Composition , Stability and Probable Structure of a Colourless Organometallic Complex ( Gd ( III )-Malic Acid )

The formation of colourless gadolinium complexes (x,y,z), between x gadolinium ions, y ligands and z protons, of some organic acids, has been studied in aqueous solution. In this work we present the results of investigations on the interaction of the gadolinium ion (Gd) with malic acid (C4H6O5, -hydroxyl dicarboxylic acid), in dilute aqueous solution for pH values between 5.5 and 7.5. Colourless gadolinium complexes of malate ions have no absorption band UV-visible, the indirect photometric detection (IPD) technique was used and studies have identified a major tri-nuclear complex of malate ion (OOC-CH2-CHOH-COO). The formation of this new colourless complex is derived from three Gd(III) ions that react with two malate ions and two hydronium ions (H3O), giving for this colourless complex, a (3,2,2) composition and apparent stability constant depends on the acidity of the medium, with logK'322 = 18.88 ± 0.05 at pH = 6.30. To complement previous results and to propose a probable structure for this new complex detected in solution, studies of IR spectroscopy have been conducted to identify the chelation sites for both ligands. The results were analysed and show that this organometallic gadolinium complex, contains two different sites, respectively, two lateral tetradentate mono-nuclear sites and a single central bidentate mono-nuclear site. From these results, the reaction of formation, the stability constant and the probable structure of this new colourless organometallic gadolinium complex are proposed.


Introduction
In the field of analysis of very dilute solutions, we developed a new detection technique for determining the compositions and stabilities of some colourless organomet-allic complexes, which have no absorption band UV-visible.This technique is the indirect photometric detection (IPD), based on competitive reactions, by ligand-ligand exchange.The method is simple, reproducible, effective and applicable to very dilute solutions.Thus, the importance of IPD technique was also revealed by its adaptation to other techniques of separation and determination, such as liquid chromatography [1][2][3], capillary electrophoresis [4] and continuous flow analysis (FIA) [5][6][7].Some studies [8,9] show that this technique is very effective in identifying some colourless tungstate complexes of sugars and organic acids.
The paramagnetic complexes of the trivalent gadolinium ion ( ), have emerged as very important agents of contrast, for many applications in Magnetic Resonance Imaging (MRI), through interest electronic and magnetic properties of this ion [10,11].Recent studies have shown that the lanthanide complexes of coumarin (1,2-benzopyrone), exhibit antiproliferative activity [12].In this work, investigations by indirect photometric detection (IPD) were carried out to study the interaction of the trivalent gadolinium ions, with malate ions (conjugate base of malic acid), detecting the majority of colourless complex formed in solution and determine its composition and stability.To elucidate the structure and the chelation sites of this major malate complex of 3 Gd  3 Gd  ions, the technique of IR spectroscopy has been used.Indeed, these two techniques (IPD and IR spectroscopy) are very useful for elucidating the formation reaction of the major complex for the system (Gd (III)-malic acid), completely, determine, the composition and stability, the nature of chelation sites for each of ligands ( − OOC-CHOH-COO − ), involved in the composition of detected complex and propose a probable structure for this major gadolinium complex of the malate ion.

Indirect Photometric Detection
A standard Helios  UV-visible spectrum-eter controlled by Vision 32 software was used for spectrometric measurements, using quartz cells of optical path length .The absorption measurements have been performed at room temperature and at wavelength L were added, using a Gilson micropipet of 0.200 mL.After each addition, the resulting solution was left at least 5 min in order to reach equilibrium (and thus a constant absorption value).Addition was repeated until a maximum volume of 2.00 mL of the organic acid solution was added.The change in the total volume was neglected.For fixed pH environments, the apparent formation constants  H O , mixing and finally adjusting the pH with concentrated HCl or NaOH and concentra nalyzed les are 10 −3 M. Analyses were performed using an infrared spectrometer, Fourier transform (FT-IR), Perkin Elmer BX, equipped with a DTGS detector, a splitter and a cesium iodide window.In this configuration, the interval of analysis is that the middle infrared, 6000 cm −1 to 250 cm −1 and analysis are conducted on small samples, whose size is less than 1 mm 3 .Liquid samples are placed between two plates of very pure salt (KBr), these plates are transparent to infrared light and the spectra relative to free ligand (malate ion) and complexed, have been plotted for frequencies from 4400 cm −1 to 400 cm −1 .

Raman Spectroscopy
The complex is precepts quickly a concentration of 10 −2 M and (Higher than the acidity constant of malic acid).The precipitate was filtered and dried in drying oven and the complex formed is insoluble in water, methanol and ethanol.The Raman spectra of the ligand (Malic acid) and its new Gd(III) complex was performed using an Fourier transformation Raman spectrometer (FT-Raman) VERTEX 70 with a range of measurement (4000 -50) cm −1 , laser source NdYag (1.064 µm), a nominal power of 500 MW, detecting Ge with high sensitivity and a resolution of 4 cm −1 (64 scan).The spectra relative to free malic acid and Gd(III)-carboxylate complex, have been plotted for frequencies from 3600 cm −1 to 200 cm −1 .

Indirect Photometric De
The complexation reaction of x gadolinium ions   , , Additionally, a conditional equilibrium constant xyz K d solu-is defined in case of constant pH value (buffere tion): The complexation studied using a sp otometric m and its detected gadoliniu ch ( 4) ectroph e reagent (malic acid) not possess a aracteristic UV-visible absorption spectrum, a second ligand (called the sacrificial ligand) is introduced.This second ligand must absorb in the UV-visible spectrum and form a colored complex with 3 Gd  ions.Based on ligand-ligand displacement, the photometric method is said to be in the indirect mode.The dissociation of this colored complex has to cause large variations in the UVvisible spectrum which allow the calculation of the concentration of the sacrificial complex.Using the formation constant of this colored complex, the concentration of the unknown complex can be obtained.Therefore, the sacrificial ligand must form a single colored complex of lower stability than the gadolinium complex under study.In this work, 3"-sulfo-2",6"-dichloro-3,3'-dimeth-yl-4'-hydroxy-fuchsone-5,5'-dicarboxylic acid, often called Chrome Azurol S and noted  ), gives a coloured reagent ( max = 545 nm) of average stability for values of pH ranging between 5.50 and 7.50.The buffer "MESH morpholino) sulphoni ane acid] was adopted to fix pH in the study of the sacrificial complex 4

Gd H Ch
 and the detected gadolinium-malate complex.We chosed this buffer because it does not present any interaction with 3 Gd  ions and so that we would be k in range of pH where the stability of the sacrificial complex is maximal. .So, we have studied the formation of this sacrificial complex at this maximum wavelength, depending on the ratio

Formation of the Colored Sacrificial Complex
as shown in the Figure 2.
The curve in Figure 2 indicates that the value of the ratio q, relative to the complete formation of this sacrifiection of the linear (positive sl cial complex is the inters ope), with the final segment (slope = 0) of the curve, this value of q is close to 1.50.This indicates that the  HCh  ions in the pH range 5.50 to 7.50, led to the formation of the colored tri-nuclear complex, by following balanced reaction: complex lues of formation, calculated from the experimental absorbance by the relation , with I A and F A which respectively represent the initial absorbance (free ligand) and final (100% of the complex).
appare constant on the formation reaction according to the balanced reaction (II) is given by the following equati Therefore, the expression of the nt on : The formation constant of this  complex is defined as: The conditional equilibrium constant for a fixed value of pH is given by: , , x y z K  from values of is calculated using a computer program w the balanced reaction (II), we obtained constant ritten for the pair   The same calcu tion progr z K  were calculated and the obtained results are summarized in the Table 1.These results show that the stability of the sacrificial complex depends on the acid of the medium.
e value When the conditional equilibrium constant 323 K  of ation of the sacrificial complex is known, the concent free gadolinium ion ( ), can be calcula Equations ( 7)- (9).
 , the concentration of the gadolinium complex under study   , , x y z     (balanced reaction I) can rmined using the gadolinium mass balance equati be det on : Gd C being the initial gadolinium concentration.In a similar way, the concentration of the free ligand is obtained by z (11) of the sacrificial colored complex is necessary.The determination of the comp the stability of the sacrificial complex and of It should be also noted that a perfect knowledge of the characteristics 3 2 Gd HCh o the precision ch experi-sition and the conditions of its formation are paramount stages to apply the indirect photometry technique.In ea ment, the ligand (malate ions), is added stepwise in order to measure the absorption at different values (at least 12) of the overall initial concentration of this studied ligand.The correct xyz K  is looked for by varying x and y in order to obtain a constant value for all values of

Determination of the Composition and the Stability Constant of the Gd-Malate
With , showed a reduction in the ab max of the solution progressively with the addition of the malate ions (Figure 4).The dissociation of the sacrificial complex, relating to the reduction in the absorbance by the addition of malate ions solution, is done in favour of the formation of the colourless complex between  2).The data-processing treatment of the preceding experimental results, shows that this tri-nuclear detected complex  formed between the gadolinium ions and malate ions, resulting from the interaction of three 3 Gd  ions equivalents with two equivalents of malate s so a molar ratio pecies, for the co lex ons (1)-( 3) allow to write the following expressions: ull.The stability constant of this formed com lex is ned by: For all pH range we obtained   The value of z can be positive, negative or n experimental pH values higher than e of ma acid, thus: se For the th and lic The evolution of

Consequently
In the light of the spectrophotometric results relating to the interaction of 3 Gd  ions with the malate ions, namely that the complexation reaction uses three hydrolyzed 3 Gd  ions for two malate species and requires fixatio o prot According to the literature [16], the ion can be presented in various hydrolyzed form ueous solution and this new tri-nuclear gadolinium complex detected in solution at experimental pH r e, is probably formed from the hydrolyzed form , according to the following reaction: In order to confirm our results, to have mo informais new re for this tri-nuclear ectroscopy investigations.These studies by IR spectroscopy carried out in the same h more concentrated solutions, help to ga n of tw ons. 3 re tion on the nature of th gadolinium complex and likely to propose a probable structu specie, we carried out IR sp pH range but wit identify the nature of the chelation sites and the probable structure of this new dolinium complex formed by interaction of malate ions with this hydrolyzed form of 3 Gd  ions.

IR Spectroscopy Investigations olinium respective
The IR spectroscopic studies can identify different groups of malic acid (ligand), which participate in chelation sites for the formation of the detected gad complex.0 −1 cm), ly, the free Three dilute solutions (1 malate ion  

pH 
, free malic acid (pH = 2.02) and detected complex of malate ion (pH = 5.60), were pre and bi hic data.
f. [17,18] gadolinium pared and their spectra recorded (Figure 6) and analyzed.Table 3 contains bibliographic data [17,18], relative to vibrations intervals of various groups: OH, C=O, COO − and C-C=O, and the intervals of vibration on the analyzed spectra (1, 2 and 3).
The experimental spectra obtained for the three analyzed samples, clearly indicate that the frequency for vibration of the groups: OH, C=O and COO − , have the intense vibration observed in the complex near 1069 The seriously reduced 75%, 80% and 66% passing the free to complexed ligand (Figure 6).So, for each of the two malate ions involved in the formation of this detected tri-nuclear complex, the four oxygen atoms of the ionized carboxylic groups, participate in chelation sites, and the OH group in  position of the ionized − weak bands [21,22], and the strong vibration of Gd(III)oxygen nitrate appeared at 187 cm −1 [23].The different vibrations (stretching and deformation) of C-C (aliphatic chains) appeared in a long field (1400 -400 cm −1 ) with intense and medium intensities [21,20,24].Bands in the 2590 ± 80 cm −1 region were assigned to the vibrations of CH and CH 2 [25,26], these bands are moderate intensities in complex spectra

FT-Raman Spectroscopy Investigations
FT-Raman spectra of free malic acid and its complex These results clearly indicate that, this new tri-nuclear gadolinium complex contains two types of sites, a single central bidentate mono-nuclear site with participation of only OH groups in  position and two lateral tetradentate mono-nuclear sites, each consisting of four oxygen atoms of two ionized carboxylic functions, belonging to the two malate ions ( − OOC-CHOH-COO − ), involved in the formation of this new detected gadolinium organometallic complex.Indeed, all these results can offer for this trinuclear Gd-Malic acid, the structure presented in Figure 8.
with Gadolinium ions , are shown in Figure 7.A detailed analysis of vibrations in Raman spectroscopy was performed on the nment [1 vibrat tected in the complex spectra and the vibration of C-C=O group is reduced from free malic acid to its gadolinium complex.So, all oxygen atoms involved in the chelation sites of this new tri-nuclear complex.
The vibration spectra (weak broad) of the complex in 1600 cm −1 , indicate the existence of water molecules [21],

Conclusion
In this work, we used some techniques to study the interactions of the trivalent Gd(III) ions with different ionic forms of malic acid and identify the composition, stability and structure of the major colourless complex, formed in solution for pH values between 5.50 and 7.50.The photometry in indirect mode (IPD) was used successfully to determine the composition and the stability of this major gadolinium complex.Only the tri-nuclear cm −1 indicate the existence of the nitrate ions [12].new band vibrations related to Gd(III)-Oxygen carboxylate functions, are located at 600 and 547 cm −1 with a

.
Stock solutions of Gd(III) nitrate and Chrome Azurol S  4  buffer of MESH (0.1 M) [2-(N-morpholino) sulphonic ethane acid].The initial solution also contained a calculated amount of 1 M NaOH in order to obtain the desired pH value , experimental pH range (5.5 -7.5).pH values are measured with a Microprocessor pH Meter HANNA 210 equipped with a combined glass electrode and calibrated with comercial buffers (pH 4.00 and 7.00).Then aliquots of an aqueous solution of the malic acid

4 H
Ch , and an excess of Gadolinium(III) (performed at pH intervals of 0.20).Assuming various integers for the Gadolinium and organic acid stoichiometry, a formation constant is calculated for each added amount of ligand and corresponding absorption value.The results are rejected when a systematic variation of log xyz K  occurs with increasing added amount of ligand or when individual values of log xyz K  differed from the

4 H
Ch , has been used as sacrificial ligand.Chrome Azurol S is a tetraprotic acid with pKa values of 2.25   series of experiments was conducted to determ composition, the the colored sacrifici (  ine, the al complex stability of  x y Gd HCh ation reac ) and its form tion. Indeed, in 50 cm −3 of the auxiliary ligand solution   3 HCh  of a concentration 4 10 M HCh C   and pH = 5.91, we in-troduc g quantities of a 3 Gd  ions solution of initial concentration 10 −2 M. The evol UVvisible spectra on of this sacrificial complex is represented by the diagram in f wing Figure 1.The UV-visible spectra show clearly that the maximum absorbance of the sacrificial complex is located at

Figure 1 .
Figure 1.UV-visible spectra for the formation of sacrificial complex   x y Gd HCh , pH = 5.91,

Figure 2 . 3 HCh
Figure 2. Formation of the sacrificial complex, depending on the ratio3           3 pH  .Under the same conditions of temperature and concen ns, several exp e performed for pH values between 5.50 and 7.50.lam was used and the results clearly indicate that only the detected sacrificial complex   3, 2, z , is formed in solution.For all studied solutions at known values of pH, the apparent constants 32 tratio a eriments wer

K
 depending on the medium pH (Figure is the equivalent number of 3), and the slop

Figure 3 .
Figure 3.The stability of the tri-nuclear complex

2 LC
 .If xyz K  is determined at different pH values, the slope of the log xyz K  .pH plot reveals the number z of protons, necessary for formation of the studied gadolinium complex by use of equation 4, since the value of the xyz K  is i endent of pH.Now the stability and the total composi the gadoliniummalate complex have been determined.

 in Figure 4 ,
clearly shows that the absorbance decreases and stabilizes.This stability indicates that all 3 Gd  ions, initially present in the solution h added malate ions.Knowing the concentration of gadolinium ions and the quantity of ligand from the added volume of the malic acid solution, necessary to reach this stage of absorption, we could determine the molar ratio q have reacted wit  3 malate Gd , involved in the complexation reaction.The preceding experiment was ca out for different values o rried f pH between 5.50 and 7.50, the way in which bsorption decreases, depends on the pH of the medium and on the formation constant of the detected comp Gd-malate), as well as on the absorp-( F A ) of the free and totally complexed Chrome Azurol S ( 3 HCh  ).Analyzing the experimental data with the computer program written from the balanced reaction (I), the results confirm the reproducibility of the m to determine the composition and apparent stability constant olar ratio q and xyz K  (Table

Table 3 . The vibration frequencies, for the studied spectra bliograp
Gd 3 ions, in fact, is the first was proposed