Dual-detection Gel Permeation Chromatography (GPC), also known as Size Exclusion Chromatography (SEC), provides an easy and effective way to measure the molar mass distribution and the amount of free, unbound polysaccharide of iron polysaccharide complexes.
Human bodies contains iron in the red blood cells (as hemoglobin) or in muscle cells (as part of myoglobin). Both proteins are necessary for oxygen transport. Thus, iron is an essential nutrient. In case of iron deficiency, complexes of a polysaccharide and iron are applied as drugs to enhance low iron levels. Suitable characterization of these complexes and their formulations are mandatory for regulatory reasons, quality control and research.
GPC/SEC provides an easy and effective way to measure the molar mass distributions of iron polysaccharide complexes. In the present investigation, iron polysaccharide complexes from different sources were analyzed on a GPC/SEC system with simultaneous UV/RI- detection.
Procedure, Results & Discussion
An advantage of this application is that the iron polysaccharide complex is selectively detected by the UV-detector operated at 254 nm. The more universal RI-detector detects the complex, unbound polysaccharide and the typical and unavoidable system peaks.
Interestingly, this possibility of selective detection of the iron complex by UV is frequently ignored. Instead evaluation of the more complex RI-trace is performed in most of the studies.
However for this application the UV-traces were used to measure the molar mass distributions, molar mass averages and polydispersities of the iron polysaccharide complexes, while for an in-depth characterization with respect to unbound polysaccharide the RI detector signal was used.
Figure 1 shows the overlay of the UV-chromatograms for the 4 different samples A, B, C and D. All complexes reveal well shaped nearly Gaussian peak shapes, indicating that neither the high molar mass exclusion limit nor the low molar mass separation limit of the PSS SUPREMA column combination is reached for any of the samples analyzed. This means that the PSS SUPREMA column combination is ideal for this molar mass separation range.
Three of the four samples can be clearly differentiated based on their chromatograms and the resulting molar masses. However, samples A and B render identical elution profiles. All UV-signals can be easily evaluated as potentially co-eluting residual components are invisible for the UV-detector at the selected wavelength.
By applying a 12 point calibration curve, established using PSS pullulan standards ranging up to 1.2×106 Da, the relative molar mass distributions as well as the molar masses averages and the polydispersities are derived. These results are summarized in Table 1.
For the two samples A and B, which render identical elution profiles in the UV, differences can be found when reviewing the simultaneously measured RI-signals (conf. Figure 2). When comparing the RI-traces, it becomes clear that sample A contains a significantly higher amount of the unbound polysaccharide.