Asymmetrical flow field-flow fractionation

5.3.1.1 Determination of the molecular weight of OSA starch by AF4-MALS

The thermogravimetry (TG) experiment yielded a thermal mass loss of OSA starch of 5.11 %, 5.11 %, and 5.12 % during the heating period from 20 °C to 150 °C. The evaporated molecules can be referred to adhering water.

Thus the OSA starch moisture content can be assumed to be approximately 5.11 %. This value was used for the determination of the refractive index increment dn/dc and thereby contributed to the determination of the molar mass of OSA starch by AF4-MALS.

Figure 33 describes the dependence of the RI signal on the OSA starch concentration. The dn/dc value which is reflected by the slope of linearly fitted graph was determined to be equal to 0.1759 cm³/g (correction of the moisture content included). The dn/dc increment served for the determination of the molar mass of OSA starch in the following section.

76 Results & discussion

Figure 33 Plot of RID signal intensity versus OSA starch concentration for the determination of the refractive index increment dn/dc

The AF4 elugrams for OSA starch are shown in Figure 34, where the obtained light scattering and differential RI signals as well as the molar mass are plotted as a function of elution time. Both the MALS as well as the RI detector signals indicate that the molar mass of OSA starch covers a wide size range. Determination of the weight average molar mass (Mw) was performed for the elution time range from 9.5 to 35.5 minutes because after that the RI signal decayed to zero. For each point of elution time a Zimm plot was generated in order to calculate the molecular weight.

Figure 34 Elugrams of an OSA starch solution showing the A: MALSD signal, and B: RI signal and molar mass By means of Equation (13) and each individual Zimm plot, which is exemplarily given in Figure 35A for the mass fraction eluting in the MALSD peak at 31.5 minutes, the weight average molar mass (M_w) of OSA starch could be obtained since it is equal to the reciprocal of K*c/R(θ) value when sin²(θ/2) = 0. OSA starch eluting at 31.5 minutes had a molar mass of 9.6x10⁶ g/mol. Figure 35B shows the differential molar mass distribution of the OSA starch sample in a logarithmic plot detected during the mentioned elution time. The sample´s molar mass ranged from 5x10⁴ to 3x10⁷ g/mol, the main fraction being sized from 10⁶ to 10⁷ g/mol. The modal score (most frequently occurred molecular weight in the data set) obtained from the frequency distribution curve in Figure 35B amounts to 2.26x10⁶ Da and can be referred to as the best description of an average molar mass determined for OSA starch.

Further investigation of the nanocapsule size 77

Figure 35 A: Zimm plot at time t=31.5 min (example). For each elution time, Mw is calculated by Kc/Rθ = 1/Mw

at sin²(θ/2) = 0. For t=31.5 min, Mw (OSA starch) = 9.6x10⁶ Da. B: Resulting differential mass distribution with an approximate average molar mass (OSA starch) of Mmodal = 2,26x10⁶ Da (modal score).

This result did not consider particles being responsible for the signals after the main MALSD peak, i.e. from 35.5 to 41 minutes because they cannot reliably be identified as OSA starch molecules due to the missing RID signal at that time range. Generating a Zimm plot over the whole range (9.5 to 41 min) was not possible due to poor data fit quality. That is why the value of the modal score can only be given approximately (2.26x10⁶ Da).

Figure 36 demonstrates the size development of the dissolved OSA starch molecules with increasing molar mass. Those with a molar mass of less than 10⁶ g/mol do not appear is this graph because their RMS radius is far smaller than 1 nm. It is shown that the fraction with a molar mass of less than approximately 2x10⁶ g/mol is smaller than 15 nm and shows a strong increase in size with increasing molar mass, whereas larger particles do not show such a strong dependence of the size on the molar mass. The RMS radius is maximum 53 nm large.

Figure 36 RMS radius versus molar mass of OSA starch

5.3.1.2 Size determination of emulsions and nanocapsules by AF4-MALS

AF4 coupled to MALSD was used to study the size of the samples which are listed below:

(i) E-OSA5-5% (v) NC-OSA-GEL-SBP-5.55%

(ii) NC-OSA-CHI-CARR-2.5% (NC3) (vi) NC-OSA-GEL-GA-5.625%

(iii) NC-OSA-CHI-CARR-CHI-CARR-1.11% (NC5) (vii) NC-OSA-CHI-GA-3.7%

(iv) NC-OSA-CHI-CARR-8.6%

78 Results & discussion

They present the selection of the successfully developed nanocapsule samples from Table 4 (p. 37) as well as their nanoemulsion template. The elugram for the sample E-OSA5-5% is shown in Figure 37.

Figure 37 AF4-MALSD elugram of E-OSA5-5% (det. 11, 90° angle) and 3D overview (detectors 4 to 18)

Best representation of the particle size is usually reflected by the given 90° angle view (detector 11). For comparison, the 3D image shows how the MALSD signal varied from detector 4 (26°) to detector 18 (163°) for the emulsion. The main peak at 16 min was detected at each scattering angle. However, at small light scattering angles such as detector 4 (26°) the signal showed further peaks at approx. 30 min and 40 min elution time which are hardly visible at 90°. They were induced by very few particles eluting later and being slightly larger than the main fraction which can be concluded later from the geometric radius distribution of Figure 38A (left). The particle size obtained for the emulsions and nanocapsules was each related to the elution time as given by Equation (8). The weight average geometric radius (Rw) was calculated from RMS radius by Equation (15) (assumption of spherical shape). This calculation showed an excellent data fit for the emulsion over the whole scattering angle width. However, for polyelectrolyte NCs poor data fit quality was observed in the Zimm plot for very small and very large scattering angles (detectors 4, 5, 6, 18). Possibly the spherical shape of the capsules has been influenced by polymer chains directing out of the particle. Another reason for poor fit data at the mentioned detectors could be interaction of the particles with the membrane. Hence, the curve fit was optimised by neglecting the mentioned scattering angles. Scattering angles No. 7 (52°) to 17 (153°) were applied for particle size determination of all samples. A summary of the size results is given in Table 12.

The AF4-MALS results of all investigated samples are given in the following figures. Figure 38 shows the elugrams of the consecutively prepared samples (OSA emulsion template, three- and five-layered NCs consisting of OSA, chitosan and carrageenan). The elugram peak maximum was reached at approx. 16 min for each of the three samples of Figure 38. In general, the elugrams look very similar. The geometric radius was calculated in the elution time range from 9 to 43 min. It mainly increased with increasing elution time in the range from 10 min to 30 min which indicates that the particles mostly eluted normally but not sterically. The region after the main peak (from 30 to 43 min) shows some variations of the size. However, these signals derived from only very few particles.

Further investigation of the nanocapsule size 79

Table 12 Size results (in nm) obtained by AF4; ^#calculated from RMS radius of scattering detectors 7-17

sample name

average geometric radius^# (Rw)

average geometric

diameter

d (0.1) from cumulative mass fraction

d (0.9) from cumulative mass

fraction

E-OSA5-5% 120.8 241.6 45.07 · 2 78.57 · 2

NC-OSA-CHI-CARR-2.5% 143.8 287.6 53.76 · 2 91.97 · 2

NC-OSA-CHI-CARR-CHI-CARR-1.11% 125.9 251.8 59.45 · 2 91.76 · 2

NC-OSA-CHI-CARR-8.6% 146.2 292.4 67.65 · 2 166.0 · 2

NC-OSA-GEL-SBP-5.55% 91.3 182.6 86.31 · 2 94.4 · 2

NC-OSA-CHI-GA-3.7% 121.4 242.8 48.6 · 2 106.8 · 2

Figure 38 Elugrams from AF4-MALSD (90°), geometric radius of the particles (both left column) and the differential mass distribution (right column) of samples A: E-OSA5-5%, B: NC-OSA-CHI-CARR-2.5% (NC3), C:

NC-OSA-CHI-CARR-CHI-CARR-1.11% (NC5).

80 Results & discussion

Moreover, their statistical relevance is not proven because for all three samples the calculated size between 30 and 43 min elution time rises and decreases again. A superimposition of normal and steric elution in this region cannot be excluded definitely. As obtained from the cumulative mass fraction (see Table 12, p. 79), the particle radius (r(0.9) = ½ · d(0.9)) is smaller than 100 nm for 90 % of the particles in the 3 samples. A narrow size distribution of the radius between 40 and 100 nm is shown in the graphs of Figure 38 (right column). In conclusion, emulsion droplets of 240 nm and NCs of 250 to 290 nm (average geometric diameter, Table 12) were detected by AF4-MALS. The emulsion droplets (Figure 38A) were slightly smaller than the subsequently prepared NCs (Figure 38B, C). This result is appropriate to confirm the fact that emulsion, three- and five-layered NCs were prepared after each other. Figure 39 shows the AF4 results of the further NC formulations with highest possible oil content (NC-OSA-CHI-CARR-8.6%; NC-OSA-GEL-SBP-5.55%; NC-OSA-CHI-GA-3.7%).

Figure 39 Elugrams from AF4-MALSD (90°), geometric radius of the particles (both left column) and the differential mass distribution (right column) of samples A: NC-OSA-CHI-CARR-8.6%, B: NC-OSA-GEL-SBP-5.55%, C: NC-OSA-CHI-GA-3.7%.

Further investigation of the nanocapsule size 81

In contrast to the three samples of Figure 38, the elugrams of Figure 39 (left column) apparently differ from each other. While particles of sample A (NC-OSA-CHI-CARR-8.6%, composed similarly to the NCs in Figure 38B and C) eluted during the whole AF4 elution step (10 to 40 min, cf. Table 7, p. 42), the main fraction of samples B (NC-OSA-GEL-SBP-5.55%) and C (NC-OSA-CHI-GA-3.7%) eluted already between 10 and 20 min.

Since the cross flow decreased from minute 10 to 40 of the separation program, larger particles should elute later than smaller ones in case of normal elution behaviour. So if eluted normal, it can be concluded that particles of sample A might be larger in average than those of sample B and C. The geometric radius (calculated from the elution range of 9 to 43 min) mainly increased during the elution of the main fraction of sample A and C (range of 9 min to 35 min) which again indicates predominant normal (but not sterical) elution behaviour.

However, sample B showed steric elution (decreasing geometric radius) in the range of its main fraction (9 to 20 min). Only the particles eluting between 20 and 40 min showed normal elution behaviour (increasing geometric radius). Hence, the elution behaviour of the particles of sample B is hard to describe because a superimposition of normal and steric elution in the whole elution step (10 to 40 min) has to be considered. As the size distribution of these particles was determined as to be very narrow, an interaction of the separation membrane with the external polyelectrolyte (sugar beet pectin, SBP) is assumed to be the reason for the steric elution behaviour of sample B. As obtained from the cumulative mass fraction (not shown, see Table 12), the radius of 90 % of the particles (r(0.9) = ½ · d(0.9)) is smaller than 166 nm in sample A, but even smaller than 95 nm and 107 nm in sample B and C. This result confirms the conclusion drawn from the elugrams, that particles of sample A would be larger than of sample B and C. A narrow size distribution of the radius of sample B between 80 and 100 nm is shown in the differential mass fraction graph of Figure 39 (right column). The particle size of sample A, however, is widely distributed between 50 and 250 nm, the radius of sample C ranges from 40 to 150 nm. In conclusion, smallest (Rw 92 nm) and most homogeneous particles eluting partly sterically were detected by in sample B (NC-OSA-GEL-SBP-5.55%). Particles of sample C (NC-OSA-CHI-GA-3.7%; Rw

121 nm) and A (NC-OSA-CHI-CARR-8.6%; Rw 146 nm) were larger and less homogenous (Rw: from Table 12, p. 79).

The elugram of sample NC-OSA-GEL-GA-5.625% is not given because it could not reliably be evaluated due to a very poor data fit. This could be a result of the superimposition of both normal and steric elution. Another reason could be a wide size distribution. Additionally, the presence of gelatin should be considered as a possible influencing factor, especially because sample NC-OSA-GEL-SBP-5.55% (Figure 39B) also showed both steric and normal elution behaviour. Gelatin chains potentially interacted with or adhered to the membrane.