Propuesta de Reforma al artículo 9 del Reglamento de Etiquetado de

HPLC analysis of the sulphonated MPc samples was performed to evaluate the degree of sulphonation of the species present in the independently synthesised mixtures. The response exhibited by CuTSPc provided a standard to which the HPLC retention times reported for the remainder of the MPc preparations could be compared, as CuTSPc sourced and purified to homogeneity by the commercial supplier. Figure 3.1 illustrates examples of the HPLC retention times demonstrated by each MPc mixture. No retention time peak was recorded for the CoTCPc sample. Due to the negative charge carried by the analytical column, one assumes the initial peak recorded to be the more sulphonated species, followed by the second highest sulphonated species, and so on [101].

63 R e la ti ve In tens ity (AU)

AlPcSmix CoPcSmix

CuTSPc GePcSmix

ZnPcSmix

Retention Time (min)

Figure 3.1. HPLC trace for MPcs indicating the degree of sulphonation of the MPc species present in the MPc preparations. The MPc preparations analysed were AlPcSmix, CoPcSmix, CuTSPc, GePcSmix and ZnPcSmix. HPCL traces are illustrated for retention time 0 to 2.5 min. Peak retention times are summarised in Table 3.1.

As Figure 3.1 indicates, five peaks were recorded for AlPcSmix, two for GePcSmix and

ZnPcSmix, and one for the CoPcSmix, and CuTSPc. Table 3.1 summarises the average peak

retention times recorded for the peaks observed in Figure 3.1, and the subsequent conclusions drawn from the study. The single peak observed for the purchased CuTSPc was assumed to be indicative of a tetra-sulphonated species, therefore suggesting peaks possessing a retention

time of 1.10 min arise from the presence of a tetra-sulphonated species in MPcmix. A single

dominant peak was recorded when examining CoPcSmix, demonstrated a similar retention

time to that of CuTSPc. It was therefore concluded that the CoPcSmix contained tetra-

AlPcSmix Minutes 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.400 660 1.117 59699 1.400 114500 1.683 33774 2.117 33081 CoPcSmix Minutes 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.450 714 0.617 3310 1.117 279679 2.083 3757 CuTSPc Minutes 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 1.100 534543 GePcSmix Minutes 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 ZnPcSmix Minutes 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 1.133 65438 2.100 332194 A B C D B B B E B F E

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sulphonated species (Table 3.1). Similarly, tetra-sulphonated species were assumed to be present in the AlPcSmix, GePcSmix, and ZnPcSmix samples, (Figure 3.1 and Table 3.1).

HPLC analysis of AlPcSmix indicated the presence of five variants of the sulphonated MPc,

observed as five peaks (Figure 3.1). The initial peak (A), demonstrated a retention time substantially less than that of the predicted tetra-sulphonated species. This lower retention time suggests the presence of species which demonstrate a greater negative charge in comparison to that of the tetra-sulphonated species, and therefore a higher degree of sulphonation. From the assumed tetra-sulphonated AlPc retention time, the responses obtained for the remainder of the peaks, namely, C, D, and E, were utilised as an indication of the retention times expected for tri-, di-, and mono- sulphonated species, respectively. It was

therefore concluded that GePcSmix, contained both tetra- and mono-sulphonated species. The

HPLC response for ZnPcSmix, indicated a broad band demonstrating a high retention time (F).

The high retention time of the species suggests the presence of MPc aggregates, possibly consisting of low charged ZnPc molecules (i.e. mono-sulphonated). According to literature, the degree of aggregation is dependent on the lipophilicity of the MPc molecules, therefore suggesting an increase in particle aggregation as the level of sulphonation decreases. [101]_.

Table 3.1. HPLC Analysis of the Degree of Metallophthalocyanine Sulphonation

MPc Ave. Retention Time (min) Degree of Sulphonationa

Peak A Peak B Peak C Peak D Peak E Peak F

AlPcSmix 0.98 1.13 ± 0.01 1.29 1.38 ±0.02 1.68 Penta-; Tetra-; Tri-; Di-; Mono-

CoPcSmix 1.11 ± 0.01 Tetra-

CuTSPc 1.10 ±0.00 Tetra-

GePcSmix 1.12 ±0.01 1.74 ±0.11 Tetra-; Mono-

ZnPcSmix 1.13 ±01.00 2.12 ±0.03 Tetra-; Aggregates

a : Determined by retention time

Number of replicants was 3. Uncertainty was standard deviation from the mean.

In the case of standard deviation being omitted, peaks were not apparent in all three replicants

In conclusion, HPLC analysis indicated; AlPcSmix contained penta-, tetra, tri-, di-, and mono-

sulphonated species; both CoPcSmix and CuTSPc contained tetra-sulphonated species;

GePcSmix contained tetra- and mono-sulphonated species; and ZnPcSmix contained tetra-

65 3.3.1.2. Spectrophotometric Analysis of MPc Aggregation

Phthalocyanine absorption spectra are characterised by two absorption bands, namely, the

Soret/B-band (~350 nm), and the Q-band (~680 nm) [95, 99]_{. According to literature,}

aggregation of Pc molecules may be evaluated through observing the Q-band. Splitting or broadening of the latter band has been reported to correspond with the presence of particle aggregates [49, 51, 95, 102]. Therefore, the focus of the spectrophotometric analysis of MPc aggregation was on the Q-band.

Absorbance spectrophotometric analysis of the MPc preparations was performed to crudely assess particle aggregation in the chosen buffer utilised for MPc immobilisation analysis via

QCM-D, namely, PBSMg/Ca (pH 7.4). Comparisons were made between the absorbance

spectra (Q-band) of the MPc preparations in PBSMg/Ca buffer, with that of the spectra obtained

where the surfactant, Triton X-100, was utilised as a dispersing agent (Figure 3.2). Similar means of investigating MPc aggregation were reported by Ogunsipe and Nyokong (2005) [101]_.

Figure 3.2 illustrates the absorbance spectra obtained for the MPc preparations, both in PBSMg/Ca buffer, and when dispersed in PBSMg/Ca using 0.2% Triton X-100. Q-band splitting

was observed in all MPc preparations. In the case of MPcs dispersed in PBSMg/Ca,the Q-band

splitting may suggest one of two occurrences. Firstly, that aggregated species are present, and/or secondly, the presence of species bearing varying degrees of sulphonation, which would therefore suggest individual Q-bands were observed, in comparison to Q-band splitting [95]_{. The addition of the surfactant, Triton X-100, was expected to result in} monomerization of any MPc aggregates present in solution. This was predicted to be

66

MPc PBSMg/Ca PBSMg/Ca (0.2% Triton X-100)

AlPcSmix A b so rb an ce ( O p ti ca l Densi ty Un its) CoPcSmix CuTSPc GePcSmix ZnPcSmix CoTCPc Wavelength (nm)

Figure 3.2. Absorbance spectrophotometric analysis of MPc aggregation. Absorbance spectral

scans of MPc preparations in PBSMg/Ca buffer (pH 7.4) and PBSMg/Ca (0.2% Triton X-100). Dashed lines indicate the MPc monomer Q-band.

67

Similar to the findings in literature, the addition of Triton X-100 resulted in an increase in

peak intensity, when assessing AlPcSmix (Figure 3.2). The latter implies monomerisation of

the MPc aggregates. However, the factor responsible for the splitting of the Q-band was concluded to be the degree of sulphonation within the mixture rather than particle

aggregation [95]_{. Spectrophotometic analysis of AlPcS}

mix (Figure 3.2) corresponds with HPLC analysis (Table 3.1), which suggests the presence of mono-, di-, tri-, tetra-, and penta- sulphonated MPc species, as MPc molecules demonstrating lower levels of sulphonation have been suggested to aggregate in aqueous solution [95, 101].

The addition of Triton X-100 to both the CoPcSmix, CuTSPc and CoTCPc preparations

resulted in a decrease in peak intensity. According to literature, a decrease in Q-band intensity is indicative of photodegradation of the MPc molecule as a result of exposure to

light [101]_{. The proposed photodegradation of CoTCPc, resulted in a decrease in absorbance}

levels, which generated the Q-band almost unidentifiable. The presence of surfactant resulted in slight peak sharpening when assessing GePcSmix preparations, therefore suggesting that majority of the MPc species were in monomer form while in aqueous solution, with a small portion are present in aggregate form. These results correspond with the HPLC analysis of the MPc preparation, which suggested high levels of tetra-sulphonated species and low levels of mono-sulphonated species. The inclusion of Triton X-100 to the ZnPcSmix preparation resulted in a considerable change in peak intensities, suggesting a large portion of the MPc molecules are in aggregated form when in aqueous solution, as suggested by HPLC analysis indicating possible aggregation (Table 3.1).

3.3.2. Surface Modification with MPcs

According to literature, MPc molecules may adopt two conformations when immobilised to a target surface, namely, edge-on and/or planar conformations [49]. For the purpose of this research, immobilisation of both sulphonated and carboxylated MPcs were carried out using the amine terminating SAM, 11-AUT. A positively charged SAM surface was utilised as it is predicted to promote MPc immobilisation through electrostatic interactions with the sulfonic acid groups present on sulphonated MPc molecules (Figure 1.6), and form a basis for covalent bonding with the carboxylic acid group of CoTCPc molecules. 11-AUT SAM

68

modification was favoured over Cyst. modification, due to the longer chain length, which has

been reported to aid in the ordering of the self assembly process [84-86]_{. Figure 3.3 provides a}

schematic illustration of the anticipated orientations of MPc molecules immobilised on an 11-AUT SAM.

Figure 3.3. Schematic representation of immobilised metallophthalocyanine orientation. Planar and edge-on MPc conformations are illustrated by I and II, respectively.