method for identifying the homogeneity of polysaccharides, for example, gel permeation chromatography and HPLC combined with differential refractive index detector RID.
The method can be used to detect the homogeneity of the polysaccharide as well as calculate the molecular weight of the polysaccharide [ 43 ]. Polysaccharides of different masses can move at different speeds in the gel column; thus, we can use an appropriate flow rate to elute different components of the polysaccharide sample.
Specifically, the absorbance curve is plotted by using the tube number and the absorbance measurement as the ordinates. In this method, if a single and symmetrical peak appears, the component is usually considered to be a homogeneous polysaccharide [ 44 , 45 ]. The polysaccharide content can also be measured by the phenol-sulfuric acid method [ 46 ].
Polyacrylamide gel electrophoresis and cellulose acetate membrane electrophoresis in conjunction with GPC are also commonly used for determining the molecular weight of polysaccharides. The two methods are carried out simultaneously to further confirm the purity of polysaccharides [ 47 , 48 , 49 ].
The physicochemical properties and the pharmacological activity of polysaccharides are closely related to the molecular weight [ 50 , 51 , 52 , 53 ]. Polysaccharides with similar structures generally have different molecular weights. The average molecular weight is obtained by dividing the molecular mass of n polymers that make up the polysaccharides into n , and the average molecular weight is calculated based on the relative molecular weight [ 54 ].
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There are many methods for determining the molecular weight of polysaccharides. Among them, the most commonly used method is HPLC.
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These techniques offer the advantages of speed, high resolution, and reproducibility and can simultaneously detect the homogeneity of polysaccharides [ 56 , 57 ]. The mobile phases include water, buffer, or aqueous organic solvent. The detectors include refractive index refractometers, evaporative light scattering, multi-angle excitation diffuser, etc. In a gel column of a certain length, the polysaccharide molecules are separated according to the relative molecular weight [ 59 ].
This method does not require calibration with a reference material and has high accuracy and precision. The collision-induced cleavage, electron transfer cleavage, electron capture cleavage, post-source decay, and other post-source cleavage techniques are not only used for determination of the molecular weight of polysaccharides but also for identification of the structural fragments [ 61 ].
In the measurement process, the matrix and the sample concentration are selected according to the structure of the polysaccharide to achieve the desired result. Solutions of high molecular weight polysaccharides generally have higher viscosity. Therefore, the viscosity method can be used to determine the molecular weight of polysaccharides.
In practice, there are some uncertainties in the method, because determination of the viscosity is generally influenced by many factors such as molecular weight and molecule shape. In general, natural polysaccharides are composed of different monosaccharides. Hydrolysis is commonly used to analyze the monosaccharide components of polysaccharides. The determination of the monosaccharide composition is helpful for predicting the core structure of the polysaccharide main chain and studying the physicochemical properties of polysaccharides. In recent research, polysaccharides were hydrolyzed to monosaccharides or subjected to complete hydrolysis for further analysis of the monosaccharide composition using various chromatographic methods.
Acid hydrolysis is the first step in the analytical process, where the process differs based on the type of polysaccharide. Trifluoroacetic acid is commonly used for hydrolyzing neutral hexose, pentose, deoxyhexose, etc. The excess acid is removed by total water distillation, and the hydrolytic product is reduced with NaBH 4. The reduced polysaccharide is acetylated in a boiling water bath using pyridine-acetic anhydride and is then analyzed by GLC, which can be used to determine the types of monosaccharides as well as to quantitatively assess the proportion of monosaccharides [ 62 ].
HPLC is a high-frequency method for detecting the composition of monosaccharides in polysaccharides [ 63 ]. First, the polysaccharide is hydrolyzed to monosaccharides, and the monosaccharides are then chemically derivatized; for example, a fluorescent group is introduced to increase the sensitivity of the detection. The commonly used derivative reagent is 1-phenylmethylpyrazolone PMP [ 36 ].
It has been reported that the polysaccharide is hydrolyzed to monosaccharides by an acid and is derivatized by PMP [ 64 ].
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The sample is then assayed by the apparatus. The mole percentage of monosaccharides can be calculated using the peak area [ 65 , 66 ].