CHAPTER 5 CRYSTALLISATION OF HYDRATED AND ANHYDROUS URIC ACID AND AMMONIUM ACID URATE CRYSTALS IN GEL

Transcription

1 61 CHAPTER 5 CRYSTALLISATION OF HYDRATED AND ANHYDROUS URIC ACID AND AMMONIUM ACID URATE CRYSTALS IN GEL 5.1 INTRODUCTION Uric acid is an important constituent of human stones. Under normal circumstances uric acid is a minor serum constituent. However, certain reptiles and birds excrete most of their nitrogenous waste in the form of uric acid. In the normal, urea is the main form of nitrogenous waste product and, except in humans, apes, new world monkeys and Dalmatian dogs almost all uric acid is converted into allantoin prior to excretion (Smith et al, 1969). Uric acid stones are known to occur in patients in whom urinary uric acid excretion is raised, due to primary or secondary gout and the incidence rises with increasing urinary uric acid (Gutman 1963). Henneman et al (1962) indicated a tendency for uric acid stone formation to be associated with the passage of an unusually acid urine. An increase of uric acid levels in blood leads to the deposition of uric acid in joints which result in gout causing pain and swelling. Uric acid is often found as a constituent of urinary calculi and has high incidence in nephrolithiasis resulting in severe kidney damage. Hence studies on nucleation and crystal growth of uric acid play a vital role in medicine and pharmaceutical research. Several workers have grown crystals of uric acids from solution (Rinaudo and Boistelle 1980, Boistelle and Rinaudo 1981). Anhydrous uric acid is known to crystallize in monoclinic system with unit cell constants a = nm, b = nm and c = nm with/3 = 65.10*. There is an increasing interest in growing urinary crystals as a model system for studying crystals deposition diseases in human beings. A method

2 62 suitable for use under practical conditions based on diffusion and crystallization in gels has been developed (Henisch 1970, Bisaillon and Tawashi 1973, Legeros and Morales 1973, Phaneuf-mimeault and Tawashi 1977). There have been several studies on the solubilities of uric acid and the degree of supersaturation attained under various conditions (Porter 1963 a, Marshall and Robertson 1976, Vermueien et al 1964). Babic-Ivacic et al (1987) studied the solubility of uric acid dihydrate (UA.2H2O) and the precipitation of UA.2H20 and anhydrous uric acid (UA) from solutions containing sodium hydroxide and hydrochloric acid. Boistelle and Rinaudo (1981) grew (0.6 x 0.4 x 0.2 mm3) crystals of anhydrous uric acid in solution. The experimental study on dissolution of uric acid by Vermeulen and Fried (1965) indicates that uric acid stone formation is also due to precipitation from a supersaturated solution. Little is known about the factors which may influence the crystallization of uric acid from gel. In the present work, crystallization of both uric acid and uric add dihydrate using the gel technique have been investigated. The conditions for growing these crystals of uric acids using silica gel and to obtain good crystals for X- ray diffraction and other studies have been enumerated. Crystals of linear dimensions ranging from 0.2 to 1 mm have been grown in the present investigation. The general growth aspects of uric acids as a contribution to the knowledge of uric acid stone growth which grow in a synovial medium (gout, joints) have also been investigated. Ammonium acid urate crystals (C5H7N5O3) have been found as one of the compositions of urinary calculi (Tsay 1961, prien 1963). Ammonium urate is a pure state of urinary calculi. The most important type of bladder stones from developing countries consists mainly of ammonium acid urate. Ammonium acid urate was the most common constituent of the stones from Dalmatians dog (Sutor and Wooley 1970 b). Teotia and Sutor (1971) have shown that excess of both uric acid and the ammonium ion is essential for the formation of Ammonia acid urate from urine over the ph range of It is chiefly found in the endemic bladder stones and which are still prevalent in certain areas of some of the developing countries such as India, Turkey etc. (Lonsdale et al 1968). Because of their interest in biological system we have studied the growth and morphology of these crystals.

3 63 Despite the biological interest in Ammonium acid urate crystals, surprisingly little is known about its growth and characterisation, although the solubility has been more interestingly studied (Marshalf and Robertson 1976, Porter 1963 b). The present work describes the growth conditions of these crystals in sodium metasilicate gel under ambient conditions. The effects due to the change of concentration of the reactions, the density of gel and the ph of the gel solution impregnated with the inner reactants on the growth of the crystals have also been investigated and discussed. In view of the varied occurrence of ammonium acid urate and its importance in endemic children s bladder stone formation, a knowledge of its precipitation from gel technique would be useful to study growth conditions of crystals found in urinary systems. 5.2 EXPERIMENTAL A sodium meta silicate solution (Baker analysed) of densities ranging from 1.03 to 1.06 g/cc were prepared from the stock solution. Uric acid solution was prepared (200mg in 100ml) in hot water. The ph of the solution was maintained between 3 to 6 by treating it with glacial acetic acid (BDH-Analar). The sodium meta silicate solution of known density 1.03 g/cc and the uric acid solutions (United States chemical) were mixed in the ratio 2:1. The gel solutions thus prepared were transferred into 25 x 300 mm Borosil test tubes adding about 30 ml mixtures to each tube. Sometimes small test tubes were also used. After the gelation along with the mixtures at room temperature (25 *C), 20 ml of 10% acetic acid is poured carefully over the gel. The tubes were covered with cork stoppers and allowed to stand at room temperature for crystallization. Completion of crystallization took about 30 to 40 days. White tabular crystals of different sizes are obtained for the above case. The crystals grown in test tubes are shown in Fig 5.1. Because of the continuous diffusion of outer solution into the gel medium white precipitate is formed just below the interface. At the same time within a period of 2 to 3 weeks tabular crystals of anhydrous uric acid are formed throughout the bottom portion of the test tube. The same result was obtained when we used U tube apparatus.

4 64 In a variation of the above procedure, uric acid-gel mixtures, ph 5, were used to set the gel as above in U tubes. Then the solution of the uric acid (20ml) prepared by taking 250mg of uric acid in 50 cc of 0.2 N NaOH solution (Merk-purified pellet) was poured on the top of the gel in one leg of the U tube, while in the other leg a 10% HC1 acid solution was poured. The reaction takes place slowly and within a fortnight, tiny crystallites are found to grow just below the gel solution interface of the leg which had uric acid solution and later in the bent portion of the U tube. In one experiment with the gel solution of density 1.03 g/cc acetone was mixed in a ratio 5:1 and the ph of the gel solution was adjusted to 4 or 4.5 with 3M acetic acid and taken in a test tube or U tube and allowed to set. The supernatant solution UA/NaOH was poured carefully on the set gel. In this case hydrated uric acid crystals are found to grow (Fig.5.2). After the completion of each experiment, crystalline products were harvested from the gels by decanting of the supernatant solution and washed in distilled water. The harvested crystals are observed by optical microscopy and separated into various groups. X-ray patterns were obtained for a portion of each group to enable identification of the phases using a Philips APD 3600 automated powder diffractometer (CuKa radiation = nm), by scanning electron microscopy and by infrared absorption spectroscopy with a P.E. 980, ratio recording dispersive instrument. In order to minimise dehydration of UA 2H2O, X- ray diffraction patterns were recorded from moist samples. In order to grow ammonium acid urate crystals in sodium meta silicate gel, a gel solution of density 1.04 gm/cc is prepared from the stock solution. In the growth of ammonium acid urates 20 ml of ammonium chloride is used as the inner reactant. The gel solutions impregnated with the inner reactant are acidified with 3M acetic acid and taken in straight tubes of 2.5 cm diameter and 23 cm length. The period of gelation varied from 12 hours to 24 hours. A solution of uric acid is used as the outer reactant. The outer

5 65 reactant diffused into the gel medium and immediately the reactions are taking place between inner and outer reactants. The expected reaction during the process of crystallization is C5H4N403 + NH4C > C5H7N5O3 + HC1 The experiment is repeated by changing various parameters like the concentration of reactants, the ph of the gel solution and the density of the gel solution. The effects of these parameters on the growth of ammonium acid urate crystals are discussed. The gel grown crystals were characterised by powder X-ray diffraction pattern and identified as C5H7N5O3 by comparison with known patterns from the ASTM index. 5.3 RESULTS AND DISCUSSION Crystal Growth The anhydrous crystals grew throughout the gel as white tabular reaching diameter up to 1 mm over 4 to 6 weeks period. The hydrated crystals of maximum size 0.8 mm are also grown in our experiment for the above period. Table 5.1 summarises the results of the experiments with and without uric acid in gel phase and dilute acids or uric acid in the overlying solution. It also gives the amounts of uric acid that are to be chosen to crystallize the hydrate or anhydrous phase. When uric acid dissolved in hot water was taken with the gel at ph=5 and UA/NaOH in one arm of the U tube and dil. HC1 (10%) in the other arm, it was observed that minute crystallites are formed first in the portion below the gel interface of UA/NaOH solution side at a temperature below 20 C. When the crystals grown with the above conditions are allowed for fairly a longer time it was found that the minute crystallites in the upper region of gel column grew into agglomerated orthorhombic uric acid crystals. At the same time, low solution concentration of the uric acid in the bent portion of the U tube facilitates the growth of anhydrous monoclinic uric acid crystals and has the similar situation as the growth of

6 Figure 5-3 Figure 5-2

7 67 anhydrous uric acid crystals grown in test or U tube at ph = 5, when UA/hot was taken as inner reactant and dil. HCI as the outer reactant. Table 5.1 Product with various reagents in U - tube experiments ph Inner reactants Outer reactants 3.5 UA/NaOH dil. HCI (on both limbs) 4.0 UA/NaOH dil. acetic (on both limbs) 4.0 Acetone UA/NaOH (both limbs) Product uric acid Anhydrous Anhydrous Hydrated Size of the crystal up to 1 mm.3 to.8 mm.2 to.6 mm 4.5 Acetone UA/NaOH Hydrated.2 to.8 mm 5.0 UA/hot dil. HCI (on both limbs) 5.0 UA/hot UA/NaOH (on one limb) dil. HCI (on the other limb) Anhydrous Hydrated Anhydrous.2 to.6 mm.2 to.6 mm.2 to.6 mm 6.0 UA/hot dil. HCI Anhydrous.2 to.8 mm UA/hot - 200mg in 100ml hot water; UA/NaOH - 250mg in 50ml 0.2 NaOH, dil NaOH = 10%, dilute acetic acid = 10%, acetone = 10ml. Thus it is observed that uric acids of both the phases are found to grow in the same experiment (Fig.5.3). Moreover, the hydrated orthorhombic uric acid crystals whose solubility is greater than anhydrous uric acid are formed first. This is true because when the formation of several phases is possible the one with the highest solubility will form first and transform sooner or later to the thermodynamically most stable phase (Ostwald, 1897, Lamerwin and Nancollas

8 ). In the present investigation, however, the author has not observed the modification of orthorhombic uric acid to monoclinic uric acid and vice versa because the crystals formed are more stable in the medium. The monoclinic anhydrous crystals that appear in the bent portion remain as the stable anhydrous uric acid because of the acidic environment (Rinaudo ef al 1984) that prevail in the bent portion of the U tube and the hydrated uric acid crystals just below the gel interface are stable in alkaline or neutral environment at low temperature (Babic-Ivancic et al 1987, Shirley 1966). X-ray diffraction analysis of crystals extracted from gel confirmed that the crystals harvested from the upper portion of the tube and bent portion of the tube are respectively the hydrated and anhydrous crystals. Thus the simultaneous growth of two phases of uric acid crystals are found in gel growth experiment (Irusan et al 1993 c). The same process occurs in human stones made up of both the uric acids (Lonsdale and Mason 1966). The spherulitic types of crystals grown in our study have been viewed by SEM (Fig.5.4) and these crystals are found to consist of number of needle like crystals showing the characteristic behaviour of the grown crystals in gel (Morse and Donnay 1936). The size and crystal habit of uric acid grown in gels are affected by temperature, concentration of reactant and ph. In the present study, at far below the room temperature (20* C) in silica gel medium, the sole crystalline products are tabular or spherulitic and at a higher temperature they are needle shaped. The crystallization of ammonium acid urate in gel is due to the chemical reaction between the inner reactant impregnated into the gel solution and the outer reactant diffused into the gel medium. In respect of ammonium acid urate due to the chemical reaction between the inner and outer reactants, pale yellow crystals are formed just below the interface. Crystals of platelet and hexagonal morphology were found to grow within two to three weeks (Irusan et al 1993 a). In the U tube method suggested by Henisch et al (1965 b), it is found that a few crystals are formed as clusters. Crystals that grow at or near the surface of the gel tend to form spherical aggregates of ammonium urate rather than growing

9 uniformly. However, at a depth ranging from 1-30 mm beneath the gel interface crystals show hexagonal morphology. The crystals are lemon yellow in colour and transparent with perfect edges as shown in Fig.5.5. Most of the crystals are prismatic and well faceted. The maximum size of the harvested crystals in straight tube is 8.9 x 2.8 x 1 mm Effects due to change of growth parameters Effect of ph ph is the key factor in gel technique producing interesting effect on the growth of uric acid systems in sodium metasilicate gel at ambient temperature. The crystallization is possible in case when the ph of the gel solution is between 3 to 6. Tabular and spherulitic types of crystals are obtained when the ph is 3.5 to 5.5 and for ph 6 twin crystals are formed. When the ph is less than 3 or greater than 6 crystallization is not at all possible. Hence in the case of uric acid, the change of ph has control over the growth phenomenon. At very low ph values only single hair-like crystals are obtained. The crystals grown at various ph values are tabulated. The ph of the gel solution impregnated with the inner reactants is varied in the range of 3.5 to 7 in steps of 0.5 in the growth of ammonium acid urate. Even though the crystallization is obtained in all the above ph ranges larger number of single crystals of smooth surfaces are obtained when the ph is 5. The crystallization is very poor at higher ph values. A number of well defined crystals of Ammonium acid urate are obtained when the ph is 4. In general there is no pronounced effect due to the variation of the ph of the gel solution impregnated with the inner reactant in the range of 4 to 5. Both the hexagonal and rhombohedral crystals are obtained in this range. Figure 5.6 shows the SEM photograph of the Ammonium acid urate crystals harvested in 30 days (ph = 4).

10 Effect of gel density The gel densities ranging from 1.02 to 1.08 were prepared from the stock solution keeping the gel ph constant at 5. When the gel density is 1.06 g/cc, the number of crystals is reduced very much. Decrease in the nucleation density has been observed when we increase the gel density. The gelation has also its effective role in the growth of uric acid crystals. The effects due to the variation of gel density on the growth of ammonium urate crystals were studied. The density of the gel solution is varied in the range of 1.02 to 1.0 gm/cc. Crystallisation of Ammonium acid urate crystals is observed in the range between 1.02 to But the most favourable gel density for obtaining good crystals is 1.04 gm/cc. Above this concentration the gel inclusions are observed and the growth rate is small. Most of the crystals formed are hexagonal and their size varies from the interface to deep inside gel column. The ageing of gels has profound effect on the quality and morphology of the crystals. The outer electrolyte was added after different ageing of the gel. Only very small crystals were seen and the growth rate is also low. Since the pore size distribution of the gel is affected by ageing: the growth rate is drastically reduced (Henisch 1970). But it is observed that the more aged gels produced more perfect crystals. Experiments on the growth of Ammonium urate results in excessive nucleation and the successful growth of these crystals depends on the supersaturation Effect of temperature The growth of uric acid crystals in gel is temperature dependent. The crystals can only be grown in gel when the temperature of the growth system is less than 38 *C. No nucleation can be observed when the temperature is above 38 *C. Growth is favourable when the temperature is between 20 C to 25 *C. Number of crystals formed becomes lesser and lesser when the temperature is increased

11 71 from 30 to 38 * C. Hydrated crystals are grown well at a temperature of 20 C. The modification from hydrated uric acid phase to anhydrous is very much influenced by the change of temperature. This investigation has shown that uric acids of both the phases can be crystallized using silica gel under proper conditions of ph, temperature and the reactant concentrations Effect of concentration of the reactants If the concentration of the inner reactant (Ammonium chloride) for the growth of ammonium urate is changed in the range of 0.25M to 1 M it is observed that the crystallization is effective. Crystals of hexagonal, rhombohedral and platelet morphology have been found to grow when the concentration of inner reactant is kept constant and the outer reactants are varied from 0.25M to 0.75M. Crystallisation is observed to be mainly depending upon the relative concentration of the outer reactant. It is noticed that when the concentration of the outer reactant (uric add) is less than 0.25M crystallization is not at all observed. A number of isolated, transparent good crystals are obtained when the concentration of inner and outer are 0.75M and 0.5M respectively. Platelet crystals were obtained when 0.25M ammonium chloride was used as the inner and 0.5M uric acid as outer reactant. Thus the concentration of inner and outer electrolyte have distinct influence on the growth of Ammonium urate crystals Effect of impurities The effect of different ions such as lead, cadmium, magnesium and pyrophosphate and juices of some medicinal plants like ocimum sanctum on the growth of ammonium urate crystals were also studied. The presence of magnesium and pyrophosphate produced an alteration of crystal habit and stunting of the growth. No appreciable effects had been noted with other ions.

12 CONCLUSION It has been proved by experiment that the difficultly soluble crystals of uric acids and ammonium acid urate can be grown from gel under ambient conditions. The uric acid crystals are found to be white in colour with spherulitic and platelet morphology. But ammonium acid urate crystal is pale yellow in colour with shining surfaces. The hydrated orthorhombic modification is obtained only when the ph and the temperatures are very low. Experiments showed that uric acid which has a low aqueous and organic solubility can be used to determine the conditions under which crystals already formed can be dissolved, modified or inhibited from the view point of devising new treatment and means to control the disease. X - ray diffraction studies have been done and the lattice parameters of respective crystals are found to be in good agreement with the reported values.