EL CONCEPTO DE PERFIL DE FUNCIONABILIDAD

The present fundamental data concerning the molecular weight distribution of different gelatin fractions was generated under the major guidance of Dr. Wolfgang Fraunhofer. Parts of the presented data, such as the ones depicted in Fig. 6 – Fig. 8, are already published (Fraunhofer et al. 2004).

Morgenstern 2003). The use of a UV detector requires previous calibration of the retention times via globular molecular weight standards resulting in more or less straight calibration lines. The accuracy of these calibrations in connection with gelatin is rather questionable, since gelatin has no globular structure. Thus, previous molecular weight findings based on UV detection alone (Coester 2000; Weber et al.

2000) have to be considered as approximations only. In the present study we additionally measured the light scattering signals of the separated gelatin fractions employing a static light scattering (SLS) detector. As soluble gelatin is below the diameter boundary of λ/20 (see 2.3.2), a 3-angle light scattering detector is sufficient for correct analysis.

Investigating gelatin with our experimental settings, the resulting chromatogram (Fig. 6) of a bulk gelatin solution demonstrated the large molecular heterogeneity of this biopolymer. MALS determined a molecular weight range from about 4 – 1000 kDa. Moreover, two distinct fractions could be identified at 280 nm wavelength. The first fraction comprised high molecular weight (hmw) components >100 kDa and the second one low molecular weight (lmw) components < 100 kDa. 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 0 5 10 15 20 25 30 35 40 45 time (min) molecul a r weight (D a) 0 1 2 3 4 5 6 7 8 UV signal ( V )

Fig. 6: SE-HPLC analysis via UV280 and MALS; flow rate: 0.7 mL/min

Adopting the previously described classification scheme for the different gelatin molecular weight fractions (Farrugia 1998; Farrugia & Groves 1999), the borderline between both fractions corresponds to the α-fraction of gelatin (80-125 kDa),

which consists of soluble tropocollagen α-chains. Consequently, the first peak summarizes the higher molecular weight fractions (β-, γ-, ε-, ζ-, δ-, and microgel- fraction) i.e. the multimers of the tropocollagen α-chain, whereas the hydrolysis products classified as sub-α and low molecular weight fraction are responsible for the second peak. The initial steepness of the first peak has to be attributed to an artifact of the separation column, because the molecular weight of the eluted protein was above the maximum separable molecular weight. Thus, these very large components were already eluted with the void volume.

Based on these chromatographic data for bulk gelatin, we further analyzed solutions of the two phases after the first desolvation step: the precipitated sediment which would be further processed for nanoparticle preparation and the supernatant that has to be discarded (Fig. 7). Comparing the chromatograms, both gelatin samples revealed high and low molecular components as well. But as supposed, a higher amount of the hmw components could be found in the sediment. The percentage of hmw components was 58% in the sediment sample compared to 38% in bulk gelatin. A percentage of only 29% of hmw components remained in the supernatant.

0 1 2 3 4 5 6 7 8 9 10 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 time (min) UV sign al (V )

Fig. 7: SE-HPLC chromatograms of bulk gelatin (black), sediment (grey) and

supernatant (blue) before the first desolvation step

Even though SE-HPLC is the method of choice to characterize the molecular weight of gelatin (Barth et al. 1994; Farrugia 1998), our experiments demonstrated

ratios between the hmw and lmw components of the samples. Lowering the flow rate led to an intense reduction of the hmw component percentage throughout the analyzed samples; e.g. a reduction of the flow rate to 0.3 mL/min reduced the hmw component content to 24% (sediment), 15% (bulk), and 13% (supernatant). Apparently, the change of the flow rate prohibits to state absolute quantitative values for the molecular weight distribution of gelatin.

Since our major interest was to get a more detailed idea about the actual molecular weight distributions of the gelatin samples, we evaluated AF4/MALS as alternative analytical tool. Applying mild cross-flow conditions (5%), a separation of the bulk gelatin into two fractions was not observed (Fig. 8). However, the molecular weight results, calculated from these experiments, differed remarkable from the results obtained with SE-HPLC. With a molecular weight distribution of 20 – 10,000 kDa, the calculated maximal molar mass was about one order of magnitude higher.

1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 0.00 5.00 10.00 15.00 20.00 25.00 time (min) molec u la r w e ight ( D a) 0 2 4 6 8 10 12 UV s ignal (V )

Fig. 8: AF4 fractogram of bulk gelatin (5% cross-flow)

However, these results seem to describe more precisely the actual molar mass distribution of gelatin solutions. It is supposable that these very hmw components could either not pass the separation column in SE-HPLC or got degraded by abrasive shear forces caused by the column package material (Barth et al. 1994). In AF4 experiments, this problem does not occur because the separation channel itself is free of package material. Additionally, the field of force that is exerted on

the sample can be minimized by reducing the cross-flow. So, even very delicate analytes can be kept from degradation during the analytical run.

If higher cross-flows are applied, the fractograms of gelatin reveal two populations analogous to the SE-HPLC chromatogram. These two separated fractions can already be generated with 40% initial cross-flow. The molecular weight borderline between these two fractions was not 100 kDa but approximately 450 kDa, thus the the hmw peak represented a lower amount of total gelatin than in SE-HPLC experiments. Nevertheless, most hmw components could be detected in the sediment (27%), followed by 17 % in bulk gelatin and 10% in the supernatant sample (Fig. 9) 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10 0.00 10.00 20.00 30.00 40.00 50.00 time ( min ) mo lecu la r wei g h t (D a ) 0 1 2 3 4 5 6 UV-signal (V)

Fig. 9: AF4 fractograms of bulk gelatin (black), sediment (grey), and supernatant

(blue) after the first desolvation step (40% cross-flow)

Even though augmentations of the cross-flow intensity influenced the absolute values of the hmw ratios, the relative proportions of the individual samples to each other concerning the hmw content remained constant (data not shown). Thus, no sample degradation occurred.

So, it can be summarized that AF-4 analysis exceeded SE-HPLC in the present study, as it delivered more accurate molecular weight distributions of the applied gelatin solutions. This can be attributed to the milder and more flexible separation conditions the samples were committed to.