Analysis of thermal degradation of mixed dye solution
HPLC-DAD-MS analysis was initially conducted on the untreated dye solution to find the retention time of the chromatogram peak, UV-visible absorption spectrum (λmax), and major molecular ion of each coloring compound. The retention times of the peaks of berberine (1), palmatine (2), alizarin (3), purpurin (4), indigotin (5), and indirubin (6) in the chromatogram of the mixed dye solution were 6.2 ~ 6.9 min, 6.4 ~ 6.9 min, 8.8 ~ 9.0 min, 10.2 min, 9.8 ~ 10.2 min, and 10.7 ~ 11.4 min, respectively (Figure 1) (Ahn et al., 2014). The chromatogram peak of indigotin and purpurin overlapped and the λmax of both compounds appeared in the UV absorption band of each compound concomitantly (Figure 2). However the two compounds were distinguishable using the differences in major molecular ion typical of each compound which were m/z 263 for indigotin and m/z 257 for purpurin (Ahn et al., 2013; Ahn et al., 2014; Ren et al., 2007). The chromatogram peaks of berberine and palmatine also overlapped and their UV absorption bands were identical due to the fact that berberine and palmatine were both protoberberine alkaloids (Ahn et al., 2013; Ahn et al., 2014; Zhang et al., 2009). But the two compounds were distinguishable by the major molecular ions [M + H2]+ of each coloring compound which are m/z 338 and m/z 354 respectively (Ahn et al., 2014; Ren et al., 2007). The details of the λmax of the UV-visible absorption bands and the major ion detected for each coloring compound follow the analytical results and discussion on the standard dyes reported previously (Ahn et al., 2013; Ahn et al., 2014).
The dye solution was thermally degraded in 100°C oven for 1 ~ 9 days and the degraded samples were analyzed using the HPLC-DAD-MS instrument. When the HPLC chromatograms of the degraded samples were examined, there was a gradual lowering of the peaks of the six coloring compounds as treatment time progressed (Figure 3). The lowering of the peaks suggested decrease in relative abundance of each coloring compound, and visually, such decrease was most prominent in purpurin (4) and indigotin (5) peaks (Figure 3).
In order to examine the change in relative concentration of six coloring compounds in the mixed dye solution by thermal degradation, ion chromatogram was generated from the mass spectrum of each ion m/z 338 (berberine), m/z 354 (palmatine), m/z 241 (alizarin), m/z 257 (purpurin), m/z 263 (indigotin), and m/z 263 (indirubin). The abundance of ion chromatogram was used as the measure of concentration of corresponding coloring compound detected in the HPLC-MS. Based on the abundance of the compound in untreated solution, the relative concentration of each coloring compound in the degraded dye solution was calculated in percentage, and the results were plotted in an Excel graph (Figure 4).
The result indicated that except for alizarin, the concentrations of all five dyes were close to zero % by 6 days of thermal treatment, meaning that most intact dye molecules were lost after 6 days of treatment (Figure 4). The decrease in concentration was more prominent in indigotin and purpurin. Purpurin in particular, lost most of its dye molecule by 2 days of thermal treatment. On the other hand, alizarin showed the least decrease in concentration and when the treatment was terminated, more than 30% of alizarin molecule survived in the dye solution. Degradation behaviors of berberine and palmatine were almost identical, and the two dyes were more resistant to degradation than indirubin, indigotin, and purpurin and less resistant than alizarin.
Analysis of silk dyed with mixed dye solution after thermal degradation
Two pieces of silk sample were dyed with berberine, palmatine, alizarin, purpurin, and indigotin consecutively, one treated with alum mordanting before each dyeing procedure and the other treated with iron sulfate mordanting before each dyeing procedure. After degrading the silk dyeings in 100°C oven up to 7 days, dye was extracted from each silk and the extracts were analyzed using the HPLC-DAD-MS analysis.
Figure 5 illustrates the HPLC chromatogram of the dye extracted from alum mordanted silk before and after the thermal degradation treatment. There was a noticeable change in the height of the peaks in HPLC chromatograms as the treatment progressed. After 7 days of thermal treatment the height of the peaks for all five dyes lowered. And such visual decrease was more dramatic in indigotin and purpurin since the peaks of these compounds almost disappeared after 7 days of thermal treatment.
Figure 6 illustrates the HPLC chromatogram of the dye extracted from iron mordanted silk before and after the thermal degradation treatment. Compare to the alum mordanted silk, there was more prominent lowering of the peaks of the five coloring compounds in iron mordanted silk. The HPLC chromatogram of alum (Figure 5) and iron (Figure 6) mordanted silk suggest that the concentration of the five dyes decreased in the silk dyeings after the thermal degradation treatment.
Figure 7 and Figure 8 illustrate the change in the relative concentration of five dyes in alum mordanted silk and iron mordanted silk, respectively. Both alum mordanted silk dyeing and iron mordanted silk dyeing showed decrease in the relative concentration of five dyes, and as expected from the HPLC chromatograms, there was a higher amount of decrease in iron mordanted silk sample. In both alum mordanted silk and iron mordanted silk, alizarin and purpurin showed lower amount of decrease compared to the other dyes. And indigotin showed a dramatic decrease during the 7 days of thermal treatment. And furthermore, the results indicated that in case of alizarin, iron mordanted silk showed a slightly lower decrease in dye concentration than the alum mordanted silk. This suggests that alizarin may be more resistant to degradation when the silk is mordanted with iron type mordant.
Effect of thermal degradation on the color values of silk dyed with individual dye
Color differences of alum mordanted silk and iron mordanted silk dyed with individual dye solution, after 0 ~ 14 days of thermal degradation treatment are illustrated in Figure 9 and Figure 10, respectively. In both alum mordanted silk and iron mordanted silk, the color different of the degraded sample relative to the untreated sample was most prominent in berberine and palmatine dyed silk and the least color difference was observed in alizarin and purpurin dyed silk. Such results were in agreement with the change in dye concentration analyzed by the HPLC-DAD-MS. However, in case of indigotin, there was a discrepancy between the two data. The color difference values indicated that indigotin dyed silk showed higher color difference in alum mordanted silk than in iron mordanted silk, whereas it was opposite when the change of dye concentration was examined using the HPLC analysis.
Alizarin and purpurin were more resistance to degradation than other coloring compounds both when in solution form and in silk dyeings. And such result was verified by the color difference measurement of the degraded silk dyed with individual dye solution. The resistance of alizarin and purpurin toward degradation treatment in silk dyeing was due to their dyeing mechanism. Being mordant dyes, alizarin and purpurin forms fiber-metal-dye chelated complex within the fiber by the metal compounds used in mordanting (Ahn et al., 2014; Khan et al., 2012; Samanta & Konar, 2011). Thus the aluminium and iron which were used in alum mordanting and iron mordanting, respectively, allow strong dye fixation by forming chelated structure (Ahn et al., 2014). Furthermore, alizarin and purpurin dye molecules will also form aggregates by the dye-metal-dye chelation (Ahn et al., 2014). And larger aggregates will allow the dye to be more resistant to degradation since the thermal oxidation will initially affect the peripheral structures of the dye molecules, possibly leaving the larger portion of dye to be unaffected (Ahn et al., 2014; Crews, 1987; Giles & Mckay, 1963).
Berberine and palmatine showed a larger degree of color difference in silk dyeings relative to the untreated samples. They are affixed to silk fibers by the ionic bond which forms between the cationic nitrogen in the dye molecule and the anionic sites of acidic amino acids in silk protein (Ahn et al., 2014). However, the size of berberine and palmatine dye particles within the fiber would be smaller since their structure do not enable the dye to form hydrogen bonding which allow the formation of larger aggregates (Ahn et al., 2014). Therefore, berberine and palmatine would be more susceptible to oxidative degradation by high temperature since larger surface area is exposed by the smaller particle size of dye molecules.
Indigotin is known to be one of the most fast natural dye since dyeing with indigo involves vat dyeing process which forms insoluble indigotin dye molecules within the fiber (Crews, 1987; Padfield & Landi, 1996). However, in a recent study on the degradation of indigotin in solution it was found that indigotin was highly susceptible to degradation by H2O2 treatment with UV radiation forming isatin as main degradation product (Ahn et al., 2014). Similar oxidative degradation would have occurred by the thermal treatment of this study. It is interesting that indigotin in silk dyeing also showed a high degree of degradation by thermal treatment when the dye is known to be one of the most fast natural dye. Although indigo dye forms insoluble indigotin molecules within the fiber, the dye molecules do not have any chemical bonding sites with fiber nor it has possible sites for forming aggregates within themselves (Ahn et al., 2014). Therefore small indigotin particles are merely trapped in the amorphous area of the fiber (Ahn et al., 2014) which enable easy attack from external oxidative degradation forces.