HEMATOXYLIN PLASMA TREATMENT

Transcription

1 HEMATOXYLIN PLASMA TREATMENT A. P. CHIRIAC, L. E. NITA, I. NEAMTU, C. M. POPESCU, A. IOANID, G. E. IOANID Petru Poni Institute of Macromolecular Chemistry Grigore Ghica Voda Alley No. 41-A, Iaºi, Romania Received December 21, 2004 The arising problems using hematoxylin as a dye, is their oxidization to hematein. This process is called ripening. The oxidation is achieved by adding a wide variety of oxidizing agents, the two most common being sodium iodate and mercuric oxide. The paper presents the possibilities of hematoxylin ripening in plasma conditions. Key words: hematoxylin, plasma treatment, oxidizing. 1. INTRODUCTION Although it is common practice to use hematoxylin as the source of dye, it is not itself the actual dye and it has to be oxidized to hematein, which is a dye, before it can be used. This process is called ripening, and can be accomplished in two distinct ways. The ripening is usually accomplished with chemical oxidizing agents, but is sometimes accomplished by atmospheric oxygen over time. Hematein is not incorporated directly in most staining solutions because it continues to oxidize in solution and forms non-staining products. The quality of hematoxylin is usually higher and more consistent than the quality of hematein, and solutions made with it are more easily standardized. Many technologists believe that natural ripening by atmospheric oxidation is the best, and gives a more reliable and longer lasting solution. Natural ripening is accomplished by putting the solution in an oversize flask, so it can be shaken, with the top plugged loosely with cotton wool, allowing air to enter. This is left in a warm, light and airy place (a windowsill) for oxidation to take place. The flask is shaken periodically. Oxidation may take several months, and is determined by testing the solution from time to time. The other way to ripen hematoxylin is to use chemical oxidizing agents. The most commonly used is sodium iodate, at about 200 mg per gram Paper presented at the 5th International Balkan Workshop on Applied Physics, 5 7 July 2004, Constanþa, Romania. Rom. Journ. Phys., Vol. 50, Nos. 9 10, P , Bucharest, 2005

2 1120 A. P. Chiriac et al. 2 hematoxylin. Others have also been suggested for particular formula as for example mercuric oxide, potassium permanganate, potassium periodate, hydrogen peroxide, but sodium iodate can be substituted for just about all of them if used at the stated amount. Half oxidation is a term used to describe partial oxidation of hematoxylin to hematein. The rationale is that during natural ripening the solution becomes usable when a significant portion (i.e. half) of the hematoxylin has been oxidized. These solutions have an extended life because they are usable before full conversion takes place. Throughout their life, oxidation of hematoxylin and further oxidation of already converted hematein continues. They do not become unusable until oxidation proceeds so far that insufficient hematein remains to give acceptable staining. Chemical half oxidation seeks to emulate this process by adding sufficient oxidant to convert only half of the hematoxylin to hematein. Oxidation will then continue as for a naturally ripened solution. Many hematein samples contain only small amounts of the dye, the rest being something else. This is most likely the oxidation products of hematein. These are not dyes and do not stain satisfactorily. They do sometimes give objectionable brown or other off-color tinges to the staining. The problem that arises is: if hematein is the dye, and if hematoxylin needs to be oxidized to it before it can stain, why not use hematein in the first place? The difficulty is in obtaining satisfactory hematein, practically to obtain a proper oxidized hematoxylin suitable for being stained. As it is well known plasma pretreatments are environmentally benign and energy efficient processes for modifying the surface as well the chemistry of materials. The major advantages of RF plasma generators are that no internal electrodes are needed and the plasma is easily sustained at a low pressure (< 50 mtorr). [1 7] Because there are no internal electrodes, no impurities are sputtered off the electrodes and incorporated in the present substances. In this type of generator, plasmas are produced when energy is transferred by an electric field to free electrons within a low-pressure environment. The free electrons collide with molecules and any other substances exposed to the plasma. Through inelastic electron collisions with molecules, more electrons, ions, free radicals, and molecules in excited states are generated, and this results in the plasma state. Cold gas oxygen plasmas contain species such as O +, O, O + 2, O, and O 3, as well as ionized ozone and meta stably-excited O 2 and free electrons [8 13]. Each of the energetic fragments is very reactive with other fragments, and any substance exposed to the plasma, mostly via free radical chemistry. In glow discharges, electrons typically have bombardment energies of 20 ev or less which is enough energy to break most covalent bonds. Practical plasmas used for surface modification have electron densities of to electrons/meter. In lowpressure cold plasmas, electrons do not achieve thermodynamic equilibrium with

3 3 Hematoxylin plasma treatment 1121 gas molecules, and thus the temperature of the plasma remains close to ambient. This important feature allows plasma treatment to be carried out without thermal degradation of reactants or products. The purpose of this paper is to report the experimental results concerning the effect of a plasma treatment upon hematoxylin in order to obtain proper oxidized structure of hematein available to stain. 2. EXPERIMENTAL 2.1. MATERIALS 7,11b-dihidrobenz[b]indeno[1,2-d]piran-3,4,6a,9,10(6H)-pentol or cis-(+)- 7,11b-dihydrobenz(b)indeno(1,2-d)pyran-3,4,6a,9,10(6H)-pentol having as commercial name Hematoxylin (Riedel de Haën provenience) was used as received. The substance was treated in plasma conditions for different period of time, respectively 1, 3, 5, 10 and 20 minutes EXPERIMENTAL SET-UP Plasma polymerization is performed in the system [17] shown in Fig. 1. Fig. 1. Schematic diagram of the plasma reactor for hematoxylin treatment: A system for gas feeding, rotation training and vacuum; B reaction vessel; C system for electrodes fixing; D HF generator; 1 rotative cylindrical reactor; 2 semicylindrical electrodes; 3 current supply.

4 1122 A. P. Chiriac et al. 4 The treatment procedure by plasma consists mainly of a high frequency source, a horizontal vacuum chamber of the reactor which is a Pyrex glass cylindrical column (20 cm 3 ), the vacuum pump and pressure gauge [14]. This vacuum chamber is evacuated before each run and subsequently filled with hematoxylin. The discharge is obtained between two semi cylindrical-shaped electrodes placed on the exterior walls of the reactor and connected to the high frequency power generator operating at 1.5 MHz. Before the plasma treatment, the chamber pressure was pumped down to less than 4 torr. During the plasma process, the irradiation power was 40 W at a maximum output voltage of 100 V/cm. The interval for hematoxylin plasma treatment was between 1 to 20 minutes. The plasma is constantly in contact with the reactor walls and with 1 g hematoxylin solid substance CHARACTERIZATION The hematoxylin (witness and plasma treated variants) was characterized using Fourier transform infrared FTIR experiment on a spectrophotometer DIGILAB Scimitar Series-USA, with 4 cm 1 resolution. The procedure intends to observe and evidence the plasma treatment effect concretized in oxidizing process upon hydroxyl groups of hematoxylin. Spectra were performed for equal quantities of samples. UV-vis spectra performed on a Specord 42 M were used also to evidence the modification brought by plasma treatment upon hematoxylin which presents the behavior of a dye. The solution concentration was 10 6 g/l. Analysis was performed quickly after the plasma treatment samples being maintained in dark vessels. Hydroxyl functional groups of hematoxylin witness sample as well as of the plasma treated hematoxylin were determined through a quantitative method based on the estherification reaction of OH groups with anhydride phtalique in the presence of pyridine. 3. RESULTS AND DISCUSSION Figs. 2 a, b and c illustrates the hematoxylin and hematein structure as well the modality to form multi molecule multi chelate complexes. As it was already mentioned used alone, hematoxylin is a poor stain, but in combination with various metallic salts, mordants, which couple it to the tissue, it is one of the best nuclear stains known. It has also been used to detect metals in tissues, to stain myelin, to stain mitotic figures, fibrin, muscle cross striations and other tissue elements. But up to being used as hematoxylin lake, this substance must be oxidized. The structures show the possibilities for oxidizing, practically for

5 5 Hematoxylin plasma treatment 1123 Fig. 2. Hematoxylin (a) and hematein (b) structure and the complex dye mordant (c). breaking the bond, which we intend to achieve in the plasma conditions in order to obtain hematein. Some typical bond for various organic species and their correspondingly dissociation energies (ev) are mentioned. Accordingly with the hematoxylin structure and with the correspondingly dissociation energies the most probably functional groups capable for oxidizing during plasma treatment are the 5 hydroxyl groups. Bond Dissociation Energy [ev] C-C 3.61 C=C 6.35 C=C (π bond) 2.74 C-H 4.3 C-O 3.74 O-H 4.83 O-O 1.52

6 1124 A. P. Chiriac et al. 6 The plasma effect upon the hematoxylin structure during the 20 minutes treatment is first evidenced by infrared spectroscopy (Fig. 3 a e). The intervened modifications, resulted after the oxidizing process, are evidenced by the following Fig. 3. FTIR spectra of hematoxylin and the correspondingly samples treated in the plasma conditions for 3, 5, 10 and 20 minutes.

7 7 Hematoxylin plasma treatment 1125 FTIR spectra of the investigated samples. It can be observed that plasma treatment of hematoxylin can be divided in two stages. Thus, the witness sample of hematoxylin which have both hydrogen bonding donors and acceptors located, so that intramolecular hydrogen bonding is favored, display slightly broaden O-H stretching absorption in the 3500 to 3600 cm 1 range. In case of the treated hematoxylin those bands are slightly diminished in the order of: hematoxylin 3 treatment > hematoxylin 10 treatment > > witness > hematoxylin 20 treatment > hematoxylin 5 treatment. The observation agrees with fact that many hematein samples contain only small amounts of the dye, the rest being something else. Practically the oxidation process, which can be performed at any of the O H or C O groups, provide products with structure having again the possibility for being oxidized or reduced. Any way it seems that first we have a process of oxidation which can be continued after that with one of reduction. The oxidizing process upon hematoxylin sample is confirmed also by the UV spectra (Fig. 4). Fig. 4. UV spectra of hematoxylin and plasma treated hematoxylin during 1, 3, 5, 10 and 20. It is also confirmed the benefic oxidation process, performed in the plasma conditions, up to 3 to 5 minutes which conduct the hematoxylin at a half oxidized substance, a hematein proper as a dye and for being stained. UV as well FTIR spectra, as well as the oxidizing process in two steps which is performed upon the hematoxylin structure in the plasma condition, are verified and validated by the analytical determination of the hydroxyl groups. Thus, for the witness sample it was determined a concentration of about 23.63% hydroxyl groups (the theoretic value being 26.46%). The correspondingly values for hydroxyl groups in case of the treated substance are: 18.63% for 1 treatment; 10.12% for 3 treatment, 9.18% for 5 treatment, 11.66% at 10 treatment, and

8 1126 A. P. Chiriac et al % for 20 treatment. The content of hydroxyl groups allow us to conclude that the oxidizing process up to 10 minutes is inverted in one of reducing. 4. CONCLUSIONS It was tried to obtain a hematein structure from a hematoxylin one through a process of oxidization in plasma condition. Up to a treatment of 5 minutes we conclude as having an optimum structure of dye. For extended period of treatment the oxidizing process is inverted in one of reducing. Unfortunately we couldn t achieve a convincing opinion concerning the processes performed in the hematoxylin structure during the plasma treatment owing to the competitive ablation and addition processes performed in the plasma condition. This aspect refers also to plasma processes that show intense system dependence, in our case a substance capable to perform oxidizing as well reducing reactions, owing to the nature of the addition and ablation processes that are occurring simultaneously. Of course, if the obtained structure will give good results at staining, the study must be improved and optimized in order to have the conditions for the obtaining of a hematoxylin structure with an optimum content of dye. REFERENCES 1. A. T. Bell, S. Morita, M. Schen, S. Ishibashi, K. Hida, H. Kawamura, S.,Ikeda, Plasmas Polymerized Ethane in a Capacitely Coupled Reactor, p. 279 in Advances in Low- Temperature Plasma Chemistry, Technology, Apllications (H. Boenig, Ed.), Technomic Publishing Comp. Inc., Lancaster, USA, C. M. Chan, T. M. Ko, H. Hiraoka, Surface Science Reports, 24, 1 (1996). 3. S. H. Cho, Z. T. Park, J. G. Kim, J. H. Boo, Surf. Coat. Technmol., (2003). 4. D. T. Clark, M. M. Abu-Skbak, J. Polym. Sci., Polym. Chem. Ed., 21, 2907 (1983); ibidem 22, 1 ºi 17 (1984). 5. J. Friedrich, Surf. Coat. Techol., 98, 879 (1998). 6. S. Gaur, G. Vergason, 43rd Annual Technical Conf. Proceed., Denver, Apr.15 20, K. Gorna, S. Gogolewski, Polym. Degrad. Stability, 79, 475 (2003). 8. N. Inagaki, Plasma Surface Modification and Plasma Polymerization Technomic Pub Co, C. M. Kim, S. H. Cho, J. G. Han, B. Y. Hong, Y. J. Kim, S. H. Yang, J. H. Boo, Surf. Coat. Technmol., 169/170, 595 (2003). 10. H. Kobayashi, M. Schen, A. T. Bell, J. Macromol. Sci., Chem., A-8, 373 ºi 1345 (1974). 11. R. March, H. Drost, Kinetics of the Methane Conversion in a Glow Discharge up to Chemical Equilibrium, p. 251 in Advances in Low-Temperature Plasma Chemistry, Technology, Apllications (H. Boenig, Ed.), Technomic Publishing Comp. Inc., Lancaster, USA, T. Matsuoka, H. Yasuda, J. Polym. Sci., Polym. Chem. Ed., 19, 2937 (1981), ibidem 20, 2633 (1982). 13. C. Oehr, M. Muller, B. Elkin, D. Hegemann, U. Vohrer, Surf. Coat. Technol., 25, 116 (1999). 14. G. E. Ioanid, C. Topor, Rom Patent 110, 537, 1997.