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Title:
A METHOD OF OXIDISING ORGANIC MOLECULES
Document Type and Number:
WIPO Patent Application WO/2019/083453
Kind Code:
A1
Abstract:
This invention relates to a method of oxidising organic molecules, comprising adding an oxidiser and an oxidising catalyst for activating the oxidiser, wherein the oxidising catalyst comprises an oligopeptide ligand complexed with a copper ion. In a preferred embodiment, the oligopeptide ligand comprises a tripeptide containing amino acids of glycine and histidine. The method of the invention may be applicable to many industrial applications such as wastewater treatment, decontamination of polluted sites, bleaching applications, and in the functioning of algaecides.

Inventors:
YANG KUN-LIN (SG)
HO WING-FAT (SG)
NGUYEN LE-TRUC (AU)
Application Number:
PCT/SG2018/050532
Publication Date:
May 02, 2019
Filing Date:
October 24, 2018
Export Citation:
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Assignee:
NAT UNIV SINGAPORE (SG)
International Classes:
B01J31/18; B01J23/72
Foreign References:
US8445431B22013-05-21
JP2009131250A2009-06-18
Other References:
MUSIE, G. ET AL.: "Alcohol oxidation catalyzed by manganese(II), nickel (II) and copper(II) complexes", BOOK OF ABSTRACTS, 216TH ACS NATIONAL MEETING, 23 August 1998 (1998-08-23), Boston
UEDA, J.-I. ET AL.: "Oxidation of linoleic acid by copper(II) complexes: effects of ligand", JOURNAL OF INORGANIC BIOCHEMISTRY, vol. 76, no. 1, 11 October 1999 (1999-10-11), pages 55 - 62, XP027300205
CHATTOPADHYAY, T. ET AL.: "Copper(II)-coordinated organic nanotube: A novel heterogeneous catalyst for various oxidation reactions", CATALYST COMMUNICATIONS, vol. 12, no. 1, 6 August 2010 (2010-08-06), pages 9 - 13, XP027305355
POGNI, R. ET AL.: "EPR and O2- scavenger activity: Cu(II)-peptide complexes and superoxide dismutase models", JOURNAL OF INORGANIC BIOCHEMISTRY, vol. 73, 1999, pages 157 - 165, XP055597571
BI, X. ET AL.: "Complexation of Copper Ions with Histidine-Containing Tripeptides Immobilized on Solid Surfaces", LANGMUIR, vol. 23, no. 22, 29 September 2007 (2007-09-29), pages 11067 - 11073, XP055597574
Attorney, Agent or Firm:
PATEL, Upasana (SG)
Download PDF:
Claims:
Claims

1. A method of oxidising organic molecules, the method comprising adding an oxidiser and an oxidising catalyst for activating the oxidiser, wherein the oxidising catalyst comprises an oligopeptide ligand complexed with a copper ion.

2. The method according to claim 1 , wherein the oxidiser is hydrogen peroxide (H202), organic peroxide, organic hydroperoxide, peroxy acid, ozone, hypochlorite, or a combination thereof.

3. The method according to claim 1 or 2, wherein the oligopeptide ligand is complexed with the copper ion through at least two peptide bonds.

4. The method according to any preceding claim, wherein the oligopeptide ligand comprises a tripeptide having a general formula (I):

wherein each of R2 and R3 may be the same or different amino acid.

5. The method according to any preceding claim, wherein the oxidising catalyst has a general formula II):

wherein each of R^ R2 and R3 may be the same or different amino acid.

6. The method according to claim 4 or 5, wherein:

R<i is H or histidine;

R2 is H; and

R3 is H or histidine,

and wherein and R3 are not both histidine.

7. The method according to claim 6, wherein each of R2 and R3 is H, such that the oxidising catalyst is Cu-GGG.

8. The method according to claim 6, wherein is histidine and each of R2 and R3 is H, such that the oxidising catalyst is Cu-HGG.

9. The method according to claim 6, wherein each of and R2 is H and R3 is histidine, such that the oxidising catalyst is Cu-GGH.

10. The method according to any preceding claim, wherein the oxidising catalyst is: encapsulated in a hydrogel bead; or immobilised on a solid substrate.

1 1. The method according to any preceding claim, wherein the method is performed at a temperature of 20-100°C.

12. The method according to any preceding claim, wherein the method is performed at a pH of 3-12.

13. An oxidising catalyst comprising an oligopeptide ligand complexed with a copper ion, wherein the oligopeptide ligand is complexed with the copper ion through at least two peptide bonds.

14. The oxidising catalyst according to claim 13, wherein the oligopeptide ligand comprises a tripeptide having a general formula (I):

wherein each of R2 and R3 may be the same or different amino acid.

15. The oxidising catalyst according to claim 13 or 14, wherein the oxidising catalyst has a general formula I I):

wherein each of R^ R2 and R3 may be the same or different amino acid.

16. The oxidising catalyst according to any of claims 13 to 15, wherein:

Ri is H or histidine;

R2 is H; and

R3 is H or histidine,

and wherein R^ and R3 are not both histidine.

17. The oxidising catalyst according to claim 16, wherein each of R^ R2 and R3 is H, such that the oxidising catalyst is Cu-GGG.

18. The oxidising catalyst according to claim 16, wherein R^ is histidine and each of R2 and R3 is H, such that the oxidising catalyst is Cu-HGG.

19. The oxidising catalyst according to claim 16, wherein each of R^ and R2 is H and R3 is histidine, such that the oxidising catalyst is Cu-GGH.

20. The oxidising catalyst according to any of claims 13 to 19, wherein the oxidising catalyst is: encapsulated in a hydrogel bead; or immobilised on a solid substrate.

Description:
A method of oxidising organic molecules

Technical Field

The present invention relates to a method of oxidising organic molecules and an oxidising catalyst.

Background

Hydrogen peroxide is an environmental friendly oxidizer because it only releases oxygen and water when it decomposes. However, oxidation reactions involving hydrogen peroxide as an oxidizer are too slow when the temperature is below 50°C. To accelerate the reaction, oxidation reactions need to be carried out at higher temperature. To aid in the oxidation process, peroxidases, which are a family of enzymes that catalyze the oxidation of organic molecules by using hydrogen peroxide as an oxidizer, are sometimes utilised. However, peroxidases are sensitive to temperature and pH and that limits their industrial applications.

Therefore, artificial peroxidases have been manufactured. These artificial peroxidases have similar or even higher activities compared to natural peroxidases, but they are far- more stable and robust, making them suitable for industrial applications. An example of an artificial peroxidases is iron-tetraamidomacrocyclic complex (Fe-TAML). Fe-TAML has been used to catalyze oxidation of azo dyes and chlorophenols with an impressive reaction rate at room temperature. However, synthesis of Fe-TAML is lengthy and very expensive. Moreover, Fe-TAML is highly soluble in water, making the recycle and reuse of Fe-TAML very difficult. Another issue is that since Fe-TAML is a synthetic molecule, residual Fe-TAML left in the product is toxic and non-biodegradable.

There is therefore a need for an improved method of oxidising organic molecules and an improved oxidising catalyst.

Summary of the invention

The present invention seeks to address these problems, and/or to provide an improved method for oxidising organic molecules and an improved oxidising catalyst.

In general terms, the invention relates to a green and cost-effective method of oxidising organic molecules. This is because the oxidising catalyst used in the method of the present invention activates hydrogen peroxide and accelerates oxidation reactions under various conditions. In particular, an oxidation reaction involving hydrogen peroxide as the oxidising agent may be conducted at a lower temperature and over a wide range of pH. Since the hydrogen peroxide can be activated at a lower temperature, less energy will be utilised in heating the reaction mixture, thereby being more environmentally friendly and cost effective. Further, the oxidising catalyst is formed from an oligopeptide. In this way, the catalyst will be non-toxic and generally biodegradable, making the method of the present invention less hazardous.

According to a first aspect, the present invention provides a method of oxidising organic molecules, the method comprising adding an oxidiser and an oxidising catalyst for activating the oxidiser, wherein the oxidising catalyst comprises an oligopeptide ligand complexed with a copper ion.

The oxidiser may be any suitable oxidiser. For example, the oxidiser may be hydrogen peroxide (H 2 0 2 ), organic peroxide, organic hydroperoxide, peroxy acid, ozone, hypochlorite, or a combination thereof.

According to a particular aspect, the oligopeptide ligand may be complexed with the copper ion through at least two peptide bonds. In particular, the oligopeptide ligand may comprise a tripeptide having a general formula (I):

wherein each of R 2 and R 3 may be the same or different amino acid. According to a particular aspect, the oxidising catalyst may have a general form wherein each of R 2 and R 3 may be the same or different amino acid. In particular, may be H or histidine; R 2 may be H; and R 3 may be H or histidine, and wherein and R 3 are not both histidine.

Even more in particular: (i) each of R^ R 2 and R 3 may be H, such that the oxidising catalyst is Cu-GGG; (ii) Ri may be histidine and each of R 2 and R 3 may be H, such that the oxidising catalyst is Cu-HGG; or (iii) each of and R 2 may be H and R 3 may be histidine, such that the oxidising catalyst is Cu-GGH.

The oxidising catalyst may be: encapsulated in a hydrogel bead; or immobilised on a solid substrate.

The method may be performed under suitable conditions. For example, the oxidising may be at a suitable temperature. According to a particular aspect, the temperature at which the method is carried out may be 20-100°C. The oxidising may be at a suitable pH. According to a particular aspect, the method may be performed at a pH of 3-12.

According to a second aspect, the present invention also provides an oxidising catalyst comprising an oligopeptide ligand complexed with a copper ion, wherein the oligopeptide ligand is complexed with the copper ion through at least two peptide bonds. The catalyst may comprise any suitable oligopeptide ligand. For example, the oligopeptide ligand may be as described above.

According to a particular aspect, the oxidising catalyst may have a general formula (II):

wherein each of R 2 and R 3 may be the same or different amino acid. In particular, may be H or histidine; R 2 may be H; and R 3 may be H or histidine, and wherein and R 3 are not both histidine.

Even more in particular: (i) each of R^ R 2 and R 3 may be H, such that the oxidising catalyst is Cu-GGG; (ii) Ri may be histidine and each of R 2 and R 3 may be H, such that the oxidising catalyst is Cu-HGG; or (iii) each of and R 2 may be H and R 3 may be histidine, such that the oxidising catalyst is Cu-GGH.

The oxidising catalyst may be: encapsulated in a hydrogel bead; or immobilised on a solid substrate.

Brief Description of the Drawings

In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments, the description being with reference to the accompanying illustrative drawings. In the drawings: Figure 1 shows time-course degradation of trypan blue when oxidised by H 2 0 2 and an oxidising catalyst according to one embodiment of the present invention;

Figure 2 shows the comparison of H 2 0 2 oxidation efficiency in the absence and presence of different oxidising catalysts according to embodiments of the present invention;

Figure 3 shows the effect of varying the concentration of H 2 0 2 on the degradation of trypan blue;

Figure 4 shows the effect of varying the concentration of an oxidising catalyst according to one embodiment of the present invention;

Figure 5 shows the effect of pH on the degradation of trypan blue by H 2 0 2 in the presence of an oxidising catalyst according to one embodiment of the present invention; and

Figure 6 shows the bleaching of chlorophyll by H 2 0 2 in the presence and absence of an oxidising catalyst according to one embodiment of the present invention.

Detailed Description

As explained above, there is a need for an improved method of oxidising organic molecules, as well as for an improved oxidising catalyst.

The method of the present invention enables oxidation of organic molecules to be performed over a wider range of conditions and without being too energy-intensive. For example, the oxidation may be carried out at a lower temperature and over a wide range of pH. This is as a result of the oxidising catalyst used in the oxidation method. The oxidising catalyst comprises an oligopeptide ligand complexed with copper ions. The oxidising catalyst of the present invention is also highly stable, may be easily prepared and has a high catalytic activity.

In particular, the oligopeptide ligand may be customised using different amino acids and peptides. Since peptides are natural molecules and can be obtained from natural sources, they are generally non-toxic and biodegradable. This makes the oxidising catalyst environmentally friendly and easy to produce. According to a first aspect, the present invention provides a method of oxidising organic molecules, the method comprising adding an oxidiser and an oxidising catalyst for activating the oxidiser, wherein the oxidising catalyst comprises an oligopeptide ligand complexed with a copper ion.

The organic molecules may be any suitable organic molecules which are required to be oxidised. For example, the organic molecules may be phenolic compounds, chlorophyll, bacteria cells and spores and/or organic molecules found in wastewater, dyes, and the like.

The oxidiser may be any suitable oxidiser. For example, the oxidiser may be, but not limited to: hydrogen peroxide (H 2 0 2 ); organic peroxide, such as but not limited to benzoyl peroxide; organic hydroperoxide; peroxy acid; ozone; hypochlorite; or a combination thereof.

The oxidising catalyst added in the method of the present invention may be any suitable oxidising catalyst. In particular, the oxidising catalyst may be a peroxidase- mimicking enzyme. Even more in particular, the oxidising catalyst may be an artificial copper enzyme in which an oligopeptide ligand may be complexed with at least one copper ion. According to a particular aspect, the oligopeptide ligand may be complexed with the copper ion through at least two peptide bonds.

The oligopeptide ligand may be any suitable oligopeptide. For example, the oligopeptide ligand may comprise different amino acids. In particular, the oligopeptide ligand may comprise a tripeptide. According to a particular aspect, the oligopeptide ligand may comprise a tripeptide having a general formula (I):

wherein each of R 2 and R 3 may be the same or different amino acid.

In particular, by replacing different amino acids in the tripeptide, different copper- tripeptide complex oxidising catalyst may be formed. According to a particular aspect,

F may be H or histidine;

R 2 may be H; and

R 3 may be H or histidine, and wherein R^ and R 3 are not both histidine.

In particular: (i) each of R^ R 2 and R 3 may be H, such that the oligopeptide ligand is triglycine (GGG); (ii) R^ may be histidine and each of R 2 and R 3 may be H, such that the oligopeptide ligand is HGG; or (iii) each of R^ and R 2 may be H and R 3 may be histidine, such that the oligopeptide ligand is GGH, wherein G represents glycine and H represents histidine.

According to a particular aspect, the oxidising catalyst may have a general formula (II):

wherein each of R^ R 2 and R 3 may be the same or different amino acid.

In particular, R^ R 2 and R 3 may be as described above. Even more in particular: (i) each of R^ R 2 and R 3 may be H, such that the oxidising catalyst is Cu-GGG; (ii) Ri may be histidine and each of R 2 and R 3 may be H, such that the oxidising catalyst is Cu- HGG; or (iii) each of R^ and R 2 may be H and R 3 may be histidine, such that the oxidising catalyst is Cu-GGH, wherein G represents glycine and H represents histidine.

The oxidising catalyst may be in any suitable form. For example, the oxidising catalyst may be unencapsulated, encapsulated or immobilised on a solid substrate. According to a particular aspect, the oxidising catalyst may be encapsulated. The oxidising catalyst may be encapsulated in any suitable manner. For example, the oxidising catalyst may be encapsulated in a hydrogel bead, membrane, film or fiber. According to another particular aspect, the oxidising catalyst may be immobilised on a solid substrate. Examples of solid substrates include, but is not limited to, membrane, film, fiber. When the oxidising catalyst is encapsulated or immobilised, the oxidising catalyst may be recovered more easily following its use in the method. In this way, the oxidising catalyst may be reused in subsequent oxidising methods.

The method of the present invention may be performed under suitable conditions. The conditions may include the temperature at which the oxidising is carried out. For example, the temperature at which the method is carried out may be 20-100°C. In particular, the temperature may be 30-90°C, 40-80°C, 50-70°C, 55-60°C. Even more in particular, the temperature may be≤60°C, preferably about 50°C. It would be clear to a person skilled in the art that by adding the oxidising catalyst in the method of the present invention, the temperature at which oxidising may take place in the presence of an oxidiser such as hydrogen peroxide is lower. For example, when the oxidiser is H 2 0 2 , in the absence of the oxidising catalyst of the present invention, the H 2 0 2 is generally activated only at temperatures ≥ 90°C. However, in the presence of the oxidising catalyst of the present invention, the temperature at which the H 2 0 2 may be activated is lowered to about 50°C.

The conditions may include the pH at which the oxidising is carried out. When the oxidiser is H 2 0 2 , in the absence of the oxidising catalyst of the present invention, the H 2 0 2 is generally activated only at specific and narrower pH ranges. For example, when the oxidising is by a Fe(ll)-catalysed Fenton reaction, the oxidising takes place at a pH of 2.6-3.4. When the oxidising is by using iron-tetraamidomacrocyclic complex (Fe-TAML) as the oxidising catalyst, the oxidising takes place at neutral pH or in alkaline conditions. In contrast, the pH at which the method of the present invention may be carried out may be 3-12. In particular, the pH may be 7-12, 8-11 , 9-10. Even more in particular, the pH may be 1 1. It would be clear to a person skilled in the art that by adding the oxidising catalyst in the method of the present invention, the pH range at which oxidising may take place in the presence of an oxidiser such as hydrogen peroxide is much wider.

The method of the present invention may be applicable to many industrial applications such as wastewater treatment, decontamination of polluted sites, bleaching applications including fabric bleaching, food bleaching and pharmaceutical product bleaching, and in the functioning of algaecides.

The present invention also provides an oxidising catalyst. According to a second aspect, there is provided an oxidising catalyst comprising an oligopeptide ligand complexed with a copper ion, wherein the oligopeptide ligand is complexed with the copper ion through at least two peptide bonds.

The catalyst may comprise any suitable oligopeptide ligand. For example, the oligopeptide ligand may be as described above.

According to a particular aspect, the oxidising catalyst may have a general formula (II):

wherein each of R 2 and R 3 may be the same or different amino acid.

In particular, R 2 and R 3 may be as described above in the first aspect of the present invention. Even more in particular: (i) each of R^ R 2 and R 3 may be H, such that the oxidising catalyst is Cu-GGG; (ii) Ri may be histidine and each of R 2 and R 3 may be H, such that the oxidising catalyst is Cu-HGG; or (iii) each of and R 2 may be H and R 3 may be histidine, such that the oxidising catalyst is Cu-GGH, wherein G represents glycine and H represents histidine.

The oxidising catalyst may be in any suitable form. For example, the oxidising catalyst may be unencapsulated, encapsulated or immobilised on a solid substrate. According to a particular aspect, the oxidising catalyst may be encapsulated. The oxidising catalyst may be encapsulated in any suitable manner. For example, the oxidising catalyst may be encapsulated in a hydrogel bead, membrane, film or fiber. According to another particular aspect, the oxidising catalyst may be immobilised on a solid substrate. Examples of solid substrates include, but is not limited to, membrane, film or fiber. When the oxidising catalyst is encapsulated or immobilised, the oxidising catalyst may be recovered more easily following its use in the method. In this way, the oxidising catalyst may be reused in subsequent oxidising methods.

The oxidising catalyst of the present invention may be prepared by any suitable method. For example, the general scheme by which the oxidising catalyst may be prepared is as follows:

Tripeptides Copper ions Cuzymes

Having now generally described the invention, the same will be more readily understood through reference to the following embodiment which is provided by way of illustration, and is not intended to be limiting.

Example

Materials

Copper acetate monohydrate, NH 2 -Gly-Gly-Glycine-COOH (GGG) and sodium hydroxide were purchased from Sigma-Aldrich (Singapore). Trypan blue solution (0.4%) was purchased from Fisher (Singapore). (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) (HEPES) buffer (1 M, pH 7.5) was purchased from 1 st BASE (Singapore). NH 2 -Histadyl-Gly-Glycine-COOH (HGG) and NH 2 -Gly-Gly-Histidine- COOH (GGH) were custom-synthesized by BACHEM (Switzerland) with a minimum purity of 98%. Wheatgrass was purchased from a local market. All materials were used as received without further purification

Formation of copper-tripeptide oxidising catalyst Tripeptide solutions (100 mM) were prepared by dissolving tripeptide powders in HEPES buffer (10 mM, pH 8.0). The buffer (10 mM, pH 8.0) was prepared by diluting HEPES buffer stock (1 M, pH 7.5) with deionized water. Sodium hydroxide (1 M solution) was used to adjust pH to 8.0. Three types of tripeptide solutions (GGG, HGG and GGH) were prepared. Copper acetate solution (100 mM) was prepared by dissolving copper acetate monohydrate in deionized water. The copper acetate solution was centrifuged at 10,000 rpm for 3 min to remove all insoluble particles. Then, copper- tripeptides artificial enzymes (Cuzymes) were prepared by mixing tripeptide solution (100 mM, 100μΙ_) with copper acetate (100 mM, 100 μΙ_) and HEPES buffer (10 mM, pH 8.0, 800 μΙ_). Oxidising catalysts Cuz-1 , Cuz-2 and Cuz-3 were prepared by using GGG, HGG, and GGH, respectively. The solutions were vortexed before use.

Degrading of trypan blue and chlorophyll by oxidising catalyst

Degradation of trypan blue in the presence of the oxidising catalysts (50 ppm) was carried out in 4 mL of aqueous solution containing 200 μΜ of trypan blue and 0.5% of H 2 0 2 . The degradation of trypan blue was carried out at 50°C for 30 min with a stirring speed of 300 rpm. Absorbance of the solution at 595 nm was determined. Degradation percentage (%) of trypan blue was defined as:

(A - A

Degradation (%) = where A 0 and A, are the absorbance values of the reaction mixture at 0 minutes and at different time intervals, respectively.

A chlorophyll solution was prepared by grinding 250 g of wheatgrass in 500 mL of deionized water. The solution was filtered by a piece of 0.45-μηι membrane before use. The final concentration of chlorophyll in the solution was 50 mg/mL. Bleaching of chlorophyll was conducted by using Cuz-1 as the oxidising agent. Briefly, 4 mL of the chlorophyll solution was mixed with Cuz-1 (50 ppm), H 2 0 2 (0.5%) and NaOH (2 g/L). The mixture was stirred at a speed of 300 rpm for 60 minutes.

Results and discussion

(i) Degradation of trypan blue Trypan blue was selected as the substrate and H 2 0 2 was used as the oxidizer, while Cuz-1 was used as the oxidising catalyst. The oxidising catalyst was added at 0 minutes, and the solution was stirred at 50°C for 30 minutes by way of a magnetic stirrer.

Absorbance of the solution at 595 nm was also measured to estimate the degradation percentage of trypan blue. As shown in Figure 1 , the colour of the trypan blue solution became lighter within 5 minutes, indicating that trypan blue was oxidized by H 2 0 2 in the presence of Cuz-1. After 30 minutes, the solution became colourless. Based on the absorbance value at 595 nm, about 98% of trypan blue was degraded in 30 minutes. In contrast, when no Cuz-1 was added, the trypan blue solution remained blue after 30 minutes. This shows that trypan blue cannot be oxidized by H 2 0 2 alone. In this case, Cuz-1 plays an important role in the degradation of trypan blue. It exhibits peroxidase- like activity and is able to activate H 2 0 2 for degradation of trypan blue.

(ii) Specificity of oxidising catalysts

To understand the effect of different oligopeptide ligands on the catalytic activities of the oxidising catalysts, the three different oxidising catalysts prepared (Cuz-1 , Cuz-2 and Cuz-3) were used as catalysts for degrading trypan blue at 50°C and using H 2 0 2 as the oxidiser. Both Cuz-1 and Cuz-2 were able to catalyse the oxidation of trypan blue effectively. In contrast, when Cuz-3 was used, it only led to partial degradation of trypan blue after 30 minutes. By using absorbance value at 595 nm, the degradation percentage of trypan blue was determined to be 98%, 83% and 42% in the presence of Cuz-1 , Cuz-2 and Cuz-3, respectively (Figure 2). These results indicate that when the amino acid at the COOH terminus of the tripeptide is histidine, the oxidising catalyst is less effective in activating H 2 0 2 . This may be because the GGH ligand forms a very stable tetradentate structure with copper ions. Accordingly, no vacant site is available at the copper ion to active H 2 0 2 . Because Cuz-1 exhibited the highest activity for degradation of trypan blue, it was used for subsequent experiments.

(iii) Effect of H 2 0 2 concentration

To understand the effect of H 2 0 2 concentration on the degradation of trypan blue, different amounts of H 2 0 2 (0 - 0.5%) were added to the reaction mixture comprising 50 ppm of Cuz-1 as the oxidising catalyst. The temperature of the reaction mixture was 50°C. Figure 3 shows that in the absence of H 2 0 2 , there was no degradation of trypan blue. This result clearly indicates the importance of H 2 0 2 in the reaction. By increasing the concentration of H 2 0 2 from 0.05% to 0.2%, the degradation of trypan blue increased proportionally. When the concentration of H 2 0 2 was 0.2%, all trypan blue was degraded in 20-25 minutes. Any higher H 2 0 2 concentration (up to 0.4%) did lead to a faster reaction rate in the degradation of trypan blue. In these cases, the reaction rate was limited by the trypan blue concentration, not by H 2 0 2 . Thus, the concentration of H 2 0 2 was fixed at 0.2% in subsequent experiments.

(iv) Effect of oxidising catalyst concentration

The concentration of oxidising catalyst Cuz-1 was varied from 0 to 100 ppm in the reaction mixture which was at a temperature of 50°C. The effect of the oxidising catalyst concentration on the degradation of trypan blue was presented in Figure 4. As expected, in the absence of Cuz-1 , H 2 0 2 alone could not lead to the degradation of trypan blue in 30 minutes. However, when the Cuz-1 concentration was 5 ppm, the reaction accelerated considerably. After 30 minutes of reaction, approximately 70% of trypan blue was degraded. When the concentration of Cuz-1 was further increased to 50 ppm, the reaction rate increased further. After 20-25 minutes, almost all the trypan blue was degraded. However, when the concentration of Cuz-1 was further increased to 100 ppm, no increase in the reaction rate was observed. Therefore, the optimal concentration of Cuz-1 was taken as 50 ppm.

(v) Effect of pH

Degradation at 90°C of 0.1 % trypan blue by using 0.2% H 2 0 2 and 50 ppm Cuz-1 was tested under different pH conditions to understand the effect of pH on the performance of the oxidising catalysts. The pH was controlled using HCI or NaOH. As shown in Figure 5, Cuz-1 was able to work over a wide range of pH from 3-1 1. In all cases, trypan blue degraded in 30 minutes.

(vi) Degradation of chlorophyll

The degradation of chlorophyll (50 mg/mL) by using the oxidising catalysts was tested. Chlorophyll, a chlorine pigment, can be bleached by H 2 0 2 in the presence of peroxidase and phenol that is a common oxidative system in plants. In this experiment, Cuz-1 was used to replace the role of peroxidase as the oxidising catalyst. The experiment was carried out in a water-based system without phenol. The aqueous solution of chlorophyll (50 mg/mL) was reacted with 50 ppm of Cuz-1 and 0.5% of H 2 0 2 for 60 minutes at 50°C. As shown in Figure 6, chlorophyll could not be bleached without Cuz-1 after 60 minutes. In contrast, in the presence of Cuz-1 , the chlorophyll solution rapidly became colourless within 30 minutes, thereby showing that chlorophyll can be effectively bleached by Cuz-1 and H 2 0 2 .

Whilst the foregoing description has described exemplary embodiments, it will be understood by those skilled in the technology concerned that many variations may be made without departing from the present invention.