Nowadays, the fact of harmful effect of reactive oxygen species on human health is well-known. The capability of natural defense systems of living organisms against excess production of these species decreases when influenced with negative environmental factors or aging, causing a number of degenerative diseases. Organic reductants that scavenge free radicals are of great value in preventing these so called "oxidative" pathologies. That is why natural products with antioxidant properties become more and more popular all over the world. As the use of bioantioxidants in food and drug industry is increasing, easy to operate, rapid, accurate and sensitive methods for their determination are needed. Many instrument-based methods, including HPLC, polarography and enzymatic methods, reported in the literature are not commonly used for routine analysis because of their inherent limitations. However, photometric methods are available for every analytical lab. In recent years the methods using chromophoric reagents immobilized on inert matrices attract the growing interest of the researchers.

BIOANTIOXIDANTS
(natural organic reductants that scavenge free radicals):
      - prevent and treat "oxidative" pathologies
               due to their antioxidant activity;
      - are widely used in pharmaceutical and food industry

To develop the solid-phase reagents we have used the algorithm at the left. The most sensitive photometric methods for the reductant determination in solution are based on Fe(III) and Cu(II) redox reactions, so we chose them as modifying agents. As a matrix, silica was chosen due to its chemical stability, negligible absorbance in visible region, and good kinetical properties. For the reagent fixing on the surface non-covalent immobilization was applied since this way is known to be both effective and time-saving.

THE MODIFYING AGENTS:
stability, contrasting colour change, steric structure

The following chelates have been studied as modifying agents: bis-bathocuproinecopper(I), phenanthroline-iron(III) and copper (II) complex with tetrabenzotetraazacyclohexadecine (CuTAAB). These complexes are stable and have intensively coloured reduced forms. Moreover, plane steric structure of the last two ones is good for paralel orientation on the surface.

IMMOBILIZATION: ADSORPTION ON SILICA
a procedure takes 1-20 min

Adsorption of the modifiers on silica has been investigated.
The L-type of copper-bathocuproine sorption isotherm testifies the complex affinity to silica surface is rather high. Copper(I)-bathocuproine complex immobilized does not interact with the reductants. To turn the modifying agent into its active form copper(I) ions on the surface were oxidized with hydrogen peroxide. However, this process was accompanied with considerable reagent desorption from the surface. We suppose that a mutually perpendicular orientation of plane bidentate ligands made it impossible for the complex to be fixed by means of two ligands, causing reagent desorption during the oxidation of Cu(I) ions. Although the obtained sorbent is rather sensitive, the instability is its principle drawback. So, when modifiying agents are being chosen, their steric structure have to be taken into account. For further investigations chelates with plane structure were used.

Phenanthroline-iron(ІІІ) complex on the surface was obtained by iron(ІІІ) sorption with phenanthroline-loaded silica gel. Under these conditions, the complexes of 1:1 ratio are formed.
CuTAAB sorption onto silica gels from aqueous solutions has been studied. Silica gel with the particles sized from hundred to two hundred fifty micrometers was chosen as a matrix due to its high values of a sorption constant and sedimentation rate. CuTAAB immobilized (CuTAAB-SG) was obtained easily by the adsorption of the complex from its aqueous solution.

The solid-phase reagents obtained in such a way are ready-to-use analytical forms (indicator powders).

THE INTERACTION WITH ORGANIC REDUCTANTS:
contrasting colour change

TECHNIQUES:

COMPARISON:
Fe(III) and Cu(II) chelates immobilized on silica gel as solid-phase redox-reagents
for the determination of bioantioxidants

APPLICATION:
determination of organic reductants in pharmaceuticals

Solid-phase spectrophotometric and visual test methods for ascorbic acid and analgin determination in standard solutions and some pharmaceuticals have been developed. The methods provide good statistical parameters; the results of the determination are strongly correlated with those obtained by a standard method. In comparison with other methods reported in literature, the proposed ones are more simple and rapid with no loss in sensitivity, so they may be recommended for routine pharmaceutical analysis.

FePhen-SG

   • determination of ascorbic acid in pharmaceuticals

The inorganic reductants, including sulfides and sulfites, and week organic reductants do not interfere with ascorbic acid determination, making this solid-phase reagent promising for the analysis of pharmaceuticals, but quite unsuitable for the determination of bioantioxidants.

CuTAAB-SG

   • determination of ascorbic acid in pharmaceuticals
   • determination of analgin in pharmaceuticals
   • evaluation of total antioxidant power of herbal products >>

CuTAAB immobilized surpasses iron-phenanthroline-silica gel in sensitivity, the range of linear response, the productivity of the analysis. In addition, it may be regenerated easily. For all these reasons and due to its ability to interact with bioantioxidants, CuTAAB-SG was used not only for the determination of individual compound, but also for the evaluation of total parameter - the antioxidant activity of herbal products (tea, wines, Echinacea phytoceuticals)

	Key publications:
	Zaporozhets O.A., Krushinskaya E.A., Lipkovskaya N.A., Sukhan V.V. Solid-phase reagent for analgin and ascorbic acid on the basis of a copper(II) complex with tetrabenzotetraazacyclohexadecine immobilized by adsorption on silica gel // Journal of Analytical Chemistry. - 2001. - V.56, №6. - P.524-529 (full-text, pdf, 46 Kb).
	Zaporozhets O.A., Krushinskaya E.A. Determination of ascorbic acid by molecular spectroscopic techniques // Journal of Analytical Chemistry. - 2002. - V.57, №4. - P.286-297 (full-text, pdf, 68 Kb).
	Zaporozhets O.A., Krushynska O.A., Khozyaeva O.O. A solid-phase redox reagent on the base of Fe(III)-phenanthroline complex immobilized on silica gel // Voprosy khimii i khimicheskoy tekhnologii (Questions of chemistry and chemical technology). – 2004. – №4. – P.23-27.
	Zaporozhets O.A., Krushynska O.A. Hyphenated spectroscopic and test determination of quality parameters of the products with antioxidant activity // Magisterium "Natural sciences". Issue 16. - Кyiv: "Kyiv Mohyla Academy" publishing house, 2005. – P.63–66.
	Krushynska O.A. Copper and iron chelates adsorbed on silica as solid-phase redox reagents for the determination of the organic reductants. The thesis for a Candidate’s degree in chemical science by speciality 02.00.02 – analytical chemistry. – Kyiv National Taras Shevchenko University, Kyiv, 2005
	to the personal page of Olena Krushynska