{ [N (CH3)4]S [Ni4 (C6H407)3 (OH) (�O)]. 1 8 �O}2' Citrate was later shown to exist in the
tetraionised state in this complex by Still and Wikberg (1980). At pH > 5-8, the tri-ionised state of
citrate predominates. It may be recalled that Hedwig ¥Ul.. (1980) were able to characterise several
Ni-citrate complexes over the pH range 4-7 in which citrate existed in the tri-ionised state. However
Still and Wikberg (1980) observed that when metal ions
arebound to citrate, the hydroxyl proton
can be removed at pH >8 thereby transforming citrate into a tetraionised state. Mastrapaolo m...al..
(1976) showed that for Cu(II), tetraionised citrate can be formed at pH 4, thus the pH at which the
hydroxyl proton is removed appears to be metal dependent
Examination of the structure of tri-ionised citrate shows that it can coordinate Ni in three possible ways (Fig. IV.8). One involves coordination between the hydroxyl group and the adjacent and tenninal carboxyl groups (FIg. IV.8a). Another involves coordination between the hydroxyl group and the two tenninal carboxyl groups (Fig. IV.8b). Coordination through the three carboxyl groups constitutes the third possibility (Fig. IV.8c). The relationship between the stability of a chelate complex to ring size and number is well documented (Rossotti,
1960;
Douglas and McDaniel,1965;
Beck,1970).
Five-membered rings are usually the most stable, with stability decreasing as ring size increases. Stability also increases with the number of chelate rings. Based on these considerations, Liddle( 1979)
proposed that coordination through the hydroxyl group and the adjacent and tenninal carboxyl groups (Fig. IV.8a) would provide the most stable structure. Despite the presence of two rings in each of the three structures, structure a is the only onecontaining a five-membered ring. The stability of the furan ring in 2-furylacetic acid will not readily facilitate coordination to nickel. The presence of only one carboxyl group and a stable keto group on 4-oxo-pentanoic acid also makes this acid unsuitable as a ligand. However tartaric acid, with its
two (terminal) carboxyl groups and adjacent hydroxyl groups may show some potential as a Ni chelator.
In
1983,
Baker et al. reported the structure of a Ni-citrate complex of fonnula�[Ni (C6Hs07) �O)2h.4 �O. The complex crystallized from solution at pH
5
which is close to the physiological pH for plants.In
the crystal, the complex exists as centrosymmetric dimers (Fig. IV.8d). Each citrate ion is coordinated to one Ni atom through its hydroxyl group and the adjacent and terminal carboxyl groups. This type .of coordination is in agreement with the proposed coordination of Liddle(1979).
The third carboxyl group is coordinated to the other Ni atom of the dimer. Two bound water molecules complete the octahedral coordination sphere of each Ni atom. The formation of a hydrogen bond between the hydroxyl proton and the carboxylate oxygen on thethird carboxyl group indicates that citrate exists in the tri-ionised state in the complex. This
corroborates the fmdings of Still and Wikberg
(1980),
and Hedwig et al.( 1980)
with respect to the ionic state of citrate at pH 5-6. The spaces between the Ni-citrate dimers are occupied byK+
ions and molecules of water of crystallization. There appears tobe no record of similar studies carried out on the Ni-malate system.a) Coordination - hydroxy b) Coordination - hydroxy
adjacent and tenninal carboxylates two tenninal carboxylates
c) Coordination -three carboxylates d) Coordination - hydroxy
In
view of the magnitude of structural detail obtained from X-ray crystallographic studies, the next step in the research was to attempt to procure a crystal from the synthetic solution of Ni, citrate and malate of mole ratio 1 :0.4: 1 , and the purified Ni-rich solid fromD.
subsp. tuberculatum. Microscopic examination of the green powder revealed the presence of aggregates of non-crystalline material. On account of the small amount of sample available, further refinement was not carried out.. Efforts were therefore dedicated to obtaining a crystal from the synthetic solution.
N. 1 0.II. of Nickel-Citrate-Malate Solution of Mole Ratio 1 :0.4: 1 .
In
order to obtain a crystal for structural elucidation, the two techniques of complete mixing and slow diffusion were applied using a range of Analar organic solvents. The solvents selected were ethanol, methanol, acetone, dimethyl sulphoxide, n-butanol, iso-butanol, n-propanol, iso-propanol, n-amyl alcohol and iso-amyl alcohol. All preparations were made in siliconized test-tubes. Siliconization was carried out in the following manner. The tubes were first rinsed with iso-propanol, then Serva Silicone solution (in propanol). Coating of the inner walls was then achieved by heating at 100- 150° C in a drying oven for one hour.For the complete mixing technique, 1 cm3 of complex was mixed with solvent in a 1 1 x 100mm test-tube. The ratio of complex to solvent was varied from 1-2.5. Two sets of mixtures
were prepared. One set was stored at room temperature while the other was refrigerated at 4° C. All tubes were stoppered with rubber bungs through which 2 mm holes had been drilled. Ethanol, methanol, acetone and dimethyl sulphoxide were used in these experiments as preliminary studies showed crystallization to be easily achieved in their presence. Crystals were observed after leaving the solutions overnight. They were removed from the mother liquor, placed on Whatman # 1 fllter paper, rinsed with the solvent used and allowed to air-dry overnight beneath an inverted funnel. Microscopic examination of the crystals showed them to be twinned in some instances and also highly aggregated. Such crystals were unsuitable for structure determination so the alternative technique was utilized.
In
applying the slow diffusion techniqiIe,3
cm3 of solvent was carefully layered over1
cm3 of complex in a
5
x125
mm test-tube. With the exception of ethanol, methanol, acetone and dimethyl sulphoxide improved crystallization was attempted using the solvents originally listed. Duplicate preparations were made. The test-tubes were sealed with Whatman Parafilm due to the unavailability of adequately-sized rubber bungs, and the temperature regime used earlier was applied. Crystals were observed two to three days after the experiment had begun. These were dried using the same procedure as before. When viewed under the microscope, there was a marked improvement in the quality of the crystals. However, some extraneous matter appeared to adhere to the crystals; these were considered impurities. Crystals formed using n-propanol at roomtemperature were the most suitable.
In
an attempt to further improve the pUrity of the crystals, diffusion was slowed down even more by placing a layer of distilled deionised water(
-0.5
cm3) between the layers of complex and n-propanol. The complex solution, distilled deionised water and n-propanol were previously filtered using MillexGV 1 (3
mm) Millipore filters of pore size0.22
mm. The rest of theexperiment was carried out as before. Mter a period of
12
days, several single (untwinned) crystalswere obtained at
4°
C. One was mounted for structural determination by X-ray crystallography.X-ray crystallography provides information regarding the type and arrangement of atoms comprising a crystal, based on the interaction between X-rays and electrons in the atoms. Due to the short wavelength (high energy) of the radiation, this interaction usually results in scatter. When X-rays are diffracted (scattered) by an ordered environment such as a crystal lattice, both
constructive and destructive interference occur owing to the fact that the distance between the
scattering centres and the wavelength of the X -rays are of the same order of magnitude. In order for the X-rays to appear to be reflected from the crystal, only constructive interference must occur.
This criterion is mathematically described by Bragg's Law :
nJ.. = 2d sin e
where n = an integer ; J.. = the wavelength of the X-ray ; d = interplanar distance of the crystal and e = the angle of the incident X-rays. The angle of incidence must satisfy the condition sin e =
nA/2d. At all other angles, destructive interference occurs. The intensity of the reflection varies depending on the distribution of electron density within the units making up the crystal.
Like the spectroscopic techniques described earlier, the application of X -ray diffraction in chemical analysis (X-ray crystallography). requires a source, a wavelength selection device, a sample holder, a radiation detector and output equipment X-ray crystallography was fIrst used to identify unknown substances during the earlier part of this century. Because the resulting X-ray diffraction pattern is dependent on the exact atomic arrangement in the sample, the technique provides unequivocal identification of the sample (Azaroff,
1968). No two crystals give rise to the
same diffraction pattern. The diffraction pattern is usually recorded by allowing the beam to strike and blacken a photographic fIlm or by measuring the diffracted radiation with some form of counter (Dent-Glasser,1977). Data analysis then furnishes information concerning the atoms present, their
arrangement (unit cell) and the dimensions of the 'arrangement, all of which are used to characterise the crystal.For this work, accurate cell dimensions were determined from the least squares analysis of the positions of 25 general reflections on an