How sweet it is! Sucrose analysis with OMNISEC
Sucrose, otherwise known as table sugar, is found in a wide assortment of food products. Its popularity spans the globe and history, as various civilizations throughout the past three millennia developed their own techniques for production. To satiate our current society’s increasing appetite for sugar, various technologies have been implemented to improve the production processes.
Sucrose is a disaccharide, comprised of one fructose molecule and one glucose molecule joined through an α-glycosidic linkage. As a discreet, small molecule, generously described as oligomeric, it has a molecular weight of 342 Da. Molecules in this size range have been typically characterized using techniques such as mass spectrometry (MS) or proton nuclear magnetic resonance (NMR). However, these techniques aren’t suited to detect the presence of larger species in the sample. This is especially critical for sucrose; as a component in foods and medicinal products (e.g. Venofer) its purification is a critical part of the refinement process. A more appropriate technique with the scope to detect both the sucrose and larger species present is size exclusion chromatography.
Gel permeation chromatography / size exclusion chromatography (GPC/SEC) is often discussed in the context of the analysis of macromolecules. Samples ideal for this technique include synthetic and natural polymers, proteins, and other biomolecules. However, recent advances in detector technology have allowed the lower limits of characterization to extend beyond what is typically thought of as polymeric into a range that can be described as oligomeric, or even small molecule. In some cases, GPC/SEC instruments like Malvern Panalytical’s OMNISEC system and OMNISEC REVEAL ULTRA possess the ability to offer comprehensive characterization for samples that lie outside the typical polymer size and molecular weight range.
This post will describe the GPC/SEC analysis of sucrose, including molecular weight and molecular size characterization. Further discussion will include the detection of a larger species present in the sample, and how the results compare to those obtained in similar sucrose studies using dynamic light scattering (DLS).
GPC/SEC analysis of sucrose
The goal of this study was to analyze two sucrose samples, 1 and 2, to determine if there was a detectable difference that might affect their relative processability. It was known that sucrose 1 was easier to process and sucrose 2 was harder to process, but there were no apparent differences in the samples that the manufacturer could quantify.
Due to their low molecular weight, the sucrose samples were prepared at concentrations of about 20 mg/mL, which is higher than normal, to ensure sufficient detector response. The samples were analyzed using a combination of A2000 & A2500 columns at a flow rate of 0.75 mL/min with injection volumes of 100 µL in a mobile phase of water with 0.05M Na2SO4. The multi-detector chromatograms for samples 1 and 2 are shown below. The RI signal is presented in red, the viscometer signal is blue, and the right angle light scattering detector is green. The limits of integration for the sucrose peak are represented by the vertical blue lines.
The chromatography of both samples showed a strong response in all three detectors around 16 mL. The resulting sample peaks were narrow, which was expected considering that sucrose is a discreet molecule, as opposed to a distribution of polymer chains with varying lengths. Despite the low molecular weight of sucrose, the sensitivity of the OMNISEC light scattering detector and increased concentration of the samples allowed for the determination of the samples’ absolute molecular weight. The calculated results for samples 1 and 2 are presented in the table below.
Employing a dn/dc value of 0.150, typical for polysaccharides in aqueous media, provided calculated molecular weights of 342 and 336 Da for samples 1 and 2, respectively. These values correspond exactly with the 342 Da molar mass of sucrose. Also calculated were the intrinsic viscosity (IV) values, 0.026 dL/g for both samples, and the hydrodynamic radius (Rh) values, 0.52 and 0.51 nm for samples 1 and 2, respectively.
Frequent Materials-Talks contributor Dr. Ulf Nobbmann was part of a team that previously reported DLS analyses of sucrose1 and determined a hydrodynamic diameter of about 1 nm, which matches the hydrodynamic radii of about 0.5 nm calculated here. These values are consistent with prior characterizations of sucrose performed by Einstein2 and others3 in the early to mid-twentieth century.
Potential aggregate in sucrose samples
Aside from the main sucrose peak, a prominent feature in the chromatograms of both samples was the presence of a larger species eluting at about 10 mL. This peak was notable for two reasons: 1) it appeared only in the light scattering response, and 2) it provided the only real difference between the two samples, as it was smaller than the sucrose light scattering peak in sample 1 and larger than the sucrose light scattering peak in sample 2.
That it produces only a light scattering response indicates that while it’s large (early elution volume) and high molecular weight (light scattering response), it has a dense molecular structure (no viscometer response) and exists in low concentration (no RI response). This combination of detector responses represents a typical profile of an aggregate in GPC/SEC samples.
The previously mentioned DLS results describe the presence of a large species within sucrose samples as well.1 This component was found to have a hydrodynamic diameter of about 200 nm, as measured by DLS. This corresponds to the maximum size of molecules passing through the OMNISEC system, as all samples are filtered through a 0.2 µm filter prior to and during analysis. Further studies by Weinbuch et. al. confirm that this early eluting peak represents nanoparticulates most likely comprised of raw materials such as dextrans, ash components, and aromatic colorants not removed during the refinement process.4 Filtration of the sucrose solution through a 0.02 µm filter was found to eliminate the aggregate material from the sample.
The difference in size of the aggregate peak relative to the sucrose peak might be an indicator of processability. Sucrose 1 is easier to process and has a smaller aggregate peak relative to the sucrose peak in the light scattering detector, while sucrose 2 is more difficult to process and exhibits a larger aggregate peak. However, while the relationship is suggestive, further studies would be required to determine if these two data points indicate a trend that would persist through multiple samples.
Final thoughts
The ability of OMINSEC to characterize relatively small, discreet molecules was demonstrated through the analysis of two sucrose samples. The calculated molecular weight corresponded with the known mass of sucrose, and the molecular size obtained matched previously reported results obtained through DLS analysis. The presence of an aggregate species, initially identified by DLS, was confirmed in the GPC/SEC data. The relative intensity of the light scattering signals of the aggregate and sucrose peak may offer insight into the processability or purity of the sucrose samples.
In conclusion, I hope this post expands your perception of OMNISEC’s ability to analyze low molecular weight materials. In addition to the post linked above and data shown here, OMNISEC has recently been shown to successfully analyze very small quantities of the peptide Bradykinin, possessing a molecular weight around 1060 Da. If you have any questions, please don’t hesitate to contact us or email me directly at kyle.williams@malvernpanalytical.com.
References
- Kaszuba, M.; McKnight, D.; Connah, M. T.; McNeil-Watson, F. K.; Nobbmann, U. Measuring Sub Nanometre Sizes Using Dynamic Light Scattering. J Nanopart Res 2008, 10, 823-829. DOI 10.1007/s11051-007-9317-4
- Einstein, A. Ann. Physik. 1906, 19, 289-306.
- Schultz, S. G.; Solomon, A. K. J Gen Physiol 1961, 44, 1189-1199.
- Weinbuch, D.; Cheung, J. K.; Ketalaars, J.; Filipe, V.; Hawe, A.; den Engelsman, J.; Jiskoot, W. Nanoparticulate Impurities in Pharmaceutical-Grade Sugars and Their Interference with Light Scattering-Based Analysis of Protein Formulations. Pharm Res 2015, 32, 2419-2427. DOI 10.1007/s11095-015-1634-1
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