Applications of asymmetrical flow field-flow fractionation for separation and characterization of polysaccharides: A review

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Highlights

  • The applications of AF4 for the analysis of polysaccharide from different sources were reviewed.

  • The separation of polysaccharide can be completed by AF4.

  • The effect of operating conditions of AF4 on analysis of polysaccharides was discussed.

  • More information on polysaccharide can be obtained by AF4 coupled with multidetector.

  • AF4 will be a state-of-the-art technology for studying polysaccharide.

Abstract

Polysaccharides are the most abundant natural biopolymers on the earth and are widely used in food, medicine, materials, cosmetics, and other fields. The physicochemical properties of polysaccharides such as particle size and molecular weight often affect their practical applications. In recent years, asymmetrical flow field-flow fractionation (AF4) has been widely used in the separation and characterization of polysaccharides because it has no stationary phases or packing materials, which reduces the risk of shear degradation of polysaccharides. In this review, the principle of AF4 was introduced briefly. The operation conditions of AF4 for the analysis of polysaccharides were discussed. The applications of AF4 for the separation and characterization of polysaccharides from different sources (plants, animals, and microorganisms) over the last decade were critically reviewed.

Introduction

Polysaccharides are carbohydrates with complex molecular structures that are widely found in plants, animals, microorganisms, and other organisms. They not only act as a source of energy, but also participate in various activities of cells in life phenomena. Due to the special properties of polysaccharides and a variety of biological activities (such as rheological properties, adhesion, antioxidation, antitumor activity, immune regulation, and antiobesity), great interest has arisen in their properties and they have been widely used in food [1,2], medicine [3], [4], [5], materials [6,7], cosmetics [8,9] and other fields [10], [11], [12].

Polysaccharides are sugar chains composed of glycosidic bonds connecting monosaccharide units. The common glycosidic bonds are α-1,4-, β-1,4- and α-1,6-glycosidic bonds, and monosaccharides can be linked into straight chains or branched chains [13,14]. Polysaccharides usually have a complex structure and most of them have ultrahigh molecular weight (MW) in their primary structure. Related studies have shown that the conformation of polysaccharides often affects their natural biological activity, thus affecting their practical applications [15,16]. Therefore, accurate characterization of polysaccharide structures is particularly important. Size exclusion chromatography (SEC) is a technique for size-based separation in complex samples. It is also used in the separation of polysaccharides. However, the range of molar mass (generally > 106 Da) of SEC is limited, and the column adsorption effect and shear effect of the stationary phase limit its application in polysaccharides [17,18]. In recent years, asymmetrical flow field-flow fractionation (AF4) has attracted increasing attention due to the wide dynamic range (approximately from 1 nm up to 1 μm in the normal mode), and the use of ‘open channels’ without stationary phases or packing materials, which creates a wide range of selectivity in the analytical carrier liquid [19,20]. These characteristics of AF4 enable the sample to maintain not only good integrity but also good biological activity in the detection process, which is especially suitable for the analysis of complex analytes with fully preserved natural properties [20]. These advantages make AF4 widely use in the analysis and detection of biological macromolecules such as polysaccharides, proteins, and nucleic acids [21], [22], [23]. Moreover, the shear force is minimized in the AF4 channel compared to the SEC columns during separation which allows for a decrease in the impact of shear scission particularly on branched macromolecules and large macromolecules. In addition to separation, the size characterization of analytes by direct measurement of retention time is one of the key features of AF4. As a gentle separation technique, AF4 coupled with multiple detectors has been widely used in the separation and characterization of polysaccharides [24], [25], [26], [27].

In this review, the separation principle and elution mode of AF4 are briefly introduced, and an introduction to AF4 theory can be found elsewhere [21,27,28]. Early works on the characterization of polysaccharides by AF4 have been reviewed [18,29]. The focus of this review is on the development of AF4 in the separation and characterization of polysaccharides from different sources over the last decade to provide a basis for further research, production, and application of polysaccharides.

Section snippets

Principle of AF4

AF4 was first introduced by Wahlund and Giddings [19]. In this review, AF4 theory is described briefly. AF4 is a gentle and fast size-based separation method. As shown in Fig. 1(a), the upper wall of the AF4 channel is an impermeable polycarbonate glass plate, and the bottom channel plate is permeable and made of porous stainless steel frit material, a polyester trapezoidal spacer, and ultrafiltration membrane in the middle. The bottom channel plate and the ultrafiltration membrane form an

Optimization of operation conditions in AF4

In the process of AF4 separation, the resolution of the sample, sample recovery, and sample information obtained are affected by several factors, such as the carrier liquid, type of membrane, flow rate, channel thickness and type of detector. Therefore, it is necessary to optimize the operating conditions of AF4 to achieve good resolution and accurate sample information. The optimization of AF4 operation conditions depends on the surface properties of the sample. This section mainly reviews the

Polysaccharides from seed

Seed starch is one of the most abundant plant polysaccharides and has been widely studied as the most important source of human food energy. It is mainly composed of amylose and amylopectin. Amylose consists of linear chains of (1→4)-α-D-glucose linked residues with a small number of long chain branches and amylopectin also contains sequences of (1→4)-α-D-glucose linked units; however, it has extensive branching via (1→6)-α-D-glucose linkages. The literature [27,32] reviewed the application of

Summary and Outlook

AF4 has been widely used in the field of polysaccharides in recent years due to its wide separation range in size. AF4 coupled with multiple detectors (UV, MALS, dRI, ICP-MS, FL, QELS) provides more information on the sample such as Rh, Rg, MW, ρ, polymer conformation and branching degree of polysaccharides. For glycoproteins, AF4-FL can also be used for semiquantitative determination of polysaccharides. Compared to SEC, AF4 is more suitable for the separation of branched polysaccharides due to

Credit Author Statement

Xue Chen and Wenhui Zhang: writing-original draft. Yuwei Dou: Table data. Tiange Song: Drawing figure. Shigang Shen: Revising the manuscript. Haiyang Dou: Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors acknowledge the support provided by the Nature Science Foundation of Hebei Province (B2016201002), the Key Project of Hebei Education Department (ZD2019009), the Medical Science Foundation of Hebei University (2020A08), and the Post-graduate's Innovation Fund Project (hbu2020ss056).

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