Size exclusion chromatography for analyses of fibroin in silk: optimization of sampling and separation conditions

Abstract

A direct goal of this paper was to improve the methods of sample preparation and separation for analyses of fibroin polypeptide with the use of size exclusion chromatography (SEC). The motivation for the study arises from our interest in natural polymers included in historic textile and paper artifacts, and is a logical response to the urgent need for developing rationale-based methods for materials conservation. The first step is to develop a reliable analytical tool which would give insight into fibroin structure and its changes caused by both natural and artificial ageing. To investigate the influence of preparation conditions, two sets of artificially aged samples were prepared (with and without NaCl in sample solution) and measured by the means of SEC with multi angle laser light scattering detector. It was shown that dialysis of fibroin dissolved in LiBr solution allows removal of the salt which destroys stacks chromatographic columns and prevents reproducible analyses. Salt rich (NaCl) water solutions of fibroin improved the quality of chromatograms. Abbreviations A 2 Second virial coefficient in Rayleigh equation c Concentration of a solution, mg/ml k Wavelength of incidental beam (in vacuum), nm M w Weight average molar mass, derived from Rayleigh equation, g/mol n 0 Refractive index of a solvent N A Avogadro constant dn/dc Refractive index increment P(h) Form factor in Rayleigh equation R(h) Excess Rayleigh ratio, the difference between Rayleigh ratio for a solution and a pure solvent g Branching ratio R 2 lin Mean square radius of linear sample R 2 br

Fibroin degradation – Critical evaluation of conventional analytical methods

“…Major changes in relative viscosity in the first 24 h of ageing could be explained by rapid radical oxidation of fibroin (indicated by DE values above) that directly effects the polymer chain. This supports prior findings on molecular weight distribution drop upon thermos-ageing described elsewhere [3]. The overall influence of relative humidity on the degradation progress monitored by viscosity is rather low and noticeable also just after 1 day (about 7% gather decrease than in dry and closed rector conditions).…”

Evaluating the impact of different exogenous factors on silk textiles deterioration with use of size exclusion chromatography

“…However, regardless of the level of relative humidity, the final degradation stage of fibroin seems to be similar which may indicate that during ageing the recrystallisation also takes place which inhibits further degradation [18,19]. The negative influence of moisture on silk fibres was noted in the literature [18,39,40]. Depending on the humidity level, the degradation dynamic is different, in VOC the most significant changes appear in the final ageing stage, while with increasing water vapour concentration degradation accelerates in the beginning of degradation.…”

“…Sample preparation procedure was described in details in previous work [39]. In the current work, dissolution of silk samples in 9.3 M LiBr (at 60°C for 3 h) was followed by dialysis in a cellulose membrane tube (molecular weight cutoff 3.5 kDa, ZelluTrans/Roth T1) against pure water (specific resistivity 18.2 MX, Simplicity 185 Merck Millipore) for 4 h in microdialysis capsules a'1 cm 3 QuixSep (Roth).…”

“…In the literature, the degradation was evaluated by the changes of silk mechanical properties [34], colour [30], crystalline structure by X-ray diffraction [14,18,35], viscosity [36,37], molecular weight of silk component by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [38] and molecular weight distribution by SEC [19,30,36,[39][40][41]. As the macroscopic properties (like mechanical endurance) of silk textiles may be reflected by the distribution of fibroin chain length, condition of the historical silk textiles can be evaluated from measurements of the molecular weight distribution of the silk [ref].…”

Absolute Molecular Weight Determination of Sodium Salt of Methacrylic Acid/Acrylic Acid‐2‐Isooctyl Ester/2‐Acrylamido‐2‐Methylpropanesulfonic Acid Terpolymer by GPC/MALLS

“…7 Gel permeation chromatography (GPC) appears as the most direct approach to determine the molecular weight based on the hydrodynamic volume of polymers. 8 However, this method requires calibration standards, which is ineffective and often yields erroneous molecular weight if the standards and the samples have different composition, structure, and conformation. 9 Multiangle laser light scattering (MALLS) technique was developed since 1980s, 10,11 and its principle has been reviewed by Wyatt.…”