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Abstract
In this study, a Size Exclusion High-Performance Liquid Chromatography (HPLC-SEC) method was evaluated for its application in determining the apparent molecular weight of biopolymers, specifically exopolysaccharide (EPS) released by the cyanobacterium Arthrospira maxima. The primary objective of this research was to develop a simple, reliable, and reproducible analytical method for measuring the molecular weight of polymers, based on a calibration curve that would characterize polymers using different polyethylene glycols (PEGs) as standards. This approach is important for two reasons, firstly, it provides an alternative to the use of dextran, and secondly, traditional methods of molecular weight measurement can be complex especially when applied to high molecular weight polymers or biopolymers with diverse and variable structures. The HPLC-SEC method was optimized to ensure the separation of various PEGs based on their retention times and to minimize any interferences that could affect the accuracy of the results. Quantification was carried out after the PEGs had been separated, each characterized by a number that typically indicates the average molecular weight of the molecules. Although PEGs are relatively high in molecular mass, the chromatographic conditions were adjusted to enable their clear resolution, which is crucial for ensuring reliable and precise quantification of these standards. Separation was achieved using an Ultra-hydrogel DP column (Waters), which specializes in size exclusion and is designed to allow effective separation of polymers of varying sizes. The mobile phase consisted of a water-acetonitrile mixture in a 65:35 ratio. This composition ensured the optimal separation and enhanced resolution of the samples. The separated PEGs were detected using a refractive index detector, which enabled highly accurate quantitative analysis of the various standards. The six PEG standards employed in this work were PEG300, PEG400, PEG1500, PEG4000, PEG6000, and PEG8000. Remarkably, all these standards were successfully separated in less than 10 minutes, demonstrating the efficiency and rapidity of the developed method. To ensure the robustness and reliability of the method, a set of validation parameters was rigorously assessed. Specificity was confirmed by verifying the absence of interferences from other components in the sample matrix. Linearity was evaluated by analyzing the detector response to different PEG standards across a concentration range, and a considerable degree of correlation was obtained. Precision and intermediate precision were confirmed through repeated analyses, with minimal variation observed between repetitions, thus ensuring the reproducibility of the method. Furthermore, accuracy of the method was validated by comparing the experimental results with theoretical molecular weight values, which showed an excellent agreement between the two, reinforcing the method’s validity. This present study makes a significant contribution to the field of analytical methods for characterizing biopolymers. The optimization of the HPLC-SEC method, coupled with a full validation, enables the precise and rapid determination of the molecular weight of the studied biopolymer, which is essential for its potential use in various industrial applications. Moreover, this methodology could be useful for the analysis of other biopolymers, paving the way for further advancements in the analysis of polymers in different contexts, including biomaterials, biotechnological products, and environmental studies.
Supplementary materials
Title
Supplemental file
Description
The supplemental file provides representative chromatograms obtained from the Size Exclusion High-performance liquid chromatography (SEC-HPLC), specifically it shows results obtained during specificity test and linearity test. Additionally, it represents a typical example of biopolymer (ESP) elution using the described analytical method. On page 1, Chromatograms of blank solution (Fig.1.S1) and reference solution (Fig.1.S2) are presented. The blank solution chromatogram demonstrates the absence of interference according to the elution time of the studied molecules. Additionally, Fig.2.S displays the calibration curve obtained from the linearity test, demonstrating the relationship between response and retention times, is also represented. Page 3 contains the typical chromatograms of Polyethylene glycol (PEG) standards used in the study, labeled from letter a to letter f. Finally, page 4 displays the chromatogram of the studied biopolymer, highlighting the successful application of the method for its analysis. These figures collectively validate the robustness and reliability of the developed HPLC-SEC method.
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